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Sample records for 4t1 mouse mammary

  1. Molecular response of 4T1-induced mouse mammary tumours and healthy tissues to zinc treatment.

    PubMed

    Sztalmachova, Marketa; Gumulec, Jaromir; Raudenska, Martina; Polanska, Hana; Holubova, Monika; Balvan, Jan; Hudcova, Kristyna; Knopfova, Lucia; Kizek, Rene; Adam, Vojtech; Babula, Petr; Masarik, Michal

    2015-04-01

    Breast cancer patients negative for the nuclear oestrogen receptor α have a particularly poor prognosis. Therefore, the 4T1 cell line (considered as a triple-negative model) was chosen to induce malignancy in mice. The aim of the present study was to assess if zinc ions, provided in excess, may significantly modify the process of mammary oncogenesis. Zn(II) ions were chosen because of their documented antitumour effects. Zn(II) is also known to induce the expression of metallothioneins (MT) and glutathion (GSH). A total dose of zinc sulphate per one gram of mouse weight used in the experiment was 0.15 mg. We studied the expression of MT1, MT2, TP53 and MTF-1 genes and also examined the effect of the tumour on antioxidant capacity. Tumour-free mice had significantly higher expression levels of the studied genes (p<0.003). Significant differences were also revealed in the gene expression between the tissues (p<0.001). The highest expression levels were observed in the liver. As compared to brain, lung and liver, significantly lower concentrations of MT protein were found in the primary tumour; an inverse trend was observed in the concentration of Zinc(II). In non-tumour mice, the amount of hepatic hydrosulphuryl groups significantly increased by the exposure to Zn(II), but the animals with tumour induction showed no similar trend. The primary tumour size of zinc-treated animals was 20% smaller (p=0.002); however, no significant effect on metastasis progression due to the zinc treatment was discovered. In conclusion, Zn(II) itself may mute the growth of primary breast tumours especially at their early stages.

  2. Distribution and inhibition effect of Seleno-L-Methionine on 4T1 mouse mammary carcinoma

    PubMed Central

    Song, Hongjiao; Ren, Xiaomin; Liu, Ping

    2015-01-01

    To identify the relationship between selenium intake and breast cancer progression, SeMet supplement was applied to 4T1 murine mammary carcinoma for 28 days and the effect of SeMet on tumor growth was assessed. Combined with SXRF mapping, trace metal distribution in tumor tissues in relation to selenium-associated protein expressions and cellular transcription factors, HIF-1α accumulation and its targeted molecule expressions involved in tumor progression, were further investigated using immunohistochemical staining. Our results showed that tumor growth was inhibited significantly and tumor cells apoptosis was promoted after SeMet supplement. High Se intakes in tumor tissues overlaid with high iron distribution were observed by SXRF mapping. SeMet supplement increased SBP1 expression and decreased GPx1 expression, and greatly inhibited VEGF expression and tumor immune suppression cells accumulation in tumor tissues. In conclusion, SeMet-mediated anti-cancer effect was likely to inhibit tumor growth through high expression of SBP1 inhibiting GPx1 activity and HIF-1α, which could be used as chemopreventive strategies in breast cancer. PMID:26330897

  3. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells.

    PubMed

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C B; Hashim, Onn H; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer. PMID:27226773

  4. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells

    PubMed Central

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C.B.; Hashim, Onn H.; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer. PMID:27226773

  5. Green tea (Camellia sinensis) extract inhibits both the metastasis and osteolytic components of mammary cancer 4T1 lesions in mice.

    PubMed

    Luo, Ke-Wang; Ko, Chun-Hay; Yue, Grace Gar-Lee; Lee, Julia Kin-Ming; Li, Kai-Kai; Lee, Michelle; Li, Gang; Fung, Kwok-Pui; Leung, Ping-Chung; Lau, Clara Bik-San

    2014-04-01

    Green tea (Camellia sinensis, CS), a kind of Chinese tea commonly consumed as a healthy beverage, has been demonstrated to have various biological activities, including antioxidation, antiobesity and anticancer. Our study aims to investigate the antitumor, antimetastasis and antiosteolytic effects of CS aqueous extract both in vitro and in vivo using metastasis-specific mouse mammary carcinoma 4T1 cells. Our results showed that treatment of 4T1 cells with CS aqueous extract resulted in significant inhibition of 4T1 cell proliferation. CS extract induced 4T1 apoptosis in a dose-dependent manner as assessed by annexin-V and propidium iodide staining and caspase-3 activity. Western blot analysis showed that CS increased the expression of Bax-to-Bcl-2 ratio and activated caspase-8 and caspase-3 to induce apoptosis. CS also inhibited 4T1 cell migration and invasion at 0.06-0.125 mg/ml. In addition, CS extract (0.6 g/kg, orally fed daily for 4 weeks) was effective in decreasing the tumor weight by 34.8% in female BALB/c mice against water treatment control (100%). Apart from the antitumor effect, CS extract significantly decreased lung and liver metastasis in BALB/c mice bearing 4T1 tumors by 54.5% and 72.6%, respectively. Furthermore, micro-computed tomography and in vitro osteoclast staining analysis suggested that CS extract was effective in bone protection against breast cancer-induced bone destruction. In conclusion, the present study demonstrated that the CS aqueous extract, which closely mimics green tea beverage, has potent antitumor and antimetastasis effects in breast cancer and could protect the bone from breast cancer-induced bone destruction.

  6. Rapamycin Promotes Mouse 4T1 Tumor Metastasis that Can Be Reversed by a Dendritic Cell-Based Vaccine

    PubMed Central

    Lin, Tien-Jen; Liang, Wen-Miin; Hsiao, Pei-Wen; M. S, Pradeep; Wei, Wen-Chi; Lin, Hsin-Ting; Yin, Shu-Yi; Yang, Ning-Sun

    2015-01-01

    Suppression of tumor metastasis is a key strategy for successful cancer interventions. Previous studies indicated that rapamycin (sirolimus) may promote tumor regression activity or enhance immune response against tumor targets. However, rapamycin also exhibits immunosuppressant effects and is hence used clinically as an organ transplantation drug. We hypothesized that the immunosuppressive activities of rapamycin might also negatively mediate host immunity, resulting in promotion of tumor metastasis. In this study, the effects of rapamycin and phytochemical shikonin were investigated in vitro and in vivo in a 4T1 mouse mammary tumor model through quantitative assessment of immunogenic cell death (ICD), autophagy, tumor growth and metastasis. Tumor-bearing mice were immunized with test vaccines to monitor their effect on tumor metastasis. We found that intraperitoneal (ip) administration of rapamycin after a tumor-resection surgery drastically increased the metastatic activity of 4T1 tumors. Possible correlation of this finding to human cancers was suggested by epidemiological analysis of data from Taiwan’s National Health Insurance Research Database (NHIRD). Since our previous studies showed that modified tumor cell lysate (TCL)-pulsed, dendritic cell (DC)-based cancer vaccines can effectively suppress metastasis in mouse tumor models, we assessed whether such vaccines may help offset this rapamycin-promoted metastasis. We observed that shikonin efficiently induced ICD of 4T1 cells in culture, and DC vaccines pulsed with shikonin-treated TCL (SK-TCL-DC) significantly suppressed rapamycin-enhanced metastasis and Treg cell expansion in test mice. In conclusion, rapamycin treatment in mice (and perhaps in humans) promotes metastasis and the effect may be offset by treatment with a DC-based cancer vaccine. PMID:26426423

  7. Rapamycin Promotes Mouse 4T1 Tumor Metastasis that Can Be Reversed by a Dendritic Cell-Based Vaccine.

    PubMed

    Lin, Tien-Jen; Liang, Wen-Miin; Hsiao, Pei-Wen; M S, Pradeep; Wei, Wen-Chi; Lin, Hsin-Ting; Yin, Shu-Yi; Yang, Ning-Sun

    2015-01-01

    Suppression of tumor metastasis is a key strategy for successful cancer interventions. Previous studies indicated that rapamycin (sirolimus) may promote tumor regression activity or enhance immune response against tumor targets. However, rapamycin also exhibits immunosuppressant effects and is hence used clinically as an organ transplantation drug. We hypothesized that the immunosuppressive activities of rapamycin might also negatively mediate host immunity, resulting in promotion of tumor metastasis. In this study, the effects of rapamycin and phytochemical shikonin were investigated in vitro and in vivo in a 4T1 mouse mammary tumor model through quantitative assessment of immunogenic cell death (ICD), autophagy, tumor growth and metastasis. Tumor-bearing mice were immunized with test vaccines to monitor their effect on tumor metastasis. We found that intraperitoneal (ip) administration of rapamycin after a tumor-resection surgery drastically increased the metastatic activity of 4T1 tumors. Possible correlation of this finding to human cancers was suggested by epidemiological analysis of data from Taiwan's National Health Insurance Research Database (NHIRD). Since our previous studies showed that modified tumor cell lysate (TCL)-pulsed, dendritic cell (DC)-based cancer vaccines can effectively suppress metastasis in mouse tumor models, we assessed whether such vaccines may help offset this rapamycin-promoted metastasis. We observed that shikonin efficiently induced ICD of 4T1 cells in culture, and DC vaccines pulsed with shikonin-treated TCL (SK-TCL-DC) significantly suppressed rapamycin-enhanced metastasis and Treg cell expansion in test mice. In conclusion, rapamycin treatment in mice (and perhaps in humans) promotes metastasis and the effect may be offset by treatment with a DC-based cancer vaccine. PMID:26426423

  8. High Resolution X-Ray Microangiography of 4T1 Tumor in Mouse Using Synchrotron Radiation

    SciTech Connect

    Sun Jianqi; Liu Ping; Gu Xiang; Liu Xiaoxia; Zhao Jun; Xiao Tiqiao; Xu, Lisa X.

    2010-07-23

    Angiogenesis is very important in tumor growth and metastasis. But in clinic, only vessels lager than 200 {mu}m in diameter, can be observed using conventional medical imaging. Synchrotron radiation (SR) phase contrast imaging, whose spatial resolution can reach as high as 1 {mu}m, has great advantages in imaging soft tissue structures, such as blood vessels and tumor tissues. In this paper, the morphology of newly formed micro-vessels in the mouse 4T1 tumor samples was firstly studied with contrast agent. Then, the angiogenesis in nude mice tumor window model was observed without contrast agent using the SR phase contrast imaging at the beamline for X-ray imaging and biomedical applications, Shanghai Synchrotron Radiation Facility (SSRF). The images of tumors showed dense, irregular and tortuous tumor micro-vessels with the smallest size of 20-30 {mu}m in diameter.

  9. Licoricidin, an Active Compound in the Hexane/Ethanol Extract of Glycyrrhiza uralensis, Inhibits Lung Metastasis of 4T1 Murine Mammary Carcinoma Cells

    PubMed Central

    Park, So Young; Kwon, Soo Jin; Lim, Soon Sung; Kim, Jin-Kyu; Lee, Ki Won; Park, Jung Han Yoon

    2016-01-01

    Licorice extracts containing glycyrrhizin exhibit anti-carcinogenic properties. Because glycyrrhizin induces severe hypokalemia and hypertension, we prepared a hexane/ethanol extract of Glycyrrhiza uralensis (HEGU) that lacks glycyrrhizin, and showed that HEGU induces apoptosis and G1 cell cycle arrest and inhibits migration of DU145 human prostate cancer cells. Our previous in vitro studies identified two active components in HEGU: isoangustone A, which induces apoptosis and G1 cycle arrest, and licoricidin, which inhibits metastasis. This study examined whether HEGU and licoricidin inhibit metastasis using the 4T1 mammary cancer model. Both HEGU and licoricidin treatment reduced pulmonary metastasis and the expression of CD45, CD31, HIF-1α, iNOS, COX-2, and VEGF-A in tumor tissues. Additionally, a decrease in protein expression of VEGF-R2, VEGF-C, VEGF-R3, and LYVE-1 was noted in tumor tissues of licoricidin-treated mice. Furthermore, the blood concentrations of MMP-9, ICAM-1, VCAM-1, and VEGF-A were decreased in HEGU-treated mice. In vitro 4T1 cell culture results showed that both HEGU and licoricidin inhibited cell migration, MMP-9 secretion, and VCAM expression. The present study demonstrates that the licoricidin in HEGU inhibits lung metastasis of 4T1 mammary carcinoma cells, which may be mediated via inhibition of cancer cell migration, tumor angiogenesis, and lymphangiogenesis. PMID:27314329

  10. Licoricidin, an Active Compound in the Hexane/Ethanol Extract of Glycyrrhiza uralensis, Inhibits Lung Metastasis of 4T1 Murine Mammary Carcinoma Cells.

    PubMed

    Park, So Young; Kwon, Soo Jin; Lim, Soon Sung; Kim, Jin-Kyu; Lee, Ki Won; Park, Jung Han Yoon

    2016-01-01

    Licorice extracts containing glycyrrhizin exhibit anti-carcinogenic properties. Because glycyrrhizin induces severe hypokalemia and hypertension, we prepared a hexane/ethanol extract of Glycyrrhiza uralensis (HEGU) that lacks glycyrrhizin, and showed that HEGU induces apoptosis and G1 cell cycle arrest and inhibits migration of DU145 human prostate cancer cells. Our previous in vitro studies identified two active components in HEGU: isoangustone A, which induces apoptosis and G1 cycle arrest, and licoricidin, which inhibits metastasis. This study examined whether HEGU and licoricidin inhibit metastasis using the 4T1 mammary cancer model. Both HEGU and licoricidin treatment reduced pulmonary metastasis and the expression of CD45, CD31, HIF-1α, iNOS, COX-2, and VEGF-A in tumor tissues. Additionally, a decrease in protein expression of VEGF-R2, VEGF-C, VEGF-R3, and LYVE-1 was noted in tumor tissues of licoricidin-treated mice. Furthermore, the blood concentrations of MMP-9, ICAM-1, VCAM-1, and VEGF-A were decreased in HEGU-treated mice. In vitro 4T1 cell culture results showed that both HEGU and licoricidin inhibited cell migration, MMP-9 secretion, and VCAM expression. The present study demonstrates that the licoricidin in HEGU inhibits lung metastasis of 4T1 mammary carcinoma cells, which may be mediated via inhibition of cancer cell migration, tumor angiogenesis, and lymphangiogenesis. PMID:27314329

  11. Humanization of the mouse mammary gland.

    PubMed

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

    2015-01-01

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

  12. The combined use of Camellia sinensis and metronomic zoledronate in 4T1 mouse carcinoma against tumor growth and metastasis.

    PubMed

    Luo, Ke-Wang; Yue, Grace Gar-Lee; Ko, Chun-Hay; Gao, Si; Lee, Julia Kin-Ming; Li, Gang; Fung, Kwok-Pui; Leung, Ping-Chung; Lau, Clara Bik-San

    2015-07-01

    In previous studies, we demonstrated that the green tea Camellia sinensis (CS) water extract had potent antitumor and antimetastatic effects on 4T1 breast cancer. The metronomic regimen (0.0125 mg/kg twice a week for 4 weeks) of zoledronate (ZOL) was found to be effective in decreasing tumor burden and metastasis as compared with conventional regimen. The aim of the present study was to investigate the antitumor, antimetastatic and anti-osteolytic effects of the combined use of CS water extract and metronomic ZOL against 4T1 breast carcinoma in vitro and in vivo. The results demonstrated that the combination of CS+ZOL exerted a more potent effect on lung and liver by decreasing tumor burden and metastasis, when compared to CS or metronomic ZOL as monotherapies. The combination of CS+ZOL demonstrated optimal bone protection against breast cancer-induced osteolysis for the three groups of CS, ZOL and CS+ZOL. The in vitro results further demonstrated that ZOL enhanced CS-induced apoptosis in 4T1 cells as assessed by the Annexin V-FITC/PI staining and caspase-3 activity assays. In addition, the combined use of CS+ZOL significantly inhibited 4T1 cell migration. Mechanistic studies showed that the enzyme levels of matrix metalloproteinases (MMP)-2 and MMP-9 were suppressed significantly by CS+ZOL. In conclusion, to the best of our knowledge, this is the first study to investigate the novel combined application of herbal extract CS and chemotherapy ZOL in 4T1 breast cancer. The combination of CS plus metronomic ZOL demonstrated significant antitumor, antimetastatic and anti-osteolytic effects against breast cancer, and suggested potential clinical application for breast cancer patients.

  13. Formulation, Characterization, and Antitumor Properties of Trans- and Cis-Citral in the 4T1 Breast Cancer Xenograft Mouse Model

    PubMed Central

    Zeng, San; Kapur, Arvinder; Patankar, Manish S.; Xiong, May P.

    2015-01-01

    Purpose Citral is composed of a random mixture of two geometric stereoisomers geranial (trans-citral) and neral (cis-citral) yet few studies have directly compared their in vivo antitumor properties. A micelle formulation was therefore developed. Methods Geranial and neral were synthesized. Commercially-purchased citral, geranial, and neral were formulated in PEG-b-PCL (block sizes of 5000:10000, Mw/Mn 1.26) micelles. In vitro degradation, drug release, cytotoxicity, flow cytometry, and western blot studies were conducted. The antitumor properties of drug formulations (40 mg/kg and 80 mg/kg based on MTD studies) were evaluated on the 4T1 xenograft mouse model and tumor tissues were analyzed by western blot. Results Micelles encapsulated drugs with >50% LE at 5-40% drug to polymer (w/w), displayed sustained release (t1/2 of 8-9 hours), and improved drug stability at pH 5.0. The IC50 of drug formulations against 4T1 cells ranged from 1.4-9.9 μM. Western blot revealed that autophagy was the main cause of cytotoxicity. Geranial at 80 mg/kg was most effective at inhibiting tumor growth. Conclusions Geranial is significantly more potent than neral and citral at 80 mg/kg (p<0.001) and western blot of tumor tissues confirms that autophagy and not apoptosis is the major mechanism of tumor growth inhibition in p53-null 4T1 cells. PMID:25673043

  14. Elimination of the chemotherapy resistant subpopulation of 4T1 mouse breast cancer by haploidentical NK cells cures the vast majority of mice.

    PubMed

    Frings, Peter W H; Van Elssen, Catharina H M J; Wieten, Lotte; Matos, Catarina; Hupperets, Pierre S J G; Schouten, Harry C; Bos, Gerard M J; van Gelder, Michel

    2011-12-01

    Metastatic breast cancer is currently incurable despite initial responsiveness, assumingly due to the presence of chemoresistant subpopulations that can be characterized as label retaining cells (LRC). In the 4T1 mouse breast cancer model, we previously achieved cure after Cyclophosphamide and Total Body Irradiation (CY + TBI) followed by haploidentical bone marrow and spleen transplantation (BMSPLT). CY + TBI without transplantation induced only transient impaired tumor growth indicating a critical role of donor immune cells. As it remained unknown if the 4T1 model resembles human disease with respect to the presence of subpopulations of chemoresistant LRC, we now demonstrate this is indeed the case. Chemoresistance of 4T1 LRC was demonstrated by in vitro co-incubation of fluorescently labeled 4T1 cells in limiting dilution with cyclophosphamide, doxorubicin or cisplatinum, after which only LRC containing colonies remained. LRC also remain in vivo after treatment with CY + TBI. Succeeding experiments set up to identify the haploidentical effector cell responsible for cure and, therefore, for the elimination of chemoresistant LRC designate donor NK cells crucial for the anti-tumor effect. NK cell depletion of the haploidentical graft fully abrogated the anti-tumor effect. Increased disease-free survival retained after transplantation of haploidentical bone marrow and NK cell-enriched spleen cell grafts, even in the absence of donor T-cells or of donor bone marrow. Tumor growth analysis indicates the anti-tumor effect being immediate (days). Based on these data, it is worthwhile to explore alloreactive adoptive NK cell therapy as consolidation for patients with metastasized breast cancer. PMID:21274621

  15. Mouse mammary tumor biology: a short history.

    PubMed

    Cardiff, Robert D; Kenney, Nicholas

    2007-01-01

    For over a century, mouse mammary tumor biology and the associated Mouse mammary tumor virus (MMTV) have served as the foundation for experimental cancer research, in general, and, in particular, experimental breast cancer research. Spontaneous mouse mammary tumors were the basis for studies of the natural history of neoplasia, oncogenic viruses, host responses, endocrinology, and neoplastic progression. However, lacking formal proof of a human mammary tumor virus, the preeminence of the mouse model faded in the 1980s. Since the late 1980s, genetically engineered mice (GEM) have proven extremely useful for studying breast cancer and have become the animal model for human breast cancer. Hundreds of mouse models of human breast cancer have been developed since the first demonstration, in 1984, that the mouse mammary gland could be molecularly targeted and used to test the oncogenicity of candidate human genes. Now, very few scientists can avoid using a mouse model to test the biology of their favorite gene. The GEM have attracted a new generation of molecular and cellular biologists eager to apply their skills to these surrogates of the human disease. Newcomers often enter the field without an appreciation of the origins of mouse mammary tumor biology and the basis for many of the prevailing concepts. Our purpose in writing this short history of mouse mammary tumor biology is to provide a historical perspective for the benefit of the newcomers. If Einstein was correct in that "we stand on the shoulders of giants," the neophytes should meet their giants.

  16. Escherichia coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumors through Expression of Azurin Protein

    PubMed Central

    Zhang, Yunlei; Zhang, Youming; Zhang, Xiangli; Ding, Xuezhi; Yan, Fu; Wu, Feng

    2012-01-01

    Many studies have demonstrated that intravenously administered bacteria can target and proliferate in solid tumors and then quickly be released from other organs. Here, we employed the tumor-targeting property of Escherichia coli Nissle 1917 to inhibit mouse B16 melanoma and 4T1 breast tumors through the expression of azurin protein. For this purpose, recombinant azurin-expressing E. coli Nissle 1917 was developed. The levels of in vitro and in vivo azurin secretion in the engineered bacterium were determined by immunochemistry. Our results demonstrated that B16 melanoma and orthotopic 4T1 breast tumor growth were remarkably restrained and pulmonary metastasis was prevented in immunocompetent mice. It is worth noting that this therapeutic effect partially resulted from the antitumor activity of neutrophils and lymphocytes due to inflammatory responses caused by bacterial infections. No toxicity was observed in the animal during the experiments. This study indicates that E. coli Nissle 1917 could be a potential carrier to deliver antitumor drugs effectively for cancer therapy. PMID:22923405

  17. Low local blood perfusion, high white blood cell and high platelet count are associated with primary tumor growth and lung metastasis in a 4T1 mouse breast cancer metastasis model

    PubMed Central

    WANG, CHUAN; CHEN, YING-GE; GAO, JIAN-LI; LYU, GUI-YUAN; SU, JIE; ZHANG, QI; JI, XIN; YAN, JI-ZHONG; QIU, QIAO-LI; ZHANG, YUE-LI; LI, LIN-ZI; XU, HAN-TING; CHEN, SU-HONG

    2015-01-01

    It was originally thought that no single routine blood test result would be able to indicate whether or not a patient had cancer; however, several novel studies have indicated that the median survival and prognosis of cancer patients were markedly associated with the systemic circulation features of cancer patients. In addition, certain parameters, such as white blood cell (WBC) count, were largely altered in malignant tumors. In the present study, routine blood tests were performed in order to observe the change of blood cells in tumor-bearing mice following the implantation of 4T1 breast cancer cells into the mammary fat pad; in addition, blood flow in breast tumor sites was measured indirectly using laser Doppler perfusion imaging (LDPI), in an attempt to explain the relevance between the blood circulation features and the growth or metastasis of breast cancer in mice model. The LDPI and blood test results indicated that the implantation of 4T1 breast cancer cells into BALB/c mice led to thrombosis as well as high WBC count, high platelet count, high plateletcrit and low blood perfusion. Following implantation of the 4T1 cells for four weeks, the lung metastatic number was determined and the Pearson correlation coefficient revealed that the number of visceral lung metastatic sites had a marked negative association with the ratio of basophils (BASO%; r=-0.512; P<0.01) and the mean corpuscular hemoglobin was significantly correlated with primary tumor weight (r=0.425; P<0.05). In conclusion, the results of the present study demonstrated that tumor growth led to thrombosis and acute anemia in mice; in addition, when blood BASO% was low, an increased number of lung metastases were observed in tumor-bearing mice. PMID:26622565

  18. Canine parvovirus NS1 protein exhibits anti-tumor activity in a mouse mammary tumor model.

    PubMed

    Gupta, Shishir Kumar; Yadav, Pavan Kumar; Gandham, Ravi Kumar; Sahoo, A P; Harish, D R; Singh, Arvind Kumar; Tiwari, A K

    2016-02-01

    Many viral proteins have the ability to kill tumor cells specifically without harming the normal cells. These proteins, on ectopic expression, cause lysis or induction of apoptosis in the target tumor cells. Parvovirus NS1 is one of such proteins, which is known to kill high proliferating tumor cells. In the present study, we assessed the apoptosis inducing ability of canine parvovirus type 2 NS1 protein (CPV2.NS1) in vitro in 4T1 cells, and found it to cause significant cell death due to induction of apoptosis through intrinsic or mitochondrial pathway. Further, we also evaluated the oncolytic activity of CPV2.NS1 protein in a mouse mammary tumor model. The results suggested that CPV2.NS1 was able to inhibit the growth of 4T1 induced mouse mammary tumor as indicated by significantly reduced tumor volume, mitotic, AgNOR and PCNA indices. Further, inhibition of tumor growth was found to be because of induction of apoptosis in the tumor cells, which was evident by a significant increase in the number of TUNEL positive cells. Further, CPV2.NS1 was also able to stimulate the immune cells against the tumor antigens as indicated by the increased CD4+ and CD8+ counts in the blood of CVP2.NS1 treated mice. Further optimization of the delivery of NS1 protein and use of an adjuvant may further enhance its anti-tumor activity.

  19. To grow mouse mammary epithelial cells in culture

    PubMed Central

    1984-01-01

    Normal mouse mammary epithelial cells from Balb/c mice were successfully cultivated on tissue culture plastic with lethally irradiated LA7 feeder cells. The feeder cells also promoted colony formation from single mouse mammary cells, and the fraction of cells that formed colonies was proportional to the density of feeder cells. The mouse mammary cells could be passaged at least 8-12 times as long as new feeder cells were added at each passage. The cells now in culture have doubled in number at least 30 times, but the in vitro lifespan is not yet known. The cultures of mouse cells maintained by this technique never became overgrown with fibroblasts and numerous domes formed in the cultures. PMID:6699079

  20. Pleiotrophin (PTN) expression and function and in the mouse mammary gland and mammary epithelial cells.

    PubMed

    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.

  1. Mouse Mammary Tumor Virus-Like Nucleotide Sequences in Canine and Feline Mammary Tumors▿

    PubMed Central

    Hsu, Wei-Li; Lin, Hsing-Yi; Chiou, Shyan-Song; Chang, Chao-Chin; Wang, Szu-Pong; Lin, Kuan-Hsun; Chulakasian, Songkhla; Wong, Min-Liang; Chang, Shih-Chieh

    2010-01-01

    Mouse mammary tumor virus (MMTV) has been speculated to be involved in human breast cancer. Companion animals, dogs, and cats with intimate human contacts may contribute to the transmission of MMTV between mouse and human. The aim of this study was to detect MMTV-like nucleotide sequences in canine and feline mammary tumors by nested PCR. Results showed that the presence of MMTV-like env and LTR sequences in canine malignant mammary tumors was 3.49% (3/86) and 18.60% (16/86), respectively. For feline malignant mammary tumors, the presence of both env and LTR sequences was found to be 22.22% (2/9). Nevertheless, the MMTV-like LTR and env sequences also were detected in normal mammary glands of dogs and cats. In comparisons of the MMTV-like DNA sequences of our findings to those of NIH 3T3 (MMTV-positive murine cell line) and human breast cancer cells, the sequence similarities ranged from 94 to 98%. Phylogenetic analysis revealed that intermixing among sequences identified from tissues of different hosts, i.e., mouse, dog, cat, and human, indicated the MMTV-like DNA existing in these hosts. Moreover, the env transcript was detected in 1 of the 19 MMTV-positive samples by reverse transcription-PCR. Taken together, our study provides evidence for the existence and expression of MMTV-like sequences in neoplastic and normal mammary glands of dogs and cats. PMID:20881168

  2. Neuropilin-2 promotes branching morphogenesis in the mouse mammary gland.

    PubMed

    Goel, Hira Lal; Bae, Donggoo; Pursell, Bryan; Gouvin, Lindsey M; Lu, Shaolei; Mercurio, Arthur M

    2011-07-01

    Although the neuropilins were characterized as semaphorin receptors that regulate axon guidance, they also function as vascular endothelial growth factor (VEGF) receptors and contribute to the development of other tissues. Here, we assessed the role of NRP2 in mouse mammary gland development based on our observation that NRP2 is expressed preferentially in the terminal end buds of developing glands. A floxed NRP2 mouse was bred with an MMTV-Cre strain to generate a mammary gland-specific knockout of NRP2. MMTV-Cre;NRP2(loxP/loxP) mice exhibited significant defects in branching morphogenesis and ductal outgrowth compared with either littermate MMTV-Cre;NRP2(+/loxP) or MMTV-Cre mice. Mechanistic insight into this morphological defect was obtained from a mouse mammary cell line in which we observed that VEGF(165), an NRP2 ligand, induces branching morphogenesis in 3D cultures and that branching is dependent upon NRP2 as shown using shRNAs and a function-blocking antibody. Epithelial cells in the mouse mammary gland express VEGF, supporting the hypothesis that this NRP2 ligand contributes to mammary gland morphogenesis. Importantly, we demonstrate that VEGF and NRP2 activate focal adhesion kinase (FAK) and promote FAK-dependent branching morphogenesis in vitro. The significance of this mechanism is substantiated by our finding that FAK activation is diminished significantly in developing MMTV-Cre;NRP2(loxP/loxP) mammary glands compared with control glands. Together, our data reveal a VEGF/NRP2/FAK signaling axis that is important for branching morphogenesis and mammary gland development. In a broader context, our data support an emerging hypothesis that directional outgrowth and branching morphogenesis in a variety of tissues are influenced by signals that were identified initially for their role in axon guidance.

  3. The role of neutralizing antibodies for mouse mammary tumor virus transmission and mammary cancer development

    NASA Astrophysics Data System (ADS)

    Finke, Daniela; Luther, Sanjiv A.; Acha-Orbea, Hans

    2003-01-01

    Mouse mammary tumor virus (MMTV) infection establishes chronic germinal centers and a lifelong neutralizing Ab response. We show that removal of the draining lymph node after establishment of the germinal center reaction led to complete loss of neutralizing Abs despite comparable infection levels in peripheral lymphocytes. Importantly, in the absence of neutralization, only the exocrine organs mammary gland, salivary gland, pancreas, and skin showed strikingly increased infection, resulting in accelerated mammary tumor development. Induction of stronger neutralization did not influence chronic infection levels of peripheral lymphoid organs but strongly inhibited mammary gland infection and virus transmission to the next generation. Taken together, we provide evidence that a tight equilibrium in virus neutralization allows limited infection of exocrine organs and controls cancer development in susceptible mouse strains. These experiments show that a strong neutralizing Ab response induced after infection is not able to control lymphoid MMTV infection. Strong neutralization, however, is capable of blocking amplification of mammary gland infection, tumor development, and virus transmission to the next generation. The results also indicate a role of neutralization in natural resistance to MMTV infection.

  4. Amplification of mouse mammary tumor virus genomes in non-mammary tumor cells.

    PubMed Central

    Racevskis, J; Beyer, H

    1989-01-01

    Extra proviral copies of mouse mammary tumor virus (MMTV) are known to be present in the genomes of certain T-cell lymphomas of mice. Analysis of additional non-mammary tumor cell types known to express MMTV transcripts and antigens revealed the presence of extra acquired MMTV proviruses in a pituitary tumor cell line, a macrophage line, and Leydig testicular tumor cells. The nature of the amplified MMTV proviruses in these various tumor cell types differed with regard to copy number and presence of alterations in the long terminal repeat region. Images PMID:2535749

  5. 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

  6. 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

  7. 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

  8. A novel non-mouse mammary tumor virus activation of the Int-3 gene in a spontaneous mouse mammary tumor.

    PubMed Central

    Kordon, E C; Smith, G H; Callahan, R; Gallahan, D

    1995-01-01

    In a mouse mammary tumor model system in which carcinogenic progression can be investigated, we have found a unique mutation of Int-3 associated with progression from premalignant lobular hyperplasia to tumor. Sequence analysis of the rearranged fragment revealed an insertion of an intracisternal type A particle (IAP) within the Int-3 gene. Int-3 is mutated frequently in mouse mammary tumor virus (MMTV)-induced mammary tumors by insertion of MMTV proviral DNA into this intragenic region. In these mutations, the insertion produces a chimeric Int-3 transcript encoding the cytoplasmic portion of the Int-3 protein driven by the MMTV long terminal repeat promoter. In this case, the IAP DNA was inserted in the opposite transcriptional orientation relative to Int-3; nevertheless, a similar chimeric RNA transcript driven by a cryptic promoter in the oppositely oriented 5' IAP long terminal repeat was generated. This is the first demonstration that an insertional mutation unrelated to MMTV activates an Int gene commonly associated with mammary tumorigenesis. PMID:7494323

  9. In vitro infectivity assay for mouse mammary tumor virus.

    PubMed

    Vacquier, J P; Cardiff, R D

    1979-08-01

    Studies of mouse mammary tumor virus (MMTV) have been impeded by the lack of an in vitro infectivity assay. We have developed a rapid, quantitative in vitro assay for MMTV infectivity based on the detection of positively staining foci by immunoperoxidase. This assay and a 50% end-point titration of MMTV infectivity gave identical virus titers. Infection of a rat hepatoma cell line, a feline kidney cell line, and a normal murine mammary gland cell line by virus from the mouse mammary tumor GR3A cell line was linear with respect to virus concentration. The infectious titers obtained in both homologous and heterologous cell lines were not significantly different, demonstrating a lack of host range specificity. Virus infectivity was inactivated by heating at 55 degrees C and by ultraviolet irradiation. Rabbit anti-MMTV serum neutralized the infectivity with a 50% neutralization end point of 1:5000. Applications of this assay to the study of the immunological, biological, and biochemical characteristics of MMTV are discussed.

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

    PubMed

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

    2016-10-01

    Programmed death ligand 1 (PD-L1) is expressed by many cancer cell types, as well as by activated T cells and antigen-presenting cells. Constitutive and inducible PD-L1 expression contributes to immune evasion by breast cancer (BC) cells. We show here that the dietary phytochemical apigenin inhibited interferon (IFN)-γ-induced PD-L1 upregulation by triple-negative MDA-MB-468 BC cells, HER2(+) SK-BR-3 BC cells, and 4T1 mouse mammary carcinoma cells, as well as human mammary epithelial cells, but did not affect constitutive PD-L1 expression by triple-negative MDA-MB-231 BC cells. IFN-β-induced expression of PD-L1 by MDA-MB-468 cells was also inhibited by apigenin. In addition, luteolin, the major metabolite of apigenin, inhibited IFN-γ-induced PD-L1 expression by MDA-MB-468 cells. Apigenin-mediated inhibition of IFN-γ-induced PD-L1 expression by MDA-MB-468 and 4T1 cells was associated with reduced phosphorylation of STAT1, which was early and transient at Tyr701 and sustained at Ser727. Apigenin-mediated inhibition of IFN-γ-induced PD-L1 expression by MDA-MB-468 cells also increased proliferation and interleukin-2 synthesis by PD-1-expressing Jurkat T cells that were co-cultured with MDA-MB-468 cells. Apigenin therefore has the potential to increase the vulnerability of BC cells to T cell-mediated anti-tumor immune responses. PMID:27378243

  11. Murine breast carcinoma 4T1 cells are more sensitive to atranorin than normal epithelial NMuMG cells in vitro: Anticancer and hepatoprotective effects of atranorin in vivo.

    PubMed

    Solár, Peter; Hrčková, Gabriela; Koptašíková, Lenka; Velebný, Samuel; Solárová, Zuzana; Bačkor, Martin

    2016-04-25

    The aim of this study was to evaluate the anticancer effect of atranorin (ATR) on murine 4T1 breast carcinoma cells and compare its sensitivity with normal mammary epithelial NMuMG cells in vitro. Anti-tumor and hepatoprotective activity of ATR-therapy was examined on mouse model of 4T1-induced cancer disease. ATR significantly reduced clonogenic ability of carcinoma 4T1 cells at the concentration of 75 μM, but clonogenicity of normal NMuMG cells was not affected by any of ATR concentrations tested. Moreover, flow cytometric and BrdU incorporation analysis did not confirm the inhibited entry into S-phase of the cell cyle after ATR incubation, and on the contrary, it induced apoptosis associated with the activation of caspase-3 and PARP cleavage in 4T1 cells. Although ATR did not cause any significant changes in Bcl-xL protein expression in NMuMG cells, an apparent depletion of Bcl-xL protein in 4T1 cells after 48 h ATR therapy was confirmed. Based on this result as well as the result of the total cell number decline, we can conclude that 4T1 cells are more sensitive to ATR therapy than NMuMG cells. ATR administration resulted in significantly longer survival time of BALB/c mice inoculated with 4T1 cells, what was associated with reduced tumor size and the higher numbers of apoptotic 4T1 cells. No differences were recorded in the number of BrdU-positive tumor cells between ATR-treated group and controls. Results indicate that ATR has rather proapoptotic than antiproliferative effect on 4T1 cells in vitro and in vivo and normal NMuMG cells are less sensitive to ATR. Furthermore, our studies revealed protective effect of ATR against oxidative stress in the livers of the tumor-bearing mice. PMID:26969521

  12. Murine breast carcinoma 4T1 cells are more sensitive to atranorin than normal epithelial NMuMG cells in vitro: Anticancer and hepatoprotective effects of atranorin in vivo.

    PubMed

    Solár, Peter; Hrčková, Gabriela; Koptašíková, Lenka; Velebný, Samuel; Solárová, Zuzana; Bačkor, Martin

    2016-04-25

    The aim of this study was to evaluate the anticancer effect of atranorin (ATR) on murine 4T1 breast carcinoma cells and compare its sensitivity with normal mammary epithelial NMuMG cells in vitro. Anti-tumor and hepatoprotective activity of ATR-therapy was examined on mouse model of 4T1-induced cancer disease. ATR significantly reduced clonogenic ability of carcinoma 4T1 cells at the concentration of 75 μM, but clonogenicity of normal NMuMG cells was not affected by any of ATR concentrations tested. Moreover, flow cytometric and BrdU incorporation analysis did not confirm the inhibited entry into S-phase of the cell cyle after ATR incubation, and on the contrary, it induced apoptosis associated with the activation of caspase-3 and PARP cleavage in 4T1 cells. Although ATR did not cause any significant changes in Bcl-xL protein expression in NMuMG cells, an apparent depletion of Bcl-xL protein in 4T1 cells after 48 h ATR therapy was confirmed. Based on this result as well as the result of the total cell number decline, we can conclude that 4T1 cells are more sensitive to ATR therapy than NMuMG cells. ATR administration resulted in significantly longer survival time of BALB/c mice inoculated with 4T1 cells, what was associated with reduced tumor size and the higher numbers of apoptotic 4T1 cells. No differences were recorded in the number of BrdU-positive tumor cells between ATR-treated group and controls. Results indicate that ATR has rather proapoptotic than antiproliferative effect on 4T1 cells in vitro and in vivo and normal NMuMG cells are less sensitive to ATR. Furthermore, our studies revealed protective effect of ATR against oxidative stress in the livers of the tumor-bearing mice.

  13. Int-6, a highly conserved, widely expressed gene, is mutated by mouse mammary tumor virus in mammary preneoplasia.

    PubMed Central

    Marchetti, A; Buttitta, F; Miyazaki, S; Gallahan, D; Smith, G H; Callahan, R

    1995-01-01

    With a unique mouse mammary tumor model system in which mouse mammary tumor virus (MMTV) insertional mutations can be detected during progression from preneoplasia to frank malignancy, including metastasis, we have discovered a new common integration site (designated Int-6) for MMTV in mouse mammary tumors. MMTV was integrated into Int-6 in a mammary hyperplastic outgrowth line, its tumors and metastases, and two independent mammary tumors arising in unrelated mice. The Int-6 gene is ubiquitously expressed as a 1.4-kb RNA species in adult tissues and is detected beginning at day 8 of embryonic development. The nucleotide sequence of Int-6 is unrelated to any of the known genes in the GenBank database. MMTV integrates within introns of the gene in the opposite transcriptional orientation. In each tumor tested, this results in the expression of a truncated Int-6/long terminal repeat (LTR) chimeric RNA species which is terminated at a cryptic termination signal in the MMTV LTR. Since the nonrearranged Int-6 alleles in these tumors contain no mutations, we favor the conclusion that truncation of the Int-6 gene product either biologically activates its function or represents a dominant-negative mutation. PMID:7853537

  14. Multiphoton intravital microscopy setup to visualize the mouse mammary gland

    NASA Astrophysics Data System (ADS)

    Adur, Javier; Herrera Torres, Ana M.; Masedunskas, Andrius; Baratti, Mariana O.; de Thomaz, Andre A.; Pelegati, Vitor B.; Carvalho, Hernandes F.; Cesar, Carlos L.

    2013-06-01

    Recently, light microscopy-based techniques have been extended to live mammalian models leading to the development of a new imaging approach called intravital microscopy (IVM). Although IVM has been introduced at the beginning of the last century, its major advancements have occurred in the last twenty years with the development of non-linear microscopy that has enabled performing deep tissue imaging. IVM has been utilized to address many biological questions in basic research and is now a fundamental tool that provide information on tissues such as morphology, cellular architecture, and metabolic status. IVM has become an indispensable tool in numerous areas. This study presents and describes the practical aspects of IVM necessary to visualize epithelial cells of live mouse mammary gland with multiphoton techniques.

  15. Stromal Integrin α11β1 Affects RM11 Prostate and 4T1 Breast Xenograft Tumors Differently

    PubMed Central

    Skogstrand, Trude; Sortland, Kristina; Schmid, Marei Caroline; Reed, Rolf K.; Stuhr, Linda

    2016-01-01

    Purpose It has been implied that the collagen binding integrin α11β1 plays a role in carcinogenesis. As still relatively little is known about how the stromal integrin α11β1 affects different aspects of tumor development, we wanted to examine the direct effects on primary tumor growth, fibrosis, tumor interstitial fluid pressure (PIF) and metastasis in murine 4T1 mammary and RM11 prostate tumors, using an in vivo SCID integrin α11-deficient mouse model. Methods Tumor growth was measured using a caliper, PIF by the wick-in-needle technique, activated fibroblasts by α-SMA immunofluorescence staining and fibrosis by transmission electron microscopy and picrosirius-red staining. Metastases were evaluated using hematoxylin and eosin stained sections. Results RM11 tumor growth was significantly reduced in the SCID integrin α11-deficient (α11-KO) compared to in SCID integrin α11 wild type (WT) mice, whereas there was no similar effect in the 4T1 tumor model. The 4T1 model demonstrated an alteration in collagen fibril diameter in the integrin α11-KO mice compared to WT, which was not found in the RM11 model. There were no significant differences in the amount of activated fibroblasts, total collagen content, collagen organization or PIF in the tumors in integrin α11-deficient mice compared to WT mice. There was also no difference in lung metastases between the two groups. Conclusion Deficiency of stromal integrin α11β1 showed different effects on tumor growth and collagen fibril diameter depending on tumor type, but no effect on tumor PIF or development of lung metastasis. PMID:26990302

  16. Comparison of mouse mammary gland imaging techniques and applications: Reflectance confocal microscopy, GFP Imaging, and ultrasound

    PubMed Central

    Tilli, Maddalena T; Parrish, Angela R; Cotarla, Ion; Jones, Laundette P; Johnson, Michael D; Furth, Priscilla A

    2008-01-01

    Background Genetically engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology. However, traditional whole mount and histological imaging modalities are only applicable to non-viable tissue. Methods We evaluated three techniques that can be quickly applied to living tissue for imaging normal and cancerous mammary gland: reflectance confocal microscopy, green fluorescent protein imaging, and ultrasound imaging. Results In the current study, reflectance confocal imaging offered the highest resolution and was used to optically section mammary ductal structures in the whole mammary gland. Glands remained viable in mammary gland whole organ culture when 1% acetic acid was used as a contrast agent. Our application of using green fluorescent protein expressing transgenic mice in our study allowed for whole mammary gland ductal structures imaging and enabled straightforward serial imaging of mammary gland ducts in whole organ culture to visualize the growth and differentiation process. Ultrasound imaging showed the lowest resolution. However, ultrasound was able to detect mammary preneoplastic lesions 0.2 mm in size and was used to follow cancer growth with serial imaging in living mice. Conclusion In conclusion, each technique enabled serial imaging of living mammary tissue and visualization of growth and development, quickly and with minimal tissue preparation. The use of the higher resolution reflectance confocal and green fluorescent protein imaging techniques and lower resolution ultrasound were complementary. PMID:18215290

  17. Protein tyrosine kinase 6 regulates mammary gland tumorigenesis in mouse models

    PubMed Central

    Peng, M; Ball-Kell, S M; Franks, R R; Xie, H; Tyner, A L

    2013-01-01

    Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed in the majority of human breast tumors and breast cancer cell lines, but its expression has not been reported in normal mammary gland. To study functions of PTK6 in vivo, we generated and characterized several transgenic mouse lines with expression of human PTK6 under control of the mouse mammary tumor virus (MMTV) long terminal repeat. Ectopic active PTK6 was detected in luminal epithelial cells of mature transgenic mammary glands. Lines expressing the MMTV-PTK6 transgene exhibited more than a two-fold increase in mammary gland tumor formation compared with nontransgenic control animals. PTK6 activates signal transducer and activator of transcription 3 (STAT3), and active STAT3 was detected in PTK6-positive mammary gland epithelial cells. Endogenous mouse PTK6 was not detected in the normal mouse mammary gland, but it was induced in mouse mammary gland tumors of different origin, including spontaneous tumors that developed in control mice, and tumors that formed in PTK6, H-Ras, ERBB2 and PyMT transgenic models. MMTV-PTK6 and MMTV-ERBB2 transgenic mice were crossed to explore crosstalk between PTK6 and ERBB2 signaling in vivo. We found no significant increase in tumor incidence, size or metastasis in ERBB2/PTK6 double transgenic mice. Although we detected increased proliferation in ERBB2/PTK6 double transgenic tumors, an increase in apoptosis was also observed. MMTV-PTK6 clearly promotes mammary gland tumorigenesis in vivo, but its impact may be underrepresented in our transgenic models because of induction of endogenous PTK6 expression. PMID:24323291

  18. Specific Medicinal Plant Polysaccharides Effectively Enhance the Potency of a DC-Based Vaccine against Mouse Mammary Tumor Metastasis

    PubMed Central

    Chang, Wei Ting; Lai, Tzung Hsien; Chyan, Yau Jan; Yin, Shu Yi; Chen, Yung Hsiang; Wei, Wen Chi; Yang, Ning-Sun

    2015-01-01

    Dendritic cell (DC) vaccines are a newly emerging immunotherapeutic approach for the treatment and prevention of cancer, but major challenges still remain particularly with respect to clinical efficacy. Engineering and optimization of adjuvant formulations for DC-based vaccines is one strategy through which more efficacious treatments may be obtained. In this study, we developed a new ex vivo approach for DC vaccine preparation. We evaluated two highly purified mixed polysaccharide fractions from the root of Astragalus membranaceus and Codonopsis pilosulae, named Am and Cp, for their use in enhancing the efficiency of a DC-based cancer vaccine against metastasis of 4T1 mammary carcinoma in mice. Mixed lymphocyte reaction showed all Am-, Cp- and [Am+Cp]-treated DCs enhanced mouse CD4+ and CD8+ T-cell proliferation. [Am+Cp]-treated DCs exhibited the strongest anti-4T1 metastasis activity in test mice. Treatments with Am, Cp and [Am+Cp] also resulted in augmented expression of CD40, CD80 and CD86 markers in test DCs. Bioinformatics analysis of the cytokine array data from treated DCs identified that [Am+Cp] is efficacious in activation of specific immune functions via mediating the expression of cytokines/chemokines involved in the recruitment and differentiation of defined immune cells. Biochemical analysis revealed that Am and Cp are composed mainly of polysaccharides containing a high level (70–95%) glucose residues, but few or no (< 1%) mannose residues. In summary, our findings suggest that the specific plant polysaccharides Am and Cp extracted from traditional Chinese medicines can be effectively used instead of bacterial LPS as a potent adjuvant in the formulation of a DC-based vaccine for cancer immunotherapies. PMID:25825910

  19. Scintillation Studies of the Mouse Mammary Tumor Virus with ^125I

    NASA Astrophysics Data System (ADS)

    Yazdi, Amir; Blue, Eric; Bradley, Eric; Majewski, Stan; Mohammed, Shira; Qian, Jianguo; Saha, Margaret; Schworer, Stephen; Sutton, Jonathan; Weisenberger, Andrew; Welsh, Robert

    2007-10-01

    We have applied the techniques of scintillation imaging to studies of the mouse mammary tumor virus (MMTV). In these studies, Sodium Iodide Symporter (NIS) transfers the radioactive ^125I to the mammary glands of lactating mice and in particular to those mammaries with visible tumors. These studies have principally been carried out using pixellated scintillators coupled to position sensitive photomultiplier tubes (PSPMTs). More recently, we have initiated such studies with a monolithic slab of LaBr3 scintillator coupled to an array of PSPMTs. Several techniques of mapping and measuring the development of such tumors have been employed. These will be discussed in detail and preliminary results will be reported.

  20. Raman spectra of normal and cancerous mouse mammary gland tissue using near infrared excitation energy

    NASA Astrophysics Data System (ADS)

    Naik, Vaman; Serhatkulu, G. K.; Dai, H.; Shukla, N.; Weber, R.; Thakur, J. S.; Freeman, D. C.; Pandya, A. K.; Auner, G. W.; Naik, R.; Miller, R. F.; Cao, A.; Klein, M. D.; Rabah, R.

    2006-03-01

    Raman spectra of normal mammary gland tissues, malignant mammary gland tumors, and lymph nodes have been recorded using fresh tissue from mice. Tumors were induced in mice by subcutaneously injecting 4T1 BALB/c mammary tumor (a highly malignant) cell line. The Raman spectra were collected using the same tissues that were examined by histopathology for determining the cancerous/normal state of the tissue. Differences in various peak intensities, peak shifts and peak ratios were analyzed to determine the Raman spectral features that differentiate mammary gland tumors from non-tumorous tissue. Tissues that were confirmed by pathology as cancerous (tumors) show several distinctive features in the Raman spectra compared to the spectra of the normal tissues. For example, the cancerous tissues show Raman peaks at 621, 642, 1004, 1032, 1175 and 1208 cm-1 that are assignable to amino acids containing aromatic side-chains such as phenylalanine, tryptophan and tyrosine. Further, the cancerous tissues show a greatly reduced level of phospholipids compared to the normal tissues. The Raman spectral regions that are sensitive to pathologic alteration in the tissue will be discussed.

  1. Magnolol inhibits the inflammatory response in mouse mammary epithelial cells and a mouse mastitis model.

    PubMed

    Wei, Wang; Dejie, Liang; Xiaojing, Song; Tiancheng, Wang; Yongguo, Cao; Zhengtao, Yang; Naisheng, Zhang

    2015-02-01

    Mastitis comprises an inflammation of the mammary gland, which is almost always linked with bacterial infection. The treatment of mastitis concerns antimicrobial substances, but not very successful. On the other hand, anti-inflammatory therapy with Chinese traditional medicine becomes an effective way for treating mastitis. Magnolol is a polyphenolic binaphthalene compound extracted from the stem bark of Magnolia sp., which has been shown to exert a potential for anti-inflammatory activity. The purpose of this study was to investigate the protective effects of magnolol on inflammation in lipopolysaccharide (LPS)-induced mastitis mouse model in vivo and the mechanism of this protective effects in LPS-stimulated mouse mammary epithelial cells (MMECs) in vitro. The damage of tissues was determined by histopathology and myeloperoxidase (MPO) assay. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA). Nuclear factor-kappa B (NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and Toll-like receptor 4 (TLR4) were determined by Western blot. The results showed that magnolol significantly inhibit the LPS-induced TNF-α, IL-6, and IL-1β production both in vivo and vitro. Magnolol declined the phosphorylation of IκBα, p65, p38, ERK, and JNK in LPS-stimulated MMECs. Furthermore, magnolol inhibited the expression of TLR4 in LPS-stimulated MMECs. In vivo study, it was also observed that magnolol attenuated the damage of mastitis tissues in the mouse models. These findings demonstrated that magnolol attenuate LPS-stimulated inflammatory response by suppressing TLR4/NF-κB/mitogen-activated protein kinase (MAPK) signaling system. Thereby, magnolol may be a therapeutic agent against mastitis.

  2. Differential roles of ERα and ERβ in normal and neoplastic development in the mouse mammary gland.

    PubMed

    Mehta, Rajendra G; Hawthorne, Michael; Mehta, Rajeshwari R; Torres, Karen E O; Peng, Xinjian; McCormick, David L; Kopelovich, Levy

    2014-01-01

    The present experiments were performed to determine the roles of estrogen receptors α and β (ERα and ERβ) in normal and neoplastic development in the mouse mammary gland. In wild-type mice, in vivo administration of estradiol (E) + progesterone (P) stimulated mammary ductal growth and alveolar differentiation. Mammary glands from mice in which the ERβ gene has been deleted (βERKO mice) demonstrated normal ductal growth and differentiation in response to E + P. By contrast, mammary glands from mice in which the ERα gene has been deleted (αERKO mice) demonstrated only rudimentary ductal structures that did not differentiate in response to E + P. EGF demonstrates estrogen-like activity in the mammary glands of αERKO mice: treatment of αERKO mice with EGF + P (without E) supported normal mammary gland development, induced expression of progesterone receptor (PR), and increased levels of G-protein-coupled receptor (GPR30) protein. Mammary gland development in βERKO mice treated with EGF + P was comparable to that of wild-type mice receiving EGF + P; EGF had no statistically significant effects on the induction of PR or expression of GPR30 in mammary glands harvested from either wild-type mice or βERKO mice. In vitro exposure of mammary glands to 7,12-dimethylbenz[a]anthracene (DMBA) induced preneoplastic mammary alveolar lesions (MAL) in glands from wild-type mice and βERKO mice, but failed to induce MAL in mammary glands from αERKO mice. Microarray analysis of DMBA-treated mammary glands identified 28 functional pathways whose expression was significantly different in αERKO mice versus both βERKO and wild-type mice; key functions that were differentially expressed in αERKO mice included cell division, cell proliferation, and apoptosis. The data demonstrate distinct roles for ERα and ERβ in normal and neoplastic development in the mouse mammary gland, and suggest that EGF can mimic the ERα-mediated effects of E in this organ.

  3. Mouse mammary tumors display Stat3 activation dependent on leukemia inhibitory factor signaling

    PubMed Central

    Quaglino, Ana; Schere-Levy, Carolina; Romorini, Leonardo; Meiss, Roberto P; Kordon, Edith C

    2007-01-01

    Introduction It has been demonstrated that leukemia inhibitory factor (LIF) induces epithelium apoptosis through Stat3 activation during mouse mammary gland involution. In contrast, it has been shown that this transcription factor is commonly activated in breast cancer cells, although what causes this effect remains unknown. Here we have tested the hypothesis that locally produced LIF can be responsible for Stat3 activation in mouse mammary tumors. Methods The studies were performed in different tumorigenic and non-tumorigenic mammary cells. The expression of LIF and LIF receptor was tested by RT-PCR analysis. In tumors, LIF and Stat3 proteins were analyzed by immunohistochemistry, whereas Stat3 and extracellular signal-regulated kinase (ERK)1/2 expression and phosphorylation were studied by Western blot analysis. A LIF-specific blocking antibody was used to determine whether this cytokine was responsible for Stat3 phosphorylation induced by conditioned medium. Specific pharmacological inhibitors (PD98059 and Stat3ip) that affect ERK1/2 and Stat3 activation were used to study their involvement in LIF-induced effects. To analyze cell survival, assays with crystal violet were performed. Results High levels of LIF expression and activated Stat3 were found in mammary tumors growing in vivo and in their primary cultures. We found a single mouse mammary tumor cell line, LM3, that showed low levels of activated Stat3. Incidentally, these cells also showed very little expression of LIF receptor. This suggested that autocrine/paracrine LIF would be responsible for Stat3 activation in mouse mammary tumors. This hypothesis was confirmed by the ability of conditioned medium of mammary tumor primary cultures to induce Stat3 phosphorylation, activity that was prevented by pretreatment with LIF-blocking antibody. Besides, we found that LIF increased tumor cell viability. Interestingly, blocking Stat3 activation enhanced this effect in mammary tumor cells. Conclusion LIF is

  4. Mouse Mammary Gland Is Refractory to the Effects of Ethanol after Natural Lactation

    PubMed Central

    Garofalo, Jennifer-Marie; Bowers, Dawn M; Browne, Richard W; MacQueen, Brian T; Mashtare, Terry; Martin, Lisa B; Masso-Welch, Patricia A

    2013-01-01

    Ethanol is a dietary factor that dose-dependently increases breast cancer risk in women. We previously have shown that ethanol increases mammary epithelial density through increased branching after dietary exposure during puberty in CD2/F1 mice. To extend these studies to parous mice in a breast cancer model, we used a transgenic mouse model of human parity-associated breast cancer, the FVB-MMTV-Her2/Neu mouse, which overexpresses wildtype EGFR2, resulting in constitutive activation of growth signaling in the mammary epithelium. Here we describe the short-term effects of ethanol feeding on progression through involution. Mice were fed diets supplemented with 0%, 0.5%, 1%, or 2% ethanol for 4, 9, or 14 d starting on day 21 of lactation (that is, at the start of natural postlactational involution). Unlike peripubertal mice exposed to ethanol, postlactational dams showed no changes in body weight; liver, spleen, and kidney weights; and pathology. Ethanol exposure had no effect on mammary gland lobular density and adipocyte size throughout involution. Likewise, the infiltration of inflammatory cells and serum oxidized lipid species were unchanged by diet, suggesting that ethanol feeding had no effect on local inflammation (leukocyte infiltration) or systemic inflammation (oxidized lipids). In conclusion, ethanol exposure of parous dams had no effect on mammary gland structure or the regression of the lactating mammary gland to a resting state. The period of involution that follows natural lactation appears to be refractory to developmental effects of ethanol on mammary epithelium. PMID:23561936

  5. Protein tyrosine kinase 6 promotes ERBB2-induced mammary gland tumorigenesis in the mouse.

    PubMed

    Peng, M; Ball-Kell, S M; Tyner, A L

    2015-01-01

    Protein tyrosine kinase 6 (PTK6) expression, activation, and amplification of the PTK6 gene have been reported in ERBB2/HER2-positive mammary gland cancers. To explore contributions of PTK6 to mammary gland tumorigenesis promoted by activated ERBB2, we crossed Ptk6-/- mice with the mouse mammary tumor virus-ERBB2 transgenic mouse line expressing activated ERBB2 and characterized tumor development and progression. ERBB2-induced tumorigenesis was significantly delayed and diminished in mice lacking PTK6. PTK6 expression was induced in the mammary glands of ERBB2 transgenic mice before tumor development and correlated with activation of signal transducer and activator of transcription 3 (STAT3) and increased proliferation. Disruption of PTK6 impaired STAT3 activation and proliferation. Phosphorylation of the PTK6 substrates focal adhesion kinase (FAK) and breast cancer anti-estrogen resistance 1 (BCAR1; p130CAS) was decreased in Ptk6-/- mammary gland tumors. Reduced numbers of metastases were detected in the lungs of Ptk6-/- mice expressing activated ERBB2, compared with wild-type ERBB2 transgenic mice. PTK6 activation was detected at the edges of ERBB2-positive tumors. These data support roles for PTK6 in both ERBB2-induced mammary gland tumor initiation and metastasis, and identify STAT3, FAK, and BCAR1 as physiologically relevant PTK6 substrates in breast cancer. Including PTK6 inhibitors as part of a treatment regimen could have distinct benefits in ERBB2/HER2-positive breast cancers.

  6. Acceleration of Mouse Mammary Tumor Virus-Induced Murine Mammary Tumorigenesis by a p53172H Transgene

    PubMed Central

    Chatterjee, Gouri; Rosner, Andrea; Han, Yi; Zelazny, Edward T.; Li, Baolin; Cardiff, Robert D.; Perkins, Archibald S.

    2002-01-01

    We previously showed that a mammary-specific dominant-negative p53 transgene (WAP-p53172H) could accelerate ErbB2-induced mammary tumorigenesis in mice, but was not tumorigenic on its own. To identify other genes that cooperate with WAP-p53172H in tumorigenesis, we performed mouse mammary tumor virus (MMTV) proviral mutagenesis. We derived F1, N2, and N4/N5 mice from p53172H transgenic FVB mice backcrossed onto MMTV+ C3H/He mice. Results show the latency of MMTV tumorigenesis is correlated with FVB contribution. F1 tumors had the shortest latency (217 days), had a higher rate of metastasis, and were less differentiated than the N2 and N4/N5 tumors. The latency was 269 days in N2 mice, and lengthened to 346 days in N4/N5 mice. p53172H significantly accelerated MMTV tumorigenesis only in N2 mice, indicating cooperativity between p53172H and MMTV in this cohort. To identify genes that may be causally involved in MMTV-induced mammary tumorigenesis, we identified 60 sites of proviral insertion in the N2 tumors. Among the insertions in p53172H transgenic tumors were 10 genes not previously found as sites of MMTV insertion including genes involved in signaling (Pdgfra, Pde1b, Cnk1), cell adhesion (Cd44), angiogenesis (Galgt1), and transcriptional regulation (Olig1, Olig2, and Uncx4.1). These may represent cellular functions that are likely not deregulated by mutation in p53. PMID:12466138

  7. Conditional knockout of fibronectin abrogates mouse mammary gland lobuloalveolar differentiation

    PubMed Central

    Liu, Keyi; Cheng, Le; Flesken-Nikitin, Andrea; Huang, Lynn; Nikitin, Alexander Y.; Pauli, Bendicht U.

    2010-01-01

    Fibronectin (Fn) plays an important part in the branching morphogenesis of salivary gland, lung, and kidney. Here, we examine the effect of the conditional knockout of Fn in the mammary epithelium [FnMEp−/−] on postnatal mammary gland development, using Cre-loxP mediated gene knockout technology. Our data show that Fn deletion causes a moderate retardation in outgrowth and branching of the ductal tree in 5-week old mice. These defects are partially compensated in virgin 16-week old mice. However, mammary glands consisting of Fn-deficient epithelial cells fail to undergo normal lobuloalveolar differentiation during pregnancy. The severity of lobuloalveolar impairment ranged from lobular hypoplasia to aplasia in some cases and was associated with the amount of Fn protein recovered from these glands. Decreased rates of mammary epithelial cell proliferation accounted for delayed ductal outgrowth in virgin and lack of alveologenesis in pregnant FnMEp−/− mice. Concomitant decreased expression of integrin β1 (Itgb1) and lack of autophosphorylation of focal adhesion kinase (Fak) suggest that this pathology might, at least in part, be mediated by disruption of the Fn/Itgb1/Fak signaling pathway. PMID:20624380

  8. Glucocorticoid modulation of casein gene transcription in mouse mammary gland.

    PubMed Central

    Ganguly, R; Mehta, N M; Ganguly, N; Banerjee, M R

    1979-01-01

    The influence of cortisol and prolactin on casein gene expression in the mammary gland of lactating BALB/c mice was measured by using a specific cDNA probe to 15S casein mRNA (cDNAcsn). Casein mRNA (mRNAcsn) level in the mammary gland was decreased by 85% 5 days after adrenal ablation, but then was increased 4.4-fold 12 hr after a single injection of hydrocortisone-21-acetate. An 80% decrease in serum prolactin level, induced by the prolactin inhibitor 2-bromo-alpha-ergocryptin (CB-154), did not alter the level of mRNAcsn in the gland. Specific transcription of the casein gene in nuclei isolated from lactating mammary glands was measured by cDNAcsn hybridization to the in vitro synthesized Hg-CTP-containing RNA (Hg-RNA), which was purified by SH-agarose chromatography. The level of the mRNAcsn in Hg-RNA synthesized in the isolated nuclei was 0.09% and this was decreased 85% by alpha-amanitin, indicating that the mRNAcsn sequences in the Hg-RNA were the products of RNA polymerase II-directed DNA-dependent RNA synthesis. Transcription of the mRNAcsn in isolated nuclei was decreased by 70% 5 days after adrenalectomy and a single injection of the glucocorticoid then increased the transcription level 2-fold at 6 hr. Essentially no alteration of the level of transcription was detectable in mammary nuclei isolated from lactating mice with 80% decreased serum prolactin level, induced by CB-154 treatment. The results thus demonstrate a glucocorticoid involvement on the modulation of casein gene expression at the transcriptional level of control. PMID:293734

  9. Social isolation induces autophagy in the mouse mammary gland: link to increased mammary cancer risk.

    PubMed

    Sumis, Allison; Cook, Katherine L; Andrade, Fabia O; Hu, Rong; Kidney, Emma; Zhang, Xiyuan; Kim, Dominic; Carney, Elissa; Nguyen, Nguyen; Yu, Wei; Bouker, Kerrie B; Cruz, Idalia; Clarke, Robert; Hilakivi-Clarke, Leena

    2016-10-01

    Social isolation is a strong predictor of early all-cause mortality and consistently increases breast cancer risk in both women and animal models. Because social isolation increases body weight, we compared its effects to those caused by a consumption of obesity-inducing diet (OID) in C57BL/6 mice. Social isolation and OID impaired insulin and glucose sensitivity. In socially isolated, OID-fed mice (I-OID), insulin resistance was linked to reduced Pparg expression and increased neuropeptide Y levels, but in group-housed OID fed mice (G-OID), it was linked to increased leptin and reduced adiponectin levels, indicating that the pathways leading to insulin resistance are different. Carcinogen-induced mammary tumorigenesis was significantly higher in I-OID mice than in the other groups, but cancer risk was also increased in socially isolated, control diet-fed mice (I-C) and G-OID mice compared with that in controls. Unfolded protein response (UPR) signaling (GRP78; IRE1) was upregulated in the mammary glands of OID-fed mice, but not in control diet-fed, socially isolated I-C mice. In contrast, expression of BECLIN1, ATG7 and LC3II were increased, and p62 was downregulated by social isolation, indicating increased autophagy. In the mammary glands of socially isolated mice, but not in G-OID mice, mRNA expressions of p53 and the p53-regulated autophagy inducer Dram1 were upregulated, and nuclear p53 staining was strong. Our findings further indicated that autophagy and tumorigenesis were not increased in Atg7(+/-) mice kept in social isolation and fed OID. Thus, social isolation may increase breast cancer risk by inducing autophagy, independent of changes in body weight. PMID:27550962

  10. Social isolation induces autophagy in the mouse mammary gland: link to increased mammary cancer risk.

    PubMed

    Sumis, Allison; Cook, Katherine L; Andrade, Fabia O; Hu, Rong; Kidney, Emma; Zhang, Xiyuan; Kim, Dominic; Carney, Elissa; Nguyen, Nguyen; Yu, Wei; Bouker, Kerrie B; Cruz, Idalia; Clarke, Robert; Hilakivi-Clarke, Leena

    2016-10-01

    Social isolation is a strong predictor of early all-cause mortality and consistently increases breast cancer risk in both women and animal models. Because social isolation increases body weight, we compared its effects to those caused by a consumption of obesity-inducing diet (OID) in C57BL/6 mice. Social isolation and OID impaired insulin and glucose sensitivity. In socially isolated, OID-fed mice (I-OID), insulin resistance was linked to reduced Pparg expression and increased neuropeptide Y levels, but in group-housed OID fed mice (G-OID), it was linked to increased leptin and reduced adiponectin levels, indicating that the pathways leading to insulin resistance are different. Carcinogen-induced mammary tumorigenesis was significantly higher in I-OID mice than in the other groups, but cancer risk was also increased in socially isolated, control diet-fed mice (I-C) and G-OID mice compared with that in controls. Unfolded protein response (UPR) signaling (GRP78; IRE1) was upregulated in the mammary glands of OID-fed mice, but not in control diet-fed, socially isolated I-C mice. In contrast, expression of BECLIN1, ATG7 and LC3II were increased, and p62 was downregulated by social isolation, indicating increased autophagy. In the mammary glands of socially isolated mice, but not in G-OID mice, mRNA expressions of p53 and the p53-regulated autophagy inducer Dram1 were upregulated, and nuclear p53 staining was strong. Our findings further indicated that autophagy and tumorigenesis were not increased in Atg7(+/-) mice kept in social isolation and fed OID. Thus, social isolation may increase breast cancer risk by inducing autophagy, independent of changes in body weight.

  11. Multivariate statistical analysis of Raman spectra to distinguish normal, tumor, lymph nodes and mastitis in mouse mammary tissues

    NASA Astrophysics Data System (ADS)

    Dai, H.; Thakur, J. S.; Serhatkulu, G. K.; Pandya, A. K.; Auner, G. W.; Naik, R.; Freeman, D. C.; Naik, V. M.; Cao, A.; Klein, M. D.; Rabah, R.

    2006-03-01

    Raman spectra ( > 680) of normal mammary gland, malignant mammary gland tumors, and lymph node tissues from mice injected with 4T1 tumor cells have been recorded using 785 nm excitation laser. The state of the tissues was confirmed by standard pathological tests. The multivariate statistical analysis methods (principle component analysis and discriminant functional analysis) have been used to categorize the Raman spectra. The statistical algorithms based on the Raman spectral peak heights, clearly separated tissues into six distinct classes, including mastitis, which is clearly separated from normal and tumor. This study suggests that the Raman spectroscopy can possibly perform a real-time analysis of the human mammary tissues for the detection of cancer.

  12. 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.

  13. Pre-irradiation of mouse mammary gland stimulates cancer cell migration and development of lung metastases

    PubMed Central

    Bouchard, G; Bouvette, G; Therriault, H; Bujold, R; Saucier, C; Paquette, B

    2013-01-01

    Background: In most patients with breast cancer, radiotherapy induces inflammation that is characterised by an increase of promigratory factors in healthy tissues surrounding the tumour. However, their role in the emergence of the migration phenotype and formation of metastases is still unclear. Methods: A single mammary gland of BALB/c mice was irradiated with four doses of 6 Gy given at a 24-h interval. After the last session of irradiation, treated and control mammary glands were either collected for quantification of promigratory and proinflammatory factors or were implanted with fluorescent ubiquitination-based cell cycle indicator (FUCCI)-expressing mouse mammary cancer D2A1 cells. The migration of cancer cells in the mammary glands was monitored by optical imaging. On day 21, mammary tumours and lungs were collected for histology analyses and the quantification of metastases. Results: Pre-irradiation of the mammary gland increased by 1.8-fold the migration of cancer cells, by 2-fold the quantity of circulating cancer cells and by 2.4-fold the number of lung metastases. These adverse effects were associated with the induction of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2). Conclusion: The emergence of the metastasis phenotype is believed to be associated with the accumulation of mutations in cancer cells. Our results suggest an alternative mechanism based on promigratory factors from irradiated mammary glands. In clinic, the efficiency of radiotherapy could be improved by anti-inflammatory agents that would prevent the stimulation of cancer cell migration induced by radiation. PMID:24002607

  14. Common Integration Sites for MMTV in Viral Induced Mouse Mammary Tumors

    PubMed Central

    Callahan, Robert

    2011-01-01

    The paradigm of mammary cancer induction by the mouse mammary tumor virus (MMTV) is used to illustrate the body of evidence that supports the hypothesis that mammary epithelial stem/progenitor cells represent targets for oncogenic transformation. It is argued that this is not a special case applicable only to MMTV-induced mammary cancer, because MMTV acts as an environmental mutagen producing random interruptions in the somatic DNA of infected cells by insertion of proviral DNA copies. In addition to disrupting the host genome, the proviral DNA also influences gene expression through its associated enhancer sequences over significant inter-genomic distances. Genes commonly affected by MMTV insertion in multiple individual tumors include, the Wnt, FGF, RSpo gene families as well as eIF3e and Notch4. All of these gene families are known to play essential roles in stem cell maintenance and behavior in a variety of organs. The MMTV-induced mutations accumulate in cells that are long-lived and possess the properties of stem cells, namely, self-renewal and the capacity to produce divergent epithelial progeny through asymmetric division. The evidence shows that epithelial cells with these properties are present in normal mammary glands, may be infected with MMTV, become transformed to produce epithelial hyperplasia through MMTV-induced mutagenesis and progress to frank mammary malignancy. Retroviral marking via MMTV proviral insertion demonstrates that this process progresses from a single mammary epithelial cell that possesses all of the features ascribed to tissue-specific stem cells. PMID:18709449

  15. Model of mouse mammary gland hyperproliferation and hyperplasia induced by a western-style diet.

    PubMed

    Xue, L; Newmark, H; Yang, K; Lipkin, M

    1996-01-01

    Mammary glands of female C57BL/6J mice were analyzed after they were fed a Western-style diet or control AIN-76A diet. The Western-style diet contained several risk factors found in human diets in geographic regions having increased risk for breast cancer: high fat and phosphate and low calcium and vitamin D. After they were fed these diets for 8, 14, and 20 weeks, mice were sacrificed, and mammary glands were removed for morphometric and radioautographic measurements. Although after the animals were fed the Western-style diet for 8 weeks the number of terminal ducts per mouse mammary gland (NTDMG) was similar in the Western-style and control diet groups, after they were fed the Western-style diet for 14 weeks (p < 0.05) and 20 weeks (p < 0.01) the NTDMG significantly increased compared with the control group. Moreover, there was a significant increase (p < 0.01) in the tritiated thymidine labeling index of mammary terminal ductal epithelial cells after 14 and 20 weeks of Western-style diet administration. Thus the Western-style diet induced increased epithelial cell proliferation and increased NTDMG in female mice when fed during young adult growth and development. The findings raise the possibility that the ingestion of a diet with Western-style fat and phosphate content and with low calcium and vitamin D may induce similar changes during the early development of the human mammary gland.

  16. A Compendium of the Mouse Mammary Tumor Biologist: From the Initial Observations in the House Mouse to the Development of Genetically Engineered Mice

    PubMed Central

    Cardiff, Robert D.; Kenney, Nicholas

    2011-01-01

    For over a century, mouse mammary tumor biology and the associated mouse mammary tumor virus (MMTV) have served as the foundation for experimental cancer research, in general, and, in particular, experimental breast cancer research. Spontaneous mouse mammary tumors were the basis for studies of the natural history of neoplasia, oncogenic viruses, host responses, endocrinology and neoplastic progression. However, lacking formal proof of a human mammary tumor virus, the preeminence of the mouse model faded in the 1980s. Since the late 1980s, genetically engineered mice (GEM) have proven extremely useful for studying breast cancer and have become the animal model for human breast cancer. Hundreds of mouse models of human breast cancer have been developed since the first demonstration in 1984. The GEM have attracted a new generation of molecular and cellular biologists eager to apply their skill sets to these surrogates of the human disease. Newcomers often enter the field without an appreciation of the origins of mouse mammary tumor biology and the basis for many of the prevailing concepts. Our purpose in writing this compendium is to extend an “olive branch” while simultaneously deepen the knowledge of the novice mouse mammary tumor biologist as they journey into a field rich in pathology and genetics spanning several centuries. PMID:20961975

  17. A compendium of the mouse mammary tumor biologist: from the initial observations in the house mouse to the development of genetically engineered mice.

    PubMed

    Cardiff, Robert D; Kenney, Nicholas

    2011-06-01

    For over a century, mouse mammary tumor biology and the associated mouse mammary tumor virus (MMTV) have served as the foundation for experimental cancer research, in general, and, in particular, experimental breast cancer research. Spontaneous mouse mammary tumors were the basis for studies of the natural history of neoplasia, oncogenic viruses, host responses, endocrinology and neoplastic progression. However, lacking formal proof of a human mammary tumor virus, the preeminence of the mouse model faded in the 1980s. Since the late 1980s, genetically engineered mice (GEM) have proven extremely useful for studying breast cancer and have become the animal model for human breast cancer. Hundreds of mouse models of human breast cancer have been developed since the first demonstration in 1984. The GEM have attracted a new generation of molecular and cellular biologists eager to apply their skill sets to these surrogates of the human disease. Newcomers often enter the field without an appreciation of the origins of mouse mammary tumor biology and the basis for many of the prevailing concepts. Our purpose in writing this compendium is to extend an "olive branch" while simultaneously deepen the knowledge of the novice mouse mammary tumor biologist as they journey into a field rich in pathology and genetics spanning several centuries. PMID:20961975

  18. A compendium of the mouse mammary tumor biologist: from the initial observations in the house mouse to the development of genetically engineered mice.

    PubMed

    Cardiff, Robert D; Kenney, Nicholas

    2011-06-01

    For over a century, mouse mammary tumor biology and the associated mouse mammary tumor virus (MMTV) have served as the foundation for experimental cancer research, in general, and, in particular, experimental breast cancer research. Spontaneous mouse mammary tumors were the basis for studies of the natural history of neoplasia, oncogenic viruses, host responses, endocrinology and neoplastic progression. However, lacking formal proof of a human mammary tumor virus, the preeminence of the mouse model faded in the 1980s. Since the late 1980s, genetically engineered mice (GEM) have proven extremely useful for studying breast cancer and have become the animal model for human breast cancer. Hundreds of mouse models of human breast cancer have been developed since the first demonstration in 1984. The GEM have attracted a new generation of molecular and cellular biologists eager to apply their skill sets to these surrogates of the human disease. Newcomers often enter the field without an appreciation of the origins of mouse mammary tumor biology and the basis for many of the prevailing concepts. Our purpose in writing this compendium is to extend an "olive branch" while simultaneously deepen the knowledge of the novice mouse mammary tumor biologist as they journey into a field rich in pathology and genetics spanning several centuries.

  19. Establishment of two hormone-responsive mouse mammary carcinoma cell lines derived from a metastatic mammary tumor.

    PubMed

    Efeyan, Alejo; Fabris, Victoria; Merani, Susana; Lanari, Claudia; Molinolo, Alfredo A

    2004-02-01

    We report the establishment of two mouse mammary cancer cell lines, MC7-2A and MC7-2B obtained from a mouse mammary carcinoma induced by medroxyprogesterone acetate (MPA) and maintained by syngeneic transplantation in BALB/c mice. They are epithelial (express cytokeratins) and express both estrogen receptors alpha (ERalpha) and progesterone receptors (PRs) isoforms A and B (western blots). In vitro, MPA inhibited 3H-thymidine uptake, starting from concentrations as low as 10(-13) M in MC7-2A and 10(10) M in MC7-2B; the antiprogestin RU 486 exerted a stimulatory effect at 10(-14) M in both cell lines; 17-beta-estradiol (E2) also exerted a stimulatory effect starting at 10(-10) M in MC7-2A and at 10(-13) M in MC7-2B. When transplanted in syngeneic mice, both cell lines originated adenocarcinomas that gave rise to lung metastases within 3 months. In in vivo studies, in MC7-2A, the antiprogestin inhibited completely tumor growth, E2 induced a slight although significant ( p < 0.05) stimulatory effect and MPA stimulated tumor growth while MC7-2B cells were unresponsive to all treatments. ER and PR were also expressed in tumors as assessed by immunohistochemistry. Two marker chromosomes were identified by FISH as translocations between chromosomes 4 and 7, and between chromosomes X and 2; the third marker chromosome remains unidentified. All these markers were also present in the parental tumor. A new marker, a centric fusion of chromosomes 2, was acquired in both cell lines. Considering that there are very few murine breast carcinoma responsive cell lines, these cells represent new tools in which the regulatory effect of hormones can be studied. PMID:14758093

  20. Reinitiation of Growth in Senescent Mouse Mammary Epithelium in Response to Cholera Toxin

    NASA Astrophysics Data System (ADS)

    Daniel, Charles W.; Silberstein, Gary B.; Strickland, Phyllis

    1984-06-01

    Several lines of mouse mammary tissue that had been serially transplanted until mitotic senescence was reached were exposed in vivo to plastic implants that slowly released cholera toxin. Gland tissue surrounding the implants displayed new end buds, indicating reinitiation of growth and morphogenesis. The ability of cholera toxin, which elevates intracellular adenosine 3',5'-monophosphate, to temporarily reverse the senescent phenotype suggests that this mitotic dysfunction results not from generalized cellular deterioration but from specific changes in cell regulation.

  1. Comparative analysis of caveolins in mouse and tammar wallaby: role in regulating mammary gland function.

    PubMed

    Kuruppath, Sanjana; Sharp, Julie A; Lefevre, Christophe; Murphy, Robyn M; Nicholas, Kevin R

    2014-11-15

    Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70-80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP. PMID:25200498

  2. Epigenetic Modifications Unlock the Milk Protein Gene Loci during Mouse Mammary Gland Development and Differentiation

    PubMed Central

    Rijnkels, Monique; Freeman-Zadrowski, Courtneay; Hernandez, Joseph; Potluri, Vani; Wang, Liguo; Li, Wei; Lemay, Danielle G.

    2013-01-01

    Background Unlike other tissues, development and differentiation of the mammary gland occur mostly after birth. The roles of systemic hormones and local growth factors important for this development and functional differentiation are well-studied. In other tissues, it has been shown that chromatin organization plays a key role in transcriptional regulation and underlies epigenetic regulation during development and differentiation. However, the role of chromatin organization in mammary gland development and differentiation is less well-defined. Here, we have studied the changes in chromatin organization at the milk protein gene loci (casein, whey acidic protein, and others) in the mouse mammary gland before and after functional differentiation. Methodology/Principal Findings Distal regulatory elements within the casein gene cluster and whey acidic protein gene region have an open chromatin organization after pubertal development, while proximal promoters only gain open-chromatin marks during pregnancy in conjunction with the major induction of their expression. In contrast, other milk protein genes, such as alpha-lactalbumin, already have an open chromatin organization in the mature virgin gland. Changes in chromatin organization in the casein gene cluster region that are present after puberty persisted after lactation has ceased, while the changes which occurred during pregnancy at the gene promoters were not maintained. In general, mammary gland expressed genes and their regulatory elements exhibit developmental stage- and tissue-specific chromatin organization. Conclusions/Significance A progressive gain of epigenetic marks indicative of open/active chromatin on genes marking functional differentiation accompanies the development of the mammary gland. These results support a model in which a chromatin organization is established during pubertal development that is then poised to respond to the systemic hormonal signals of pregnancy and lactation to achieve the

  3. 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.

  4. Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

    PubMed Central

    Römer, Winfried; Sonnleitner, Alois

    2015-01-01

    Several studies have revealed that aquaporins play a role in tumor progression and invasion. In breast carcinomas, high levels of aquaporin 5 (AQP5), a membrane protein involved in water transport, have been linked to increased cell proliferation and migration, thus facilitating tumor progression. Despite the potential role of AQP5 in mammary oncogenesis, the mechanisms controlling mammary AQP5 expression are poorly understood. In other tissues, AQP5 expression has been correlated with its promoter methylation, yet, very little is known about AQP5 promoter methylation in the mammary gland. In this work, we used the mouse mammary gland cell line EpH4, in which we controlled AQP5 expression via the steroid hormone dexamethasone (Dex) to further investigate mechanisms regulating AQP5 expression. In this system, we observed a rapid drop of AQP5 mRNA levels with a delay of several hours in AQP5 protein, suggesting transcriptional control of AQP5 levels. Yet, AQP5 expression was independent of its promoter methylation, or to the presence of negative glucocorticoid receptor elements (nGREs) in its imminent promoter region, but was rather influenced by the cell proliferative state or cell density. We conclude that AQP5 promoter methylation is not a universal mechanism for AQP5 regulation and varies on cell and tissue type. PMID:25767807

  5. Reconstruction of 3-dimensional histology volume and its application to study mouse mammary glands.

    PubMed

    Shojaii, Rushin; Bacopulos, Stephanie; Yang, Wenyi; Karavardanyan, Tigran; Spyropoulos, Demetri; Raouf, Afshin; Martel, Anne; Seth, Arun

    2014-07-26

    Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs(1,2,3) is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution. This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

  6. Primary 4T1 tumor resection provides critical "window of opportunity" for immunotherapy.

    PubMed

    Ghochikyan, Anahit; Davtyan, Arpine; Hovakimyan, Armine; Davtyan, Hayk; Poghosyan, Anna; Bagaev, Alexander; Ataullakhanov, Ravshan I; Nelson, Edward L; Agadjanyan, Michael G

    2014-02-01

    It is believed that primary tumor resection modulates host-tumor immune interaction, but this has not been characterized in a stringent breast cancer tumor model. This report, using the 4T1 murine mammary tumor model, characterizes for the first time the dynamic longitudinal changes in immunosuppressive and effector components of the immune system after resection of an established orthotopic primary tumor with a defined natural history of developing lung metastases. More specifically, we analyzed changes of absolute numbers and frequencies of MDSC, regulatory T cells (Treg), as well as activated CD4 and CD8 positive T cells in spleens and, in some studies, lungs of 4T1 tumor-bearing mice and mice after primary tumor resection. Importantly, using mathematical analyses we established that primary resection of an orthotopic tumor had created a "window of opportunity" with decreased tumor-associated immune suppression that existed for approximately 10 days. Although tumor resection did slightly prolong survival, it did not affect the ultimate development of metastatic disease since animals with resected tumors or intact primary tumors eventually died by day 47 and 43, respectively. This window of opportunity likely occurs in humans providing a rationale and parameters for integration and testing of immunotherapeutic strategies in this critical "window of opportunity" to combat the development of metastatic disease. PMID:24096737

  7. Primary 4T1 tumor resection provides critical "window of opportunity" for immunotherapy.

    PubMed

    Ghochikyan, Anahit; Davtyan, Arpine; Hovakimyan, Armine; Davtyan, Hayk; Poghosyan, Anna; Bagaev, Alexander; Ataullakhanov, Ravshan I; Nelson, Edward L; Agadjanyan, Michael G

    2014-02-01

    It is believed that primary tumor resection modulates host-tumor immune interaction, but this has not been characterized in a stringent breast cancer tumor model. This report, using the 4T1 murine mammary tumor model, characterizes for the first time the dynamic longitudinal changes in immunosuppressive and effector components of the immune system after resection of an established orthotopic primary tumor with a defined natural history of developing lung metastases. More specifically, we analyzed changes of absolute numbers and frequencies of MDSC, regulatory T cells (Treg), as well as activated CD4 and CD8 positive T cells in spleens and, in some studies, lungs of 4T1 tumor-bearing mice and mice after primary tumor resection. Importantly, using mathematical analyses we established that primary resection of an orthotopic tumor had created a "window of opportunity" with decreased tumor-associated immune suppression that existed for approximately 10 days. Although tumor resection did slightly prolong survival, it did not affect the ultimate development of metastatic disease since animals with resected tumors or intact primary tumors eventually died by day 47 and 43, respectively. This window of opportunity likely occurs in humans providing a rationale and parameters for integration and testing of immunotherapeutic strategies in this critical "window of opportunity" to combat the development of metastatic disease.

  8. Presence of hyperplastic pectoral mammary glands in a white-footed mouse (Peromyscus leucopus) from a Superfund Site in Oklahoma, USA.

    PubMed

    Hays, Kimberly A; Breshears, Melanie A

    2011-01-01

    Laboratory experiments have documented the effects of hormones and endocrine-disrupting compounds on mammary development in mammals. However, few observations of mammary hyperplasia have been presented for wild rodents. We describe hyperplastic mammary glands in a wild-caught white-footed mouse (Peromyscus leucopus) from an area contaminated with heavy metals. PMID:21270018

  9. Presence of hyperplastic pectoral mammary glands in a white-footed mouse (Peromyscus leucopus) from a Superfund Site in Oklahoma, USA.

    PubMed

    Hays, Kimberly A; Breshears, Melanie A

    2011-01-01

    Laboratory experiments have documented the effects of hormones and endocrine-disrupting compounds on mammary development in mammals. However, few observations of mammary hyperplasia have been presented for wild rodents. We describe hyperplastic mammary glands in a wild-caught white-footed mouse (Peromyscus leucopus) from an area contaminated with heavy metals.

  10. The chromosomal integration site determines the tissue-specific methylation of mouse mammary tumour virus proviral genes.

    PubMed Central

    Günzburg, W H; Groner, B

    1984-01-01

    Multiple endogenous mouse mammary tumour virus (MMTV) proviral genes are present at different chromosomal locations in inbred mouse strains. Proviral DNA methylation is location and tissue specific. The methylation patterns are stably inherited and appear to be conferred upon the viral DNA by the flanking mouse genomic DNA. In transformed cells, either mammary carcinoma cells, or cells immortalized by SV40 in vitro, the stable pattern of methylation is lost. Although hypomethylation of proviral genes, both in normal and in transformed tissue, accompanies MMTV-specific RNA expression, it is also observed in non-expressing tissues. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6329738

  11. Neoplastic transformation of mouse mammary epithelial cells by deregulated myc expression.

    PubMed Central

    Telang, N T; Osborne, M P; Sweterlitsch, L A; Narayanan, R

    1990-01-01

    A spontaneously immortalized, nontumorigenic mouse mammary epithelial cell line (MMEC) was transfected with an activated myc construct by electroporation. Constitutive expression of myc in MMEC resulted in anchorage independence in soft agar and tumorigenicity in nude mice. The myc-expressing MMEC showed higher saturation density, faster growth rate, and partial abrogation of serum-derived growth factor(s) requirement compared with parent MMEC. Epidermal growth factor or transforming growth factor alpha stimulated the anchorage-independent growth, but not the anchorage-dependent growth, of MMEC-myc cells. Type 1 transforming growth factor beta, on the other hand, inhibited both the anchorage-independent and anchorage-dependent growth of MMEC-myc cells. These results demonstrate that deregulated expression of myc results in neoplastic transformation iin mammary epithelial cells. Accompanying the transformation is altered sensitivity to polypeptide growth factors. Images PMID:2088530

  12. Selection of Novel Peptides Homing the 4T1 CELL Line: Exploring Alternative Targets for Triple Negative Breast Cancer

    PubMed Central

    Nobrega, Franklin L.; Martins, Ivone M.

    2016-01-01

    The use of bacteriophages to select novel ligands has been widely explored for cancer therapy. Their application is most warranted in cancer subtypes lacking knowledge on how to target the cancer cells in question, such as the triple negative breast cancer, eventually leading to the development of alternative nanomedicines for cancer therapeutics. Therefore, the following study aimed to select and characterize novel peptides for a triple negative breast cancer murine mammary carcinoma cell line– 4T1. Using phage display, 7 and 12 amino acid random peptide libraries were screened against the 4T1 cell line. A total of four rounds, plus a counter-selection round using the 3T3 murine fibroblast cell line, was performed. The enriched selective peptides were characterized and their binding capacity towards 4T1 tissue samples was confirmed by immunofluorescence and flow cytometry analysis. The selected peptides (4T1pep1 –CPTASNTSC and 4T1pep2—EVQSSKFPAHVS) were enriched over few rounds of selection and exhibited specific binding to the 4T1 cell line. Interestingly, affinity to the human MDA-MB-231 cell line was also observed for both peptides, promoting the translational application of these novel ligands between species. Additionally, bioinformatics analysis suggested that both peptides target human Mucin-16. This protein has been implicated in different types of cancer, as it is involved in many important cellular functions. This study strongly supports the need of finding alternative targeting systems for TNBC and the peptides herein selected exhibit promising future application as novel homing peptides for breast cancer therapy. PMID:27548261

  13. Selection of Novel Peptides Homing the 4T1 CELL Line: Exploring Alternative Targets for Triple Negative Breast Cancer.

    PubMed

    Silva, Vera L; Ferreira, Debora; Nobrega, Franklin L; Martins, Ivone M; Kluskens, Leon D; Rodrigues, Ligia R

    2016-01-01

    The use of bacteriophages to select novel ligands has been widely explored for cancer therapy. Their application is most warranted in cancer subtypes lacking knowledge on how to target the cancer cells in question, such as the triple negative breast cancer, eventually leading to the development of alternative nanomedicines for cancer therapeutics. Therefore, the following study aimed to select and characterize novel peptides for a triple negative breast cancer murine mammary carcinoma cell line- 4T1. Using phage display, 7 and 12 amino acid random peptide libraries were screened against the 4T1 cell line. A total of four rounds, plus a counter-selection round using the 3T3 murine fibroblast cell line, was performed. The enriched selective peptides were characterized and their binding capacity towards 4T1 tissue samples was confirmed by immunofluorescence and flow cytometry analysis. The selected peptides (4T1pep1 -CPTASNTSC and 4T1pep2-EVQSSKFPAHVS) were enriched over few rounds of selection and exhibited specific binding to the 4T1 cell line. Interestingly, affinity to the human MDA-MB-231 cell line was also observed for both peptides, promoting the translational application of these novel ligands between species. Additionally, bioinformatics analysis suggested that both peptides target human Mucin-16. This protein has been implicated in different types of cancer, as it is involved in many important cellular functions. This study strongly supports the need of finding alternative targeting systems for TNBC and the peptides herein selected exhibit promising future application as novel homing peptides for breast cancer therapy. PMID:27548261

  14. Transcription factors link mouse WAP-T mammary tumors with human breast cancer.

    PubMed

    Otto, Benjamin; Streichert, Thomas; Wegwitz, Florian; Gevensleben, Heidrun; Klätschke, Kristin; Wagener, Christoph; Deppert, Wolfgang; Tolstonog, Genrich V

    2013-03-15

    Mouse models are important tools to decipher the molecular mechanisms of mammary carcinogenesis and to mimic the respective human disease. Despite sharing common phenotypic and genetic features, the proper translation of murine models to human breast cancer remains a challenging task. In a previous study we showed that in the SV40 transgenic WAP-T mice an active Met-pathway and epithelial-mesenchymal characteristics distinguish low- and high-grade mammary carcinoma. To assign these murine tumors to corresponding human tumors we here incorporated the analysis of expression of transcription factor (TF) coding genes and show that thereby a more accurate interspecies translation can be achieved. We describe a novel cross-species translation procedure and demonstrate that expression of unsupervised selected TFs, such as ELF5, HOXA5 and TFCP2L1, can clearly distinguish between the human molecular breast cancer subtypes--or as, for example, expression of TFAP2B between yet unclassified subgroups. By integrating different levels of information like histology, gene set enrichment, expression of differentiation markers and TFs we conclude that tumors in WAP-T mice exhibit similarities to both, human basal-like and non-basal-like subtypes. We furthermore suggest that the low- and high-grade WAP-T tumor phenotypes might arise from distinct cells of tumor origin. Our results underscore the importance of TFs as common cross-species denominators in the regulatory networks underlying mammary carcinogenesis.

  15. 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

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

    PubMed

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

    2016-09-20

    Cancer genomics has 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 genetically engineered mouse models (GEMMs). 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 has established a robust in vivo platform for functional cancer genomics and has discovered functional breast cancer mutations. PMID:27653681

  17. Decrease in an Inwardly Rectifying Potassium Conductance in Mouse Mammary Secretory Cells after Forced Weaning

    PubMed Central

    Kamikawa, Akihiro; Sugimoto, Shota; Ichii, Osamu; Kondoh, Daisuke

    2015-01-01

    Mammary glands are physiologically active in female mammals only during nursing. Immediately after weaning, most lactation-related genes are downregulated and milk production ceases. In our previous study, we have detected an inwardly rectifying potassium channel (Kir) 2.1-like current in mammary secretory (MS) cells freshly isolated from lactating mice. This current is highly sensitive to external Ba2+. The potassium permeability of the Kir channels may contribute to the secretion and/or preservation of ions in milk. We hypothesized that the functions of the Kir channels in MS cells are regulated after weaning. To test this hypothesis, we examined the effect of forced weaning on the Ba2+-sensitive Kir current and Kir2.1 expression in the mouse mammary glands. Twenty-four hours after weaning, the lumina of mammary acini were histologically enlarged by milk accumulation. The whole-cell patch-clamp analyses showed that the Ba2+-sensitive Kir current in the post-weaning MS cells was smaller than in the lactating MS cells. The inward conductances of the current in the lactating and post-weaning cells were 4.25 ± 0.77 and 0.93 ± 0.34 nS, respectively. Furthermore, real-time PCR and Western blot analyses showed that Kir2.1 mRNA and protein expression decreased in the post-weaning mammary gland (mRNA, 90% reduction; protein, 47% reduction). Moreover, the local milk accumulation caused by teat sealing decreased Kir conductance in MS cells (2.74 ± 0.45 and 0.36 ± 0.27 nS for control and sealed mammary glands, respectively). This was concomitant with the reduction in the Kir2.1 mRNA expression. Our results suggest that milk stasis after weaning immediately decreases the Kir conductance in MS cells. This decrease in the Kir conductance may be partly caused by the reduction in the Kir2.1 mRNA and protein expression. These alterations during the post-weaning period may be involved in the cessation of ion secretion and/or preservation in the milk. PMID:26484867

  18. Regulated expression of mouse mammary tumor proviral genes in cells of the B lineage

    PubMed Central

    1991-01-01

    We evaluated the expression of mouse mammary tumor proviral (MMTV) transcripts during B cell ontogeny and compared levels of RNA in B lymphocytes and B cell lines with levels in other cells of the hematopoietic lineage and in a mammary cell line. We demonstrate that MMTV transcripts are expressed as early as the pro-B cell stage in ontogeny and are expressed at basal constitutive levels throughout most of the B cell developmental pathway. The level of MMTV expression in B cells is similar to constitutive levels in mammary tissues and two to three orders of magnitude greater than in activated T cells. Levels of MMTV transcripts in B cells are not solely due to positional effects. Transient transfection assays showed that MMTV upregulation resulted from transcriptional activation of the viral LTR, indicating that there are specific and inducible transcription factors that regulate MMTV expression in B cells. MMTV transcripts could not be upregulated in pre- B cell lines but could be induced in some mature B cell lines. There was a correlation between the ability to stimulate B cells to secrete antibody and the ability to induce upregulated MMTV expression. Evidence is presented that suggests that the principal transcription factors involved in MMTV expression do not include the B cell factors OTF-2 or NF-kappa B, but rather are likely to be novel factors that are induced during differentiation to antibody secretion. A hypothesis for why mammary tumor viruses are well adapted for expression in cells of the B lineage is proposed, and the implications of this for the documented influence of MMTV gene products on the T cell repertoire are discussed. PMID:1660524

  19. PTEN deficiency in a luminal ErbB-2 mouse model results in dramatic acceleration of mammary tumorigenesis and metastasis.

    PubMed

    Schade, Babette; Rao, Trisha; Dourdin, Nathalie; Lesurf, Robert; Hallett, Michael; Cardiff, Robert D; Muller, William J

    2009-07-10

    Overexpression and/or amplification of the ErbB-2 oncogene as well as inactivation of the PTEN tumor suppressor are two important genetic events in human breast carcinogenesis. To address the biological impact of conditional inactivation of PTEN on ErbB-2-induced mammary tumorigenesis, we generated a novel transgenic mouse model that utilizes the murine mammary tumor virus (MMTV) promoter to directly couple expression of activated ErbB-2 and Cre recombinase to the same mammary epithelial cell (MMTV-NIC). Disruption of PTEN in the mammary epithelium of the MMTV-NIC model system dramatically accelerated the formation of multifocal and highly metastatic mammary tumors, which exhibited homogenous pathology. PTEN-deficient/NIC-induced tumorigenesis was associated with an increase in angiogenesis. Moreover, inactivation of PTEN in the MMTV-NIC mouse model resulted in hyperactivation of the phosphatidylinositol 3'-kinase/Akt signaling pathway. However, like the parental strain, tumors obtained from PTEN-deficient/NIC mice displayed histopathological and molecular features of the luminal subtype of primary human breast cancer. Taken together, our findings provide important implications in understanding the molecular determinants of mammary tumorigenesis driven by PTEN deficiency and ErbB-2 activation and could provide a valuable tool for testing the efficacy of therapeutic strategies that target these critical signaling pathways.

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

    PubMed

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

    2015-11-01

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

  1. In-silico QTL mapping of postpubertal mammary ductal development in the mouse uncovers potential human breast cancer risk loci

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic background plays a dominant role in mammary gland development and breast cancer (BrCa). Despite this, the role of genetics is only partially understood. This study used strain-dependent variation in an inbred mouse mapping panel, to identify quantitative trait loci (QTL) underlying structura...

  2. Deletion of Cd151 reduces mammary tumorigenesis in the MMTV/PyMT mouse model

    PubMed Central

    2014-01-01

    Background Tetraspanins are transmembrane proteins that serve as scaffolds for multiprotein complexes containing, for example, integrins, growth factor receptors and matrix metalloproteases, and modify their functions in cell adhesion, migration and transmembrane signaling. CD151 is part of the tetraspanin family and it forms tight complexes with β1 and β4 integrins, both of which have been shown to be required for tumorigenesis and/or metastasis in transgenic mouse models of breast cancer. High levels of the tetraspanin CD151 have been linked to poor patient outcome in several human cancers including breast cancer. In addition, CD151 has been implicated as a promoter of tumor angiogenesis and metastasis in various model systems. Methods Here we investigated the effect of Cd151 deletion on mammary tumorigenesis by crossing Cd151-deficient mice with a spontaneously metastasising transgenic model of breast cancer induced by the polyoma middle T antigen (PyMT) driven by the murine mammary tumor virus promoter (MMTV). Results Cd151 deletion did not affect the normal development and differentiation of the mammary gland. While there was a trend towards delayed tumor onset in Cd151−/− PyMT mice compared to Cd151+/+ PyMT littermate controls, this result was only approaching significance (Log-rank test P-value =0.0536). Interestingly, Cd151 deletion resulted in significantly reduced numbers and size of primary tumors but did not appear to affect the number or size of metastases in the MMTV/PyMT mice. Intriguingly, no differences in the expression of markers of cell proliferation, apoptosis and blood vessel density was observed in the primary tumors. Conclusion The findings from this study provide additional evidence that CD151 acts to enhance tumor formation initiated by a range of oncogenes and strongly support its relevance as a potential therapeutic target to delay breast cancer progression. PMID:25012362

  3. Leukocyte populations and cytokine expression in the mammary gland in a mouse model of Streptococcus agalactiae mastitis.

    PubMed

    Trigo, Gabriela; Dinis, Márcia; França, Angela; Bonifácio Andrade, Elva; Gil da Costa, Rui M; Ferreira, Paula; Tavares, Delfina

    2009-07-01

    Streptococcus agalactiae is a contagious, mastitis-causing pathogen that is highly adapted to survive in the bovine mammary gland. This study used a BALB/c mouse model of Streptococcus agalactiae mastitis to evaluate leukocyte populations in regional lymph nodes and cytokine expression in the mammary gland involved in the immune response against Streptococcus agalactiae. It was found that the bacteria replicated efficiently in the mammary gland, peaking after 24 h and increasing by 100-fold. Dissemination of bacteria to systemic organs was observed 6 h after infection. At the same time, a massive infiltration of polymorphonuclear cells and an increase in the inflammatory cytokines interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha were detected in mammary glands, indicating an early inflammatory response. A decrease in the levels of inflammatory cytokines in mammary glands was observed 72 h after infection, accompanied by an increase in the levels of IL-12 and IL-10, which were related to a gradual decrease in bacterial load. An increase in the number of macrophages and B220(+) lymphocytes and similar increases in both CD4(+) and CD8(+) T cells in regional lymph nodes were observed, being most pronounced 5 days after infection. Moreover, increased levels of anti-Streptococcus agalactiae antibodies in the mammary gland were observed 10 days after infection. Overall, these data suggest that the host exhibits both innate and acquired immune responses in response to Streptococcus agalactiae mastitis.

  4. Comparison of virulence of ovine respiratory mycoplasmas in the mouse mammary gland.

    PubMed

    Buddle, B M; Herceg, M; Davies, D H

    1984-08-01

    The virulence of isolates of Mycoplasma ovipneumoniae and M. arginini from pneumonic and unaffected ovine lungs was compared in a mouse mammary gland model. The isolates varied in their ability to induce a neutrophilic response in the mammary gland. A moderate to severe form of mastitis was induced by 3 M. ovipneumoniae isolates recovered from pneumonic lungs, while the remaining M. ovipneumoniae isolates from pneumonic lungs and those from unaffected lungs induced a very mild histopathological response. The severity of the mastitis could not be increased by the simultaneous inoculation of a mixture of 5 mycoplasma isolates. Mycoplasma arginini isolates induced only a very mild histopathological response despite having been isolated from pneumonic lungs. The finding that the 3 most virulent M. ovipneumoniae isolates were initially recovered from pneumonic ovine lungs suggested that these virulent isolates may contribute to ovine pneumonia. However, the isolation of M. ovipneumoniae from pneumonic ovine lungs does not necessarily imply that these organisms are the causal agents, since M. ovipneumoniae isolates may vary in virulence.

  5. Interaction of mouse mammary epithelial cells with collagen substrata: regulation of casein gene expression and secretion

    SciTech Connect

    Lee, E.Y.H.P.; Lee, W.H.; Kaetzel, C.S.; Parry, G.; Bissell, M.J.

    1985-03-01

    Mouse mammary epithelial cells (MMEC) secrete certain milk proteins only when cultured on floating collagen gels. The authors demonstrate that modulation of milk proteins by substrata is manifested at several regulatory levels; (i) cells cultured on floating collagen gels have 3- to 10-fold more casein mRNA than cells cultured on plastic or attached collagen gels. (ii) Cells on the latter two flat substrata, nevertheless, synthesize a significant amount of caseins, indicating that the remaining mRNA is functional. (iii) Cells on all substrata are inducible for casein mRNA and casein proteins by prolactin, but the extent of induction is greater on collagen than that on plastic - i.e., the substratum confers an altered degree of inducibility. (iv) Cells on all substrata synthesize casein proteins at rates proportional to the amount of casein mRNA, but the newly synthesized caseins in cells on plastic are degraded intracellularly, whereas those synthesized by cells on floating gels are secreted into the medium. (v) Cells on all substrata examined lose virtually all mRNA for whey acidic protein despite the fact that this mRNA is abundant in the mammary gland itself; the authors conclude that additional, as-yet-unknown, factors are necessary for synthesis and secretion of whey acidic protein in culture.

  6. A new immunization and treatment strategy for mouse mammary tumor virus (MMTV) associated cancers

    PubMed Central

    Braitbard, Ori; Roniger, Maayan; Bar-Sinai, Allan; Rajchman, Dana; Gross, Tamar; Abramovitch, Hillel; Ferla, Marco La; Franceschi, Sara; Lessi, Francesca; Naccarato, Antonio Giuseppe; Mazzanti, Chiara M.; Bevilacqua, Generoso; Hochman, Jacob

    2016-01-01

    Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma or lymphoma in mice. An increasing body of evidence in recent years supports its involvement also in human sporadic breast cancer. It is thus of importance to develop new strategies to impair the development, growth and metastasis of MMTV-associated cancers. The signal peptide of the envelope precursor protein of this virus: MMTV-p14 (p14) is an excellent target for such strategies, due to unique characteristics distinct from its regular endoplasmic reticulum targeting function. These include cell surface expression in: murine cancer cells that harbor the virus, human breast cancer (MCF-7) cells that ectopically express p14, as well as cultured human cells derived from an invasive ductal breast carcinoma positive for MMTV sequences. These findings support its use in signal peptide-based immune targeting. Indeed, priming and boosting mice with p14 elicits a specific anti-signal peptide immune response sufficient for protective vaccination against MMTV-associated tumors. Furthermore, passive immunization using a combination of anti-p14 monoclonal antibodies or the transfer of T-cells from immunized mice (Adoptive Cell Transfer) is also therapeutically effective. With reports demonstrating involvement of MMTV in human breast cancer, we propose the immune-mediated targeting of p14 as a strategy for prevention, treatment and diagnosis of MMTV-associated cancers. PMID:26934560

  7. Overexpression of miR-30b in the Developing Mouse Mammary Gland Causes a Lactation Defect and Delays Involution

    PubMed Central

    Laubier, Johann; Passet, Bruno; Vilotte, Marthe; Castille, Johan; Laloë, Denis; Polyte, Jacqueline; Bouet, Stéphan; Jaffrézic, Florence; Cribiu, Edmond-Paul; Vilotte, Jean-Luc; Le Provost, Fabienne

    2012-01-01

    Background MicroRNA (miRNA) are negative regulators of gene expression, capable of exerting pronounced influences upon the translation and stability of mRNA. They are potential regulators of normal mammary gland development and of the maintenance of mammary epithelial progenitor cells. This study was undertaken to determine the role of miR-30b on the establishment of a functional mouse mammary gland. miR-30b is a member of the miR-30 family, composed of 6 miRNA that are highly conserved in vertebrates. It has been suggested to play a role in the differentiation of several cell types. Methodology/Principal Findings The expression of miR-30b was found to be regulated during mammary gland development. Transgenic mice overexpressing miR-30b in mammary epithelial cells were used to investigate its role. During lactation, mammary histological analysis of the transgenic mice showed a reduction in the size of alveolar lumen, a defect of the lipid droplets and a growth defect of pups fed by transgenic females. Moreover some mammary epithelial differentiated structures persisted during involution, suggesting a delay in the process. The genes whose expression was affected by the overexpression of miR-30b were characterized by microarray analysis. Conclusion/Significance Our data suggests that miR-30b is important for the biology of the mammary gland and demonstrates that the deregulation of only one miRNA could affect lactation and involution. PMID:23029204

  8. Transcription factor access is mediated by accurately positioned nucleosomes on the mouse mammary tumor virus promoter.

    PubMed Central

    Archer, T K; Cordingley, M G; Wolford, R G; Hager, G L

    1991-01-01

    A fragment of the mouse mammary tumor virus (MMTV) promoter was reconstituted from pure histones into a dinucleosome with uniquely positioned octamer cores. Core boundaries for the in vitro-assembled dinucleosome corresponded to the observed in vivo phasing pattern for long terminal repeat nucleosomes A and B. Nuclear factor 1 (NF1), a constituent of the MMTV transcription initiation complex, was excluded from the assembled dinucleosome, whereas the glucocorticoid receptor was able to bind. During transcription of MMTV in vivo, displacement of nucleosome B was necessary to permit assembly of the initiation complex. These results indicate that the nucleoprotein structure of the promoter can provide differential access to sequence-specific DNA-binding proteins and that active chromatin remodeling can occur during transcription activation. Images PMID:1846670

  9. Selection of early-occurring mutations dictates hormone-independent progression in mouse mammary tumor lines.

    PubMed

    Gattelli, Albana; Zimberlin, María N; Meiss, Roberto P; Castilla, Lucio H; Kordon, Edith C

    2006-11-01

    Mice harboring three mouse mammary tumor virus (MMTV) variants develop pregnancy-dependent (PD) tumors that progress to pregnancy-independent (PI) behavior through successive passages. Herein, we identified 10 predominant insertions in PI transplants from 8 independent tumor lines. These mutations were also detected in small cell populations in the early PD passages. In addition, we identified a new viral insertion upstream of the gene Rspo3, which is overexpressed in three of the eight independent tumor lines and codes for a protein very similar to the recently described protein encoded by Int7. This study suggests that during progression towards hormone independence, clonal expansion of cells with specific mutations might be more relevant than the occurrence of new MMTV insertions.

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

    PubMed

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

    2015-07-30

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

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

    PubMed Central

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

    2015-01-01

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

  12. Presence of mouse mammary tumour‐like virus gene sequences may be associated with morphology of specific human breast cancer

    PubMed Central

    Lawson, J S; Tran, D D; Carpenter, E; Ford, C E; Rawlinson, W D; Whitaker, N J; Delprado, W

    2006-01-01

    Background Mouse mammary tumour virus (MMTV) has a proven role in breast carcinogenesis in wild mice and genetically susceptible in‐bred mice. MMTV‐like env gene sequences, which indicate the presence of a replication‐competent MMTV‐like virus, have been identified in some human breast cancers, but rarely in normal breast tissues. However, no evidence for a causal role of an MMTV‐like virus in human breast cancer has emerged, although there are precedents for associations between specific histological characteristics of human cancers and the presence of oncogenic viruses. Aim To investigate the possibility of an association between breast cancer and MMTV‐like viruses. Methods Histological characteristics of invasive ductal human breast cancer specimens were compared with archival MMTV‐associated mammary tumours from C3H experimental mice. The presence of MMTV‐like env DNA sequences in the human breast cancer specimens was determined by polymerase chain reaction and confirmed by Southern hybridisation. Results MMTV‐like env gene sequences were identified in 22 of 59 (37.3%) human breast cancer specimens. Seventeen of 43 (39.5%) invasive ductal carcinoma breast cancer specimens and 4 of 16 (25%) ductal carcinoma in situ specimens had some histological characteristics, which were similar to MMTV‐associated mouse mammary tumours. However, these similarities were not associated with the presence or absence of MMTV‐like gene sequences in the human breast tumour specimens. A significant (p = 0.05) correlation was found between the grade of the human breast cancer and similarity to the mouse mammary tumours. The lower the grade, the greater the similarity. Conclusion Some human breast cancer specimens, in which MMTV‐like env DNA sequences have been identified, were shown to have histological characteristics (morphology) similar to MMTV‐associated mouse mammary tumours. These observations are compatible with, but not conclusive of, an

  13. Three dimensional multiphoton imaging of fresh and whole mount developing mouse mammary glands

    PubMed Central

    2013-01-01

    Background The applications of multiphoton microscopy for deep tissue imaging in basic and clinical research are ever increasing, supplementing confocal imaging of the surface layers of cells in tissue. However, imaging living tissue is made difficult by the light scattering properties of the tissue, and this is extraordinarily apparent in the mouse mammary gland which contains a stroma filled with fat cells surrounding the ductal epithelium. Whole mount mammary glands stained with Carmine Alum are easily archived for later reference and readily viewed using bright field microscopy to observe branching architecture of the ductal network. Here, we report on the advantages of multiphoton imaging of whole mount mammary glands. Chief among them is that optical sectioning of the terminal end bud (TEB) and ductal epithelium allows the appreciation of abnormalities in structure that are very difficult to ascertain using either bright field imaging of the stained gland or the conventional approach of hematoxylin and eosin staining of fixed and paraffin-embedded sections. A second advantage is the detail afforded by second harmonic generation (SHG) in which collagen fiber orientation and abundance can be observed. Methods GFP-mouse mammary glands were imaged live or after whole mount preparation using a Zeiss LSM510/META/NLO multiphoton microscope with the purpose of obtaining high resolution images with 3D content, and evaluating any structural alterations induced by whole mount preparation. We describe a simple means for using a commercial confocal/ multiphoton microscope equipped with a Ti-Sapphire laser to simultaneously image Carmine Alum fluorescence and collagen fiber networks by SHG with laser excitation set to 860 nm. Identical terminal end buds (TEBs) were compared before and after fixation, staining, and whole mount preparation and structure of collagen networks and TEB morphologies were determined. Flexibility in excitation and emission filters was explored

  14. The in vivo Therapeutic Effect of Free Wanderer Powder ( xiāo yáo sǎn, Xiaoyaosan) on Mice with 4T1 Cell Induced Breast Cancer Model.

    PubMed

    Chen, Wen-Fang; Xu, Li; Yu, Chung-Him; Ho, Chui-Kwan; Wu, Ka; Leung, Gina Cw; Wong, Man-Sau

    2012-01-01

    In the present study, we investigated the therapeutic effect of a classical TCM formula, Free Wanderer Powder ( xiāo yáo sǎn), in a breast cancer mouse model induced with estrogen-insensitive breast cancer 4T1 cells. Ovariectomized Balb/c mice (6-8 weeks) or sham mice were injected into the fourth mammary fat pad with 4T1 cells in which tumors were palpable 7 days after injection. On the eighth day, the mice were divided into 4 groups and tubefed daily with vehicle, Free Wanderer Powder ( xiāo yáo sǎn) formula or tamoxifen for 28 days. Tumor growth inhibition and the decrease of the average tumor mass were most evident in mice treated with Free Wanderer Powder ( xiāo yáo sǎn). Free Wanderer Powder ( xiāo yáo sǎn) treatment significantly reduced Bcl-2 and elevated Bax and p53 protein expressions in breast cancer tumor. These results were further confirmed by immunohistochemisty. Tamoxifen could decrease spleen mass and Bcl-2 protein expression, increase the Bax protein expression as well as exert uterotrophic effects by increasing uterus index and inducing the gene expressions in the uterus. Taken together, these results show that Free Wanderer Powder ( xiāo yáo sǎn) treatment induced apoptosis at protein level and inhibited the tumor growth in 4T1-induced ovariectomized Balb/c female mice, indicating the possibility of its future use for treatment of estrogen-insensitive breast caner.

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

    SciTech Connect

    Lee, E.Y.H.; Parry, G.; Bissell, M.J.

    1984-01-01

    It has been shown previously that cultures of mouse mammary epithelial cells retain their characteristic morphology and their ability to produce ..gamma..-casein, a member of the casein gene family, only if they are maintained on floating collagen gels. In this paper we show: (a) Cells on floating collagen gels secrete not only ..gamma..-casein but also ..cap alpha../sub 1/-, ..cap alpha../sub 2/-, and ..beta..-caseins. These are not secreted by cells on plastic and are secreted to only a very limited extent by cells on attached collagen gels. (b) The floating collagen gel regulates at the level of synthesis and/or stabilization of the caseins rather than at the level of secretion alone. Contraction of the floating gel is important in that cells cultured on floating glutaraldehyde cross-linked gels do not secrete any of the caseins. (c) The secretion of an 80,000-mol-wt protein, most probably transferrin, and a 67,000-mol-wt protein, probably butyrophilin, a major protein of the milk fat globule membrane, are partially modulated by substrata. However, in contrast to the caseins, these are always detectable in media from cells cultured on plastic and attached gels. (d) Whey acidic protein, a major whey protein, is actively secreted by freshly isolated cells but is secreted in extremely limited quantities in cultured cells regardless of the nature of the substratum used. Lactalbumin secretion is also decreased significantly in cultured cells. (e) A previously unreported set of proteins, which may be minor milk proteins, are prominently secreted by the mammary cells on all substrata tested. We conclude that while the substratum profoundly influences the secretion of the caseins, it does not regulate the expression of every milk-specific protein in the same way. The mechanistic implications of these findings are discussed.

  16. Dissecting the dynamics of dysregulation of cellular processes in mouse mammary gland tumor

    PubMed Central

    2009-01-01

    Background Elucidating the sequence of molecular events underlying breast cancer formation is of enormous value for understanding this disease and for design of an effective treatment. Gene expression measurements have enabled the study of transcriptome-wide changes involved in tumorigenesis. This usually occurs through identification of differentially expressed genes or pathways. Results We propose a novel approach that is able to delineate new cancer-related cellular processes and the nature of their involvement in tumorigenesis. First, we define modules as densely interconnected and functionally enriched areas of a Protein Interaction Network. Second, 'differential expression' and 'differential co-expression' analyses are applied to the genes in these network modules, allowing for identification of processes that are up- or down-regulated, as well as processes disrupted (low co-expression) or invoked (high co-expression) in different tumor stages. Finally, we propose a strategy to identify regulatory miRNAs potentially responsible for the observed changes in module activities. We demonstrate the potential of this analysis on expression data from a mouse model of mammary gland tumor, monitored over three stages of tumorigenesis. Network modules enriched in adhesion and metabolic processes were found to be inactivated in tumor cells through the combination of dysregulation and down-regulation, whereas the activation of the integrin complex and immune system response modules is achieved through increased co-regulation and up-regulation. Additionally, we confirmed a known miRNA involved in mammary gland tumorigenesis, and present several new candidates for this function. Conclusions Understanding complex diseases requires studying them by integrative approaches that combine data sources and different analysis methods. The integration of methods and data sources proposed here yields a sensitive tool, able to pinpoint new processes with a role in cancer, dissect

  17. Polyproteins related to the major core protein of mouse mammary tumor virus.

    PubMed Central

    Dickson, C; Atterwill, M

    1978-01-01

    The mouse mammary tumor virus (MuMTV) contains several low-molecular-weight proteins which, together with the genomic RNA, constitute the core structure of the virion. The most abundant protein in the core is the 27,000-dalton protein (p27), and, by analogy to the type C viruses, this protein probably forms the core shell. In mouse mammary tumor cell lines (GR and Mm5MT) producing MuMTV the major p57 antigenic specificity resides in a large protein, which migrates in polyacrylamide gels as a doublet of 77,000 and 75,000 daltons (p 77/75). A series of lower-molecular-weight proteins, p61, p48, p38, and p34, is also present in small amounts and is probably derived by proteolytic cleavage of the p 77/75. These proteins have been identified by immunoprecipitation with monospecific antiserum, and their sequence relatedness to p27 has been determined by an analysis of the peptides after trypsin digestion. After a 15-min pulse with [35S]-methionine, all of the p27-related proteins in these cell lines were labelled and, during a subsequent chase, progressively disappeared. The p27 was labeled poorly during the pulse, but the amount of label in this protein increased during the chase. A quantitation of these experiments suggested that the majority of the p27-related proteins were quite rapidly turned over in these cell lines. Hence, if p27 is derived by a progressive proteolytic cleavage mechanism, then the process is inefficient in the GR cells and only moderately efficient in the Mm5MT cells. When MuMTV was isolated from the culture medium of these cells harvested at 5-min intervals, the major p27-related protein was p34. The p27 accounted for only 29% of the anti-p27 serum immunoprecipitable proteins compared to 95% in virus isolated from an 18-h harvest. Incubation of the rapid-harvest virus at 37 degrees C for 2 h resulted in some conversion of p34 to p27. These results suggest that some of the p27 in MuMTV is formed in the virions by proteolytic cleavage of p34

  18. Regulation of lipoprotein lipase activity and mRNA in the mammary gland of the lactating mouse.

    PubMed Central

    Jensen, D R; Gavigan, S; Sawicki, V; Witsell, D L; Eckel, R H; Neville, M C

    1994-01-01

    We examined the effects of reproductive stage and fasting on lipoprotein lipase (LPL) activity and mRNA in the mouse mammary gland. Heparin-releasable and cell-associated LPL activity rose immediately after birth, followed 1-2 days later by an increase in LPL mRNA. Fasting decreased LPL activity in the mammary gland at all reproductive stages. During lactation, both milk and heparin-releasable LPL were substantially decreased by an overnight fast, whereas cell-associated LPL was less affected and LPL mRNA did not change. These studies indicate that the extracellular, heparin-releasable, fraction of mammary LPL activity responds most rapidly to alterations in physiological state, usually accompanied by smaller changes in cellular enzyme activity. Changes in the level of LPL mRNA were seen only during the transition from pregnancy to lactation, and these tended to follow, rather than precede, changes in enzyme activity. We conclude that in the mammary gland as in adipose tissue, LPL is regulated primarily at the translational and post-translational level. Images Figure 1 PMID:8135737

  19. Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module.

    PubMed

    McIlrath, Victoria; Trye, Alice; Aguanno, Ann

    2015-06-18

    Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

  20. Nuclear, optical, and magnetic resonance imaging in a mouse mammary window chamber model

    NASA Astrophysics Data System (ADS)

    Leung, Hui Min; Schafer, Rachel; Gmitro, Arthur F.

    2014-09-01

    An orthotopic mouse mammary window chamber (MWC) model has been developed for multimodal in-vivo functional and anatomical imaging of breast cancer xenografts. Capabilities to image numerous physiological aspects of the same tumor microenvironment over time has important applications such as in experiments studying the efficacies of therapeutic interventions, improvement of cancer detection and investigating basic cancer biology. The compatibility of this MWC model with optical, nuclear and magnetic resonance imaging (MRI) makes it possible to perform a multitude of studies ranging from cellular imaging to whole body imaging. Thus, the MWC represents a powerful tool for breast cancer research. Here, two imaging applications are highlighted, namely the nuclear imaging of glycolytic metabolism with 18FFDG and MRI of tissue perfusion. Nuclear imaging is performed with the use of a 3μm thin phosphor scintillator placed directly in contact with the tissue and visible light from the scintillation is directly detected in a low noise, light tight imaging system. Tissue perfusion is imaged either qualitatively with a dynamic contrast enhancement (DCE) MRI technique or quantitatively with an arterial spin labeling flow-sensitive alternating inversion recovery-rapid acquisition with relaxation enhancement (FAIR-RARE) technique.

  1. Endogenization of mouse mammary tumor virus (MMTV)-like elements in genomes of pikas (Ochotona sp.).

    PubMed

    Lemos de Matos, Ana; de Sousa-Pereira, Patrícia; Lissovsky, Andrey A; van der Loo, Wessel; Melo-Ferreira, José; Cui, Jie; Esteves, Pedro J

    2015-12-01

    Despite the finding in European rabbit and other leporid genomes of the first ever described endogenous lentivirus and of a European rabbit exclusive endogenous gammaretrovirus, until now no exogenous retroviruses have been isolated in Lagomorpha species. Nevertheless, looking for the presence of endogenous retroviruses (ERVs) in the species genomes could lead to the discovery of retroviral lineages yet to be found in Lagomorpha. Different mammalian genomes harbor endogenous viral sequences phylogenetically close to the betaretrovirus mouse mammary tumor virus (MMTV), propelling us to look for such retroviral "fossil" in American pika (Ochotona princeps) and European rabbit (Oryctolagus cuniculus) genomes. By performing genomic mining using MMTV gag and LTR as query sequences, we found that such viral elements were absent from the European rabbit genome. Oppositely, significant matches were found in American pika, and more importantly, a nearly complete MMTV-like virus (Pika-BERV) was identified. Using Pika-BERV gag and LTR as templates, we found similar sequences endogenized in different pika (Ochotona sp.) species. The orthology of the LTR flanking region between some pika species supported shared ancestry of specific endogenous betaretroviruses, while in other pika species similar sequences, but not orthologous, should have resulted from independent insertions. Our study supports the possible existence of infecting exogenous betaretroviruses for a long term, after the divergence of Ochotonidae from Leporidae, but yet to be identified. PMID:26151606

  2. Green tea polyphenols as potent enhancers of glucocorticoid-induced mouse mammary tumor virus gene expression.

    PubMed

    Abe, I; Umehara, K; Morita, R; Nemoto, K; Degawa, M; Noguchi, H

    2001-02-16

    The effect of natural and synthetic galloyl esters on glucocorticoid-induced gene expression was evaluated by using rat fibroblast 3Y1 cells stably transfected with a luciferase reporter gene under the transcriptional regulation of the mouse mammary tumor virus promoter. The glucocorticoid-induced gene transcription was strongly suppressed by synthetic alkyl esters; n-dodecyl gallate showed the most potent inhibition (66% inhibition at 10 microM), which was far more potent than that of crude tannic acid. n-Octyl and n-cetyl gallate also showed good inhibition, while gallic acid itself was not so active, suggesting that the presence of hydrophobic side chain is important for the suppressive effect. On the other hand, surprisingly, green tea gallocatechins, (-)-epigallocatechin-3-O-gallate and theasinensin A, potently enhanced the promoter activity (182 and 247% activity at 1 microM, respectively). The regulation of the level of the glucocorticoid-induced gene expression by the antioxidative gallates is of great interest from a therapeutic point of view.

  3. ENDOTOXIN AS ADJUVATOR TO THE TRANSPLANTATION OF A MOUSE MAMMARY TUMOR

    PubMed Central

    Henderson, James Stuart

    1968-01-01

    The mouse mammary tumor MT 296 was used in a further series of experiments on the implantation of tumor, plated out in vivo, from suspensions of individual cells. Lipopolysaccharide from S. typhosa was shown to exert an adjuvator influence. But this adjuvator, an endotoxin, had no direct effect on the suspended tumor cells, unlike the liver preparations previously reported. Lipopolysaccharide from S. typhosa was shown to act on the host. It made the host's connective tissue expanses more susceptible to successful implantation by the tumor cells. It did this only if present at the time these connective tissue expanses were split. The increased susceptibility, caused by splitting the connective tissue expanses in the presence of lipopolysaccharide, declined quickly after 24 hr. The structural changes wrought upon the connective tissues by splitting them in the presence of lipopolysaccharide are described. They show kinship to a Schwartzman reaction of the local type. Their possible role in the adjuvator effect on the plating of single cell suspensions of this tumor is discussed. PMID:5688080

  4. Students Investigating the Antiproliferative Effects of Synthesized Drugs on Mouse Mammary Tumor Cells

    PubMed Central

    2005-01-01

    The potential for personalized cancer management has long intrigued experienced researchers as well as the naïve student intern. Personalized cancer treatments based on a tumor's genetic profile are now feasible and can reveal both the cells' susceptibility and resistance to chemotherapeutic agents. In a weeklong laboratory investigation that mirrors current cancer research, undergraduate and advanced high school students determine the efficacy of common pharmacological agents through in vitro testing. Using mouse mammary tumor cell cultures treated with “unknown” drugs historically recommended for breast cancer treatment, students are introduced to common molecular biology techniques from in vitro cell culture to fluorescence microscopy. Student understanding is assessed through laboratory reports and the successful identification of the unknown drug. The sequence of doing the experiment, applying logic, and constructing a hypothesis gives the students time to discover the rationale behind the cellular drug resistance assay. The breast cancer experiment has been field tested during the past 5 yr with more than 200 precollege/undergraduate interns through the Gains in the Education of Mathematics and Science program hosted by the Walter Reed Army Institute of Research. PMID:16220143

  5. Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

    PubMed Central

    Abt, Melissa A.; Grek, Christina L.; Ghatnekar, Gautam S.; Yeh, Elizabeth S.

    2016-01-01

    Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3–6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue. PMID:26862835

  6. Riboflavin uptake transporter Slc52a2 (RFVT2) is upregulated in the mouse mammary gland during lactation.

    PubMed

    Wu, Alex Man Lai; Dedina, Liana; Dalvi, Pooja; Yang, Mingdong; Leon-Cheon, John; Earl, Brian; Harper, Patricia A; Ito, Shinya

    2016-04-01

    While it is well recognized that riboflavin accumulates in breast milk as an essential vitamin for neonates, transport mechanisms for its milk excretion are not well characterized. The multidrug efflux transporter ABCG2 in the apical membrane of milk-producing mammary epithelial cells (MECs) is involved with riboflavin excretion. However, it is not clear whether MECs possess other riboflavin transport systems, which may facilitate its basolateral uptake into MECs. We report here that transcripts encoding the second (SLC52A2) and third (SLC52A3) member of the recently discovered family of SLC52A riboflavin uptake transporters are expressed in milk fat globules from human breast milk. Furthermore, Slc52a2 and Slc52a3 mRNA are upregulated in the mouse mammary gland during lactation. Importantly, the induction ofSlc52a2, which was the major Slc52a riboflavin transporter in the lactating mammary gland, was also observed at the protein level. Subcellular localization studies showed that green fluorescent protein-tagged mouse SLC52A2 mainly localized to the cell membrane, with no preferential distribution to the apical or basolateral membrane in polarized kidney MDCK cells. These results strongly implicate a potential role for SLC52A2 in riboflavin uptake by milk-producing MECs, a critical step in the transfer of riboflavin into breast milk.

  7. Identification of reliable reference genes for qRT-PCR studies of the developing mouse mammary gland

    PubMed Central

    van de Moosdijk, Anoeska Agatha Alida; van Amerongen, Renée

    2016-01-01

    Cell growth and differentiation are often driven by subtle changes in gene expression. Many challenges still exist in detecting these changes, particularly in the context of a complex, developing tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) allows relatively high-throughput evaluation of multiple genes and developmental time points. Proper quantification of gene expression levels by qRT-PCR requires normalization to one or more reference genes. Traditionally, these genes have been selected based on their presumed “housekeeping” function, with the implicit assumption that they are stably expressed over the entire experimental set. However, this is rarely tested empirically. Here we describe the identification of novel reference genes for the mouse mammary gland based on their stable expression in published microarray datasets. We compared eight novel candidate reference genes (Arpc3, Clock, Ctbp1, Phf7, Prdx1, Sugp2, Taf11 and Usp7) to eight traditional ones (18S, Actb, Gapdh, Hmbs, Hprt, Rpl13a, Sdha and Tbp) and analysed all genes for stable expression in the mouse mammary gland from pre-puberty to adulthood using four different algorithms (GeNorm, DeltaCt, BestKeeper and NormFinder). Prdx1, Phf7 and Ctbp1 were validated as novel and reliable, tissue-specific reference genes that outperform traditional reference genes in qRT-PCR studies of postnatal mammary gland development. PMID:27752147

  8. Measuring oxygen tension modulation, induced by a new pre-radiotherapy therapeutic, in a mammary window chamber mouse model

    NASA Astrophysics Data System (ADS)

    Schafer, Rachel; Gmitro, Arthur F.

    2015-03-01

    Tumor regions under hypoxic or low oxygen conditions respond less effectively to many treatment strategies, including radiation therapy. A novel investigational therapeutic, NVX-108 (NuvOx Pharma), has been developed to increase delivery of oxygen through the use of a nano-emulsion of dodecofluoropentane. By raising pO2 levels prior to delivering radiation, treatment efficacy may be improved. To aid in evaluating the novel drug, oxygen tension was quantitatively measured, spatially and temporally, to record the effect of administrating NVX-108 in an orthotopic mammary window chamber mouse model of breast cancer. The oxygen tension was measured through the use of an oxygen-sensitive coating, comprised of phosphorescent platinum porphyrin dye embedded in a polystyrene matrix. The coating, applied to the surface of the coverslip of the window chamber through spin coating, is placed in contact with the mammary fat pad to record the oxygenation status of the surface tissue layer. Prior to implantation of the window chamber, a tumor is grown in the SCID mouse model by injection of MCF-7 cells into the mammary fat pad. Two-dimensional spatial distributions of the pO2 levels were obtained through conversion of measured maps of phosphorescent lifetime. The resulting information on the spatial and temporal variation of the induced oxygen modulation could provide valuable insight into the optimal timing between administration of NVX-108 and radiation treatment to provide the most effective treatment outcome.

  9. 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

  10. Impact of diethylhexyl phthalate on gene expression and development of mammary glands of pregnant mouse.

    PubMed

    Li, Lan; Liu, Jing-Cai; Zhao, Yong; Lai, Fang-Nong; Yang, Fan; Ge, Wei; Dou, Cheng-Li; Shen, Wei; Zhang, Xi-Feng; Chen, Hong

    2015-10-01

    The widely used diethylhexyl phthalate (DEHP) is a known endocrine disruptor that causes persistent alterations in the structure and function of female reproductive system, including ovaries, uterus and oviducts. To explore the molecular mechanism of the effect of DEHP on the development of mammary glands, we investigated the cell cycle, growth, proliferation and gene expression of mammary gland cells of pregnant mice exposed to DEHP. It was demonstrated, for the first time, that the mammary gland cells of pregnant mice treated with DEHP for 0.5-3.5 days post-coitum had increased proliferation, growth rate and number of cells in the G2/S phase. The expression of cell proliferation-related genes was significantly altered after short time and low-dose DEHP treatment of mammary gland cells in vivo and in vitro. These findings showed adverse effects of DEHP on mammary gland cells in pregnant mice. PMID:26170149

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

    PubMed Central

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

    2016-01-01

    Depletion of Brca1 leads to defects in mouse mammary gland development and mammary tumors in humans and mice. To explore the role of microRNAs (miRNAs) in this process, we examined the mammary glands of MMTV-Cre Brca1Co/Co mice for differential miRNA expression using a candidate approach. Several miRNAs were differentially expressed in mammary tissue at day 1 of lactation and in mammary epithelial cell lines in which Brca1 messenger RNA (mRNA) levels have been reduced. Functional studies revealed that several of these miRNAs regulate mammary epithelial cell function in vitro, including miR-206. Creation and analysis of MMTV-miR-206 transgenic mice showed no effect on lactational mammary development and no tumors, but indicates a role in mammary tissue remodeling in mature mice, potentially involving Igf-1 and Sfrp1. These results indicate the potential of miRNAs to mediate the consequences of Brca1 loss and suggest a novel function for miR-206. PMID:27043663

  12. The Rac Inhibitor EHop-016 Inhibits Mammary Tumor Growth and Metastasis in a Nude Mouse Model

    PubMed Central

    Castillo-Pichardo, Linette; Humphries-Bickley, Tessa; De La Parra, Columba; Forestier-Roman, Ingrid; Martinez-Ferrer, Magaly; Hernandez, Eliud; Vlaar, Cornelis; Ferrer-Acosta, Yancy; Washington, Anthony V.; Cubano, Luis A.; Rodriguez-Orengo, Jose; Dharmawardhane, Suranganie

    2014-01-01

    Metastatic disease still lacks effective treatments, and remains the primary cause of cancer mortality. Therefore, there is a critical need to develop better strategies to inhibit metastatic cancer. The Rho family GTPase Rac is an ideal target for anti-metastatic cancer therapy, because Rac is a key molecular switch that is activated by a myriad of cell surface receptors to promote cancer cell migration/invasion and survival. Previously, we reported the design and development of EHop-016, a small molecule compound, which inhibits Rac activity of metastatic cancer cells with an IC50 of 1 μM. EHop-016 also inhibits the activity of the Rac downstream effector p21-activated kinase (PAK), lamellipodia extension, and cell migration in metastatic cancer cells. Herein, we tested the efficacy of EHop-016 in a nude mouse model of experimental metastasis, where EHop-016 administration at 25 mg/kg body weight (BW) significantly reduced mammary fat pad tumor growth, metastasis, and angiogenesis. As quantified by UPLC MS/MS, EHop-016 was detectable in the plasma of nude mice at 17 to 23 ng/ml levels at 12 h following intraperitoneal (i.p.) administration of 10 to 25 mg/kg BW EHop-016. The EHop-016 mediated inhibition of angiogenesis In Vivo was confirmed by immunohistochemistry of excised tumors and by In Vitro tube formation assays of endothelial cells. Moreover, EHop-016 affected cell viability by down-regulating Akt and Jun kinase activities and c-Myc and Cyclin D expression, as well as increasing caspase 3/7 activities in metastatic cancer cells. In conclusion, EHop-016 has potential as an anticancer compound to block cancer progression via multiple Rac-directed mechanisms. PMID:25389450

  13. Thermochemotherapy in vivo of a C3H mouse mammary carcinoma: thermotolerant tumours.

    PubMed

    Monge, O R; Rofstad, E K

    1989-01-01

    The ability of cyclophosphamide (CTX) and mitomycin C (MMC) to modify the expression of thermotolerance in vivo at 43.5 degrees C was investigated in a transplantable C3H mouse mammary carcinoma grown s.c. in feet of C3D2F1/Bom mice. Dose-effect curves subjected to linear regression analysis were constructed for single-fraction treatment and for a second treatment 24 h after a priming heat treatment of 43.5 degrees C for 30 min. Tumour volume doubling time during regrowth showed no significant variation among treatment groups, justifying the use of tumour growth time as effect parameter. Thermotolerance ratio for heat alone was 11.6 +/- 2.3. Drug enhancement ratio in thermotolerant tumours was 6.2 +/- 1.4 for CTX (100 mg/kg) and 3.5 +/- 0.9 for MMC (3 mg/kg). These values are about 4 and 3 times larger than the corresponding enhancement ratios found for previously untreated tumours. Thermotolerance ratio for thermochemotherapy was 2.6 +/- 0.3 for CTX and 4.4 +/- 0.7 for MMC, i.e. the degree of thermotolerance was substantially reduced by both drugs, but not completely overcome. Thermal enhancement ratio for CTX and MMC was about equally large in thermotolerant and previously untreated tumours. Thermotolerant tumours showed a tendency for increased resistance to drug treatment alone. Both CTX and MMC may be used clinically to reduce the expression of thermotolerance, particularly in situations with inhomogeneous heating and short fractionation intervals.

  14. Effect of glycogen synthase kinase-3 inactivation on mouse mammary gland development and oncogenesis

    PubMed Central

    Dembowy, J; Adissu, H A; Liu, J C; Zacksenhaus, E; Woodgett, J R

    2015-01-01

    Many components of the Wnt/β-catenin signaling pathway have critical functions in mammary gland development and tumor formation, yet the contribution of glycogen synthase kinase-3 (GSK-3α and GSK-3β) to mammopoiesis and oncogenesis is unclear. Here, we report that WAP-Cre-mediated deletion of GSK-3 in the mammary epithelium results in activation of Wnt/β-catenin signaling and induces mammary intraepithelial neoplasia that progresses to squamous transdifferentiation and development of adenosquamous carcinomas at 6 months. To uncover possible β-catenin-independent activities of GSK-3, we generated mammary-specific knockouts of GSK-3 and β-catenin. Squamous transdifferentiation of the mammary epithelium was largely attenuated, however, mammary epithelial cells lost the ability to form mammospheres suggesting perturbation of stem cell properties unrelated to loss of β-catenin alone. At 10 months, adenocarcinomas that developed in glands lacking GSK-3 and β-catenin displayed elevated levels of γ-catenin/plakoglobin as well as activation of the Hedgehog and Notch pathways. Collectively, these results establish the two isoforms of GSK-3 as essential integrators of multiple developmental signals that act to maintain normal mammary gland function and suppress tumorigenesis. PMID:25195860

  15. Molecular cloning and characterization of the mouse carboxyl ester lipase gene and evidence for expression in the lactating mammary gland

    SciTech Connect

    Lidmer, A.S.; Lundberg, L.; Kannius, M.; Bjursell, G.

    1995-09-01

    DNA hybridization was used to isolate a 2.04-kb cDNA encoding carboxyl ester lipase (CEL) from a mouse lactating mammary gland, {lambda}gt10 cDNA library. The cDNA sequence translated into a protein of 599 amino acids, including 20 amino acids of a putative signal peptide. Comparison of the deduced amino acid sequence of the mouse CEL with CEL from five other species revealed that there is a high degree of a homology between the different species. The mouse CEL gene was also isolated and found to span approximately 7.2 kb and to include 11 exons. This organization is similar to those of the recently reported human and rat CEL genes. We have also analyzed expression of the CEL gene in the mammary glands from other species by performing a Northern blot analysis with RNA from goat and cow. The results show that the gene is expressed in both species. 36 refs., 6 figs., 1 tab.

  16. TRAM-Derived Decoy Peptides inhibits the inflammatory response in mouse mammary epithelial cells and a mastitis model in mice.

    PubMed

    Hu, Xiaoyu; Tian, Yuan; Wang, Tiancheng; Zhang, Wenlong; Wang, Wei; Gao, Xuejiao; Qu, Shihui; Cao, Yongguo; Zhang, Naisheng

    2015-10-01

    It has been proved that TRAM-Derived Decoy peptides have anti-inflammatory properties. In this study, we synthesized a TRAM-Derived decoy peptide (TM6), belongs to TRAM TIR domain, of which sequence is "N"-RQIKIWFQNRRMKWK, KENFLRDTWCNFQFY-"C" and evaluated the effects of TM6 on lipopolysaccharide-induced mastitis in mice. In vivo, LPS-induced mice mastitis model was established by injection of LPS through the duct of mammary gland. TM6 was injected 1h before or after LPS treatment. In vitro, primary mouse mammary epithelial cells were used to investigate the effects of TM6 on LPS-induced inflammatory responses. The results showed that TM6 inhibited LPS-induced mammary gland histopathologic changes, MPO activity, and TNF-α, IL-1β and IL-6 production in mice. In vitro, TM6 significantly inhibited LPS-induced TNF-α and IL-6 production, as well as NF-κB and MAPKs activation. In conclusion, this study demonstrated that TM6 had protective effects on LPS-mastitis and may be a promising therapeutic reagent for mastitis treatment.

  17. Endocrine hormones and local signals during the development of the mouse mammary gland.

    PubMed

    Brisken, Cathrin; Ataca, Dalya

    2015-01-01

    Most of mammary gland development occurs postnatally under the control of female reproductive hormones, which in turn interact with other endocrine factors. While hormones impinge on many tissues and trigger very complex biological responses, tissue recombination experiments with hormone receptor-deficient mammary epithelia revealed eminent roles for estrogens, progesterone, and prolactin receptor (PrlR) signaling that are intrinsic to the mammary epithelium. A subset of the luminal mammary epithelial cells expresses the estrogen receptor α (ERα), the progesterone receptor (PR), and the PrlR and act as sensor cells. These cells convert the detected systemic signals into local signals that are developmental stage-dependent and may be direct, juxtacrine, or paracrine. This setup ensures that the original input is amplified and that the biological responses of multiple cell types can be coordinated. Some key mediators of hormone action have been identified such as Wnt, EGFR, IGFR, and RANK signaling. Multiple signaling pathways such as FGF, Hedgehog, and Notch signaling participate in driving different aspects of mammary gland development locally but how they link to the hormonal control remains to be elucidated. An increasing number of endocrine factors are appearing to have a role in mammary gland development, the adipose tissue is increasingly recognized to play a role in endocrine regulation, and a complex role of the immune system with multiple different cell types is being revealed. For further resources related to this article, please visit the WIREs website.

  18. Flor-Essence® herbal tonic does not inhibit estrogen receptor negative mammary tumor development in a transgenic mouse model

    PubMed Central

    Bennett, L. Michelle; Montgomery, Jennifer L.; Collins, N. Keith; Steinberg, Seth M.; Kulp, Kristen S.

    2012-01-01

    Women who are diagnosed with breast cancer often self-administer complementary and alternative medicines to augment their conventional treatments, improve health, or prevent recurrence. Flor-Essence® herbal tonic is a complex mixture of eight herbal extracts used by cancer patients because of anecdotal evidence that it can treat or prevent disease. In this study four experimental groups of female MMTV-Neu mice were left untreated or treated with 3% Flor-Essence® in utero, from birth until 5 weeks of age, or throughout their lifetime. Palpable mammary tumor incidence and body weight was determined weekly for each group. The mice were sacrificed at 28 weeks of age and mammary tumors were enumerated to determine average tumor incidence and multiplicity for each group. Female mice exposed to Flor-Essence® herbal tonic in utero weighed significantly more than the control group (p < 0.001). The average tumor incidence and tumor multiplicity in the experimental mice treated with Flor-Essence® herbal tonic did not differ from the control animals. Flor-Essence® does not inhibit mammary tumor incidence or mammary tumor multiplicity in MMTV-Neu transgenic mice. Flor-Essence® exposure in utero causes increased body weight in experimental animals. This conclusion challenges widely available anecdotal information as well as the hopes of the consumer that this product will inhibit or suppress tumor development. Lay Abstract Flor-Essence® herbal tonic is a complex mixture of eight herbal extracts often used by women with breast cancer in hopes that it will help cure disease or prevent recurrence. There is currently very little scientific data to support or refute its self-administration. We tested whether Flor-Essence® would influence tumor development in the mammary glands of a mouse model of Her2/neu breast cancer. The tonic was given at different life stages to determine if timing of the exposure influenced the response to treatment. This report shows that Flor

  19. High resolution 3D MRI of mouse mammary glands with intra-ductal injection of contrast media.

    PubMed

    Markiewicz, Erica; Fan, Xiaobing; Mustafi, Devkumar; Zamora, Marta; Roman, Brian B; Jansen, Sanaz A; Macleod, Kay; Conzen, Suzanne D; Karczmar, Gregory S

    2015-01-01

    The purpose of this study was to use high resolution three-dimensional (3D) magnetic resonance imaging (MRI) to study mouse mammary gland ductal architecture based on intra-ductal injection of contrast agents. Female FVB/N mice age 12-20 weeks (n=12), were used in this study. A 34G, 45° tip Hamilton needle with a 25μL Hamilton syringe was inserted into the tip of the nipple. Approximately 20-25μL of a Gadodiamide/Trypan blue/saline solution was injected slowly over one minute into the nipple and duct. To prevent washout of contrast media from ducts due to perfusion, and maximize the conspicuity of ducts on MRI, mice were sacrificed one minute after injection. High resolution 3D T1-weighted images were acquired on a 9.4T Bruker scanner after sacrifice to eliminate motion artifacts and reduce contrast media leakage from ducts. Trypan blue staining was well distributed throughout the ductal tree. MRI showed the mammary gland ductal structure clearly. In spoiled gradient echo T1-weighted images, the signal-to-noise ratio of regions identified as enhancing mammary ducts following contrast injection was significantly higher than that of muscle (p<0.02) and significantly higher than that of contralateral mammary ducts that were not injected with contrast media (p<0.0001). The methods described here could be adapted for injection of specialized contrast agents to measure metabolism or target receptors in normal ducts and ducts with in situ cancers.

  20. Induction of IL-25 secretion from tumour-associated fibroblasts suppresses mammary tumour metastasis

    PubMed Central

    Yin, Shu-Yi; Jian, Feng-Yin; Chen, Yung-Hsiang; Chien, Shih-Chang; Hsieh, Mao-Chih; Hsiao, Pei-Wen; Lee, Wen-Hwa; Yang, Ning-Sun

    2016-01-01

    Tumour-associated fibroblasts (TAFs), as a functionally supportive microenvironment, play an essential role in tumour progression. Here we investigate the role of IL-25, an endogenous anticancer factor secreted from TAFs, in suppression of mouse 4T1 mammary tumour metastasis. We show that a synthetic dihydrobenzofuran lignan (Q2-3), the dimerization product of plant caffeic acid methyl ester, suppresses 4T1 metastasis by increasing fibroblastic IL-25 activity. The secretion of IL-25 from treated human or mouse fibroblasts is enhanced in vitro, and this activity confers a strong suppressive effect on growth activity of test carcinoma cells. Subsequent in vivo experiments showed that the anti-metastatic effects of Q2-3 on 4T1 and human MDA-MD-231 tumour cells are additive when employed in combination with the clinically used drug, docetaxel. Altogether, our findings reveal that the release of IL-25 from TAFs may serve as a check point for control of mammary tumour metastasis and that phytochemical Q2-3 can efficiently promote such anticancer activities. PMID:27089063

  1. Insertion mutation of the int-1 and int-2 loci by mouse mammary tumor virus in premalignant and malignant neoplasms from the GR mouse strain.

    PubMed Central

    Morris, D W; Barry, P A; Bradshaw, H D; Cardiff, R D

    1990-01-01

    Mouse mammary tumor virus (MMTV)-induced mammary adenocarcinomas can develop from several different premalignant precursors common in GR mice. Insertion mutagenesis of the mammary protooncogenes int-1 and int-2 was studied in this multistep system by analyzing samples from various stages of neoplastic development for novel int-1 and int-2 restriction fragments generated by MMTV provirus integration. int-1 and int-2 insertion mutations were observed in both premalignant lesions and malignant tumors. Some of the tumors with insertion mutations were experimentally derived from insertion mutation-free premalignant precursors. Each class of neoplasm examined had a characteristic frequency of int-1 and int-2 insertion mutations; however, no correspondence was observed between neoplasm morphology and mutation of either gene. These results indicate that insertion mutation of the int-1 and int-2 loci by MMTV provirus can be involved in the earliest identifiable stages of neoplastic development as well as during progression of premalignant lesions to tumors. Insertion mutation of int-1 and int-2 is therefore not stage specific in this system. Images PMID:2157060

  2. Rejection of metastatic 4T1 breast cancer by attenuation of Treg cells in combination with immune stimulation.

    PubMed

    Chen, Li; Huang, Tian-Gui; Meseck, Marcia; Mandeli, John; Fallon, John; Woo, Savio L C

    2007-12-01

    4T1 breast carcinoma is a highly malignant and poorly immunogenic murine tumor model that resembles advanced breast cancer in humans, and is refractory to most immune stimulation-based treatments. We hypothesize that the ineffectiveness of immune stimulatory treatment is mediated by the suppressive effects of CD4(+)CD25(+) regulatory T (Treg) cells, which can be attenuated by engaging the glucocorticoid-induced tumor necrosis factor receptor family-related protein with its natural ligand (GITRL); further, combination treatment with existing immune stimulation regimens will augment anti-tumor immunity and eradicate metastatic 4T1 tumors in mice.A soluble homodimeric form of mouse GITRL (mIg-mGITRLs) was molecularly constructed and used to treat orthotopic 4T1 tumors established in immune-competent, syngeneic Balb/c mice. When applied in combination with adenovirus-mediated intratumoral murine granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-12 (IL-12) gene delivery plus systemic 4-1BB activation, mIg-mGITRLs attenuated the immune-suppressive function of splenic Treg cells, which led to elevated interferon-gamma (IFN-gamma) production, tumor-specific cytolytic T-cell activities, tumor rejection and long-term survival in 65% of the animals without apparent toxicities. The results demonstrate that addition of mIg-mGITRLs to an immune-stimulatory treatment regimen significantly improved long-term survival without apparent toxicity, and could potentially be clinically translated into an effective and safe treatment modality for metastatic breast cancer in patients. PMID:17968355

  3. Quantitative Assessment of Mouse Mammary Gland Morphology Using Automated Digital Image Processing and TEB Detection.

    PubMed

    Blacher, Silvia; Gérard, Céline; Gallez, Anne; Foidart, Jean-Michel; Noël, Agnès; Péqueux, Christel

    2016-04-01

    The assessment of rodent mammary gland morphology is largely used to study the molecular mechanisms driving breast development and to analyze the impact of various endocrine disruptors with putative pathological implications. In this work, we propose a methodology relying on fully automated digital image analysis methods including image processing and quantification of the whole ductal tree and of the terminal end buds as well. It allows to accurately and objectively measure both growth parameters and fine morphological glandular structures. Mammary gland elongation was characterized by 2 parameters: the length and the epithelial area of the ductal tree. Ductal tree fine structures were characterized by: 1) branch end-point density, 2) branching density, and 3) branch length distribution. The proposed methodology was compared with quantification methods classically used in the literature. This procedure can be transposed to several software and thus largely used by scientists studying rodent mammary gland morphology. PMID:26910307

  4. Stably integrated mouse mammary tumor virus long terminal repeat DNA requires the octamer motifs for basal promoter activity.

    PubMed Central

    Buetti, E

    1994-01-01

    In the mouse mammary tumor virus promoter, a tandem of octamer motifs, recognized by ubiquitous and tissue-restricted Oct transcription factors, is located upstream of the TATA box and next to a binding site for the transcription factor nuclear factor I (NF-I). Their function was investigated with mutant long terminal repeats under different transfection conditions in mouse Ltk- cells and quantitative S1 nuclease mapping of the transcripts. In stable transfectants, which are most representative of the state of proviral DNA with respect to both number of integrated DNA templates and chromatin organization, a long terminal repeat mutant of both octamer sites showed an average 50-fold reduction of the basal transcription level, while the dexamethasone-stimulated level was unaffected. DNase I in vitro footprinting assays with L-cell nuclear protein extracts showed that the mutant DNA was unable to bind octamer factors but had a normal footprint in the NF-I site. I conclude that mouse mammary tumor virus employs the tandem octamer motifs of the viral promoter, recognized by the ubiquitous transcription factor Oct-1, for its basal transcriptional activity and the NF-I binding site, as previously shown, for glucocorticoid-stimulated transcription. A deletion mutant with only one octamer site showed a marked base-level reduction at high copy number but little reduction at low copies of integrated plasmids. The observed transcription levels may depend both on the relative ratio of transcription factors to DNA templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting. Images PMID:8289800

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

    SciTech Connect

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

    1987-09-01

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

  6. Epigenetic modifications unlock the milk protein gene loci during mouse mammary gland development and differentiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Unlike with other tissues, development and differentiation of the mammary gland occur mostly after birth. The roles of systemic hormones and local growth factors important for this development and functional differentiation are well-studied. In other tissues, it has been shown that chromatin organiz...

  7. 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.

  8. Proteomics of Genetically Engineered Mouse Mammary Tumors Identifies Fatty Acid Metabolism Members as Potential Predictive Markers for Cisplatin Resistance*

    PubMed Central

    Warmoes, Marc; Jaspers, Janneke E.; Xu, Guotai; Sampadi, Bharath K.; Pham, Thang V.; Knol, Jaco C.; Piersma, Sander R.; Boven, Epie; Jonkers, Jos; Rottenberg, Sven; Jimenez, Connie R.

    2013-01-01

    In contrast to various signatures that predict the prognosis of breast cancer patients, markers that predict chemotherapy response are still elusive. To detect such predictive biomarkers, we investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes who have a differential knock-out status for the breast cancer 1, early onset (Brca1) gene. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared with that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma. The analyses were performed 24 h after administration of the maximum tolerable dose of cisplatin. At this time point, drug-sensitive BRCA1-deficient tumors showed DNA damage, but cells were largely viable. By applying paired statistics and quantitative filtering, we identified highly discriminatory markers for the sensitive and resistant model. Proteins up-regulated in the sensitive model are involved in centrosome organization, chromosome condensation, homology-directed DNA repair, and nucleotide metabolism. Major discriminatory markers that were up-regulated in the resistant model were predominantly involved in fatty acid metabolism, such as fatty-acid synthase. Specific inhibition of fatty-acid synthase sensitized resistant cells to cisplatin. Our data suggest that exploring the functional link between the DNA damage response and cancer metabolism shortly after the initial treatment may be a useful strategy to predict the efficacy of cisplatin. PMID:23397111

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

    SciTech Connect

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

    1990-12-01

    Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-2D has been developed in which more than 35% of the cells express {beta}-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete {beta}-casein undirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine {beta}-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency. This regulation occurered primarily at the transcriptional level and was dependent on the length of the 5{prime} flanking region of the {beta}-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous {beta}-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.

  10. No association between Epstein-Barr Virus and Mouse Mammary Tumor Virus with Breast Cancer in Mexican Women

    NASA Astrophysics Data System (ADS)

    Morales-Sánchez, Abigail; Molina-Muñoz, Tzindilú; Martínez-López, Juan L. E.; Hernández-Sancén, Paulina; Mantilla, Alejandra; Leal, Yelda A.; Torres, Javier; Fuentes-Pananá, Ezequiel M.

    2013-10-01

    Breast cancer is the most frequent malignancy affecting women worldwide. It has been suggested that infection by Epstein Barr Virus (EBV), Mouse Mammary Tumor Virus or a similar virus, MMTV-like virus (MMTV-LV), play a role in the etiology of the disease. However, studies looking at the presence of these viruses in breast cancer have produced conflicting results, and this possible association remains controversial. Here, we used polymerase chain reaction assay to screen specific sequences of EBV and MMTV-LV in 86 tumor and 65 adjacent tissues from Mexican women with breast cancer. Neither tumor samples nor adjacent tissue were positive for either virus in a first round PCR and only 4 tumor samples were EBV positive by a more sensitive nested PCR. Considering the study's statistical power, these results do not support the involvement of EBV and MMTV-LV in the etiology of breast cancer.

  11. The 5' leader sequence of mouse mammary tumor virus enhances expression of the envelope and reporter genes.

    PubMed

    Hohenadl, Christine; Gunzburg, Walter H; Salmons, Brian; Indik, Stanislav

    2012-02-01

    Mouse mammary tumor virus (MMTV) is a complex betaretrovirus, which utilizes a Rev-like auxiliary protein Rem to export the unspliced viral RNA from the nucleus. MMTV env mRNA appears to be exported via a distinct, Rem-independent, mechanism. Here, we analysed the effect of an extensively folded region coinciding with the 5' leader sequence on env gene expression. We found that the presence of the 5' leader stimulates expression of the envelope protein. Enhanced Env production was accompanied by increased cytoplasmic levels of env mRNA. The 5' leader promotes nucleocytoplasmic translocation and increases stability of env mRNA. The region responsible for this effect was mapped to the distal part of the 5' leader. Furthermore, the 5' leader inserted in the sense orientation into a heterologous luciferase expression construct increased luciferase activity. PMID:22113011

  12. Interaction of the TGGCA-binding protein with upstream sequences is required for efficient transcription of mouse mammary tumor virus.

    PubMed Central

    Miksicek, R; Borgmeyer, U; Nowock, J

    1987-01-01

    A high-affinity binding site for the TGGCA-binding protein, also known as nuclear factor I, has previously been shown to reside within the mouse mammary tumor virus (MMTV) long terminal repeat. We have introduced mutations into this binding site to test the importance of this ubiquitous nuclear protein in MMTV transcription. Mutations which abolish the binding of the TGGCA protein in vitro are shown to impair strongly glucocorticoid-induced transcription from this promoter in vivo. These data demonstrate that the TGGCA-binding protein is a multifunctional DNA-binding protein, capable of serving a transcriptional role in the case of MMTV, in addition to its known involvement in the replication of adenovirus. Images Fig. 2. Fig. 3. Fig. 4. PMID:3038519

  13. No association between Epstein-Barr Virus and Mouse Mammary Tumor Virus with Breast Cancer in Mexican Women

    PubMed Central

    Morales-Sánchez, Abigail; Molina-Muñoz, Tzindilú; Martínez-López, Juan L. E.; Hernández-Sancén, Paulina; Mantilla, Alejandra; Leal, Yelda A.; Torres, Javier; Fuentes-Pananá, Ezequiel M.

    2013-01-01

    Breast cancer is the most frequent malignancy affecting women worldwide. It has been suggested that infection by Epstein Barr Virus (EBV), Mouse Mammary Tumor Virus or a similar virus, MMTV-like virus (MMTV-LV), play a role in the etiology of the disease. However, studies looking at the presence of these viruses in breast cancer have produced conflicting results, and this possible association remains controversial. Here, we used polymerase chain reaction assay to screen specific sequences of EBV and MMTV-LV in 86 tumor and 65 adjacent tissues from Mexican women with breast cancer. Neither tumor samples nor adjacent tissue were positive for either virus in a first round PCR and only 4 tumor samples were EBV positive by a more sensitive nested PCR. Considering the study's statistical power, these results do not support the involvement of EBV and MMTV-LV in the etiology of breast cancer. PMID:24131889

  14. Stabilin-1 is expressed in human breast cancer and supports tumor growth in mammary adenocarcinoma mouse model

    PubMed Central

    Riabov, Vladimir; Yin, Shuiping; Song, Bin; Avdic, Aida; Schledzewski, Kai; Ovsiy, Ilja; Gratchev, Alexei; Verdiell, Maria Llopis; Sticht, Carsten; Schmuttermaier, Christina; Schönhaber, Hiltrud; Weiss, Christel; Fields, Alan P.; Simon-Keller, Katja; Pfister, Frederick; Berlit, Sebastian; Marx, Alexander; Arnold, Bernd; Goerdt, Sergij; Kzhyshkowska, Julia

    2016-01-01

    Stabilin-1 is a multifunctional scavenger receptor expressed on alternatively-activated macrophages. Stabilin-1 mediates phagocytosis of “unwanted-self” components, intracellular sorting, and endocytic clearance of extracellular ligands including SPARC that modulates breast cancer growth. The expression of stabilin-1 was found on tumor-associated macrophages (TAM) in mouse and human cancers including melanoma, lymphoma, glioblastoma, and pancreatic insulinoma. Despite its tumor-promoting role in mouse models of melanoma and lymphoma the expression and functional role of stabilin-1 in breast cancer was unknown. Here, we demonstrate that stabilin-1 is expressed on TAM in human breast cancer, and its expression is most pronounced on stage I disease. Using stabilin-1 knockout (ko) mice we show that stabilin-1 facilitates growth of mouse TS/A mammary adenocarcinoma. Endocytosis assay on stabilin-1 ko TAM demonstrated impaired clearance of stabilin-1 ligands including SPARC that was capable of inducing cell death in TS/A cells. Affymetrix microarray analysis on purified TAM and reporter assays in stabilin-1 expressing cell lines demonstrated no influence of stabilin-1 expression on intracellular signalling. Our results suggest stabilin-1 mediated silent clearance of extracellular tumor growth-inhibiting factors (e.g. SPARC) as a mechanism of stabilin-1 induced tumor growth. Silent clearance function of stabilin-1 makes it an attractive candidate for delivery of immunomodulatory anti-cancer therapeutic drugs to TAM. PMID:27105498

  15. Stem and progenitor cell division kinetics during postnatal mouse mammary gland development.

    PubMed

    Giraddi, Rajshekhar R; Shehata, Mona; Gallardo, Mercedes; Blasco, Maria A; Simons, Benjamin D; Stingl, John

    2015-01-01

    The cycling properties of mammary stem and progenitor cells is not well understood. To determine the division properties of these cells, we administered synthetic nucleosides for varying periods of time to mice at different stages of postnatal development and monitored the rate of uptake of these nucleosides in the different mammary cell compartments. Here we show that most cell division in the adult virgin gland is restricted to the oestrogen receptor-expressing luminal cell lineage. Our data also demonstrate that the oestrogen receptor-expressing, milk and basal cell subpopulations have telomere lengths and cell division kinetics that are not compatible with these cells being hierarchically organized; instead, our data indicate that in the adult homeostatic gland, each cell type is largely maintained by its own restricted progenitors. We also observe that transplantable stem cells are largely quiescent during oestrus, but are cycling during dioestrus when progesterone levels are high.

  16. Obesity is associated with inflammation and elevated aromatase expression in the mouse mammary gland

    PubMed Central

    Subbaramaiah, Kotha; Howe, Louise R.; Bhardwaj, Priya; Du, Baoheng; Gravaghi, Claudia; Yantiss, Rhonda K.; Zhou, Xi Kathy; Blaho, Victoria A.; Hla, Timothy; Yang, Peiying; Kopelovich, Levy; Hudis, Clifford A.; Dannenberg, Andrew J.

    2011-01-01

    Elevated circulating estrogen levels are associated with increased risk of breast cancer in obese postmenopausal women. Following menopause, the biosynthesis of estrogens through CYP19 (aromatase)-mediated metabolism of androgen precursors occurs primarily in adipose tissue, and the resulting estrogens are then secreted into the systemic circulation. The potential links between obesity, inflammation and aromatase expression are unknown. In both dietary and genetic models of obesity, we observed necrotic adipocytes surrounded by macrophages forming crown-like structures (CLS) in the mammary glands and visceral fat. The presence of CLS was associated with activation of NF-κB and increased levels of pro-inflammatory mediators (TNF-α, IL-1β, Cox-2), which were paralleled by elevated levels of aromatase expression and activity in the mammary gland and visceral fat of obese mice. Analyses of the stromal-vascular and adipose fractions of the mammary gland suggested that macrophage-derived pro-inflammatory mediators induced aromatase and estrogen-dependent gene expression (PR, pS2) in adipocytes. Saturated fatty acids, which have been linked to obesity-related inflammation, stimulated NF-κB activity in macrophages leading to increased levels of TNF-α, IL-1β and Cox-2, each of which contributed to the induction of aromatase in preadipocytes. The discovery of the obesity→inflammation→aromatase axis in the mammary gland and visceral fat and its association with CLS, may provide insight into mechanisms underlying the increased risk of hormone receptor-positive breast cancer in obese postmenopausal women, the reduced efficacy of aromatase inhibitors in the treatment of breast cancer in these women, and their generally worse outcomes. The presence of CLS may be a biomarker of increased breast cancer risk or poor prognosis. PMID:21372033

  17. Thyroid hormone regulation of epidermal growth factor receptor levels in mouse mammary glands

    SciTech Connect

    Vonderhaar, B.K.; Tang, E.; Lyster, R.R.; Nascimento, M.C.

    1986-08-01

    The specific binding of iodinated epidermal growth factor ((/sup 125/I)iodo-EGF) to membranes prepared from the mammary glands and spontaneous breast tumors of euthyroid and hypothyroid mice was measured in order to determine whether thyroid hormones regulate the EGF receptor levels in vivo. Membranes from hypothyroid mammary glands of mice at various developmental ages bound 50-65% less EGF than those of age-matched euthyroid controls. Treatment of hypothyroid mice with L-T4 before killing restored binding to the euthyroid control level. Spontaneous breast tumors arising in hypothyroid mice also bound 30-40% less EGF than tumors from euthyroid animals even after in vitro desaturation of the membranes of endogenous growth factors with 3 M MgCl2 treatment. The decrease in binding in hypothyroid membranes was due to a decrease in the number of binding sites, not to a change in affinity of the growth factor for its receptor, as determined by Scatchard analysis of the binding data. Both euthyroid and hypothyroid membranes bound EGF primarily to a single class of high affinity sites (dissociation constant (Kd) = 0.7-1.8 nM). Euthyroid membranes bound 28.4 +/- (SE) 0.6 fmol/mg protein, whereas hypothyroid membranes bound 15.5 +/- 1.0 fmol/mg protein. These data indicate that EGF receptor levels in normal mammary glands and spontaneous breast tumors in mice are subject to regulation by thyroid status.

  18. Impaired CK1 Delta Activity Attenuates SV40-Induced Cellular Transformation In Vitro and Mouse Mammary Carcinogenesis In Vivo

    PubMed Central

    Hirner, Heidrun; Günes, Cagatay; Bischof, Joachim; Wolff, Sonja; Grothey, Arnhild; Kühl, Marion; Oswald, Franz; Wegwitz, Florian; Bösl, Michael R.; Trauzold, Anna; Henne-Bruns, Doris; Peifer, Christian; Leithäuser, Frank; Deppert, Wolfgang; Knippschild, Uwe

    2012-01-01

    Simian virus 40 (SV40) is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP) gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T) mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA repair. Our data

  19. Inhibitory effect of iron withdrawal by chelation on the growth of human and murine mammary carcinoma and fibrosarcoma cells.

    PubMed

    Power Coombs, Melanie R; Grant, Taryn; Greenshields, Anna L; Arsenault, Daniel J; Holbein, Bruce E; Hoskin, David W

    2015-10-01

    Since iron uptake is essential for cell growth, rapidly dividing cancer cells are sensitive to iron depletion. To explore the effect of iron withdrawal on cancer cell growth, mouse and human mammary carcinoma cells (4T1 and MDA-MB-468, respectively) and mouse and human fibrosarcoma cells (L929 and HT1080, respectively) were cultured in the absence or presence of DIBI, a novel iron-chelating polymer containing hydroxypyridinone iron-ligand functionality. Cell growth was measured by a colorimetric assay for cell metabolic activity. DIBI-treated 4T1, MDA-MB-468, L929 and HT1080 cells, as well as their normal counterparts, showed a dose- and time-dependent reduction in growth that was selective for human cancer cells and mouse fibrosarcoma cells. The inhibitory effect of DIBI on fibrosarcoma and mammary carcinoma cell growth was reversed by addition of exogenous iron in the form of iron (III) citrate, confirming the iron selectivity of DIBI and that its inhibitory activity was iron-related. Fibrosarcoma and mammary carcinoma cell growth inhibition by DIBI was associated with S-phase cell cycle arrest and low to moderate levels of cell death by apoptosis. Consistent with apoptosis induction following DIBI-mediated iron withdrawal, fibrosarcoma and mammary carcinoma cells exhibited mitochondrial membrane permeabilization. A comparison of DIBI to other iron chelators showed that DIBI was superior to deferiprone and similar to or better than deferoxamine for inhibition of fibrosarcoma and mammary carcinoma cell growth. These findings suggest that iron withdrawal from the tumor microenvironment with a selective and potent iron chelator such as DIBI may prevent or inhibit tumor progression.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  3. Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells

    PubMed Central

    1986-01-01

    The cell surface proteoglycan on normal murine mammary gland mouse mammary epithelial cells consists of an ectodomain bearing heparan and chondroitin sulfate chains and a lipophilic domain that is presumed to be intercalated into the plasma membrane. Because the ectodomain binds to matrix components produced by stromal cells with specificity and high affinity, we have proposed that the cell surface proteoglycan is a matrix receptor that binds epithelial cells to their underlying basement membrane. We now show that the proteoglycan surrounds cells grown in subconfluent or newly confluent monolayers, but becomes restricted to the basolateral surface of cells that have been confluent for a week or more; Triton X-100 extraction distinguishes three fractions of cell surface proteoglycan: a fraction released by detergent and presumed to be free in the membrane, a fraction bound via a salt-labile linkage, and a nonextractable fraction; the latter two fractions co-localize with actin filament bundles at the basal cell surface; and when proteoglycans at the apical cell surface are cross- linked by antibodies, they initially assimilate into detergent- resistant, immobile clusters that are subsequently aggregated by the cytoskeleton. These findings suggest that the proteoglycan, initially present on the entire surface and free in the plane of the membrane, becomes sequestered at the basolateral cell surface and bound to the actin-rich cytoskeleton as the cells become polarized in vitro. Binding of matrix components may cross-link proteoglycans at the basal cell surface and cause them to associate with the actin cytoskeleton, providing a mechanism by which the cell surface proteoglycan acts as a matrix receptor to stabilize the morphology of epithelial sheets. PMID:3025223

  4. Selenium Deficiency Facilitates Inflammation Following S. aureus Infection by Regulating TLR2-Related Pathways in the Mouse Mammary Gland.

    PubMed

    Gao, Xuejiao; Zhang, Zecai; Li, Ying; Shen, Peng; Hu, Xiaoyu; Cao, Yongguo; Zhang, Naisheng

    2016-08-01

    Selenium (Se) is an essential micronutrient affecting various aspects of health. Se deficiency has been associated with inflammation and immune responses. Mastitis poses a serious problem for humans and animals in the postpartum period. Staphylococcus aureus (S. aureus) is the most common infectious bacterial pathogen associated with mastitis. The present study sought to determine the effects and underlying mechanism of dietary Se on S. aureus-induced inflammation using a model of mouse mastitis. ELISA and Western blotting were performed to detect protein levels. Quantitative PCR (qPCR) was performed to detect messenger RNA (mRNA) levels. The histopathological changes indicated that Se deficiency resulted in increased inflammatory lesions in S. aureus mastitis, whereas Se deficiency did not induce inflammatory lesions in the mammary gland. Myeloperoxidase (MPO) activity was increased in Se-deficient mice with S. aureus mastitis. Analysis of cytokine mRNA and protein showed that Se deficiency leads to increased TNF-α, IL-1β, and IL-6 production in S. aureus mastitis. In addition, Se deficiency enhanced the mRNA and protein expressions of toll-like receptor 2 (TLR2), which were originally upregulated by S. aureus in the mammary gland tissues and human embryonic kidney 293 (HEK293)-mTLR2 cells. When Se-deficient mice were infected with S. aureus, the phosphorylation of IκB, nuclear factor-κB (NF-κB), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 was greatly increased. The results indicate that Se deficiency could intensify the inflammatory reaction in S. aureus mastitis. This work contributes to the exploration of new methods of preventing or treating of S. aureus mastitis and other infectious diseases.

  5. Mls-1-like superantigen in the MA/MyJ mouse is encoded by a new mammary tumor provirus that is distinct from Mtv-7

    PubMed Central

    1992-01-01

    Mls-1 is an endogenous superantigen that leads to in vivo deletion and in vitro stimulation of T cell receptor (TCR) V beta 6-, 7-, 8.1-, and 9-expressing cells. The MA/MyJ mouse deletes the identical set of TCR from its mature T cell repertoire; however, it does not contain Mtv-7, the murine mammary tumor provirus (MMTV), whose sag gene encodes Mls-1. Interestingly, the superantigen activity of this mouse strain segregates with a new mammary tumor provirus, Mtv-43, not seen in other inbred strains. The predicted amino acid sequence of the sag gene of Mtv-43 was compared with that of Mtv-7. Strikingly, the COOH terminus of the two molecules is very similar, while all other MMTV-encoded superantigens differ 100% in this segment. PMID:1316931

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

    SciTech Connect

    Lochter, A.; Srebrow, A.; Sympson, C.J.; Terracio, N.; Werb, Z.; Bissell, M.J.

    1997-02-21

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

  7. Effects of lysostaphin on Staphylococcus aureus infections of the mouse mammary gland.

    PubMed

    Bramley, A J; Foster, R

    1990-07-01

    A 5 to 6 log10 reduction in the viable count of Staphylococcus aureus was produced in vitro with 10 micrograms lysostaphin ml-1 milk. Infusion of the lactating murine mammary gland with 10 micrograms lysostaphin, immediately following inoculation with 10(8) colony forming units of S aureus, resulted in a significant 2 to 3 log10 reduction in viable S aureus (P less than 0.02) within 30 minutes. Pre-infusion with 10 micrograms lysostaphin either immediately before or one hour before staphylococcal challenge reduced the recovery of S aureus by more than 6 log10 and greatly reduced pathological changes typical of S aureus mastitis. This clearly demonstrates that lysostaphin has considerable potential for the therapeutic or prophylactic control of staphylococcal mastitis.

  8. Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

    SciTech Connect

    Ehrhart, E.J.; Gillette, E.L.; Barcellos-Hoff, M.H.

    1996-02-01

    High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by {gamma} irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin inummoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation. 40 refs., 3 figs.

  9. Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

    NASA Technical Reports Server (NTRS)

    Ehrhart, E. J.; Gillette, E. L.; Barcellos-Hoff, M. H.; Chaterjee, A. (Principal Investigator)

    1996-01-01

    High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by gamma irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin immunoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid-remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation.

  10. Prevention of Metastasis in a 4T1 Murine Breast Cancer Model by Doxorubicin Carried by Folate Conjugated pH Sensitive Polymeric Micelles

    PubMed Central

    Gao, Zhong-Gao; Tian, Li; Hu, Jun; Park, In-Suh; Bae, You Han

    2011-01-01

    This study primarily focused on the anti-metastatic activity of doxorubicin (DOX) loaded in a pH-sensitive mixed polymeric micelle formed from two block polymers: poly(L-lactide) (PLLA) (Mn 3000)-b-poly(ethylene glycol) (PEG) (Mn 2000)-folate and poly(L-histidine) (PHis) (Mn 4700)-b-PEG (Mn 2000). Tumor formation and metastasis in mice were examined using a murine mammary carcinoma cell of 4T1 which is one of the most aggressive metastatic cancer cell lines. The efficacy was evaluated by tumor size, body weight change, survival rate, dorsal skin fold window chamber model, and histological observation of the lung, heart, liver and spleen after treatment with various DOX formulations. When the tumor reached 50–100 mm3 in size, the mice were treated by 4 times at a 3-day interval at a dose of 10 mg DOX/kg. The mixed micelle formulation resulted in retarded tumor growth, no weight loss, and no death for 4–5 weeks. In another set of the in vivo test for histological evaluation of the organs, the mice were similarly treated but the formulations were injected one day after 4T1 cell inoculation. The treatment by DOX loaded mixed micelle showed no apparent metastasis till 28 days. However, significant metastasis to the lung and heart was observed on Day 28 when the mice were treated with DOX carried by PBS, PLLA-b-PEG micelle and PHis-b-PEG micelle. PMID:21295088

  11. Tetrandrine Suppresses Cancer Angiogenesis and Metastasis in 4T1 Tumor Bearing Mice

    PubMed Central

    Gao, Jian-Li; He, Tong-Chuan; He, Kai; Chen, Su-Hong; Lv, Gui-Yuan

    2013-01-01

    Metastasis remains the most deadly aspect of cancer and still evades direct treatment. Thus, there is a great need to develop new treatment regimens to suppress tumor cells that have escaped surgical removal or that may have already disseminated. We have found that tetrandrine (TET) exhibits anticolon cancer activity. Here, we investigate the inhibition effect of TET to breast cancer metastasis, angiogenesis and its molecular basis underlying TET's anticancer activity. We compare TET with chemotherapy drug doxorubicin in 4T1 tumor bearing BALB/c mice model and find that TET exhibits an anticancer metastatic and antiangiogenic activities better than those of doxorubicin. The lung metastatic sites were decreased by TET, which is confirmed by bioluminescence imaging in vivo. On the other hand, laser doppler perfusion imaging (LDI) was used for measuring the blood flow of tumor in 4T1-tumor bearing mice. As a result, the local blood perfusion of tumor was markedly decreased by TET after 3 weeks. Mechanistically, TET treatment leads to a decrease in p-ERK level and an increase in NF-κB levels in HUVECs. TET also regulated metastatic and angiogenic related proteins, including vascular endothelial growth factor, hypoxia-inducible factor-1α, integrin β5, endothelial cell specific molecule-1, and intercellular adhesion molecule-1 in vivo. PMID:23762115

  12. Mouse mammary tumor proviruses from a T-cell lymphoma are associated with the retroposon L1Md.

    PubMed Central

    Dudley, J P

    1988-01-01

    Four Charon 4A clones containing mouse mammary tumor virus (MMTV) proviruses and their cellular flanking sequences were obtained from partial EcoRI libraries of a C57BL/6 T-cell lymphoma with both endogenous and newly acquired MMTV proviruses. The cellular flanking sequences of three of four MMTV proviruses contained DNA homologous to the 3' end of the long interspersed retroposon L1Md. Two of the three proviruses were newly acquired in the lymphoma DNA, and these MMTV proviruses appeared to be 5 kilobases downstream and in the same transcriptional orientation as the L1 sequence. The third provirus was endogenous Mtv-9 and was located less than 500 base pairs from the 3' end of L1. Seven additional clones containing MMTV proviruses were isolated from partial MboI libraries of a B6 T-cell lymphoma. Five of the seven clones contained L1 elements in the cellular DNA flanking MMTV DNA. At least two clones (including one with the Mtv-8 provirus) had multiple L1 copies flanking the MMTV provirus, and one clone contained a single MMTV long terminal repeat directly integrated into a truncated L1 sequence. Although the frequencies of B1 and L1 in random library clones were similar, only one MMTV-containing clone hybridized to the abundant repetitive element B1. These data suggest a nonrandom association between MMTV and L1Md. Images PMID:2826809

  13. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

    SciTech Connect

    Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

    1998-06-29

    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

  14. Basement-membrane heparan sulphate with high affinity for antithrombin synthesized by normal and transformed mouse mammary epithelial cells.

    PubMed Central

    Pejler, G; David, G

    1987-01-01

    Basement-membrane proteoglycans, biosynthetically labelled with [35S]sulphate, were isolated from normal and transformed mouse mammary epithelial cells. Proteoglycans synthesized by normal cells contained mainly heparan sulphate and, in addition, small amounts of chondroitin sulphate chains, whereas transformed cells synthesized a relatively higher proportion of chondroitin sulphate. Polysaccharide chains from transformed cells were of lower average Mr and of lower anionic charge density compared with chains isolated from the untransformed counterparts, confirming results reported previously [David & Van den Berghe (1983) J. Biol. Chem. 258, 7338-7344]. A large proportion of the chains isolated from normal cells bound with high affinity to immobilized antithrombin, and the presence of 3-O-sulphated glucosamine residues, previously identified as unique markers for the antithrombin-binding region of heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555], could be demonstrated. A significantly lower proportion of the chains derived from transformed cells bound with high affinity to antithrombin, and a corresponding decrease in the amount of incorporated 3-O-sulphate was observed. PMID:2963617

  15. Global Gene Expression Profiling in Interleukin-12-Induced Activation of CD8+ Cytotoxic T Lymphocytes against Mouse Mammary Carcinoma

    PubMed Central

    Cao, Shanjin; Xiang, Zhaoying; Ma, Xiaojing

    2010-01-01

    Interleukin-12 (IL-12) is a critical cytokine representing the link between the cellular and humoral branches of host immune defense apparatus. IL-12-induced cytotoxic lymphocyte (CTL) development is a central mechanism in immune responses against intracellular infectious agents as well as malignant growth. However, the molecular basis of tumor-specific CTL responses mediated by IL-12 remains poorly defined. In this study, we addressed this issue in a comprehensive manner to probe into IL-12-induced anti-tumor responses by global gene expression profiling of mRNA expression in CD8+T cells in a transplantable syngeneic mouse mammary carcinoma model treated or not with recombinant IL-12. A strong tumor regression was induced by the IL-12 treatment. An introspection of differential gene expression at an early stage of the IL-12-initiated CTL activation reveals interesting genes and molecular pathways that may account for the marked tumor regression, and is likely to provide a rich source of potential targets for further research and development of effective therapeutic modalities. PMID:16285895

  16. [Regulatory T cell depletion increases the number of CD8 cells during mouse mammary tumor virus infection].

    PubMed

    Cabrera, Gabriel; Mundiñano, Juliana; Camicia, Gabriela; Costa, Héctor; Nepomnaschy, Irene; Piazzon, Isabel

    2011-01-01

    Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. We have shown in a natural model of MMTV infection that the virus causes early and progressive increases in superantigen (Sag)-specific CD4+ CD25+ Foxp3+ regulatory T cells (Treg) in Peyer's patches. Herein, we evaluated whether the depletion of Treg cells affects the CD8+ population during milk-borne MMTV infection. At day 6 of infection, the depletion of Treg cells increased the percentage and absolute number of CD8+ cells in lymph nodes as well as the mean intensity fluorescence of the CD44 activation marker. The absolute number of CD8+ cells was increased in cells bearing both Sag reactive and non-reactive TCR Vβ chains. We have previously shown that regulatory T cell depletion at day 6 of infection decrease viral load. Results reported herein suggest that at least after day 6 of MMTV infection Treg cells play an inhibiting role on CD8 antiviral response. PMID:21745773

  17. Mouse mammary tumor virus proviruses in T-cell lymphomas lack a negative regulatory element in the long terminal repeat.

    PubMed Central

    Hsu, C L; Fabritius, C; Dudley, J

    1988-01-01

    The nucleotide sequences of long terminal repeats (LTRs) from several mouse mammary tumor virus (MMTV) proviruses acquired in mouse T-cell lymphomas were determined. All MMTV proviruses cloned from a C57BL/6 lymphoma contained an identical LTR deletion of 491 base pairs (approximately -655 to -165), whereas an MMTV provirus from a BALB/c T-cell lymphoma had a 430-base-pair deletion in the same U3 region. MMTV proviruses with LTR deletions were acquired in these tumors 10 times more frequently than proviruses with intact LTRs. Because the deletions removed a portion of the glucocorticoid response element or "regulated" enhancer, the transcriptional activity of the deleted MMTV LTRs was assessed in both transient expression and stable transfection experiments. Plasmids were constructed in which the deleted or full-length MMTV LTRs were placed upstream of the chloramphenicol acetyltransferase gene. Results from transfection experiments with these constructs showed that the basal expression of the deleted MMTV LTR in the absence of glucocorticoids was higher than that of the full-length Mtv-17 or C3H MMTV LTRs under the same conditions. Moreover, the C3H LTR with a similar deletion (-637 to -255) also promoted high basal levels of chloramphenicol acetyltransferase activity. These results, coupled with the observation in lymphomas of high basal levels of transcription from MMTV proviruses with deleted LTRs, suggested that these proviruses lack negative regulatory elements in their LTRs. Loss of the negative regulatory element may contribute to the selective propagation of proviruses with deleted LTRs. Images PMID:2846876

  18. Cell-free synthesis of mouse mammary tumor virus Pr77 from virion and intracellular mRNA.

    PubMed Central

    Dahl, H H; Dickson, C

    1979-01-01

    Mouse mammary tumor virus (MuMTV) was purified from two cell lines (GR and Mm5MT/c1), and the genomic RNA was isolated and translated in vitro in cell-free systems derived from mouse L cells and rabbit reticulocytes. The major translation product in both systems was a protein with the molecular weight 77,000. Several other products were also detected, among them a 110,000-dalton and in minor amounts a 160,000-dalton protein. All three polypeptides were specifically immunoprecipitated by antiserum raised against the major core protein of MuMTV (p27), but they were not precipitated by antiserum against the virion glycoprotein gp52. Analysis of the in vitro products by tryptic peptide mapping established their relationship to the virion non-glycosylated structural proteins. The 77,000-dalton polypeptide was found to be similar, if not identical, to an analogous precursor isolated from MuMTV-producing cells. Peptide mapping of the 110,000-dalton protein shows that it contains all of the methionine-labeled peptides found in the 77,000-dalton protein plus some additional peptides. We conclude that the products synthesized in vitro from the genomic MuMTV RNA are related to the non-glycosylated virion structural proteins. Polyadenylic acid-containing RNA from MuMTV-producing cells also directed the synthesis of the 77,000-dalton polypeptide in the L-cell system. If this RNA preparation was first fractionated by sucrose gradient centrifugation the 77,000-dalton protein appeared to be synthesized from mRNA with a sedimentation coefficient between 25 and 35S. Images PMID:221668

  19. Mouse mammary tumor virus-like gene sequences are present in lung patient specimens

    PubMed Central

    2011-01-01

    Background Previous studies have reported on the presence of Murine Mammary Tumor Virus (MMTV)-like gene sequences in human cancer tissue specimens. Here, we search for MMTV-like gene sequences in lung diseases including carcinomas specimens from a Mexican population. This study was based on our previous study reporting that the INER51 lung cancer cell line, from a pleural effusion of a Mexican patient, contains MMTV-like env gene sequences. Results The MMTV-like env gene sequences have been detected in three out of 18 specimens studied, by PCR using a specific set of MMTV-like primers. The three identified MMTV-like gene sequences, which were assigned as INER6, HZ101, and HZ14, were 99%, 98%, and 97% homologous, respectively, as compared to GenBank sequence accession number AY161347. The INER6 and HZ-101 samples were isolated from lung cancer specimens, and the HZ-14 was isolated from an acute inflammatory lung infiltrate sample. Two of the env sequences exhibited disruption of the reading frame due to mutations. Conclusion In summary, we identified the presence of MMTV-like gene sequences in 2 out of 11 (18%) of the lung carcinomas and 1 out of 7 (14%) of acute inflamatory lung infiltrate specimens studied of a Mexican Population. PMID:21943279

  20. Estradiol and tamoxifen regulate NRF-1 and mitochondrial function in mouse mammary gland and uterus.

    PubMed

    Ivanova, Margarita M; Radde, Brandie N; Son, Jieun; Mehta, Fabiola F; Chung, Sang-Hyuk; Klinge, Carolyn M

    2013-10-01

    Nuclear respiratory factor-1 (NRF-1) stimulates the transcription of nuclear-encoded genes that regulate mitochondrial (mt) genome transcription and biogenesis. We reported that estradiol (E2) and 4-hydroxytamoxifen (4-OHT) stimulate NRF-1 transcription in an estrogen receptor α (ERα)- and ERβ-dependent manner in human breast cancer cells. The aim of this study was to determine whether E2 and 4-OHT increase NRF-1 in vivo. Here, we report that E2 and 4-OHT increase NRF-1 expression in mammary gland (MG) and uterus of ovariectomized C57BL/6 mice in a time-dependent manner. E2 increased NRF-1 protein in the uterus and MG; however, in MG, 4-OHT increased Nrf1 mRNA but not protein. Chromatin immunoprecipitation assays revealed increased in vivo recruitment of ERα to the Nrf1 promoter and intron 3 in MG and uterus 6 h after E2 and 4-OHT treatment, commensurate with increased NRF-1 expression. E2- and 4-OHT-induced increases in NRF-1 and its target genes Tfam, Tfb1m, and Tfb2m were coordinated in MG but not in uterus due to uterine-selective inhibition of the expression of the NRF-1 coactivators Ppargc1a and Ppargc1b by E2 and 4-OHT. E2 transiently increased NRF-1 and PGC-1α nuclear staining while reducing PGC-1α in uterus. E2, not 4-OHT, activates mt biogenesis in MG and uterus in a time-dependent manner. E2 increased mt outer membrane Tomm40 protein levels in MG and uterus whereas 4-OHT increased Tomm40 only in uterus. These data support the hypothesis of tissue-selective regulation of NRF-1 and its downstream targets by E2 and 4-OHT in vivo.

  1. Developmental regulation of mitochondrial biogenesis and function in the mouse mammary gland during a prolonged lactation cycle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The regulation of mitochondrial biogenesis and function in the lactating mammary cell is poorly understood. The goal of this study was to use proteomics to relate temporal changes in mammary cell mitochondrial function during lactation to changes in the proteins that make up this organelle. The hypo...

  2. Bisphenol A (BPA) Exposure In Utero Leads to Immunoregulatory Cytokine Dysregulation in the Mouse Mammary Gland: A Potential Mechanism Programming Breast Cancer Risk.

    PubMed

    Fischer, Catha; Mamillapalli, Ramanaiah; Goetz, Laura G; Jorgenson, Elisa; Ilagan, Ysabel; Taylor, Hugh S

    2016-08-01

    Bisphenol-A (BPA) is a ubiquitous estrogen-like endocrine disrupting compound (EDC). BPA exposure in utero has been linked to breast cancer and abnormal mammary gland development in mice. The recent rise in incidence of human breast cancer and decreased age of first detection suggests a possible environmental etiology. We hypothesized that developmental programming of carcinogenesis may involve an aberrant immune response. Both innate and adaptive immunity play a role in tumor suppression through cytolytic CD8, NK, and Th1 T-cells. We hypothesized that BPA exposure in utero would lead to dysregulation of both innate and adaptive immunity in the mammary gland. CD1 mice were exposed to BPA in utero during gestation (days 9-21) via osmotic minipump. At 6 weeks, the female offspring were ovariectomized and estradiol was given at 8 weeks. RNA and protein were extracted from the posterior mammary glands, and the mRNA and protein levels were measured by PCR array, qRT-PCR, and western blot. In mouse mammary tissue, BPA exposure in utero significantly decreased the expression of members of the chemokine CXC family (Cxcl2, Cxcl4, Cxcl14, and Ccl20), interleukin 1 (Il1) gene family (Il1β and Il1rn), interleukin 2 gene family (Il7 receptor), and interferon gene family (interferon regulatory factor 9 (Irf9), as well as immune response gene 1 (Irg1). Additionally, BPA exposure in utero decreased Esr1 receptor gene expression and increased Esr2 receptor gene expression. In utero exposure of BPA resulted in significant changes to inflammatory modulators within mammary tissue. We suggest that dysregulation of inflammatory cytokines, both pro-inflammatory and anti-inflammatory, leads to a microenvironment that may promote disordered cell growth through inhibition of the immune response that targets cancer cells.

  3. Epstein-Barr Virus, Human Papillomavirus and Mouse Mammary Tumour Virus as Multiple Viruses in Breast Cancer

    PubMed Central

    Glenn, Wendy K.; Heng, Benjamin; Delprado, Warick; Iacopetta, Barry; Whitaker, Noel J.; Lawson, James S.

    2012-01-01

    Background The purpose of this investigation is to determine if Epstein Barr virus (EBV), high risk human papillomavirus (HPV), and mouse mammary tumour viruses (MMTV) co-exist in some breast cancers. Materials and Methods All the specimens were from women residing in Australia. For investigations based on standard PCR, we used fresh frozen DNA extracts from 50 unselected invasive breast cancers. For normal breast specimens, we used DNA extracts from epithelial cells from milk donated by 40 lactating women. For investigations based on in situ PCR we used 27 unselected archival formalin fixed breast cancer specimens and 18 unselected archival formalin fixed normal breast specimens from women who had breast reduction surgery. Thirteen of these fixed breast cancer specimens were ductal carcinoma in situ (dcis) and 14 were predominantly invasive ductal carcinomas (idc). Results EBV sequences were identified in 68%, high risk HPV sequences in 50%, and MMTV sequences in 78% of DNA extracted from 50 invasive breast cancer specimens. These same viruses were identified in selected normal and breast cancer specimens by in situ PCR. Sequences from more than one viral type were identified in 72% of the same breast cancer specimens. Normal controls showed these viruses were also present in epithelial cells in human milk – EBV (35%), HPV, 20%) and MMTV (32%) of 40 milk samples from normal lactating women, with multiple viruses being identified in 13% of the same milk samples. Conclusions We conclude that (i) EBV, HPV and MMTV gene sequences are present and co-exist in many human breast cancers, (ii) the presence of these viruses in breast cancer is associated with young age of diagnosis and possibly an increased grade of breast cancer. PMID:23183846

  4. Synergistic Action of GA-Binding Protein and Glucocorticoid Receptor in Transcription from the Mouse Mammary Tumor Virus Promoter

    PubMed Central

    Aurrekoetxea-Hernández, Koldo; Buetti, Elena

    2000-01-01

    B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position −139 to −146 from the cap site) and fp2 (at −157 to −164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways. PMID:10799572

  5. Nucleoprotein structure influences the response of the mouse mammary tumor virus promoter to activation of the cyclic AMP signalling pathway.

    PubMed Central

    Pennie, W D; Hager, G L; Smith, C L

    1995-01-01

    Recent studies have provided evidence of crosstalk between steroid receptors and cyclic AMP (cAMP) signalling pathways in the regulation of gene expression. A synergism between intracellular phosphorylation inducers and either glucocorticoids or progestins has been shown to occur during activation of the mouse mammary tumor virus (MMTV) promoter. We have investigated the effect of 8-Br-cAMP and okadaic acid, modulators of cellular kinases and phosphatases, on the hormone-induced activation of the MMTV promoter in two forms: a transiently transfected template with a disorganized, accessible nucleoprotein structure and a stably replicating template with an ordered, inaccessible nucleoprotein structure. Both okadaic acid and 8-Br-cAMP synergize significantly with either glucocorticoids or progestins in activating the transiently transfected MMTV template. In contrast, 8-Br-cAMP, but not okadaic acid, is antagonistic to hormone-induced activation of the stably replicating MMTV template. Nuclear run-on experiments demonstrate that this inhibition is a transcriptional effect on both hormone-induced transcription and basal transcription. Surprisingly, 8-Br-cAMP does not inhibit glucocorticoid-induced changes in restriction enzyme access and nuclear factor 1 binding. However, association of a complex with the TATA box region is inhibited in the presence of 8-Br-cAMP. Thus, cAMP treatment interferes with the initiation process but does not inhibit interaction of the receptor with the template. Since the replicated, ordered MMTV templates and the transfected, disorganized templates show opposite responses to 8-Br-cAMP treatment, we conclude that chromatin structure can influence the response of a promoter to activation of the cAMP signalling pathway. PMID:7891707

  6. Effects of Perfluorooctanoic Acid on Mouse Mammary Gland Development and Differentiation Resulting from Cross-Foster and Restricted Gestational Exposures

    EPA Science Inventory

    The adverse consequences of developmental exposures to perfluorooctanoic acid (PFOA) have been established, and include impaired development of the offspring mammary gland (MG). However, the relationship between the timing or route of exposure, and the phenotypic consequences in ...

  7. Targeted DNA Methylation Screen in the Mouse Mammary Genome Reveals a Parity-Induced Hypermethylation of Igf1r That Persists Long after Parturition.

    PubMed

    Katz, Tiffany A; Liao, Serena G; Palmieri, Vincent J; Dearth, Robert K; Pathiraja, Thushangi N; Huo, Zhiguang; Shaw, Patricia; Small, Sarah; Davidson, Nancy E; Peters, David G; Tseng, George C; Oesterreich, Steffi; Lee, Adrian V

    2015-10-01

    The most effective natural prevention against breast cancer is an early first full-term pregnancy. Understanding how the protective effect is elicited will inform the development of new prevention strategies. To better understand the role of epigenetics in long-term protection, we investigated parity-induced DNA methylation in the mammary gland. FVB mice were bred or remained nulliparous and mammary glands harvested immediately after involution (early) or 6.5 months following involution (late), allowing identification of both transient and persistent changes. Targeted DNA methylation (109 Mb of Ensemble regulatory features) analysis was performed using the SureSelectXT Mouse Methyl-seq assay and massively parallel sequencing. Two hundred sixty-nine genes were hypermethylated and 128 hypomethylated persistently at both the early and late time points. Pathway analysis of the persistently differentially methylated genes revealed Igf1r to be central to one of the top identified signaling networks, and Igf1r itself was one of the most significantly hypermethylated genes. Hypermethylation of Igf1r in the parous mammary gland was associated with a reduction of Igf1r mRNA expression. These data suggest that the IGF pathway is regulated at multiple levels during pregnancy and that its modification might be critical in the protective role of pregnancy. This supports the approach of lowering IGF action for prevention of breast cancer, a concept that is currently being tested clinically.

  8. Mono- and Combined Therapy of Metastasizing Breast Carcinoma 4T1 with Zoledronic Acid and Doxorubicin.

    PubMed

    Baklaushev, V P; Grinenko, N F; Yusubalieva, G M; Gubskii, I L; Burenkov, M S; Rabinovich, E Z; Ivanova, N V; Chekhonin, V P

    2016-08-01

    The efficiency of monotherapy with zoledronic acid (Resorba), doxorubicin, and their combination was studied on the model of metastasizing breast carcinoma in BALB/c mice. Doxorubicin monotherapy was accompanied by a significant increase in median survival up to 57 days (vs. 34 and 35 days in control groups); 27% animals survived for 90 days (duration of the study). Bioluminescence area of the primary tumor significantly decreased on days 21 and 28; the total number of visceral metastases also decreased according to magnetic-resonance imaging data. Resorba monotherapy produced no general toxic effect, the median survival increased to 64 days, and 90-day survival was 33%. Imaging techniques (magnetic-resonance imaging, microtomography, bioluminescent analysis) showed that Resorba delayed the development of the primary tumor (regression of luminescence area on days 21 and 28, regression of standardized bioluminescence intensity on day 28) and significantly reduced the number of visceral metastases in comparison with the control. Combination therapy was less effective than monotherapy with the same medications. Median survival was 55 days, 90-day survival was 13%, but magnetic-resonance imaging and bioluminescence analysis after combination therapy also showed delayed growth of the primary tumor and reduced number of visceral metastases. Microtomography revealed bone metastases in ~30% animals of the control group; in experimental groups, no bone metastases were found. The experiment with periosteal (distal epiphysis of the femur) injection of 4T1-Luc2 tumor cells demonstrated pronounced selective effectiveness of Resorba in relation to bone metastases. Monotherapy with Resorba can prevent the development of not only bone, but also visceral metastases of breast cancer. PMID:27590765

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

    SciTech Connect

    Miura, Yuka; Hagiwara, Natsumi; Radisky, Derek C.; Hirai, Yohei

    2014-09-10

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

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

    PubMed

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

    2016-08-23

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

  11. Theranostic probe for simultaneous in vivo photoacoustic imaging and confined photothermolysis by pulsed laser at 1064 nm in 4T1 breast cancer model

    PubMed Central

    Zhou, Min; Ku, Geng; Pageon, Laura; Li, Chun

    2015-01-01

    Here, we report that polyethylene glycol (PEG)-coated copper(II) sulfide nanoparticles (PEG-CuS NPs) with their peak absorption tuned to 1064 nm could be used both as a contrast agent for photoacoustic tomographic imaging of mouse tumor vasculature and as a mediator for confined phothermolysis of tumor cells in an orthotopic syngeneic 4T1 breast tumor model. PEG-CuS NPs showed stronger photoacoustic signal than hollow gold nanospheres and single-wall carbon nanotubes at 1064 nm. MicroPET imaging of 4T1 tumor-bearing mice showed a gradual accumulation of the NPs in the tumor over time. About 6.5% of injected dose were taken up in each gram of tumor tissue at 24 h after intravenous injection of 64Cu-labeled PEG-CuS NPs. For both photoacoustic imaging and therapeutic studies, nanosecond (ns)-pulsed laser was delivered with Q-switched Nd:YAG at a wavelength of 1064 nm. Unlike conventional photothermal ablation therapy mediated by continuous wave laser with which heat could spread to the surrounding normal tissue, interaction of CuS NPs with short pulsed laser deliver heat rapidly to the treatment volume keeping the thermal damage confined to the target tissues. Our data demonstrated that it is possible to use a single-compartment nanoplatform to achieve both photoacoustic tomography and highly selective tumor destruction at 1064 nm in small animals. PMID:25379880

  12. ROLES OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF-A) IN MEDIATION OF DIOXIN (TCDD)-INDUCED DELAYS IN DEVELOPMENT OF THE MOUSE MAMMARY GLAND

    EPA Science Inventory

    Roles of Epidermal Growth Factor (EGF) and Transforming Growth Factor-alpha (TGF-a) in Mediation of Dioxin (TCDD)-Induced Delays in Development of the Mouse Mammary Gland.
    Suzanne E. Fenton, Barbara Abbott, Lamont Bryant, and Angela Buckalew. U.S. EPA, NHEERL, Reproductive Tox...

  13. GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development.

    PubMed

    Kuwahara, Kazuhiko; Yamamoto-Ibusuki, Mutsuko; Zhang, Zhenhuan; Phimsen, Suchada; Gondo, Naomi; Yamashita, Hiroko; Takeo, Toru; Nakagata, Naomi; Yamashita, Daisuke; Fukushima, Yoshimi; Yamamoto, Yutaka; Iwata, Hiroji; Saya, Hideyuki; Kondo, Eisaku; Matsuo, Keitaro; Takeya, Motohiro; Iwase, Hirotaka; Sakaguchi, Nobuo

    2016-04-01

    Human chromosome 21 is known to be associated with the high risk of hematological malignancy but with resistance to breast cancer in the study of Down syndrome. In human cancers, we previously observed the significant alterations of the protein expression encoded by the ganp/MCM3AP gene on human chromosome 21q22.3. Here, we investigated GANP protein alterations in human breast cancer samples (416 cases) at various stages by immunohistochemical analysis. This cohort study clearly showed that expression of GANP is significantly decreased in human breast cancer cases with poor prognosis as an independent risk factor (relapse-free survival, hazard ratio = 2.37, 95% confidence interval, 1.27-4.42, P = 0.007 [univariate analysis]; hazard ratio = 2.70, 95% confidence interval, 1.42-5.13, P = 0.002 [multivariate analysis]). To investigate whether the altered GANP expression is associated with mammary tumorigenesis, we created mutant mice that were conditionally deficient in the ganp/MCM3AP gene using wap-cre recombinase transgenic mice. Mammary gland tumors occurred at a very high incidence in female mammary gland-specific GANP-deficient mice after severe impairment of mammary gland development during pregnancy. Moreover, tumor development also occurred in female post parous GANP-heterodeficient mice. GANP has a significant role in the suppression of DNA damage caused by estrogen in human breast cancer cell lines. These results indicated that the GANP protein is associated with breast cancer resistance. PMID:26749495

  14. RUNX2 correlates with subtype-specific breast cancer in a human tissue microarray, and ectopic expression of Runx2 perturbs differentiation in the mouse mammary gland.

    PubMed

    McDonald, Laura; Ferrari, Nicola; Terry, Anne; Bell, Margaret; Mohammed, Zahra M; Orange, Clare; Jenkins, Alma; Muller, William J; Gusterson, Barry A; Neil, James C; Edwards, Joanne; Morris, Joanna S; Cameron, Ewan R; Blyth, Karen

    2014-05-01

    RUNX2, a master regulator of osteogenesis, is oncogenic in the lymphoid lineage; however, little is known about its role in epithelial cancers. Upregulation of RUNX2 in cell lines correlates with increased invasiveness and the capacity to form osteolytic disease in models of breast and prostate cancer. However, most studies have analysed the effects of this gene in a limited number of cell lines and its role in primary breast cancer has not been resolved. Using a human tumour tissue microarray, we show that high RUNX2 expression is significantly associated with oestrogen receptor (ER)/progesterone receptor (PR)/HER2-negative breast cancers and that patients with high RUNX2 expression have a poorer survival rate than those with negative or low expression. We confirm RUNX2 as a gene that has a potentially important functional role in triple-negative breast cancer. To investigate the role of this gene in breast cancer, we made a transgenic model in which Runx2 is specifically expressed in murine mammary epithelium under the control of the mouse mammary tumour virus (MMTV) promoter. We show that ectopic Runx2 perturbs normal development in pubertal and lactating animals, delaying ductal elongation and inhibiting lobular alveolar differentiation. We also show that the Runx2 transgene elicits age-related, pre-neoplastic changes in the mammary epithelium of older transgenic animals, suggesting that elevated RUNX2 expression renders such tissue more susceptible to oncogenic changes and providing further evidence that this gene might have an important, context-dependent role in breast cancer.

  15. Study of agglutination of mouse mammary carcinoma (FM3A) cell induced by egg agglutinin of Rana catesbiana. II. Phytohemagglutinin P and protamine.

    PubMed

    Takeda, S; Kubota, K; Endo, Y; Matsuzawa, T

    1981-01-01

    When mouse mammary carcinoma (FM3A) cells were treated with egg agglutinin of Rana catesbiana for 15 min at 25 degrees C, percent total particle number of both cell aggregates and single cells was in direct proportion to the cell electrophoretic mobility. Phytohemagglutinin P mediated agglutination proceeded with biphasic kinetics: the higher the concentration of phytohemagglutinin P the shorter was the lag period between the first and second stages of agglutination. In protamine-mediated agglutination, the percent total particle number was reduced at low concentrations, while the electrophoretic mobility reduced only at high concentrations. Agglutinating and cytotoxic activities of these three reagents were in an intimate relation: the higher the agglutinating activity, the greater was their cytotoxic activity. PMID:6969671

  16. Use of the immune adherence hemagglutination test for titration of breast cancer patients' sea cross-reacting with purified mouse mammary tumor virus.

    PubMed

    Nagayoshi, S; Imai, M; Tsutsui, Y; Saga, S; Takahashi, M; Hoshino, M

    1981-02-01

    Ninety-two sera from patients with breast cancer, 42 sera from patients with neoplastic diseases other than breast cancer and 59 sera from apparently healthy women were examined by means of the immune adherence hemagglutination (IAHA) test using purified mouse mammary tumor virus (MMTV) fron RII mouse milk. It was found that 36.4% (34/96) of the sera from breast cancer patients, 7.1% (3/42) of the sera from patients with other neoplastic diseases and 5.1% (3/59) of the sera from apparently healthy women showed a positive reaction. Among the IAHA positive sera from breast cancer patients, 82.9% (29/35) showed a titer of more than 1:16. On the other hand, none of the positive sera from patients with cancers other than breast cancer showed a titer of more than 1:16. The sera from 4 breast cancer patients, which showed a positive reaction with RII MMTV in the IAHA test, were tested to examine the specificity of the reaction by using milk samples from sources other than RII mice, including C57BL mice, dogs, cattle and humans. None of the 4 sera showed a positive reaction with milk samples from sources other than the RIII mouse.

  17. Transcriptomic Analysis of the Mouse Mammary Gland Reveals New Insights for the Role of Serotonin in Lactation.

    PubMed

    Laporta, Jimena; Peñagaricano, Francisco; Hernandez, Laura L

    2015-01-01

    Serotonin regulates numerous processes in the mammary gland. Our objective was to discover novel genes, pathways and functions which serotonin modulates during lactation. The rate limiting enzyme in the synthesis of non-neuronal serotonin is tryptophan-hydroxylase (TPH1). Therefore, we used TPH1 deficient dams (KO; serotonin deficient, n = 4) and compared them to wild-type (WT; n = 4) and rescue (RC; KO + 100 mg/kg 5-hydroxytryptophan injected daily, n = 4) dams. Mammary tissues were collected on day 10 of lactation. Total RNA extraction, amplification, library preparation and sequencing were performed following the Illumina mRNA-Seq. Overall, 97 and 204 genes (false discovery rate, FDR ≤ 0.01) exhibited a minimum of a 2-fold expression difference between WT vs. KO and WT vs. RC dams, respectively. Most differentially expressed genes were related to calcium homeostasis, apoptosis regulation, cell cycle, cell differentiation and proliferation, and the immune response. Additionally, gene set enrichment analysis using Gene Ontology and Medical Subject Headings databases revealed the alteration of several biological processes (FDR ≤ 0.01) including fat cell differentiation and lipid metabolism, regulation of extracellular signal-related kinase and mitogen-activated kinase cascades, insulin resistance, nuclear transport, membrane potential regulation, and calcium release from the endoplasmic reticulum into the cytosol. The majority of the biological processes and pathways altered in the KO dams are central for mammary gland homeostasis. Increasing peripheral serotonin in the RC dams affects specific pathways that favor lactation. Our data confirms the importance of serotonin during lactation in the mammary gland. PMID:26470019

  18. Computerized segmentation algorithm with personalized atlases of murine MRIs in a SV40 large T-antigen mouse mammary cancer model

    NASA Astrophysics Data System (ADS)

    Sibley, Adam R.; Markiewicz, Erica; Mustafi, Devkumar; Fan, Xiaobing; Conzen, Suzanne; Karczmar, Greg; Giger, Maryellen L.

    2016-03-01

    Quantities of MRI data, much larger than can be objectively and efficiently analyzed manually, are routinely generated in preclinical research. We aim to develop an automated image segmentation and registration pipeline to aid in analysis of image data from our high-throughput 9.4 Tesla small animal MRI imaging center. T2-weighted, fat-suppressed MRIs were acquired over 4 life-cycle time-points [up to 12 to 18 weeks] of twelve C3(1) SV40 Large T-antigen mice for a total of 46 T2-weighted MRI volumes; each with a matrix size of 192 x 256, 62 slices, in plane resolution 0.1 mm, and slice thickness 0.5 mm. These image sets were acquired with the goal of tracking and quantifying progression of mammary intraepithelial neoplasia (MIN) to invasive cancer in mice, believed to be similar to ductal carcinoma in situ (DCIS) in humans. Our segmentation algorithm takes 2D seed-points drawn by the user at the center of the 4 co-registered volumes associated with each mouse. The level set then evolves in 3D from these 2D seeds. The contour evolution incorporates texture information, edge information, and a statistical shape model in a two-step process. Volumetric DICE coefficients comparing the automatic with manual segmentations were computed and ranged between 0.75 and 0.58 for averages over the 4 life-cycle time points of the mice. Incorporation of these personalized atlases with intra and inter mouse registration is expected to enable locally and globally tracking of the morphological and textural changes in the mammary tissue and associated lesions of these mice.

  19. Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells

    SciTech Connect

    Emerman, J.T.; Bartley, J.C.; Bissel, M.J.

    1981-01-01

    In the mammary gland of non-ruminant animals, glucose is utilized in a characteristic and unique way during lacation. By measuring the incorporation of glucose carbon from (U-/sup 14/C)glucose into intermediary metabolitees and metabolic products in mammary epithelia cells from virgin, pregnant, and lacating mice, we domonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate are important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells isolated from pregnant mice and cultured on collagen gels have a pattern similar to that of their freshly isolated counter-parts. When isolated from lacating mice, the metabolite patterns of cells cultured on collagen gels are different from that of the cells of origin, and resembles that of freshly isolated cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state.

  20. GLUCOSE METABOLITE PATTERNS AS MARKERS OF FUNCTIONAL DIFFERENTIATION IN FRESHLY ISOLATED AND CULTURED MOUSE MAMMARY EPITHELIAL CELLS

    SciTech Connect

    Emerman, J.T.; Bartley, J.C.; Bissell, M.J.

    1980-06-01

    In the mammary gland of nonruminant animals, glucose is utilized in a characteristic and unique way during lactation. We have measured the incorporation of glucose carbon from [U-{sup 14}C] glucose into intermediary metabolites and metabolic products in mammary epithelial cells from virgin, pregnant, and lactating mice and demonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate were important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells from pregnant mice have a pattern similar to freshly isolated cells from pregnant mice. The pattern of cells from lactating mice is different from that of the cells of origin, and resembles that of the cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state.

  1. Theranostic probe for simultaneous in vivo photoacoustic imaging and confined photothermolysis by pulsed laser at 1064 nm in 4T1 breast cancer model

    NASA Astrophysics Data System (ADS)

    Zhou, Min; Ku, Geng; Pageon, Laura; Li, Chun

    2014-11-01

    Here, we report that polyethylene glycol (PEG)-coated copper(ii) sulfide nanoparticles (PEG-CuS NPs) with their peak absorption tuned to 1064 nm could be used both as a contrast agent for photoacoustic tomographic imaging of mouse tumor vasculature and as a mediator for confined photothermolysis of tumor cells in an orthotopic syngeneic 4T1 breast tumor model. PEG-CuS NPs showed stronger photoacoustic signal than hollow gold nanospheres and single-wall carbon nanotubes at 1064 nm. MicroPET imaging of 4T1 tumor-bearing mice showed a gradual accumulation of the NPs in the tumor over time. About 6.5% of injected dose were taken up in each gram of tumor tissue at 24 h after intravenous injection of 64Cu-labeled PEG-CuS NPs. For both photoacoustic imaging and therapeutic studies, nanosecond (ns)-pulsed laser was delivered with Q-switched Nd:YAG at a wavelength of 1064 nm. Unlike conventional photothermal ablation therapy mediated by continuous wave laser with which heat could spread to the surrounding normal tissue, interaction of CuS NPs with short pulsed laser deliver heat rapidly to the treatment volume keeping the thermal damage confined to the target tissues. Our data demonstrated that it is possible to use a single-compartment nanoplatform to achieve both photoacoustic tomography and highly selective tumor destruction at 1064 nm in small animals.Here, we report that polyethylene glycol (PEG)-coated copper(ii) sulfide nanoparticles (PEG-CuS NPs) with their peak absorption tuned to 1064 nm could be used both as a contrast agent for photoacoustic tomographic imaging of mouse tumor vasculature and as a mediator for confined photothermolysis of tumor cells in an orthotopic syngeneic 4T1 breast tumor model. PEG-CuS NPs showed stronger photoacoustic signal than hollow gold nanospheres and single-wall carbon nanotubes at 1064 nm. MicroPET imaging of 4T1 tumor-bearing mice showed a gradual accumulation of the NPs in the tumor over time. About 6.5% of injected dose were

  2. Anticancer Activity of Saponins from Allium chinense against the B16 Melanoma and 4T1 Breast Carcinoma Cell

    PubMed Central

    Yu, Zhihui; Zhang, Tong; Zhou, Fengjuan; Xiao, Xiuqing; Ding, Xuezhi; He, Hao; Rang, Jie; Quan, Meifang; Wang, Ting; Zuo, Mingxing; Xia, Liqiu

    2015-01-01

    The cytotoxic substance of A. chinense saponins (ACSs) was isolated using ethanol extraction and purified with the D101 macroporous adsorption resin approach. We investigated the anticancer activity of ACSs in the B16 melanoma and 4T1 breast carcinoma cell lines. Methylthioninium chloride and hematoxylin-eosin staining with Giemsa dyestuff were used when the cells were treated with ACSs. The results showed that the cells morphologies changed significantly; ACSs induced cell death in B16 and 4T1 cells based on acridine orange/ethidium bromide double fluorescence staining, with the number and degree of apoptotic tumor cells increasing as ACS concentration increased. ACSs inhibited the proliferation of B16 and 4T1 cells in a dose-dependent manner. They also inhibited cell migration and colony formation and exhibited a concentration-dependent effect. In addition, ACSs apparently inhibited the growth of melanoma in vivo. The preliminary antitumor in vivo assay revealed that early medication positively affected tumor inhibition action and effectively protected the liver and spleen of C57 BL/6 mice from injury. This study provides evidence for the cytotoxicity of ACSs and a strong foundation for further research to establish the theoretical basis for cell death and help in the design and development of new anticancer drugs. PMID:26146506

  3. Detection of in situ mammary cancer in a transgenic mouse model: in vitro and in vivo MRI studies demonstrate histopathologic correlation

    NASA Astrophysics Data System (ADS)

    Jansen, S. A.; Conzen, S. D.; Fan, X.; Krausz, T.; Zamora, M.; Foxley, S.; River, J.; Newstead, G. M.; Karczmar, G. S.

    2008-10-01

    Improving the prevention and detection of preinvasive ductal carcinoma in situ (DCIS) is expected to lower both morbidity and mortality from breast cancer. Transgenic mouse models can be used as a 'test bed' to develop new imaging methods and to evaluate the efficacy of candidate preventive therapies. We hypothesized that despite its microscopic size, early murine mammary cancer, including DCIS, might be accurately detected by MRI. C3(1) SV40 TAg female mice (n = 23) between 10 and 18 weeks of age were selected for study. Eleven mice were subjected to in vitro imaging using a T2-weighted spin echo sequence and 12 mice were selected for in vivo imaging using a T1-weighted gradient echo, a T2-weighted spin echo and high spectral and spatial resolution imaging sequences. The imaged glands were carefully dissected, formalin fixed and paraffin embedded, and then H&E stained sections were obtained. The ratio of image-detected versus histologically detected cancers was obtained by reviewing the MR images and H&E sections independently and using histology as the gold standard. MR images were able to detect 12/12 intramammary lymph nodes, 1/1 relatively large (~5 mm) tumor, 17/18 small (~1 mm) tumors and 13/16 ducts distended with DCIS greater than 300 µm. Significantly, there were no false positives—i.e., image detection always corresponded to a histologically detectable cancer in this model. These results indicate that MR imaging can reliably detect both preinvasive in situ and early invasive mammary cancers in mice with high sensitivity. This technology is an important step toward the more effective use of non-invasive imaging in pre-clinical studies of breast cancer prevention, detection and treatment.

  4. Staphylococcus aureus and Lipopolysaccharide Modulate Gene Expressions of Drug Transporters in Mouse Mammary Epithelial Cells Correlation to Inflammatory Biomarkers

    PubMed Central

    Yagdiran, Yagmur; Tallkvist, Jonas; Artursson, Karin

    2016-01-01

    Inflammation in the mammary gland (mastitis) is the most common disease in dairy herds worldwide, often caused by the pathogens Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Little is known about the effects of mastitis on drug transporters and the impact on transporter-mediated excretion of drugs into milk. We used murine mammary epithelial HC11 cells, after lactogenic differentiation into a secreting phenotype, and studied gene expressions of ABC- and SLC- transporters after treatment of cells with S. aureus and lipopolysaccharide, an endotoxin secreted by E. coli. The studied transporters were Bcrp, Mdr1, Mrp1, Oatp1a5, Octn1 and Oct1. In addition, Csn2, the gene encoding β-casein, was analyzed. As biomarkers of the inflammatory response, gene expressions of the cytokines Il6 and Tnfα and the chemokine Cxcl2 were determined. Our results show that S. aureus and LPS treatment of cells, at non-cytotoxic concentrations, induced an up-regulation of Mdr1 and of the inflammatory biomarkers, except that Tnfα was not affected by lipopolysaccharide. By simple regression analysis we could demonstrate statistically significant positive correlations between each of the transporters with each of the inflammatory biomarkers in cells treated with S. aureus. The coefficients of determination (R2) were 0.7–0.9 for all but one correlation. After treatment of cells with lipopolysaccharide, statistically significant correlations were only found between Mdr1 and the two parameters Cxcl2 and Il6. The expression of Csn2 was up-regulated in cells treated with S. aureus, indicating that the secretory function of the cells was not impaired. The strong correlation in gene expressions between transporters and inflammatory biomarkers may suggest a co-regulation and that the transporters have a role in the transport of cytokines and chemokines. Our results demonstrate that transporters in mammary cells can be affected by infection, which may have an impact on

  5. Staphylococcus aureus and Lipopolysaccharide Modulate Gene Expressions of Drug Transporters in Mouse Mammary Epithelial Cells Correlation to Inflammatory Biomarkers.

    PubMed

    Yagdiran, Yagmur; Tallkvist, Jonas; Artursson, Karin; Oskarsson, Agneta

    2016-01-01

    Inflammation in the mammary gland (mastitis) is the most common disease in dairy herds worldwide, often caused by the pathogens Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Little is known about the effects of mastitis on drug transporters and the impact on transporter-mediated excretion of drugs into milk. We used murine mammary epithelial HC11 cells, after lactogenic differentiation into a secreting phenotype, and studied gene expressions of ABC- and SLC- transporters after treatment of cells with S. aureus and lipopolysaccharide, an endotoxin secreted by E. coli. The studied transporters were Bcrp, Mdr1, Mrp1, Oatp1a5, Octn1 and Oct1. In addition, Csn2, the gene encoding β-casein, was analyzed. As biomarkers of the inflammatory response, gene expressions of the cytokines Il6 and Tnfα and the chemokine Cxcl2 were determined. Our results show that S. aureus and LPS treatment of cells, at non-cytotoxic concentrations, induced an up-regulation of Mdr1 and of the inflammatory biomarkers, except that Tnfα was not affected by lipopolysaccharide. By simple regression analysis we could demonstrate statistically significant positive correlations between each of the transporters with each of the inflammatory biomarkers in cells treated with S. aureus. The coefficients of determination (R2) were 0.7-0.9 for all but one correlation. After treatment of cells with lipopolysaccharide, statistically significant correlations were only found between Mdr1 and the two parameters Cxcl2 and Il6. The expression of Csn2 was up-regulated in cells treated with S. aureus, indicating that the secretory function of the cells was not impaired. The strong correlation in gene expressions between transporters and inflammatory biomarkers may suggest a co-regulation and that the transporters have a role in the transport of cytokines and chemokines. Our results demonstrate that transporters in mammary cells can be affected by infection, which may have an impact on transport

  6. Cyclic AMP suppresses expression of v-rasH oncogene linked to the mouse mammary tumor virus promoter.

    PubMed

    Najam, N; Clair, T; Bassin, R H; Cho-Chung, Y S

    1986-01-14

    Clone 433.3 of NIH 3T3 cells is a stable carrier of the MMTV LTR:v-rasH chimeric DNA. Only in the presence of dexamethasone (a synthetic glucocorticoid), 433.3 cells exhibit an induced level of p21 transforming protein and phenotypic transformation. N6,O2'-dibutyryl cAMP (DBcAMP) antagonized the effect of dexamethasone in a time - and concentration - dependent manner. DBcAMP (5 X 10(-4)M) added 18 hr prior to the addition of dexamethasone (10(-7)M) almost completely blocked the hormone effect: cells contained levels of p21 20% of that in the cells treated with dexamethasone alone, and formed flat, contact inhibited monolayers. On the basis of these results together with our previous data on mammary carcinomas in vivo, we postulate that cAMP may be an intracellular suppressor acting at a regulatory locus of both cellular and viral ras genes.

  7. Genomic DNA of MCF-7 breast cancer cells not an ideal choice as positive control for PCR amplification based detection of Mouse Mammary Tumor Virus-Like Sequences.

    PubMed

    Kulkarni, Bhushan B; Hiremath, Shivaprakash V; Kulkarni, Suyamindra S; Hallikeri, Umesh R; Patil, Basavaraj R; Gai, Pramod B

    2013-11-01

    The identification of the etiology of breast cancer is a crucial research issue for the development of an effective preventive and treatment strategies. Researchers are exploring the possible involvement of Mouse Mammary Tumor Virus (MMTV) in causing human breast cancer. Hence, it becomes very important to use a consistent positive control agent in PCR amplification based detection of MMTV-Like Sequence (MMTV-LS) in human breast cancer for accurate and reproducible results. This study was done to investigate the feasibility of using genomic DNA of MCF-7 breast cancer cells to detect MMTV-LS using PCR amplification based detection. MMTV env and SAG gene located at the 3' long terminal repeat (LTR) sequences were targeted for the PCR based detection. No amplification was observed in case of the genomic DNA of MCF-7 breast cancer cells. However, the 2.7 kb DNA fragment comprising MMTV env and SAG LTR sequences yielded the products of desired size. From these results it can be concluded that Genomic DNA of MCF-7 cell is not a suitable choice as positive control for PCR or RT-PCR based detection of MMTV-LS. It is also suggested that plasmids containing the cloned genes or sequences of MMTV be used as positive control for detection of MMTV-LS.

  8. HAdV-2-suppressed growth of SV40 T antigen-transformed mouse mammary epithelial cell-induced tumours in SCID mice.

    PubMed

    Wu, Chengjun; Cao, Xiaofang; Yu, Di; Huijbers, Elisabeth J M; Essand, Magnus; Akusjärvi, Göran; Johansson, Staffan; Svensson, Catharina

    2016-02-01

    Human adenovirus (HAdV) vectors are promising tools for cancer therapy, but the shortage of efficient animal models for productive HAdV infections has restricted the evaluation of systemic effects to mainly immunodeficient mice. Previously, we reported a highly efficient replication of HAdV-2 in a non-tumorigenic mouse mammary epithelial cell line, NMuMG. Here we show that HAdV-2 gene expression and progeny formation in NMuMG cells transformed with the SV40 T antigen (NMuMG-T cells) were as efficient as in the parental NMuMG cells. Injection of HAdV-2 into tumours established by NMuMG-T in SCID mice caused reduced tumour growth and signs of intratumoural lesions. HAdV-2 replicated within the NMuMG-T-established tumours, but not in interspersed host-derived tissues within the tumours. The specific infection of NMuMG-T-derived tumours was verified by the lack of viral DNA in kidney, lung or spleen although low levels of viral DNA was occasionally found in liver.

  9. Adaptation of Laser Microdissection Technique for the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model by NanoString Technologies.

    PubMed

    Castro, Nadia P; Merchant, Anand S; Saylor, Karen L; Anver, Miriam R; Salomon, David S; Golubeva, Yelena G

    2016-01-01

    Laser capture microdissection (LCM) of tissue is an established tool in medical research for collection of distinguished cell populations under direct microscopic visualization for molecular analysis. LCM samples have been successfully analyzed in a number of genomic and proteomic downstream molecular applications. However, LCM sample collection and preparation procedure has to be adapted to each downstream analysis platform. In this present manuscript we describe in detail the adaptation of LCM methodology for the collection and preparation of fresh frozen samples for NanoString analysis based on a study of a model of mouse mammary gland carcinoma and its lung metastasis. Our adaptation of LCM sample preparation and workflow to the requirements of the NanoString platform allowed acquiring samples with high RNA quality. The NanoString analysis of such samples provided sensitive detection of genes of interest and their associated molecular pathways. NanoString is a reliable gene expression analysis platform that can be effectively coupled with LCM. PMID:27077656

  10. Adaptation of Laser Microdissection Technique for the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model by NanoString Technologies

    PubMed Central

    Saylor, Karen L.; Anver, Miriam R.; Salomon, David S.; Golubeva, Yelena G.

    2016-01-01

    Laser capture microdissection (LCM) of tissue is an established tool in medical research for collection of distinguished cell populations under direct microscopic visualization for molecular analysis. LCM samples have been successfully analyzed in a number of genomic and proteomic downstream molecular applications. However, LCM sample collection and preparation procedure has to be adapted to each downstream analysis platform. In this present manuscript we describe in detail the adaptation of LCM methodology for the collection and preparation of fresh frozen samples for NanoString analysis based on a study of a model of mouse mammary gland carcinoma and its lung metastasis. Our adaptation of LCM sample preparation and workflow to the requirements of the NanoString platform allowed acquiring samples with high RNA quality. The NanoString analysis of such samples provided sensitive detection of genes of interest and their associated molecular pathways. NanoString is a reliable gene expression analysis platform that can be effectively coupled with LCM. PMID:27077656

  11. HAdV-2-suppressed growth of SV40 T antigen-transformed mouse mammary epithelial cell-induced tumours in SCID mice.

    PubMed

    Wu, Chengjun; Cao, Xiaofang; Yu, Di; Huijbers, Elisabeth J M; Essand, Magnus; Akusjärvi, Göran; Johansson, Staffan; Svensson, Catharina

    2016-02-01

    Human adenovirus (HAdV) vectors are promising tools for cancer therapy, but the shortage of efficient animal models for productive HAdV infections has restricted the evaluation of systemic effects to mainly immunodeficient mice. Previously, we reported a highly efficient replication of HAdV-2 in a non-tumorigenic mouse mammary epithelial cell line, NMuMG. Here we show that HAdV-2 gene expression and progeny formation in NMuMG cells transformed with the SV40 T antigen (NMuMG-T cells) were as efficient as in the parental NMuMG cells. Injection of HAdV-2 into tumours established by NMuMG-T in SCID mice caused reduced tumour growth and signs of intratumoural lesions. HAdV-2 replicated within the NMuMG-T-established tumours, but not in interspersed host-derived tissues within the tumours. The specific infection of NMuMG-T-derived tumours was verified by the lack of viral DNA in kidney, lung or spleen although low levels of viral DNA was occasionally found in liver. PMID:26707269

  12. An obesity-dependent lactation defect in the viable yellow agouti mouse is associated with mammary inflammation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maternal obesity is known to delay lactogenesis in breast-feeding women, as well as negatively impact lactation in other species. Obesity is also understood to be associated with inflammation. Work with the viable yellow agouti (Avy) mouse in our laboratory has documented a lactation defect in obese...

  13. Mechanical tension induces lateral movement of intramembrane components of the tight junction: studies on mouse mammary cells in culture

    PubMed Central

    1983-01-01

    Occluding junctions of mammary epithelial cells in nonproliferating primary culture occasionally display an atypical pattern of intramembrane strands oriented predominantly perpendicular, instead of roughly parallel, to the apical border of the junction. To test whether the orienting influence was a centripetal cytoskeletal tension often observed in epithelial sheets on fixed substrates, we seeded cells at low density; this allows them to spread maximally while forming a barely confluent pavement. The result was a fourfold increase in the percentage of junctions with the strongly aligned, atypical pattern. Closely similar configurations were observed as the earliest detectable effect of chelation of extracellular Ca++, which induced pronounced centripetal contraction of the cell body. Externally imposed tension, applied so as to stretch cells in one direction only, affected the positions of strands in stretched junctions as might be predicted, by flattening their undulations, increasing their alignment parallel to the apical border. Thus mechanical tension alone, whether inherent in the cytoskeleton or imposed on the cell surface by exogenous force, can cause coordinate lateral displacement of macromolecular assemblies within the membranes of both joined cells. PMID:6682108

  14. Downregulation of CD73 in 4T1 breast cancer cells through siRNA-loaded chitosan-lactate nanoparticles.

    PubMed

    Jadidi-Niaragh, Farhad; Atyabi, Fatemeh; Rastegari, Ali; Mollarazi, Esmail; Kiani, Melika; Razavi, Alireza; Yousefi, Mehdi; Kheshtchin, Nasim; Hassannia, Hadi; Hadjati, Jamshid; Shokri, Fazel

    2016-06-01

    The immunosuppressive factors in tumor microenvironment enhance tumor growth and suppress anti-tumor immune responses. Adenosine is an important immunosuppressive factor which can be secreted by both tumor and immune cells trough action of two cell surface ecto-nucleotidase molecules CD39 and CD73. Blocking the adenosine generating molecules has emerged as an effective immunotherapeutic approach for treatment of cancer. In this study, CD73-siRNA encapsulated into chitosan-lactate (ChLa) nanoparticles (NPs) was employed to suppress the expression of CD73 molecule on 4T1 breast tumor cells, in vitro. ChLa NPs were generated through ionic gelation of ChLa by tripolyphosphate (TPP). Small interfering RNA (SiRNA)-loaded NPs had about 100 nm size with a polydispersive index below 0.3 and a zeta potential about 13. Our results showed that ChLa NPs with Ch 50 kDa exhibit the best physicochemical features with the high siRNA encapsulation capacity. Synthesized NPs were able to fully bind with siRNA, protect them against serum and heparin degradation, and promote the transfection process. While the NPs exhibited low toxicity during 72 h cell culture, the transfection of Ch-plasmid expressing green fluorescent protein (pEGFP) NPs was efficient in 4T1 cells with a transfection rate of 53.6 % as detected by flow cytometry. In addition, CD73-siRNA-loaded ChLa NPs could efficiently suppress the expression of CD73 as assayed by real-time polymerase chain reaction and flow cytometry. As a conclusion, CD73-siRNA-loaded ChLa NPs may be considered as a promising therapeutic tool for cancer therapy; however, further in vivo investigations are necessary.

  15. Tumorigenic WAP-T Mouse Mammary Carcinoma Cells: A Model for a Self-Reproducing Homeostatic Cancer Cell System

    PubMed Central

    Otto, Benjamin; Gruner, Katharina; Heinlein, Christina; Kühl, Marion; Warnecke, Gabriele; Schumacher, Udo; Deppert, Wolfgang; Tolstonog, Genrich V.

    2010-01-01

    Background In analogy to normal stem cell differentiation, the current cancer stem cell (CSC) model presumes a hierarchical organization and an irreversible differentiation in tumor tissue. Accordingly, CSCs should comprise only a small subset of the tumor cells, which feeds tumor growth. However, some recent findings raised doubts on the general applicability of the CSC model and asked for its refinement. Methodology/Principal Findings In this study we analyzed the CSC properties of mammary carcinoma cells derived from transgenic (WAP-T) mice. We established a highly tumorigenic WAP-T cell line (G-2 cells) that displays stem-like traits. G-2 cells, as well as their clonal derivates, are closely related to primary tumors regarding histology and gene expression profiles, and reflect heterogeneity regarding their differentiation states. G-2 cultures comprise cell populations in distinct differentiation states identified by co-expression of cytoskeletal proteins (cytokeratins and vimentin), a combination of cell surface markers and a set of transcription factors. Cellular subsets sorted according to expression of CD24a, CD49f, CD61, Epcam, Sca1, and Thy1 cell surface proteins, or metabolic markers (e.g. ALDH activity) are competent to reconstitute the initial cellular composition. Repopulation efficiency greatly varies between individual subsets and is influenced by interactions with the respective complementary G-2 cellular subset. The balance between differentiation states is regulated in part by the transcription factor Sox10, as depletion of Sox10 led to up-regulation of Twist2 and increased the proportion of Thy1-expressing cells representing cells in a self-renewable, reversible, quasi-mesenchymal differentiation state. Conclusions/Significance G-2 cells constitute a self-reproducing cancer cell system, maintained by bi- and unidirectional conversion of complementary cellular subsets. Our work contributes to the current controversial discussion on the existence

  16. 2D immunoblots show differential response of mouse IgG and IgM antibodies to antigens of mammary carcinoma 4 T1 cells

    PubMed Central

    2014-01-01

    Background Immunosuppression in breast cancer has been reported in women and in the highly metastatic mouse mammary tumor model 4 T1. The immunosuppressive environment complicates the use of the humoral response against the tumor as an immunodiagnostic tool. IgM has not been used in immunodiagnostic in part because its antitumor responses, both innate and adaptive, have not been studied in function of time in breast cancer. We show a new approach to analyzing the mouse humoral immune response, and compare the evolution with time of IgG and IgM responses against the antigens of 4 T1 cells. Methods The study is based on 2-dimensional immunoblotting detection of antigens from 4 T1 cells by the IgG and IgM antibodies in the serum of female mice injected with 4 T1 cells. Results There was a high variability in the intra-and inter-mouse response. Variability in the IgM response was manifested as a pattern of spots that could become a multibinomial variable of 0 and 1, which could represent a signature of the immune response. Different numbers of spots was found in the IgG and IgM responses from week 1 to 5. On average, the IgM had more but the IgG response decrease with the time. The natural IgM at t = 0 responds stronger than w1; the adaptive response of both IgM and IgG were elicited where, with the former being stronger better than the latter. Antigens that are recognized by some female mice in the first week are also recognized by other female mice at time 0. Contamination of the natural IgM makes difficult use the adaptive IgM as a tool for immunodiagnostic. Conclusions IgM and IgG response varied with the time and individuals. Spot variation in 2D pattern for the natural IgM could be expressed as a binomial signature, which opens up the way to correlate a particular pattern with resistance or susceptibility. This uncovers a battery of IgMs for each individual to confront cancer or infections. The possibility to differentiate between adaptive IgM antibodies

  17. In vivo antitumor and antimetastatic effects of flavokawain B in 4T1 breast cancer cell-challenged mice

    PubMed Central

    Abu, Nadiah; Mohamed, Nurul Elyani; Yeap, Swee Keong; Lim, Kian Lam; Akhtar, M Nadeem; Zulfadli, Aimi Jamil; Kee, Beh Boon; Abdullah, Mohd Puad; Omar, Abdul Rahman; Alitheen, Noorjahan Banu

    2015-01-01

    Flavokawain B (FKB) is a naturally occurring chalcone that can be isolated through the root extracts of the kava-kava plant (Piper methysticum). It can also be synthesized chemically to increase the yield. This compound is a promising candidate as a biological agent, as it is reported to be involved in a wide range of biological activities. Furthermore, FKB was reported to have antitumorigenic effects in several cancer cell lines in vitro. However, the in vivo antitumor effects of FKB have not been reported on yet. Breast cancer is one of the major causes of cancer-related deaths in the world today. Any potential treatment should not only impede the growth of the tumor, but also modulate the immune system efficiently and inhibit the formation of secondary tumors. As presented in our study, FKB induced apoptosis in 4T1 tumors in vivo, as evidenced by the terminal deoxynucleotidyl transferase dUTP nick end labeling and hematoxylin and eosin staining of the tumor. FKB also regulated the immune system by increasing both helper and cytolytic T-cell and natural killer cell populations. In addition, FKB also enhanced the levels of interleukin 2 and interferon gamma but suppressed interleukin 1B. Apart from that, FKB was also found to inhibit metastasis, as evaluated by clonogenic assay, bone marrow smearing assay, real-time polymerase chain reaction, Western blot, and proteome profiler analysis. All in all, FKB may serve as a promising anticancer agent, especially in treating breast cancer. PMID:25834398

  18. PG545, a heparan sulfate mimetic, reduces heparanase expression in vivo, blocks spontaneous metastases and enhances overall survival in the 4T1 breast carcinoma model.

    PubMed

    Hammond, Edward; Brandt, Ralf; Dredge, Keith

    2012-01-01

    PG545 is a clinically relevant heparan sulfate (HS) mimetic which, in addition to possessing anti-angiogenic properties, also acts as a heparanase inhibitor which may differentiate its mechanism(s) of action from approved angiogenesis inhibitors. The degradation of HS by heparanase has been strongly implicated in cell dissemination and the metastatic process. Thus, the anti-metastatic activity of PG545 has been linked to the enzymatic function of heparanase - the only endoglycosidase known to cleave HS, an important component of the extracellular matrix (ECM) which represents a potential avenue for therapeutic intervention for certain metastatic cancer indications. Recent concerns raised about the paucity of overall survival as an endpoint in mouse models of clinically relevant metastasis led us to examine the effect of PG545 on the progression of both primary tumor growth and the spontaneously metastasizing disease in the 4T1 syngeneic breast carcinoma model in a non-surgical and surgical (mastectomy) setting. PG545 significantly inhibited primary tumor growth but importantly also inhibited lung metastasis in treated mice, an effect not observed with the tyrosine kinase inhibitor sorafenib. Importantly, PG545 significantly enhanced overall survival compared to vehicle control and the sorafenib group, suggesting PG545's inhibitory effect on heparanase is indeed a critical attribute to induce anti-metastatic activity. In addition to blocking a common angiogenic signalling pathway in tumor cells, the expression of heparanase in the primary tumor and lung was also significantly reduced by PG545 treatment. These results support the ongoing development of PG545 and highlight the potential utility in metastatic disease settings. PMID:23300607

  19. INDUCTION OF MAMMARY GLAND DEVELOPMENT IN ESTROGEN RECEPTOR-ALPHA KNOCKOUT MICE

    EPA Science Inventory

    Mammary glands from the estrogen receptor knockout ( ERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted Er signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute t...

  20. Thymol inhibits LPS-stimulated inflammatory response via down-regulation of NF-κB and MAPK signaling pathways in mouse mammary epithelial cells.

    PubMed

    Liang, Dejie; Li, Fengyang; Fu, Yunhe; Cao, Yongguo; Song, Xiaojing; Wang, Tiancheng; Wang, Wei; Guo, Mengyao; Zhou, Ershun; Li, Depeng; Yang, Zhengtao; Zhang, Naisheng

    2014-02-01

    Thymol is a natural monoterpene phenol primarily found in thyme, oregano, and tangerine peel. It has been shown to possess anti-inflammatory property both in vivo and in vitro. In the present paper, we studied the anti-inflammatory effect of thymol in lipopolysaccharide (LPS)-stimulated mouse mammary epithelial cells (mMECs). The mMECs were stimulated with LPS in the presence or absence of thymol (10, 20, 40 μg/mL). The concentrations of tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-1β in the supernatants of culture were determined using enzyme-linked immunosorbent assay. Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), nuclear factor-κB (NF-κB), and inhibitor protein of NF-κB (IκBα) were measured using western blot. The results showed that thymol markedly inhibited the production of TNF-α and IL-6 in LPS-stimulated mMECs. The expression of iNOS and COX-2 was also suppressed by thymol in a dose-dependent manner. Furthermore, thymol blocked the phosphorylation of IκBα, NF-κB p65, ERK, JNK, and p38 mitogen-activated protein kinases (MAPKs) in LPS-stimulated mMECs. These results indicate that thymol exerted anti-inflammatory property in LPS-stimulated mMECs by interfering the activation of NF-κB and MAPK signaling pathways. Thereby, thymol may be a potential therapeutic agent against mastitis. PMID:24057926

  1. Umbelliprenin is Potentially Toxic Against the HT29, CT26, MCF-7, 4T1, A172, and GL26 Cell Lines, Potentially Harmful Against Bone Marrow-Derived Stem Cells, and Non-Toxic Against Peripheral Blood Mononuclear Cells

    PubMed Central

    Rashidi, Mohsen; Ziai, Seyed Ali; Moini Zanjani, Taraneh; Khalilnezhad, Ahad; Jamshidi, Hamidreza; Amani, Davar

    2016-01-01

    Background Resistance to chemotherapy is a growing concern, thus natural anticancer agents are drawing the attention of many scientists and clinicians. One natural anticancer agent, umbelliprenin, is a coumarin produced by many species of Ferula. Objectives We aimed to examine the inhibitory effect of umbelliprenin on human and mouse bone marrow-derived stem cells (BMDSCs), peripheral blood mononuclear cells (PBMCs), and different cancer cell lines. Materials and Methods In this in vitro experimental study, the HT29, CT26, MCF-7, 4T1, A172, and GL26 cancer cells and human and mouse BMDSCs and PBMCs were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), incubated at 37°C for 24 hours in a 5% CO2 atmosphere, and then were treated with different concentrations of umbelliprenin dissolved in dimethyl sulfoxide (DMSO) (3, 6, 12, 25, 50, 100, and 200 µg/mL) for 24, 48, and 72 hours at 37°C. Each experiment was performed in triplicate. Finally, the cell survival rate was assessed by MTT assay. The IC50 values were calculated based on the log values using GraphPad Prism version 5 software for windows (La Jolla CA, USA) and were expressed as mean ± SEM. Results Umbelliprenin inhibited the cancer cells in a concentration-dependent (P < 0.05) but not time-dependent manner (P > 0.05). The most sensitive and resistant cell lines at the 24-hour incubation time were 4T1 (IC50, 30.9 ± 3.1 µg/mL) and A172 (IC50, 51.9 ± 6.7 µg/mL); at the 48-hour incubation time: 4T1 (IC50, 30.6 ± 2.6 µg/mL) and CT26 (IC50, 53.2 ± 3.6 µg/mL); and at the 72-hour incubation time: HT29 (IC50, 37.1 ± 1.4 µg/mL) and 4T1 (IC50, 62.2 ± 4.8 µg/mL). Both human and mouse BMDSCs showed the highest resistance at the 24-hour incubation time (IC50s, 254.7 ± 21 and 204.4 ± 4.5 µg/mL, respectively) and the highest sensitivity at the 72-hour incubation time (IC50s, 120.4 ± 5 and 159.0 ± 7.3 µg/mL, respectively). The PBMCs of both human and mouse origin revealed very

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

    SciTech Connect

    Stubbs, J.D.; Bui, A. San Francisco State Univ., CA ); Lekutis, C.; Singer, K.L.; Srinivasan, U.; Parry, G. ); Yuzuki, D. )

    1990-11-01

    A 2.1-kilobase cDNA coding for a surface protein of mammary epithelial cells has been isolated from a mouse mammary gland {lambda}gt11 cDNA library. Sequence analysis of this cDNA reveals an open reading frame of 1,389 base pairs that defines a protein with a molecular mass of 51.5 dKa. Structural analysis of the predicted sequence identifies two putative functional domains of the protein: (i) an N-terminal cysteine-rich region that is similar to epidermal growth factor-like domains of Drosophila Notch-1 protein and (ii) a large segment of the sequence that exhibited 54.5% identify with C-terminal domains of human coagulation factors VIII and V. These similarities in structure are used to predict the possible functions of the protein and its means of interaction with the cell surface. mRNA expression was detectable in mammary tissue from nonpregnant animals but was maximal in the lactating gland. In cultured cells, mRNA levels also correlated with the degree of cellular differentiation.

  3. Effective chemoimmunotherapy with anti-TGFβ antibody and cyclophosphamide in a mouse model of breast cancer.

    PubMed

    Chen, Xin; Yang, Yuan; Zhou, Qiong; Weiss, Jonathan M; Howard, Olamae Zack; McPherson, John M; Wakefield, Lalage M; Oppenheim, Joost J

    2014-01-01

    TGFβ is reportedly responsible for accumulation of CD4(+)Foxp3(+) regulatory T cells (Tregs) in tumor. Thus, we treated mouse 4T1 mammary carcinoma with 1D11, a neutralizing anti-TGFβ (1,2,3) antibody. The treatment delayed tumor growth, but unexpectedly increased the proportion of Tregs in tumor. In vitro, 1D11 enhanced while TGFβ potently inhibited the proliferation of Tregs. To enhance the anti-tumor effects, 1D11 was administered with cyclophosphamide which was reported to eliminate intratumoral Tregs. This combination resulted in long term tumor-free survival of up to 80% of mice, and the tumor-free mice were more resistant to re-challenge with tumor. To examine the phenotype of tumor infiltrating immune cells, 4T1-tumor bearing mice were treated with 1D11 and a lower dose of cyclophosphamide. This treatment markedly inhibited tumor growth, and was accompanied by massive infiltration of IFNγ-producing T cells. Furthermore, this combination markedly decreased the number of splenic CD11b(+)Gr1(+) cells, and increased their expression levels of MHC II and CD80. In a spontaneous 4T1 lung metastasis model with resection of primary tumor, this combination therapy markedly increased the survival of mice, indicating it was effective in reducing lethal metastasis burden. Taken together, our data show that anti-TGFβ antibody and cyclophosphamide represents an effective chemoimmunotherapeutic combination. PMID:24416401

  4. Aspirin Breaks the Crosstalk between 3T3-L1 Adipocytes and 4T1 Breast Cancer Cells by Regulating Cytokine Production.

    PubMed

    Hsieh, Chia-Chien; Huang, Yu-Shan

    2016-01-01

    Breast cancer is one of the most common cancers in women worldwide. The obesity process is normally accompanied by chronic, low-grade inflammation. Infiltration by inflammatory cytokines and immune cells provides a favorable microenvironment for tumor growth, migration, and metastasis. Epidemiological evidence has shown that aspirin is an effective agent against several types of cancer. The aim of this study is to investigate the anti-inflammatory and anti-cancer effects of aspirin on 3T3-L1 adipocytes, 4T1 murine breast cancer cells, and their crosstalk. The results showed that aspirin treatment inhibited differentiation and lipid accumulation by 3T3-L1 preadipocytes, and decreased the secretion of the inflammatory adipokine MCP-1 after stimulation with tumor necrosis factor (TNF)-α or conditioned medium from RAW264.7 cells. In 4T1 cells, treatment with aspirin decreased cell viability and migration, possibly by suppressing MCP-1 and VEGF secretion. Subsequently, culture of 4T1 cells in 3T3-L1 adipocyte-conditioned medium (Ad-CM) and co-culture of 3T3-L1 and 4T1 cells using a transwell plate were performed to clarify the relationship between these two cell lines. Aspirin exerted its inhibitory effects in the transwell co-culture system, as well as the conditioned-medium model. Aspirin treatment significantly inhibited the proliferation of 4T1 cells, and decreased the production of MCP-1 and PAI-1 in both the Ad-CM model and co-culture system. Aspirin inhibited inflammatory MCP-1 adipokine production by 3T3-L1 adipocytes and the cell growth and migration of 4T1 cells. It also broke the crosstalk between these two cell lines, possibly contributing to its chemopreventive properties in breast cancer. This is the first report that aspirin's chemopreventive activity supports the potential application in auxiliary therapy against obesity-related breast cancer development. PMID:26794215

  5. Aspirin Breaks the Crosstalk between 3T3-L1 Adipocytes and 4T1 Breast Cancer Cells by Regulating Cytokine Production.

    PubMed

    Hsieh, Chia-Chien; Huang, Yu-Shan

    2016-01-01

    Breast cancer is one of the most common cancers in women worldwide. The obesity process is normally accompanied by chronic, low-grade inflammation. Infiltration by inflammatory cytokines and immune cells provides a favorable microenvironment for tumor growth, migration, and metastasis. Epidemiological evidence has shown that aspirin is an effective agent against several types of cancer. The aim of this study is to investigate the anti-inflammatory and anti-cancer effects of aspirin on 3T3-L1 adipocytes, 4T1 murine breast cancer cells, and their crosstalk. The results showed that aspirin treatment inhibited differentiation and lipid accumulation by 3T3-L1 preadipocytes, and decreased the secretion of the inflammatory adipokine MCP-1 after stimulation with tumor necrosis factor (TNF)-α or conditioned medium from RAW264.7 cells. In 4T1 cells, treatment with aspirin decreased cell viability and migration, possibly by suppressing MCP-1 and VEGF secretion. Subsequently, culture of 4T1 cells in 3T3-L1 adipocyte-conditioned medium (Ad-CM) and co-culture of 3T3-L1 and 4T1 cells using a transwell plate were performed to clarify the relationship between these two cell lines. Aspirin exerted its inhibitory effects in the transwell co-culture system, as well as the conditioned-medium model. Aspirin treatment significantly inhibited the proliferation of 4T1 cells, and decreased the production of MCP-1 and PAI-1 in both the Ad-CM model and co-culture system. Aspirin inhibited inflammatory MCP-1 adipokine production by 3T3-L1 adipocytes and the cell growth and migration of 4T1 cells. It also broke the crosstalk between these two cell lines, possibly contributing to its chemopreventive properties in breast cancer. This is the first report that aspirin's chemopreventive activity supports the potential application in auxiliary therapy against obesity-related breast cancer development.

  6. The Mammary Glands of Macaques

    PubMed Central

    Cline, J. Mark; Wood, Charles E.

    2009-01-01

    This review describes the normal biology and physiology of the mammary gland in macaques, including the typical histologic appearance across the life span (development, reproductive maturity, lactation, and senescence). The molecular events regulating breast morphogenesis are described, as well as systemic and local hormonal regulators of mammary gland proliferation, differentiation, and function. Similarities and differences to the human breast are described. Regulatory events are illuminated by discussion of genetically modified mouse models. Tissue response markers, including immunohistochemical markers of proliferation and other hormonally induced changes and studies to date, regarding the effects of exogenous hormones, are briefly summarized. In general, estrogens stimulate progesterone receptor expression and proliferation in the mammary gland, and combinations of estrogens and progestogens cause greater proliferation than estrogens alone. Evaluation of novel chemical agents in macaques requires careful evaluation of age and hormonal context to avoid the confounding effects of mammary gland development, past reproductive history, and other influences on mammary gland morphology. The expression of proliferation markers and progesterone receptors may be used as biomarkers to measure chemically induced hormonal effects. PMID:21475638

  7. Administration of probiotics Lactobacillus rhamnosus GG and Lactobacillus gasseri K7 during pregnancy and lactation changes mouse mesenteric lymph nodes and mammary gland microbiota.

    PubMed

    Treven, P; Mrak, V; Bogovič Matijašić, B; Horvat, S; Rogelj, I

    2015-04-01

    The milk and mammary gland (MG) microbiome can be influenced by several factors, such as mode of delivery, breastfeeding, maternal lifestyle, health status, and diet. An increasing number of studies show a variety of positive effects of consumption of probiotics during pregnancy and breastfeeding on the mother and the newborn. The aim of this study was to investigate the effect of oral administration of probiotics Lactobacillus gasseri K7 (LK7) and Lactobacillus rhamnosus GG (LGG) during pregnancy and lactation on microbiota of the mouse mesenteric lymph nodes (MLN), MG, and milk. Pregnant FVB/N mice were fed skim milk or probiotics LGG or LK7 resuspended in skim milk during gestation and lactation. On d 3 and 8 postpartum, blood, feces, MLN, MG, and milk were analyzed for the presence of LGG or LK7. The effects of probiotics on MLN, MG, and milk microbiota was evaluated by real-time PCR and by 16S ribosomal DNA 454-pyrosequencing. In 5 of 8 fecal samples from the LGG group and in 5 of 8 fecal samples from the LK7 group, more than 1 × 10(3) of live LGG or LK7 bacterial cells were detected, respectively, whereas no viable LGG or LK7 cells were detected in the control group. Live lactic acid bacteria but no LGG or LK7 were detected in blood, MLN, and MG. Both probiotics significantly increased the total bacterial load as assessed by copies of 16S ribosomal DNA in MLN, and a similar trend was observed in MG. Metagenomic sequencing revealed that both probiotics increased the abundance of Firmicutes in MG, especially the abundance of lactic acid bacteria. The Lactobacillus genus appeared exclusively in MG from probiotic groups. Both probiotics influenced MLN microbiota by decreasing diversity (Chao1) and increasing the distribution of species (Shannon index). The LGG probiotic also affected the MG microbiota as it increased diversity and distribution of species and proportions of the genera Lactobacillus and Bifidobacterium. These results provide evidence that

  8. Administration of probiotics Lactobacillus rhamnosus GG and Lactobacillus gasseri K7 during pregnancy and lactation changes mouse mesenteric lymph nodes and mammary gland microbiota.

    PubMed

    Treven, P; Mrak, V; Bogovič Matijašić, B; Horvat, S; Rogelj, I

    2015-04-01

    The milk and mammary gland (MG) microbiome can be influenced by several factors, such as mode of delivery, breastfeeding, maternal lifestyle, health status, and diet. An increasing number of studies show a variety of positive effects of consumption of probiotics during pregnancy and breastfeeding on the mother and the newborn. The aim of this study was to investigate the effect of oral administration of probiotics Lactobacillus gasseri K7 (LK7) and Lactobacillus rhamnosus GG (LGG) during pregnancy and lactation on microbiota of the mouse mesenteric lymph nodes (MLN), MG, and milk. Pregnant FVB/N mice were fed skim milk or probiotics LGG or LK7 resuspended in skim milk during gestation and lactation. On d 3 and 8 postpartum, blood, feces, MLN, MG, and milk were analyzed for the presence of LGG or LK7. The effects of probiotics on MLN, MG, and milk microbiota was evaluated by real-time PCR and by 16S ribosomal DNA 454-pyrosequencing. In 5 of 8 fecal samples from the LGG group and in 5 of 8 fecal samples from the LK7 group, more than 1 × 10(3) of live LGG or LK7 bacterial cells were detected, respectively, whereas no viable LGG or LK7 cells were detected in the control group. Live lactic acid bacteria but no LGG or LK7 were detected in blood, MLN, and MG. Both probiotics significantly increased the total bacterial load as assessed by copies of 16S ribosomal DNA in MLN, and a similar trend was observed in MG. Metagenomic sequencing revealed that both probiotics increased the abundance of Firmicutes in MG, especially the abundance of lactic acid bacteria. The Lactobacillus genus appeared exclusively in MG from probiotic groups. Both probiotics influenced MLN microbiota by decreasing diversity (Chao1) and increasing the distribution of species (Shannon index). The LGG probiotic also affected the MG microbiota as it increased diversity and distribution of species and proportions of the genera Lactobacillus and Bifidobacterium. These results provide evidence that

  9. Hybrid paclitaxel and gold nanorod-loaded human serum albumin nanoparticles for simultaneous chemotherapeutic and photothermal therapy on 4T1 breast cancer cells.

    PubMed

    Peralta, Donna V; Heidari, Zahra; Dash, Srikanta; Tarr, Matthew A

    2015-04-01

    The use of human serum albumin nanoparticles (HSAPs) as a drug carrier system for cancer treatment has proven successful through current marketable clinical formulations. Despite this success, there is a current lack of multifunctional HSAPs, which offer combinational therapies of more than one proven technique. Gold nanorods (AuNRs) have also shown medicinal promise due to their photothermal therapy capabilities. In this study, a desolvation and cross-linking approach was employed to successfully encapsulate gold nanorods into HSAPs simultaneously with the chemotherapeutic drug paclitaxel (PAC); forming PAC-AuNR-HSAPs with desirable overall particle sizes of 299 ± 6 nm. The loading efficiency of paclitaxel into PAC-AuNR-HSAPs reached up to 3 μg PAC/mg HSA. The PAC-AuNR-HSAPs experienced photothermal heating; with the bulk particle solution reaching up to 46 °C after 15 min of near-IR laser exposure. This heat increase marked the successful attainment of the temperature necessary to cause severe cellular hyperthermia and necrosis. The encasement strategy facilitated a colloidal hybrid treatment system capable of enhanced permeability and retention effects, photothermal ablation of cancer cells, and release of the active paclitaxel of up to 188 ng (from PAC-AuNR-HSAPs created with 30 mg HSA) in a single 15 min irradiation session. When treated with PAC-AuNR-HSAPs containing 20 μg PAC/mL particle solution, 4T1 mouse breast cancer cells experienced ∼82% cell death without irradiation and ∼94% cell death after just one irradiation session. The results for PAC-AuNR-HSAPs were better than that of free PAC, which only killed ∼77% of the cells without irradiation and ∼80% with irradiation. The hybrid particle system also lends itself to future customizable external functionalities via conjugated targeting ligands, such as antibodies. Internal entrapment of patient tailored medication combinations are also possible with this combination treatment platform, which

  10. Positional variations in mammary gland development and cancer.

    PubMed

    Veltmaat, Jacqueline M; Ramsdell, Ann F; Sterneck, Esta

    2013-06-01

    Most mammals develop their mammary glands in pairs of which the two counterparts are symmetrically displaced away from the ventral midline. Based on this symmetry and the same functional outcome as a milk-producing organ, the mammary glands are easily presumed to be mere copies of one another. Based on our analysis of published data with inclusion of new results related to mammary development and pathology in mice, we argue that this presumption is incorrect: Between and within pairs, mammary glands differ from one another, and tumor incidence and biology depend on the position along the anterior-posterior and the left-right axis as well. This insight has implications for experimental designs with mouse models and for data extrapolation between mammary glands within and between species. We suggest that improved documentation of location-specific mammary gland features will lead to more insights into the molecular mechanisms of mammary gland development and cancer biology in both mice and humans.

  11. An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis.

    PubMed

    Paschall, Amy V; Liu, Kebin

    2016-01-01

    Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 10(4) cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo. PMID:27584043

  12. The MAPKERK-1,2 pathway integrates distinct and antagonistic signals from TGF alpha and FGF7 in morphogenesis of mouse mammary epithelium

    SciTech Connect

    Fata, Jimmie E; Mori, Hidetoshi; Ewald, Andrew J; Zhang, Hui; Yao, Evelyn; Werb, Zena; Bissell, Mina J

    2006-10-03

    Transforming growth factor-{alpha} (TGF{alpha}) and fibroblast growth factor-7 (FGF7) exhibit distinct expression patterns in the mammary gland. Both factors signal through mitogen-activated kinase/extracellular regulated kinase-1,2 (MAPK{sup ERK1,2}); however, their unique and/or combined contributions to mammary morphogenesis have not been examined. In ex vivo mammary explants, we show that a sustained activation of MAPK{sup ERK1,2} for 1 h, induced by TGF{alpha}, was necessary and sufficient to initiate branching morphogenesis, whereas a transient activation (15 min) of MAPK{sup ERK1,2}, induced by FGF7, led to growth without branching. Unlike TGF{alpha}, FGF7 promoted sustained proliferation as well as ectopic localization of, and increase in, keratin-6 expressing cells. The response of the explants to FGF10 was similar to that to FGF7. Simultaneous stimulation by FGF7 and TGF{alpha} indicated that the FGF7-induced MAPK{sup ERK1,2} signaling and associated phenotypes were dominant: FGF7 may prevent branching by suppression of two necessary TGF{alpha}-induced morphogenetic effectors, matrix metalloproteinase-3 (MMP-3/stromelysin-1), and fibronectin. Our findings indicate that expression of morphogenetic effectors, proliferation, and cell-type decisions during mammary organoid morphogenesis are intimately dependent on the duration of activation of MAPK{sup ERK1,2} activation.

  13. High-Fat, High-Calorie Diet Enhances Mammary Carcinogenesis and Local Inflammation in MMTV-PyMT Mouse Model of Breast Cancer

    PubMed Central

    Cowen, Sarah; McLaughlin, Sarah L.; Hobbs, Gerald; Coad, James; Martin, Karen H.; Olfert, I. Mark; Vona-Davis, Linda

    2015-01-01

    Epidemiological studies provide strong evidence that obesity and the associated adipose tissue inflammation are risk factors for breast cancer; however, the molecular mechanisms are poorly understood. We evaluated the effect of a high-fat/high-calorie diet on mammary carcinogenesis in the immunocompetent MMTV-PyMT murine model. Four-week old female mice (20/group) were randomized to receive either a high-fat (HF; 60% kcal as fat) or a low-fat (LF; 16% kcal) diet for eight weeks. Body weights were determined, and tumor volumes measured by ultrasound, each week. At necropsy, the tumors and abdominal visceral fat were weighed and plasma collected. The primary mammary tumors, adjacent mammary fat, and lungs were preserved for histological and immunohistochemical examination and quantification of infiltrating macrophages, crown-like structure (CLS) formation, and microvessel density. The body weight gains, visceral fat weights, the primary mammary tumor growth rates and terminal weights, were all significantly greater in the HF-fed mice. Adipose tissue inflammation in the HF group was indicated by hepatic steatosis, pronounced macrophage infiltration and CLS formation, and elevations in plasma monocyte chemoattractant protein-1 (MCP-1), leptin and proinflammatory cytokine concentrations. HF intake was also associated with higher tumor-associated microvascular density and the proangiogenic factor MCP-1. This study provides preclinical evidence in a spontaneous model of breast cancer that mammary adipose tissue inflammation induced by diet, enhances the recruitment of macrophages and increases tumor vascular density suggesting a role for obesity in creating a microenvironment favorable for angiogenesis in the progression of breast cancer. PMID:26132316

  14. Loss of one Tgfbr2 allele in fibroblasts promotes metastasis in MMTV: polyoma middle T transgenic and transplant mouse models of mammary tumor progression

    PubMed Central

    Fang, Wei Bin; Jokar, Iman; Chytil, Anna; Moses, Harold L.; Abel, Ty; Cheng, Nikki

    2012-01-01

    Accumulation of fibroblasts is a phenomenon that significantly correlates with formation of aggressive cancers. While studies have shown that the TGF-β signaling pathway is an important regulator of fibroblast activation, the functional contribution of TGF-β signaling in fibroblasts during multi-step tumor progression remains largely unclear. In previous studies, we used a sub-renal capsule transplantation model to demonstrate that homozygous knockout of the Tgfbr2 gene (Tgbr2FspKO) enhanced mammary tumor growth and metastasis. Here, we show for the first time a significant role for loss of one Tgfbr2 allele during multi-step mammary tumor progression. Heterozygous deletion of Tgfbr2 in stromal cells in MMTV–PyVmT transgenic mice (PyVmT/Tgfbr2hetFspKO mice) resulted in earlier tumor formation and increased stromal cell accumulation. In contrast to previous studies of Tgbr2FspKO fibroblasts, Tgfbr2hetFspKO fibroblasts did not significantly increase tumor growth, but enhanced lung metastasis in PyVmT transgenic mice and in co-transplantation studies with PyVmT mammary carcinoma cells. Furthermore, Tgfbr2hetFspKO fibroblasts enhanced mammary carcinoma cell invasiveness associated with expression of inflammatory cytokines including CXCL12 and CCL2. Analyses of Tgbr2FspKO and Tgfbr2hetFspKO fibroblasts revealed differences in the expression of factors associated with metastatic spread, indicating potential differences in the mechanism of action between homozygous and heterozygous deletion of Tgfbr2 in stromal cells. In summary, these studies demonstrate for the first time that loss of one Tgfbr2 allele in fibroblasts enhances mammary metastases in a multi-step model of tumor progression, and demonstrate the importance of clarifying the functional contribution of genetic alterations in stromal cells in breast cancer progression. PMID:21374085

  15. Molecular changes in bone marrow, tumor and serum after conductive ablation of murine 4T1 breast carcinoma.

    PubMed

    Przybyla, Beata D; Shafirstein, Gal; Vishal, Sagar J; Dennis, Richard A; Griffin, Robert J

    2014-02-01

    Thermal ablation of solid tumors using conductive interstitial thermal therapy (CITT) produces coagulative necrosis in the center of ablation. Local changes in homeostasis for surviving tumor and systemic changes in circulation and distant organs must be understood and monitored in order to prevent tumor re-growth and metastasis. The purpose of this study was to use a mouse carcinoma model to evaluate molecular changes in the bone marrow and surviving tumor after CITT treatment by quantification of transcripts associated with cancer progression and hyperthermia, serum cytokines, stress proteins and the marrow/tumor cross-talk regulator stromal-derived factor 1. Analysis of 27 genes and 22 proteins with quantitative PCR, ELISA, immunoblotting and multiplex antibody assays revealed that the gene and protein expression in tissue and serum was significantly different between ablated and control mice. The transcripts of four genes (Cxcl12, Sele, Fgf2, Lifr) were significantly higher in the bone marrow of treated mice. Tumors surviving ablation showed significantly lower levels of the Lifr and Sele transcripts. Similarly, the majority of transcripts measured in tumors decreased with treatment. Surviving tumors also contained lower levels of SDF-1α and HIF-1α proteins whereas HSP27 and HSP70 were higher. Of 16 serum chemokines, IFNγ and GM-CSF levels were lower with treatment. These results indicate that CITT ablation causes molecular changes which may slow cancer cell proliferation. However, inhibition of HSP27 may be necessary to control aggressiveness of surviving cancer stem cells. The changes in bone marrow are suggestive of possible increased recruitment of circulatory cancer cells. Therefore, the possibility of heightened bone metastasis after thermal ablation needs to be further investigated and inhibition strategies developed, if warranted.

  16. Exposure to Ionizing Radiation Causes Long-Term Increase in Serum Estradiol and Activation of PI3K-Akt Signaling Pathway in Mouse Mammary Gland

    SciTech Connect

    Suman, Shubhankar; Johnson, Michael D.; Fornace, Albert J.; Datta, Kamal

    2012-10-01

    Purpose: Exposure to ionizing radiation is an established risk factor for breast cancer. Radiation exposure during infancy, childhood, and adolescence confers the highest risk. Although radiation is a proven mammary carcinogen, it remains unclear where it acts in the complex multistage process of breast cancer development. In this study, we investigated the long-term pathophysiologic effects of ionizing radiation at a dose (2 Gy) relevant to fractionated radiotherapy. Methods and Materials: Adolescent (6-8 weeks old; n = 10) female C57BL/6J mice were exposed to 2 Gy total body {gamma}-radiation, the mammary glands were surgically removed, and serum and urine samples were collected 2 and 12 months after exposure. Molecular pathways involving estrogen receptor-{alpha} (ER{alpha}) and phosphatidylinositol-3-OH kinase (PI3K)-Akt signaling were investigated by immunohistochemistry and Western blot. Results: Serum estrogen and urinary levels of the oncogenic estrogen metabolite (16{alpha}OHE1) were significantly increased in irradiated animals. Immunostaining for the cellular proliferative marker Ki-67 and cyclin-D1 showed increased nuclear accumulation in sections of mammary glands from irradiated vs. control mice. Marked increase in p85{alpha}, a regulatory sub-unit of the PI3K was associated with increase in Akt, phospho-Akt, phospho-BAD, phospho-mTOR, and c-Myc in irradiated samples. Persistent increase in nuclear ER{alpha} in mammary tissues 2 and 12 months after radiation exposure was also observed. Conclusions: Taken together, our data not only support epidemiologic observations associating radiation and breast cancer but also, specify molecular events that could be involved in radiation-induced breast cancer.

  17. Reduced nursing frequency during prolonged lactation in the mouse decreases milk production and increases mammary expression of tryptophan hydroxylase 1 (TPH1), but does not accelerate mammary gland remodeling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have observed that lactating mouse dams nursed 4 times per day (4X) maintained lactation, but had lower milk yields by the weigh-suckle-weigh method, than dams nursed ad libitum (AL). Therefore, we hypothesized that decreased nursing frequency would also decrease lactation persistence, increase m...

  18. Enhancement of maternal lactation performance during prolonged lactation in the mouse by mouse GH and long-R3-IGF-I is linked to changes in mammary signaling and gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    GH, prolactin (PRL), and IGF-I stimulate lactation-related metabolic processes in mammary epithelial cells. However, the ability of these factors to stimulate milk production in animals varies depending on species and experimental variables. Previous work in our laboratory demonstrated that transgen...

  19. Phospholipid makeup of the breast adipose tissue is impacted by obesity and mammary cancer in the mouse: Results of a pilot study.

    PubMed

    Margolis, Michael; Perez, Osvaldo; Martinez, Mitchell; Santander, Ana M; Mendez, Armando J; Nadji, Mehrdad; Nayer, Ali; Bhattacharya, Sanjoy; Torroella-Kouri, Marta

    2015-01-01

    Obesity, an established risk factor for breast cancer (BC), is associated with systemic inflammation. The breast contains adipose tissue (bAT), yet whether it plays a role in BC progression in obese females is being intensively studied. There is scarce knowledge on the lipid composition of bAT in health and disease. The purpose of this pilot study was: 1) to determine whether obesity and BC are associated with inflammatory changes in bAT 2) to analyze for the first time the lipid profile of bAT in obese and lean mammary tumor-bearing and normal mice. Syngeneic E0771 mammary tumor cells were implanted into the mammary fat pad of lean and diet-induced obese C57BL/6 mice. BATs were analyzed four weeks after tumor cell inoculation by immunohistochemistry and mass spectrometry. Phospholipids were identified and subjected to ratiometric quantification using a TSQ Quantum Access Max triple quadrupole mass spectrometer utilizing precursor ion scan or neutral ion loss scan employing appropriate class specific lipid standards in a two step quantification process. Four main classes of phospholipids were analyzed: phosphatidylcholines phosphatidylserines, phosphatidylethanolamines and phosphatidylinositols. Our results showed that bAT in obese (normal and tumor-bearing) mice contained hypertrophic adipocytes compared with their corresponding samples in lean mice; higher numbers of macrophages and crown-like structures were observed in obese tumor bearers compared to obese normal mice. BAT from normal obese mice revealed higher concentrations of phosphatidylethanolamines. Furthermore, bAT from tumor-bearing mice expressed higher phosphatidylcholines than that from non-tumor bearing mice, suggesting the presence of the tumor is associated with phosphatidylcholines. Conversion of phosphatidylethanolamines to phosphatidylcholines will be investigated in E0771 cells. Additional studies are projected to investigate macrophage activation by these specific classes of phospholipids

  20. 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.

  1. Suppressive effects of a proton beam on tumor growth and lung metastasis through the inhibition of metastatic gene expression in 4T1 orthotopic breast cancer model.

    PubMed

    Kwon, Yun-Suk; Lee, Kyu-Shik; Chun, So-Young; Jang, Tae Jung; Nam, Kyung-Soo

    2016-07-01

    A proton beam is a next generation tool to treat intractable cancer. Although the therapeutic effects of a proton beam are well known, the effect on tumor metastasis is not fully described. Here, we investigated the effects of a proton beam on metastasis in highly invasive 4T1 murine breast cancer cells and their orthotopic breast cancer model. Cells were irradiated with 2, 4, 8 or 16 Gy proton beam, and changes in cell proliferation, survival, and migration were observed by MTT, colony forming and wound healing assays. 4T1 breast cancer cell-implanted BALB/c mice were established and the animals were randomly divided into 4 groups when tumor size reached 200 mm3. Breast tumors were selectively irradiated with 10, 20 or 30 Gy proton beam. Breast tumor sizes were measured twice a week, and breast tumor and lung tissues were pathologically observed. Metastasis-regulating gene expression was assessed with quantitative RT-PCR. A proton beam dose-dependently decreased cell proliferation, survival and migration in 4T1 murine breast cancer cells. Also, growth of breast tumors in the 4T1 orthotopic breast cancer model was significantly suppressed by proton beam irradiation without significant change of body weight. Furthermore, fewer tumor nodules metastasized from breast tumor into lung in mice irradiated with 30 Gy proton beam, but not with 10 and 20 Gy, than in control. We observed correspondingly lower expression levels of urokinase plasminogen activator (uPA), uPA receptor, cyclooxygenase (COX)-2, and vascular endothelial growth factor (VEGF), which are important factors in cancer metastasis, in breast tumor irradiated with 30 Gy proton beam. Proton beam irradiation did not affect expressions of matrix metalloproteinase (MMP)-9 and MMP-2. Taken together, the data suggest that, although proton beam therapy is an effective tool for breast cancer treatment, a suitable dose is necessary to prevent metastasis-linked relapse and poor prognosis. PMID:27176787

  2. Roles of Fas and Fas ligand during mammary gland remodeling

    PubMed Central

    Song, Joon; Sapi, Eva; Brown, Wendi; Nilsen, Jon; Tartaro, Karrie; Kacinski, Barry M.; Craft, Joseph; Naftolin, Frederick; Mor, Gil

    2000-01-01

    Mammary involution is associated with degeneration of the alveolar structure and programmed cell death of mammary epithelial cells. In this study, we evaluated the expression of Fas and Fas ligand (FasL) in the mammary gland tissue and their possible role in the induction of apoptosis of mammary cells. FasL-positive cells were observed in normal mammary epithelium from pregnant and lactating mice, but not in nonpregnant/virgin mouse mammary tissue. Fas expression was observed in epithelial and stromal cells in nonpregnant mice but was absent during pregnancy. At day 1 after weaning, high levels of both Fas and FasL proteins and caspase 3 were observed and coincided with the appearance of apoptotic cells in ducts and glands. During the same period, no apoptotic cells were found in the Fas-deficient (MRL/lpr) and FasL-deficient (C3H/gld) mice. Increase in Fas and FasL protein was demonstrated in human (MCF10A) and mouse (HC-11) mammary epithelial cells after incubation in hormone-deprived media, before apoptosis was detected. These results suggest that the Fas-FasL interaction plays an important role in the normal remodeling of mammary tissue. Furthermore, this autocrine induction of apoptosis may prevent accumulation of cells with mutations and subsequent neoplastic development. Failure of the Fas/FasL signal could contribute to tumor development. PMID:11086022

  3. Phospholipid makeup of the breast adipose tissue is impacted by obesity and mammary cancer in the mouse: results of a pilot study

    PubMed Central

    Margolis, Michael; Perez, Osvaldo; Martinez, Mitchel; Santander, Ana M.; Mendez, Armando J.; Nadji, Mehrdad; Nayer, Ali; Bhattacharya, Sanjoy; Torroella-Kouri, Marta

    2014-01-01

    Obesity, an established risk factor for breast cancer (BC), is associated with systemic inflammation. The breast contains adipose tissue (bAT), yet whether it plays a role in BC progression in obese females is being intensively studied. There is scarce knowledge on the lipid composition of bAT in health and disease. The purpose of this pilot study was: 1) to determine whether obesity and BC are associated with inflammatory changes in bAT 2) to analyze for the first time the lipid profile of bAT in obese and lean mammary tumor-bearing and normal mice. Syngeneic E0771 mammary tumor cells were implanted into the mammary fat pad of lean and diet-induced obese C57BL/6 mice. BATs were analyzed four weeks after tumor cell inoculation by immunohistochemistry and mass spectrometry. Phospholipids were identified and subjected to ratiometric quantification using a TSQ Quantum Access Max triple quadrupole mass spectrometer utilizing precursor ion scan or neutral ion loss scan employing appropriate class specific lipid standards in a two step quantification process. Four main classes of phospholipids were analyzed: phosphatidylcholines phosphatidylserines, phosphatidylethanolamines and phosphatidylinositols. Our results showed that bAT in obese (normal and tumor-bearing) mice contained hypertrophic adipocytes compared with their corresponding samples in lean mice; higher numbers of macrophages and crown-like structures were observed in obese tumor bearers compared to obese normal mice. BAT from normal obese mice revealed higher concentrations of phosphatidylethanolamines. Furthermore, bAT from tumor-bearing mice expressed higher phosphatidylcholines than that from non-tumor bearing mice, suggesting the presence of the tumor is associated with phosphatidylcholines. Conversion of phosphatidylethanolamines to phosphatidylcholines will be investigated in E0771 cells. Additional studies are projected to investigate macrophage activation by these specific classes of phospholipids

  4. Phospholipid makeup of the breast adipose tissue is impacted by obesity and mammary cancer in the mouse: Results of a pilot study.

    PubMed

    Margolis, Michael; Perez, Osvaldo; Martinez, Mitchell; Santander, Ana M; Mendez, Armando J; Nadji, Mehrdad; Nayer, Ali; Bhattacharya, Sanjoy; Torroella-Kouri, Marta

    2015-01-01

    Obesity, an established risk factor for breast cancer (BC), is associated with systemic inflammation. The breast contains adipose tissue (bAT), yet whether it plays a role in BC progression in obese females is being intensively studied. There is scarce knowledge on the lipid composition of bAT in health and disease. The purpose of this pilot study was: 1) to determine whether obesity and BC are associated with inflammatory changes in bAT 2) to analyze for the first time the lipid profile of bAT in obese and lean mammary tumor-bearing and normal mice. Syngeneic E0771 mammary tumor cells were implanted into the mammary fat pad of lean and diet-induced obese C57BL/6 mice. BATs were analyzed four weeks after tumor cell inoculation by immunohistochemistry and mass spectrometry. Phospholipids were identified and subjected to ratiometric quantification using a TSQ Quantum Access Max triple quadrupole mass spectrometer utilizing precursor ion scan or neutral ion loss scan employing appropriate class specific lipid standards in a two step quantification process. Four main classes of phospholipids were analyzed: phosphatidylcholines phosphatidylserines, phosphatidylethanolamines and phosphatidylinositols. Our results showed that bAT in obese (normal and tumor-bearing) mice contained hypertrophic adipocytes compared with their corresponding samples in lean mice; higher numbers of macrophages and crown-like structures were observed in obese tumor bearers compared to obese normal mice. BAT from normal obese mice revealed higher concentrations of phosphatidylethanolamines. Furthermore, bAT from tumor-bearing mice expressed higher phosphatidylcholines than that from non-tumor bearing mice, suggesting the presence of the tumor is associated with phosphatidylcholines. Conversion of phosphatidylethanolamines to phosphatidylcholines will be investigated in E0771 cells. Additional studies are projected to investigate macrophage activation by these specific classes of phospholipids

  5. 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.

  6. Radio-photothermal therapy mediated by a single compartment nanoplatform depletes tumor initiating cells and reduces lung metastasis in the orthotopic 4T1 breast tumor model

    NASA Astrophysics Data System (ADS)

    Zhou, Min; Zhao, Jun; Tian, Mei; Song, Shaoli; Zhang, Rui; Gupta, Sanjay; Tan, Dongfeng; Shen, Haifa; Ferrari, Mauro; Li, Chun

    2015-11-01

    Tumor Initiating Cells (TICs) are resistant to radiotherapy and chemotherapy, and are believed to be responsible for tumor recurrence and metastasis. Combination therapies can overcome the limitation of conventional cancer treatments, and have demonstrated promising application in the clinic. Here, we show that dual modality radiotherapy (RT) and photothermal therapy (PTT) mediated by a single compartment nanosystem copper-64-labeled copper sulfide nanoparticles ([64Cu]CuS NPs) could suppress breast tumor metastasis through eradication of TICs. Positron electron tomography (PET) imaging and biodistribution studies showed that more than 90% of [64Cu]CuS NPs was retained in subcutaneously grown BT474 breast tumor 24 h after intratumoral (i.t.) injection, indicating the NPs are suitable for the combination therapy. Combined RT/PTT therapy resulted in significant tumor growth delay in the subcutaneous BT474 breast cancer model. Moreover, RT/PTT treatment significantly prolonged the survival of mice bearing orthotopic 4T1 breast tumors compared to no treatment, RT alone, or PTT alone. The RT/PTT combination therapy significantly reduced the number of tumor nodules in the lung and the formation of tumor mammospheres from treated 4T1 tumors. No obvious side effects of the CuS NPs were noted in the treated mice in a pilot toxicity study. Taken together, our data support the feasibility of a therapeutic approach for the suppression of tumor metastasis through localized RT/PTT therapy.Tumor Initiating Cells (TICs) are resistant to radiotherapy and chemotherapy, and are believed to be responsible for tumor recurrence and metastasis. Combination therapies can overcome the limitation of conventional cancer treatments, and have demonstrated promising application in the clinic. Here, we show that dual modality radiotherapy (RT) and photothermal therapy (PTT) mediated by a single compartment nanosystem copper-64-labeled copper sulfide nanoparticles ([64Cu]CuS NPs) could suppress

  7. Selenium Induces an Anti-tumor Effect Via Inhibiting Intratumoral Angiogenesis in a Mouse Model of Transplanted Canine Mammary Tumor Cells.

    PubMed

    Li, Wenyu; Guo, Mengyao; Liu, Yuzhu; Mu, Weiwei; Deng, Ganzhen; Li, Chengye; Qiu, Changwei

    2016-06-01

    Selenium (Se) has been widely reported to possess anti-tumor effects. Angiogenesis is the formation of new blood vessels and is required to supply oxygen, nutrients, and growth factors for tumor growth, progression, and metastasis. To explore whether the anti-tumor effect of Se was associated with angiogenesis in vivo, we studied the effects of sodium selenite (Sel) and methylseleninic acid (MSA) on tumors induced by canine mammary tumor cells (CMT1211) in mice; cyclophosphamide (CTX) served as a positive control. The results showed that the Se content was significantly increased in the Sel and MSA groups. Se significantly inhibited the tumor weights and volumes. Large necrotic areas and scattered and abnormal small necrotic areas were observed in the Se treatment group. Immunofluorescence double staining showed a reduction in the microvessel density (MVD) and increment in the vessel maturation index (VMI) compared with the untreated control group. As expected, the protein and mRNA levels of the angiogenesis factors angiopoietin-2 (Ang-2), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) were decreased in the Se-treated tumors by IHC, as shown by western blotting and RT-QPCR. We also found that organic Se MSA provided stronger inhibition of tumor growth compared with inorganic sodium selenite (Sel). Altogether, our results indicated that Se exerted anti-tumor effects in vivo at least partially by inhibiting angiogenic factors. PMID:26507439

  8. Pim-1 kinase expression during murine mammary development

    SciTech Connect

    Gapter, Leslie A.; Magnuson, Nancy S.; Ng, Ka-yun; Hosick, Howard L. . E-mail: hosick@wsu.edu

    2006-07-07

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

  9. Induction of mammary gland development in estrogen receptor-alpha knockout mice.

    PubMed

    Bocchinfuso, W P; Lindzey, J K; Hewitt, S C; Clark, J A; Myers, P H; Cooper, R; Korach, K S

    2000-08-01

    Mammary glands from the estrogen receptor-a knockout (alphaERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted ERalpha signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute to the alphaERKO mammary phenotype. We report that circulating PRL is reduced in the female alphaERKO mouse. Implantation of an age-matched, heterozygous ERalpha pituitary isograft under the renal capsule of 25-day-old or 12-week-old alphaERKO mice increased circulating PRL and progesterone levels, and induced mammary gland development. Grafted alphaERKO mice also possessed hypertrophied corpora lutea demonstrating that PRL is luteotropic in the alphaERKO ovary. By contrast, ovariectomy at the time of pituitary grafting prevented mammary gland development in alphaERKO mice despite elevated PRL levels. Hormone replacement using pellet implants demonstrated that pharmacological doses of estradiol induced limited mammary ductal elongation, and estradiol in combination with progesterone stimulated lobuloalveolar development. PRL alone or in combination with progesterone or estradiol did not induce alphaERKO mammary growth. Estradiol and progesterone are required for the structural development of the alphaERKO mammary gland, and PRL contributes to this development by inducing ovarian progesterone levels. Therefore, the manifestation of the alphaERKO mammary phenotype appears due to the lack of direct estrogen action at the mammary gland and an indirect contributory role of estrogen signaling at the hypothalamic/pituitary axis.

  10. Zeeman spectroscopy of the internal transition 4T1 to 6A1 of Fe3+ ions in wurtzite GaN

    NASA Astrophysics Data System (ADS)

    Neuschl, B.; Gödecke, M. L.; Thonke, K.; Lipski, F.; Klein, M.; Scholz, F.; Feneberg, M.

    2015-12-01

    Internal transitions of Fe3+ ions in wurtzite gallium nitride were analyzed by means of photoluminescence, Zeeman, and transmission spectroscopy in order to investigate the fine structure. Magnetic fields up to 14 T were applied perpendicular or parallel to the crystal c-axis, causing a characteristic splitting pattern of the luminescence related to the transition from the 4T1 excited state to the 6A1 ground state of Fe3+. The complete Hamiltonian matrix is constructed taking into account the crystal field in cubic and trigonal symmetry, spin-orbit interaction, and the influence of external magnetic fields. Numerical solution yields the exact energy level scheme of the excited state 4G of Fe3+ ions in GaN, which partly revises assumptions based on a qualitative treatment considering group theory only and invoking the influence of a Jahn-Teller effect. The coincidence of the calculated energy levels with the experimental data verifies the derived fine structure of the 3d metal ion.

  11. Effects of letrozole on breast cancer micro-metastatic tumor growth in bone and lung in mice inoculated with murine 4T1 cells.

    PubMed

    Wang, Wendan; Belosay, Aashvini; Yang, Xujuan; Hartman, James A; Song, Huaxin; Iwaniec, Urszula T; Turner, Russell T; Churchwell, Mona I; Doerge, Daniel R; Helferich, William G

    2016-06-01

    Breast cancer (BC) is the leading cancer in women worldwide. Metastasis occurs in stage IV BC with bone and lung being common metastatic sites. Here we evaluate the effects of the aromatase inhibitor letrozole on BC micro-metastatic tumor growth in bone and lung metastasis in intact and ovariectomized (OVX) mice with murine estrogen receptor negative (ER-) BC cells inoculated in tibia. Forty-eight BALB/c mice were randomly assigned to one of four groups: OVX, OVX + Letrozole, Intact, and Intact + Letrozole, and injected with 4T1 cells intra-tibially. Letrozole was subcutaneously injected daily for 23 days at a dose of 1.75 µg/g body weight. Tumor progression was monitored by bioluminescence imaging (BLI). Following necropsy, inoculated tibiae were scanned via µCT and bone response to tumor was scored from 0 (no ectopic mineralization/osteolysis) to 5 (extensive ectopic mineralization/osteolysis). OVX mice had higher tibial pathology scores indicative of more extensive bone destruction than intact mice, irrespective of letrozole treatment. Letrozole decreased serum estradiol levels and reduced lung surface tumor numbers in intact animals. Furthermore, mice receiving letrozole had significantly fewer tumor colonies and fewer proliferative cells in the lung than OVX and intact controls based on H&E and Ki-67 staining, respectively. In conclusion, BC-inoculated OVX animals had higher tibia pathology scores than BC-inoculated intact animals and letrozole reduced BC metastases to lungs. These findings suggest that, by lowering systemic estrogen level and/or by interacting with the host organ, the aromatase inhibitor letrozole has the potential to reduce ER- BC metastasis to lung. PMID:27209469

  12. Enhancement of fibroblast activation protein α-based vaccines and adenovirus boost immunity by cyclophosphamide through inhibiting IL-10 expression in 4T1 tumor bearing mice.

    PubMed

    Xia, Qiu; Geng, Fei; Zhang, Fang-Fang; Liu, Chen-Lu; Xu, Ping; Lu, Zhen-Zhen; Zhang, Hai-Hong; Kong, Wei; Yu, Xiang-Hui

    2016-08-31

    Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts (CAFs) of more than 90% of malignant epithelia carcinomas. CAFs are the main type of cells in the tumor microenvironment which offer nutrition and protection to the tumor and regulate immunosuppression. To eliminate CAFs, a vaccine targeting FAPα may be used with a heterologous prime-boost strategy to enhance the FAPα-specific cellular immunity. Here, a FAP vaccine using a recombinant adenovirus (rAd) vector was constructed as well as a DNA vaccine reported in our previous work. Although the DNA prime-rAd boost strategy enhanced FAPα-specific immune responses, improvement of anti-tumor immunity effects was not observed. Examination of immunosuppressive factors revealed that high expression of the IL-10 cytokine was considered the main cause of the failure of the prime-boost strategy. However, heterologous vaccination in combination with a low-dose of cyclophosphamide (CY), which was reported to reduce IL-10 production and promote a shift from immunosuppression to immunopotentiation, resulted in enhanced effects in terms of numbers of effector T cells and tumor growth inhibition rates, compared to the CY alone or DNA alone group. Tumor growth was inhibited markedly when the prime-boost strategy was combined with CY in both the prophylactic and therapeutic settings and the survival time of 4T1 tumor bearing mice was also prolonged significantly. With the reduction of IL-10, enhancement of the anti-tumor effect by the prime-boost strategy was observed. These results suggest that FAPα-targeted rAd boosting in combination with CY is an attractive approach to overcoming immunosuppression in cancer vaccines. PMID:27498213

  13. 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.

  14. I. Embryonal vasculature formation recapitulated in transgenic mammary tumor spheroids implanted pseudo-orthotopicly into mouse dorsal skin fold: the organoblasts concept

    PubMed Central

    Witkiewicz, Halina

    2013-01-01

    Inadequate understanding of cancer biology is a problem. This work focused on cellular mechanisms of tumor vascularization. According to earlier studies, the tumor vasculature derives from host endothelial cells (angiogenesis) or their precursors of bone marrow origin circulating in the blood (neo-vasculogenesis) unlike in embryos. In this study, we observed the neo-vasculature form in multiple ways from local precursor cells. Recapitulation of primitive as well as advanced embryonal stages of vasculature formation followed co-implantation of avascular ( in vitro cultured) N202 breast tumor spheroids and homologous tissue grafts into mouse dorsal skin chambers. Ultrastructural and immunocytochemical analysis of tissue sections exposed the interactions between the tumor and the graft tissue stem cells. It revealed details of vasculature morphogenesis not seen before in either tumors or embryos. A gradual increase in complexity of the vascular morphogenesis at the tumor site reflected a range of steps in ontogenic evolution of the differentiating cells. Malignant- and surgical injury repair-related tissue growth prompted local cells to initiate extramedullar erythropoiesis and vascular patterning. The new findings included: interdependence between the extramedullar hematopoiesis and assembly of new vessels (both from the locally differentiating precursors); nucleo-cytoplasmic conversion (karyolysis) as the mechanism of erythroblast enucleation; the role of megakaryocytes and platelets in vascular pattern formation before emergence of endothelial cells; lineage relationships between hematopoietic and endothelial cells; the role of extracellular calmyrin in tissue morphogenesis; and calmyrite, a new ultrastructural entity associated with anaerobic energy metabolism. The central role of the extramedullar erythropoiesis in the formation of new vasculature (blood and vessels) emerged here as part of the tissue building process including the lymphatic system and nerves

  15. II. Capsular vaso-mimicry formed by transgenic mammary tumor spheroids implanted ectopically into mouse dorsal skin fold: implications for cellular mechanisms of metastasis

    PubMed Central

    Witkiewicz, Halina

    2013-01-01

    Most cancer patients die of metastatic disease, not primary tumors, while biological mechanisms leading to metastases remain unclear and effective therapies are missing. Using a mouse dorsal skin chamber model we had observed that tumor growth and vasculature formation could be influenced by the way in vitro cultured (avascular) spheroids of N202 breast tumor cells were implanted; co-implantation of lactating breast tissue created stimulating microenvironment, whereas the absence of the graft resulted in temporary tumor dormancy. This report addressed the issue of cellular mechanisms of the vasculogenic switch that ended the dormancy. In situ ultrastructural analysis revealed that the tumors survived in ectopic microenvironment until some of host and tumor stem cells evolved independently into cells initiating the vasculogenic switch. The tumor cells that survived and proliferated under hypoxic conditions for three weeks were supported by erythrogenic autophagy of others. However, the host microenvironment first responded as it would to non-immunogenic foreign bodies, i.e., by encapsulating the tumor spheroids with collagen-producing fibroblasts. That led to a form of vaso-mimicry consisting of tumor cells amid tumor-derived erythrosomes (synonym of erythrocytes), megakaryocytes and platelets, and encapsulating them all, the host fibroblasts. Such capsular vaso-mimicry could potentially facilitate metastasis by fusing with morphologically similar lymphatic vessels or veins. Once incorporated into the host circulatory system, tumor cells could be carried away passively by blood flow, regardless of their genetic heterogeneity. The fake vascular segment would have permeability properties different from genuine vascular endothelium. The capsular vaso-mimicry was different from vasculogenic mimicry earlier observed in metastases-associated malignant tumors where channels formed by tumor cells were said to contain circulating blood. Structures similar to the vasculogenic

  16. Mammary Gland Development

    PubMed Central

    Macias, Hector

    2012-01-01

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

  17. Role of phospholipids in the actions of prolactin in the mammary gland

    SciTech Connect

    Etindi, R.O.N.

    1987-01-01

    These studies were designed to determine the role of phospholipid turnover in the mechanism of action of prolactin in mammary gland explants derived from 12-14 day pregnant mice. Prolactin stimulates phospholipid biosynthesis 12-16h after cultured mouse mammary tissues are exposed to it. Prolactin stimulates phospholipid biosynthesis at physiological concentrations and the response is maximal at all PRL concentrations above 25 ng/ml. p-Bromphenacyl bromide (BPB) at concentrations of 50 ..mu..M and above and quinacrine (50 ..mu..M) abolish the actions of prolactin on casein and lipid biosynthesis in cultured mouse mammary gland explants. In mouse mammary gland explants, binding of prolactin to its receptor leads to a phospholipase C type hydrolysis of inositol phospholipids, but this effect is transient and does not occur immediately after hormone exposure. Prolactin significantly stimulated the accumulation of (/sup 3/H)label in inositol monophosphate (IP/sub 1/), inositol bisphosphate (IP/sub 2/) and inositol trisphosphate (IP/sub 3/) 1-3 hours after addition of prolactin. Gossypol, a drug which has been shown to be an inhibitor of kinase C activity in mouse mammary tissues, is shown to abolish several of the actions of prolactin in cultured mouse mammary gland expalants.

  18. Two monoclonal antibodies selective for human mammary carcinoma.

    PubMed

    White, C A; Dulbecco, R; Allen, R; Bowman, M; Armstrong, B

    1985-03-01

    Mouse myeloma cells were fused with spleen cells from BALB/c mice immunized with the MCF-7 human mammary carcinoma cell line. Among hybridomas, two (3B18 and 15A8) were selected and cloned. Hybridoma 3B18 produces kappa-IgG1 antibodies that react with a cytoplasmic component of MCF-7 cells. In immunoperoxidase assays, 3B18 reacts with 27 of 31 specimens of human mammary carcinoma. It reacts most consistently with poorly differentiated and infiltrating ductal breast cancers, but it also reacts with isolated cells in 3 of 5 benign mammary pathological lesions with a variable distribution. The antibody does not react with normal mammary epithelium. It does not react with any normal human tissues, and it reacts with only one of 19 other cancers tested. Hybridoma 15A8 produces kappa-IgG1 antibodies that react with the surface membranes of the cells of two human breast cancer cell lines but not with a human fibroblast cell line. In immunoperoxidase assays, the antibody reacted with 28 out of 31 human mammary carcinomas. The antibody also reacts more weakly with normal human epithelial cells of breast, renal proximal tubule, skin, esophagus, and salivary gland, but no other normal tissue. The antibody was unreactive with 14 of 18 other malignant tissues tested. Since 3B18 and 15A8 detect antigens found predominantly in human mammary carcinomas and, possibly, distinguish overlapping categories of human mammary carcinomas, they may prove useful in determining the cellular lineage from which human mammary carcinomas arise, or they may have other clinical applications in breast cancer.

  19. Mechanism of irradiation-induced mammary cancer metastasis: A role for SAP-dependent Mkl1 signaling.

    PubMed

    Asparuhova, Maria B; Secondini, Chiara; Rüegg, Curzio; Chiquet-Ehrismann, Ruth

    2015-10-01

    Radiotherapy is a standard treatment after conservative breast cancer surgery. However, cancers relapsing within a previously irradiated area have an increased probability to metastasize. The mechanisms responsible for this aggressiveness remain unclear. Here, we used the clinically relevant 4T1 breast cancer model mimicking aggressive local relapse after radiotherapy to identify differences between tumors grown in untreated versus preirradiated mammary glands. Tumors grown within preirradiated beds were highly enriched in transcripts encoding collagens and other proteins building or modifying the extracellular matrix, such as laminin-332, tenascins, lysyl oxidases and matrix metalloproteinases. Type I collagen, known to directly contribute to tissue stiffening, and the pro-metastatic megakaryoblastic leukemia-1 (Mkl1) target gene tenascin-C were further investigated. Mammary tissue preirradiation induced Mkl1 nuclear translocation in the tumor cells in vivo, indicating activation of Mkl1 signaling. Transcript profiling of cultured 4T1 cells revealed that the majority of the Mkl1 target genes, including tenascin-C, required serum response factor (SRF) for their expression. However, application of dynamic strain or matrix stiffness to 4T1 cells converted the predominant SRF/Mkl1 action into SAP domain-dependent Mkl1 signaling independent of SRF, accompanied by a switch to SAP-dependent tumor cell migration. 4T1 tumors overexpressing intact Mkl1 became more metastatic within preirradiated beds, while tumors expressing Mkl1 lacking the SAP domain exhibited impaired growth and metastatic spread, and decreased Mkl1 target gene expression. Thus, we identified SAP-dependent Mkl1 signaling as a previously unrecognized mediator of aggressive progression of mammary tumors locally relapsing after radiotherapy, and provide a novel signaling pathway for therapeutic intervention.

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

    PubMed

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

    2014-10-15

    Vitamin D₃ receptor (VDR) signaling within the mammary gland regulates various postnatal stages of glandular development, including puberty, pregnancy, involution, and tumorigenesis. Previous studies have shown that vitamin D₃ treatment induces cell-autonomous growth inhibition and differentiation of mammary epithelial cells in culture. Furthermore, mammary adipose tissue serves as a depot for vitamin D₃ storage, and both epithelial cells and adipocytes are capable of bioactivating vitamin D₃. Despite the pervasiveness of VDR in mammary tissue, individual contributions of epithelial cells and adipocytes, as well as the VDR-regulated cross-talk between these two cell types during pubertal mammary development, have yet to be investigated. To assess the cell-type specific effect of VDR signaling during pubertal mammary development, novel mouse models with mammary epithelial- or adipocyte-specific loss of VDR were generated. Interestingly, loss of VDR in either cellular compartment accelerated ductal morphogenesis with increased epithelial cell proliferation and decreased apoptosis within terminal end buds. Conversely, VDR signaling specifically in the mammary epithelium modulated hormone-induced alveolar growth, as ablation of VDR in this cell type resulted in precocious alveolar development. In examining cellular cross-talk ex vivo, we show that ligand-dependent VDR signaling in adipocytes significantly inhibits mammary epithelial cell growth in part through the vitamin D₃-dependent production of the cytokine IL-6. Collectively, these studies delineate independent roles for vitamin D₃-dependent VDR signaling in mammary adipocytes and epithelial cells in controlling pubertal mammary gland development.

  1. Site-specific inductive and inhibitory activities of MMP-2 and MMP-3 orchestrate mammary gland branching morphogenesis

    PubMed Central

    Wiseman, Bryony S.; Sternlicht, Mark D.; Lund, Leif R.; Alexander, Caroline M.; Mott, Joni; Bissell, Mina J.; Soloway, Paul; Itohara, Shigeyoshi; Werb, Zena

    2003-01-01

    During puberty, mouse mammary epithelial ducts invade the stromal mammary fat pad in a wave of branching morphogenesis to form a complex ductal tree. Using pharmacologic and genetic approaches, we find that mammary gland branching morphogenesis requires transient matrix metalloproteinase (MMP) activity for invasion and branch point selection. MMP-2, but not MMP-9, facilitates terminal end bud invasion by inhibiting epithelial cell apoptosis at the start of puberty. Unexpectedly, MMP-2 also represses precocious lateral branching during mid-puberty. In contrast, MMP-3 induces secondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy. Nevertheless, the mammary gland is able to develop lactational competence in MMP mutant mice. Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during mammary gland branching morphogenesis. PMID:12975354

  2. In vitro activity of a Solanum tuberosum extract against mammary carcinoma cells.

    PubMed

    De Lorenzo, M S; Lorenzano Menna, P L; Alonso, D F; Gomez, D E

    2001-03-01

    We investigated the antitumor properties of a Solanum tuberosum extract (STE) on F3II mouse mammary carcinoma cells. STE significantly inhibited adhesion on fibronectin-coated surfaces and blocked migration of tumor cells in vitro. A major gelatinolytic activity (gelatinase) of 82 kD was identified in STE by zymographic analysis and characterized by exposure to different experimental conditions. Proteolytic activity of STE may be responsible, at least in part, for the in vitro effects on mammary carcinoma cells.

  3. Androgen dependent mammary gland virilism in rats given the selective estrogen receptor modulator LY2066948 hydrochloride.

    PubMed

    Rudmann, Daniel G; Cohen, Ilene R; Robbins, Michelle R; Coutant, David E; Henck, Judith W

    2005-01-01

    A selective estrogen receptor modulator (SERM) is a nonsteroidal compound with tissue specific estrogen receptor (ER) agonist or antagonist activities. In animals, SERMs may produce morphologic changes in hormonally-sensitive tissues like the mammary gland. Mammary glands from female rats given the SERM LY2066948 hydrochloride (LY2066948) for 1 month at >or= 175 mg/kg had intralobular ducts and alveoli lined by multiple layers of vacuolated, hypertrophied epithelial cells, resembling in part the morphology of the normal male rat mammary gland. We hypothesized that these SERM-mediated changes represented an androgen-dependent virilism of the female rat mammary gland. To test this hypothesis, the androgen receptor antagonist flutamide was co-administered with LY2066948 (175 mg/kg) to female rats for 1 month. Female rats given SERM alone had hyperandrogenemia and the duct and alveolar changes described here. Flutamide cotreatment did not affect serum androgen levels but completely blocked the SERM-mediated mammary gland change. In the mouse, a species that does not have the sex-specific differences in the mammary gland observed in the rat, SERM treatment resulted in hyperandrogenemia but did not alter mammary gland morphology. These studies demonstrate that LY2066948 produces species-specific, androgen-dependent mammary gland virilism in the female rat.

  4. Host Cxcr2-dependent regulation of mammary tumor growth and metastasis

    PubMed Central

    Sharma, Bhawna; Nannuru, Kalyan C.; Varney, Michelle L.

    2016-01-01

    Host-derived angiogenic and inflammatory tumor supportive microenvironment regulates progression and metastasis, but the molecular mechanism(s) underlying host-tumor interactions remains unclear. Tumor expression of CXCR2 and its ligands have been shown to regulate angiogenesis, invasion, tumor growth, and metastasis. In this report, we hypothesized that host-derived Cxcr2-dependent signaling plays an important role in breast cancer growth and metastasis. Two mammary tumor cell lines Cl66 and 4T1 cells were orthotopically implanted into the mammary fat pad of wild-type and Cxcr2−/− female BALB/c mice. Tumor growth and spontaneous lung metastasis were monitored. Immunohistochemical analyses of the tumor tissues were performed to analyze proliferation, angiogenesis, apoptosis and immune cell infiltration. Our results demonstrated that knock-down of host Cxcr2 decreases tumor growth and metastasis by reducing angiogenesis, proliferation and enhancing apoptosis. Host Cxcr2 plays an important role in governing the pro-inflammatory response in mammary tumors as evaluated by decreased Gr1+ tumor-associated granulocytes, F4/80+ tumor associated macrophages, and CD11b+Gr1+ myeloid derived suppressor cells in Cxcr2−/− mice as compared to control wild-type mice. Together, these results demonstrate that host Cxcr2-dependent signaling regulates mammary tumor growth and metastasis by promoting angiogenesis and pro-inflammatory responses. PMID:25511644

  5. Ductal barriers in mammary epithelium

    PubMed Central

    Owens, Mark B; Hill, Arnold DK; Hopkins, Ann M

    2013-01-01

    Tissue barriers play an integral role in the biology and pathobiology of mammary ductal epithelium. In normal breast physiology, tight and adherens junctions undergo dynamic changes in permeability in response to hormonal and other stimuli, while several of their proteins are directly involved in mammary tumorigenesis. This review describes first the structure of mammary ductal epithelial barriers and their role in normal mammary development, examining the cyclical changes in response to puberty, pregnancy, lactation and involution. It then examines the role of adherens and tight junctions and the participation of their constituent proteins in mammary tumorigenic functions such as migration, invasion and metastasis. Finally, it discusses the potential of these adhesion proteins as both prognostic biomarkers and potential therapeutic targets in breast cancer. PMID:24665412

  6. Lx2-32c–loaded polymeric micelles with small size for intravenous drug delivery and their inhibitory effect on tumor growth and metastasis in clinically associated 4T1 murine breast cancer

    PubMed Central

    Chen, Li-qing; Huang, Wei; Gao, Zhong-gao; Fang, Wei-shuo; Jin, Ming-ji

    2016-01-01

    Lx2-32c is a novel taxane derivative with a strong antitumor activity. In this study, we developed Lx2-32c–loaded polymeric micelles (Lx2-32c-PMs) with small size and investigated their antitumor efficacy against tumor growth and metastasis on 4T1 murine breast cancer cell line with Cremophor EL–based Lx2-32c solution as the control. In this study, copolymer monomethoxy polyethylene glycol2000–polylactide1300 was used to prepare Lx2-32c-PMs by film hydration method, and their physicochemical properties were characterized as well, according to morphology, particle size, zeta potential, in vitro drug release, and reconstitution stability. Under confocal laser scanning microscopy, it was observed that Lx2-32c-PMs could be effectively taken up by 4T1 cells in a time-dependent manner. Cell Counting Kit-8 assay showed that the IC50 of Lx2-32c-PMs was 0.3827 nM. Meanwhile, Lx2-32c-PMs had better ability to promote apoptosis and induce G2/M cycle block and polyploidy formation, compared with Lx2-32c solution. More importantly, in vivo animal studies showed that compared to Lx2-32c solution, Lx2-32c-PMs possessed better ability not only to effectively inhibit the tumor growth, but also to significantly suppress spontaneous and postoperative metastasis to distant organs in 4T1 orthotopic tumor-bearing mice. Consequently, Lx2-32c-PMs have significantly prolonged the survival lifetime of tumor-bearing mice. Thus, our study reveals that Lx2-32c-PMs had favorable antitumor activity and exhibited a good prospect for application in the field of antitumor therapy. PMID:27799769

  7. Curcumin improves the therapeutic efficacy of Listeria(at)-Mage-b vaccine in correlation with improved T-cell responses in blood of a triple-negative breast cancer model 4T1.

    PubMed

    Singh, Manisha; Ramos, Ilyssa; Asafu-Adjei, Denise; Quispe-Tintaya, Wilber; Chandra, Dinesh; Jahangir, Arthee; Zang, Xingxing; Aggarwal, Bharat B; Gravekamp, Claudia

    2013-08-01

    Success of cancer vaccination is strongly hampered by immune suppression in the tumor microenvironment (TME). Interleukin (IL)-6 is particularly and highly produced by triple-negative breast cancer (TNBC) cells, and has been considered as an important contributor to immune suppression in the TME. Therefore, we hypothesized that IL-6 reduction may improve efficacy of vaccination against TNBC cancer through improved T-cell responses. To prove this hypothesis, we investigated the effect of curcumin, an inhibitor of IL-6 production, on vaccination of a highly attenuated Listeria monocytogenes (Listeria(at)), encoding tumor-associated antigens (TAA) Mage-b in a TNBC model 4T1. Two therapeutic vaccination strategies with Listeria(at)-Mage-b and curcumin were tested. The first immunization strategy involved all Listeria(at)-Mage-b vaccinations and curcumin after tumor development. As curcumin has been consumed all over the world, the second immunization strategy involved curcumin before and all therapeutic vaccinations with Listeria(at)-Mage-b after tumor development. Here, we demonstrate that curcumin significantly improves therapeutic efficacy of Listeria(at)-Mage-b with both immunization strategies particularly against metastases in a TNBC model (4T1). The combination therapy was slightly but significantly more effective against the metastases when curcumin was administered before compared to after tumor development. With curcumin before tumor development in the combination therapy, the production of IL-6 was significantly decreased and IL-12 increased by myeloid-derived suppressor cells (MDSC), in correlation with improved CD4 and CD8 T-cell responses in blood. Our study suggests that curcumin improves the efficacy of Listeria(at)-Mage-b vaccine against metastases in TNBC model 4T1 through reversal of tumor-induced immune suppression. PMID:24156030

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

    PubMed Central

    2013-01-01

    Introduction Cancer is often suggested to result from development gone awry. Links between normal embryonic development and cancer biology have been postulated, but no defined genetic basis has been established. We recently published the first transcriptomic analysis of embryonic mammary cell populations. Embryonic mammary epithelial cells are an immature progenitor cell population, lacking differentiation markers, which is reflected in their very distinct genetic profiles when compared with those of their postnatal descendents. Methods We defined an embryonic mammary epithelial signature that incorporates the most highly expressed genes from embryonic mammary epithelium when compared with the postnatal mammary epithelial cells. We looked for activation of the embryonic mammary epithelial signature in mouse mammary tumors that formed in mice in which Brca1 had been conditionally deleted from the mammary epithelium and in human breast cancers to determine whether any genetic links exist between embryonic mammary cells and breast cancers. Results Small subsets of the embryonic mammary epithelial signature were consistently activated in mouse Brca1-/- tumors and human basal-like breast cancers, which encoded predominantly transcriptional regulators, cell-cycle, and actin cytoskeleton components. Other embryonic gene subsets were found activated in non-basal-like tumor subtypes and repressed in basal-like tumors, including regulators of neuronal differentiation, transcription, and cell biosynthesis. Several embryonic genes showed significant upregulation in estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and/or grade 3 breast cancers. Among them, the transcription factor, SOX11, a progenitor cell and lineage regulator of nonmammary cell types, is found highly expressed in some Brca1-/- mammary tumors. By using RNA interference to silence SOX11 expression in breast cancer cells, we found evidence that SOX11 regulates breast cancer cell

  9. Postnatal and postpartal morphology of the mammary gland in nude mice.

    PubMed

    Militzer, K; Schwalenstöcker, H

    1996-08-01

    The object of this work was to compare the postnatal and postpartal morphology of the mammary gland of nu/nu with that of nu/(+)-mice. All studies were carried out on groups of female (athymic) nude mice with NMRI genetic background, their nu/(+)-siblings and dams. The various age groups (3, 21, 40, 55, 70 and 120 days) each consisted of 6 nu/nu- and 6 heterozygous nu/(+)-mice respectively. The morphological examination of the mammary gland tissue were made on histological sections and whole mounts. Body weights, total areas of the mammary glands and the number of the terminal end buds were compared. The mammary gland of the athymic nude mouse exhibited no essential morphological differences from the normal developing mammary gland of the hairy euthymic nu/(+)-animal. The area of the mammary gland increased with increasing body weight. Both collectives of mice differed only in their rate of mammary gland development. As a result, the terminal end buds appeared numerously as growth points of mammary gland in nu/(+)-animals as early as the 21st day of life. The athymic nude mice showed a maximum only on the 40th day of life and a lower degree of density and differentiation of specific mammary gland structures (lateral buds, lobulo-alveolar glandular endings) until the 70th day of life. The mammary gland of 120-day-old animals and dams of both animal groups reached the same state of maturity. Thus it is not the rate of development of the dam, but other, yet unidentified factors, which determine, if successful breeding of nude mice with homozygous parents is possible.

  10. SHIP represses lung inflammation and inhibits mammary tumor metastasis in BALB/c mice

    PubMed Central

    Hamilton, Melisa J.; Halvorsen, Elizabeth C.; LePard, Nancy E.; Bosiljcic, Momir; Ho, Victor W.; Lam, Vivian; Banáth, Judit

    2016-01-01

    SH2-containing-inositol-5′-phosphatase (SHIP) is a negative regulator of the phosphatidylinositol-3-kinase pathway in hematopoietic cells and limits the development of leukemias and lymphomas. The potential role of SHIP in solid tumor development and metastasis remains unknown. While SHIP restricts the aberrant development of myeloid cells in C57BL/6 mice, there are conflicting reports regarding the effect of SHIP deletion in BALB/c mice with important consequences for determining the influence of SHIP in different model tumor systems. We generated SHIP−/− BALB/c mice and challenged them with syngeneic non-metastatic 67NR or metastatic 4T1 mammary tumors. We demonstrate that SHIP restricts the development, alternative-activation, and immunosuppressive function of myeloid cells in tumor-free and tumor-bearing BALB/c mice. Tumor-free SHIP−/− BALB/c mice exhibited pulmonary inflammation, myeloid hyperplasia, and M2-polarized macrophages and this phenotype was greatly exacerbated by 4T1, but not 67NR, tumors. 4T1-bearing SHIP−/− mice rapidly lost weight and died from necrohemorrhagic inflammatory pulmonary disease, characterized by massive infiltration of pulmonary macrophages and myeloid-derived suppressor cells that were more M2-polarized and immunosuppressive than wild-type cells. Importantly, while SHIP loss did not affect primary tumor growth, 4T1-bearing SHIP−/− mice had 7.5-fold more metastatic tumor cells in their lungs than wild-type mice, consistent with the influence of immunosuppressive myeloid cells on metastatic growth. Our findings identify the hematopoietic cell-restricted protein SHIP as an intriguing target to influence the development of solid tumor metastases, and support development of SHIP agonists to prevent the accumulation of immunosuppressive myeloid cells and tumor metastases in the lungs to improve treatment of metastatic breast cancer. PMID:26683227

  11. SHIP represses lung inflammation and inhibits mammary tumor metastasis in BALB/c mice.

    PubMed

    Hamilton, Melisa J; Halvorsen, Elizabeth C; LePard, Nancy E; Bosiljcic, Momir; Ho, Victor W; Lam, Vivian; Banáth, Judit; Bennewith, Kevin L; Krystal, Gerald

    2016-01-26

    SH2-containing-inositol-5'-phosphatase (SHIP) is a negative regulator of the phosphatidylinositol-3-kinase pathway in hematopoietic cells and limits the development of leukemias and lymphomas. The potential role of SHIP in solid tumor development and metastasis remains unknown. While SHIP restricts the aberrant development of myeloid cells in C57BL/6 mice, there are conflicting reports regarding the effect of SHIP deletion in BALB/c mice with important consequences for determining the influence of SHIP in different model tumor systems. We generated SHIP-/- BALB/c mice and challenged them with syngeneic non-metastatic 67NR or metastatic 4T1 mammary tumors. We demonstrate that SHIP restricts the development, alternative-activation, and immunosuppressive function of myeloid cells in tumor-free and tumor-bearing BALB/c mice. Tumor-free SHIP-/- BALB/c mice exhibited pulmonary inflammation, myeloid hyperplasia, and M2-polarized macrophages and this phenotype was greatly exacerbated by 4T1, but not 67NR, tumors. 4T1-bearing SHIP-/- mice rapidly lost weight and died from necrohemorrhagic inflammatory pulmonary disease, characterized by massive infiltration of pulmonary macrophages and myeloid-derived suppressor cells that were more M2-polarized and immunosuppressive than wild-type cells. Importantly, while SHIP loss did not affect primary tumor growth, 4T1-bearing SHIP-/- mice had 7.5-fold more metastatic tumor cells in their lungs than wild-type mice, consistent with the influence of immunosuppressive myeloid cells on metastatic growth. Our findings identify the hematopoietic cell-restricted protein SHIP as an intriguing target to influence the development of solid tumor metastases, and support development of SHIP agonists to prevent the accumulation of immunosuppressive myeloid cells and tumor metastases in the lungs to improve treatment of metastatic breast cancer.

  12. Immunobiology of the mammary gland.

    PubMed

    Sordillo, L M; Shafer-Weaver, K; DeRosa, D

    1997-08-01

    The mammary gland is a complex organ that provides neonatal offspring with milk for nourishment and disease resistance. Specific and innate immune factors associated with mammary gland tissues and secretion also play a vital role in protecting the gland from infectious disease. Through genetic selection and technological advances in milk removal, the bovine mammary gland yields for more milk than is needed to nourish the newborn calf. This excess is the basis of the dairy industry. Factors associated with the intense management of dairy cattle can profoundly affect mammary gland immunity and the ability of the host to resist mastitis. Technological advances in immunology have led to the availability of new research tools that can facilitate the study of mammary gland immunity and disease pathogenesis. In recent years, considerable research effort has focused on enhancing the natural defense mechanisms of the mammary gland during periods of heightened susceptibility to disease. This paper provides a comprehensive overview of mammary gland immunity with special emphasis on the bovine system. The underlying mechanisms of disease susceptibility and development of potential immunoregulatory strategies to control mastitis are discussed.

  13. Suppression of beta-casein gene expression by inhibition of protein synthesis in mouse mammary epithelial cells is associated with stimulation of NF-kappaB activity and blockage of prolactin-Stat5 signaling.

    PubMed

    Beaton, Angela; Broadhurst, Marita K; Wilkins, Richard J; Wheeler, Thomas T

    2003-02-01

    The protein synthesis inhibitor cycloheximide (Chx) suppresses prolactin-induced beta-casein gene expression in the mammary epithelial cell line COMMA-D. As the mechanism underlying this effect is unclear, the effects of protein synthesis inhibitors on interactions of transcription factors with the beta-casein promoter were examined. Suppression of prolactin-induced beta-casein gene expression occurred in both COMMA-D cells and primary mammary cell cultures with as little as 2 h protein synthesis inhibition. This was associated with changes in transcription factors interacting at a response element in the proximal region of the rat beta-casein promoter. Inhibition of protein synthesis was associated with NF-kappaB binding at a site immediately 3' to the Stat5-binding site at position 97-89 of the beta-casein promoter, suppression of Stat5 DNA-binding activity, and inhibition of Stat5 tyrosine phosphorylation. Treatment with the NF-kappaB inhibitor parthenolide failed to restore prolactin responsiveness. These results show that protein synthesis inhibition is associated with both blockage of prolactin-Stat5 signaling and NF-kappaB binding to the beta-casein promoter, but that the latter is not necessary for the suppression of beta-casein expression.

  14. Ovariectomy is associated with metabolic impairments and enhanced mammary tumor growth in MKR mice.

    PubMed

    Ben-Shmuel, Sarit; Scheinman, Eyal J; Rashed, Rola; Orr, Zila Shen; Gallagher, Emily J; LeRoith, Derek; Rostoker, Ran

    2015-12-01

    Obesity and type 2 diabetes (T2D) are associated with an increased risk of breast cancer incidence and mortality. Common features of obesity and T2D are insulin resistance and hyperinsulinemia. A mammary tumor promoting effect of insulin resistance and hyperinsulinemia was demonstrated in the transgenic female MKR mouse model of pre-diabetes inoculated with mammary cancer cells. Interestingly, in MKR mice, as well as in other diabetic mouse models, males exhibit severe hyperglycemia, while females display insulin resistance and hyperinsulinemia with only a mild increase in blood glucose levels. This gender-specific protection from hyperglycemia may be attributed to estradiol, a key player in the regulation of the metabolic state, including obesity, glucose homeostasis, insulin resistance, and lipid profile. The aim of this study was to investigate the effects of ovariectomy (including the removal of endogenous estradiol) on the metabolic state of MKR female mice and subsequently on the growth of Mvt-1 mammary cancer cells, inoculated into the mammary fat pad of ovariectomized mice, compared with sham-operated mice. The results showed an increase in body weight, accompanied by increased fat mass, elevated blood glucose levels, and hypercholesterolemia, in ovariectomized MKR mice. In addition, mammary tumor growth was significantly higher in these mice. The results suggest that ovarian hormone deficiency may promote impaired metabolic homeostasis in the hyperinsulinemic MKR female mice, which in turn is associated with an increased growth of mammary tumors. PMID:26383532

  15. Aquaporin water channels in the mammary gland: from physiology to pathophysiology and neoplasia.

    PubMed

    Mobasheri, Ali; Barrett-Jolley, Richard

    2014-03-01

    Aquaporins are membrane proteins that play fundamental roles in water and small solute transport across epithelial and endothelial barriers. Recent studies suggest that several aquaporin proteins are present in the mammary gland. Immunohistochemical techniques have confirmed the presence of aquaporin 1 (AQP1) and AQP3 water channels in rat, mouse, bovine and human mammary glands. Studies suggest that in addition to AQP1 and AQP3 AQP4, AQP5 and AQP7 proteins are expressed in different locations in the mammary gland. Aquaporins play key roles in tumor biology and are involved in cell growth, migration and formation of ascites via increased water permeability of micro-vessels. Emerging evidence suggests that expression of these proteins is altered in mammary tumors and in breast cancer cell lines although it is not yet clear whether this is a cause or a consequence of neoplastic development. This review analyzes the expression and potential functional roles of aquaporin water channels in the mammary gland. The physiological mechanisms involved in the transport of water and small solutes across mammary endothelial and epithelial barriers are discussed in the context of milk production and lactation. This paper also reviews papers from the recent cancer literature that implicate aquaporins in mammary neoplasia.

  16. Perfluorooctanoic acid effects on ovaries mediate its inhibition of peripubertal mammary gland development in Balb/c and C57Bl/6 mice

    EPA Science Inventory

    Exposure to perfluorooctanoic acid (PFOA), a synthetic perfluorinated compound and an agonist of peroxisomes proliferator-activated receptor α (PPARα), causes stunted mouse mammary gland development in various developmental stages. However, the underlying mechanisms remain poorly...

  17. RANK Signaling Amplifies WNT-Responsive Mammary Progenitors through R-SPONDIN1

    PubMed Central

    Joshi, Purna A.; Waterhouse, Paul D.; Kannan, Nagarajan; Narala, Swami; Fang, Hui; Di Grappa, Marco A.; Jackson, Hartland W.; Penninger, Josef M.; Eaves, Connie; Khokha, Rama

    2015-01-01

    Summary Systemic and local signals must be integrated by mammary stem and progenitor cells to regulate their cyclic growth and turnover in the adult gland. Here, we show RANK-positive luminal progenitors exhibiting WNT pathway activation are selectively expanded in the human breast during the progesterone-high menstrual phase. To investigate underlying mechanisms, we examined mouse models and found that loss of RANK prevents the proliferation of hormone receptor-negative luminal mammary progenitors and basal cells, an accompanying loss of WNT activation, and, hence, a suppression of lobuloalveologenesis. We also show that R-spondin1 is depleted in RANK-null progenitors, and that its exogenous administration rescues key aspects of RANK deficiency by reinstating a WNT response and mammary cell expansion. Our findings point to a novel role of RANK in dictating WNT responsiveness to mediate hormone-induced changes in the growth dynamics of adult mammary cells. PMID:26095608

  18. Stem cell marker prominin-1 regulates branching morphogenesis, but not regenerative capacity, in the mammary gland.

    PubMed

    Anderson, Lisa H; Boulanger, Corinne A; Smith, Gilbert H; Carmeliet, Peter; Watson, Christine J

    2011-03-01

    Prominin-1 (Prom1) is recognized as a stem cell marker in several tissues, including blood, neuroepithelium, and gut, and in human and mouse embryos and many cancers. Although Prom1 is routinely used as a marker for isolating stem cells, its biological function remains unclear. Here we use a knockout model to investigate the role of Prom1 in the mammary gland. We demonstrate that complete loss of Prom1 does not affect the regenerative capacity of the mammary epithelium. Surprisingly, we also show that in the absence of Prom1, mammary glands have reduced ductal branching, and an increased ratio of luminal to basal cells. The effects of Prom1 loss in the mammary gland are associated with decreased expression of prolactin receptor and matrix metalloproteinase-3. These experiments reveal a novel, functional role for Prom1 that is not related to stem cell activity, and demonstrate the importance of tissue-specific characterization of putative stem cell markers.

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

    SciTech Connect

    Streuli, Charles H; Schmidhauser, Christian; Bailey, Nina; Yurchenco, Peter; Skubitz, Amy P. N.; Roskelley, Calvin; Bissell, Mina J

    1995-04-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

  20. Laminin mediates tissue-specific gene expression in mammary epithelia

    PubMed Central

    1995-01-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta- casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain. PMID:7730398

  1. Mammary Analogue Secretory Carcinoma.

    PubMed

    Stevens, Todd M; Parekh, Vishwas

    2016-09-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor that shares the same histologic appearance and ETV6 gene (12p13) rearrangement as secretory carcinoma of the breast. Prior to its recognition, MASC cases were commonly labeled acinic cell carcinoma and adenocarcinoma, not otherwise specified. Despite distinctive histologic features, MASC may be difficult to distinguish from other salivary gland tumors, in particular zymogen-poor acinic cell carcinoma and low-grade salivary duct carcinoma. Although characteristic morphologic and immunohistochemical features form the basis of a diagnosis of MASC, the presence of an ETV6-NTRK3 gene fusion is confirmatory. Given its recent recognition the true prognostic import of MASC is not yet clearly defined. PMID:27575269

  2. Hypoxia-inducible factor-1α induces ErbB4 signaling in the differentiating mammary gland.

    PubMed

    Paatero, Ilkka; Seagroves, Tiffany N; Vaparanta, Katri; Han, Wen; Jones, Frank E; Johnson, Randall S; Elenius, Klaus

    2014-08-01

    Conditional knock-out of Hif1a in the mouse mammary gland impairs lobuloalveolar differentiation during lactation. Here, we demonstrate that expression of ErbB4 was reduced in the lobulalveoli of mice with mammary gland-specific deletion of Hif1a. Erbb4 was not, however, a direct target gene for transcriptional regulation by HIF-1α in vitro. HIF-1α overexpression or HIF accumulating prolyl hydroxylase inhibitors reduced ErbB4 endocytosis, promoted transcriptional co-regulatory activity of ErbB4, and stimulated ErbB4-induced differentiation of mammary carcinoma cells. Consistently, RNA interference-mediated down-regulation of HIF-1α resulted in reduced ErbB4 protein amount and reduced mammary carcinoma cell differentiation. These findings indicate that HIF-1α is a physiologically relevant regulator of ErbB4 and that ErbB4 is involved in HIF-regulated differentiation of the mammary gland.

  3. Curcumin improves the therapeutic efficacy of Listeriaat-Mage-b vaccine in correlation with improved T-cell responses in blood of a triple-negative breast cancer model 4T1

    PubMed Central

    Singh, Manisha; Ramos, Ilyssa; Asafu-Adjei, Denise; Quispe-Tintaya, Wilber; Chandra, Dinesh; Jahangir, Arthee; Zang, Xingxing; Aggarwal, Bharat B; Gravekamp, Claudia

    2013-01-01

    Abstract Success of cancer vaccination is strongly hampered by immune suppression in the tumor microenvironment (TME). Interleukin (IL)-6 is particularly and highly produced by triple-negative breast cancer (TNBC) cells, and has been considered as an important contributor to immune suppression in the TME. Therefore, we hypothesized that IL-6 reduction may improve efficacy of vaccination against TNBC cancer through improved T-cell responses. To prove this hypothesis, we investigated the effect of curcumin, an inhibitor of IL-6 production, on vaccination of a highly attenuated Listeria monocytogenes (Listeriaat), encoding tumor-associated antigens (TAA) Mage-b in a TNBC model 4T1. Two therapeutic vaccination strategies with Listeriaat-Mage-b and curcumin were tested. The first immunization strategy involved all Listeriaat-Mage-b vaccinations and curcumin after tumor development. As curcumin has been consumed all over the world, the second immunization strategy involved curcumin before and all therapeutic vaccinations with Listeriaat-Mage-b after tumor development. Here, we demonstrate that curcumin significantly improves therapeutic efficacy of Listeriaat-Mage-b with both immunization strategies particularly against metastases in a TNBC model (4T1). The combination therapy was slightly but significantly more effective against the metastases when curcumin was administered before compared to after tumor development. With curcumin before tumor development in the combination therapy, the production of IL-6 was significantly decreased and IL-12 increased by myeloid-derived suppressor cells (MDSC), in correlation with improved CD4 and CD8 T-cell responses in blood. Our study suggests that curcumin improves the efficacy of Listeriaat-Mage-b vaccine against metastases in TNBC model 4T1 through reversal of tumor-induced immune suppression. This study is focused on improving cancer vaccination by reducing immune suppression. Here we demonstrate that curcumin improves vaccine

  4. Chronic social isolation is associated with metabolic gene expression changes specific to mammary adipose tissue.

    PubMed

    Volden, Paul A; Wonder, Erin L; Skor, Maxwell N; Carmean, Christopher M; Patel, Feenalie N; Ye, Honggang; Kocherginsky, Masha; McClintock, Martha K; Brady, Matthew J; Conzen, Suzanne D

    2013-07-01

    Chronic social isolation is linked to increased mammary tumor growth in rodent models of breast cancer. In the C3(1)/SV40 T-antigen FVB/N (TAg) mouse model of "triple-negative" breast cancer, the heightened stress response elicited by social isolation has been associated with increased expression of metabolic genes in the mammary gland before invasive tumors develop (i.e., during the in situ carcinoma stage). To further understand the mechanisms underlying how accelerated mammary tumor growth is associated with social isolation, we separated the mammary gland adipose tissue from adjacent ductal epithelial cells and analyzed individual cell types for changes in metabolic gene expression. Specifically, increased expression of the key metabolic genes Acaca, Hk2, and Acly was found in the adipocyte, rather than the epithelial fraction. Surprisingly, metabolic gene expression was not significantly increased in visceral adipose depots of socially isolated female mice. As expected, increased metabolic gene expression in the mammary adipocytes of socially isolated mice coincided with increased glucose metabolism, lipid synthesis, and leptin secretion from this adipose depot. Furthermore, application of media that had been cultured with isolated mouse mammary adipose tissue (conditioned media) resulted in increased proliferation of mammary cancer cells relative to group-housed-conditioned media. These results suggest that exposure to a chronic stressor (social isolation) results in specific metabolic reprogramming in mammary gland adipocytes that in turn contributes to increased proliferation of adjacent preinvasive malignant epithelial cells. Metabolites and/or tumor growth-promoting proteins secreted from adipose tissue could identify biomarkers and/or targets for preventive intervention in breast cancer.

  5. The differential actions of cortisol on the synthesis and turnover of alpha-lactalbumin and casein and on accumulation of their mRNA in mouse mammary gland in organ culture.

    PubMed Central

    Nagamatsu, Y; Oka, T

    1983-01-01

    Cortisol was previously shown to exert different, concentration-dependent, effects on the accumulation of casein and alpha-lactalbumin in mammary glands from mid-pregnant mice cultured in the presence of insulin and prolactin [Ono & Oka (1980) Cell 19, 473-480]. The present study demonstrated that the addition of 30nM-cortisol to the medium containing insulin and prolactin resulted in a marked enhancement of the rate of synthesis of both alpha-lactalbumin and casein in cultured tissue. The addition of 3 microM-cortisol in combination with insulin and prolactin caused a marked decrease in the rate of alpha-lactalbumin synthesis, but increased casein synthesis substantially. Similar changes were also observed in the amount of translatable mRNA for alpha-lactalbumin and casein in mammary explants cultured with insulin, prolactin and the two concentrations of cortisol. The study of the turnover of the milk proteins in cultured explants showed that virtually all of the casein synthesized remained intact in tissue explants cultured with 3 microM cortisol, whereas about 45% of casein disappeared in 40h from explants cultured with 30nM-cortisol. In contrast, the two concentrations of cortisol did not differentially affect the disappearance of alpha-lactalbumin, which was about 55% in 40h. These results indicate that the concentration-dependent differential actions of cortisol on the accumulation of alpha-lactalbumin and casein are exerted through its effects on the rate of synthesis and turnover of the two proteins as well as on the accumulation of their mRNA species. PMID:6349620

  6. From genes to milk: genomic organization and epigenetic regulation of the mammary transcriptome.

    PubMed

    Lemay, Danielle G; Pollard, Katherine S; Martin, William F; Freeman Zadrowski, Courtneay; Hernandez, Joseph; Korf, Ian; German, J Bruce; Rijnkels, Monique

    2013-01-01

    Even in genomes lacking operons, a gene's position in the genome influences its potential for expression. The mechanisms by which adjacent genes are co-expressed are still not completely understood. Using lactation and the mammary gland as a model system, we explore the hypothesis that chromatin state contributes to the co-regulation of gene neighborhoods. The mammary gland represents a unique evolutionary model, due to its recent appearance, in the context of vertebrate genomes. An understanding of how the mammary gland is regulated to produce milk is also of biomedical and agricultural importance for human lactation and dairying. Here, we integrate epigenomic and transcriptomic data to develop a comprehensive regulatory model. Neighborhoods of mammary-expressed genes were determined using expression data derived from pregnant and lactating mice and a neighborhood scoring tool, G-NEST. Regions of open and closed chromatin were identified by ChIP-Seq of histone modifications H3K36me3, H3K4me2, and H3K27me3 in the mouse mammary gland and liver tissue during lactation. We found that neighborhoods of genes in regions of uniquely active chromatin in the lactating mammary gland, compared with liver tissue, were extremely rare. Rather, genes in most neighborhoods were suppressed during lactation as reflected in their expression levels and their location in regions of silenced chromatin. Chromatin silencing was largely shared between the liver and mammary gland during lactation, and what distinguished the mammary gland was mainly a small tissue-specific repertoire of isolated, expressed genes. These findings suggest that an advantage of the neighborhood organization is in the collective repression of groups of genes via a shared mechanism of chromatin repression. Genes essential to the mammary gland's uniqueness are isolated from neighbors, and likely have less tolerance for variation in expression, properties they share with genes responsible for an organism's survival.

  7. Shh expression is required for embryonic hair follicle but not mammary gland development.

    PubMed

    Michno, Kinga; Boras-Granic, Kata; Mill, Pleasantine; Hui, C C; Hamel, Paul A

    2003-12-01

    The embryonic mammary gland and hair follicle are both derived from the ventral ectoderm, and their development depends on a number of common fundamental developmental pathways. While the Hedgehog (Hh) signaling pathway is required for hair follicle morphogenesis, the role of this pathway during embryonic mammary gland development remains undetermined. We demonstrate here that, unlike the hair follicle, both Shh and Ihh are expressed in the developing embryonic mouse mammary rudiment as early as E12.5. In Shh(-/-) embryos, hair follicle development becomes arrested at an early stage, while the mammary rudiment, which continues to express Ihh, develops in a manner indistinguishable from that of wild-type littermates. The five pairs of mammary buds in Shh(-/-) female embryos exhibit normal branching morphogenesis at E16.5, forming a rudimentary ductal structure identical to wild-type embryonic mammary glands. We further demonstrate that loss of Hh signaling causes altered cyclin D1 expression in the embryonic dermal mesenchyme. Specifically, cyclin D1 is expressed at E14.5 principally in the condensed mesenchymal cells of the presumptive hair follicles and in both mesenchymal and epithelial cells of the mammary rudiments in wild-type and Shh-deficient embryos. By E18.5, robust cyclin D1 expression is maintained in mammary rudiments of both wild-type and Shh-deficient embryos. In hair follicles of wild-type embryos by E18.5, cyclin D1 expression switches to follicular epithelial cells. In contrast, strong cyclin D1 expression is observed principally in the mesenchymal cells of arrested hair follicles in Shh(-/-) embryos at E18.5. These data reveal that, despite the common embryonic origin of hair follicles and mammary glands, distinct patterns of Hh-family expression occur in these two tissues. Furthermore, these data suggest that cyclin D1 expression in the embryonic hair follicle is mediated by both Hh-independent and Hh-dependent mechanisms.

  8. Correlations between nuclear and fluorescent Imaging of mammary tumors in mice

    NASA Astrophysics Data System (ADS)

    Carroll, Robin; Stone, John; Blue, Eric; Bradley, Eric; Qian, Jianguo; Saha, Margaret; Welsh, Robert

    2008-10-01

    Progress with new imaging technologies permits the study of biological processes both in vivo and noninvasively. Two systems, a position-sensitive gamma camera and a cooled-CCD camera have been applied in this work. A C3H strain of mouse carrying the Mouse Mammary Tumor Virus (MMTV) was imaged using 800 nm Q-tracker fluorescent dots conjugated to a peptide targeting integrin αυβ C a mammary marker for angiogenesis. We subsequently imaged with the gamma camera to detect low levels of ^125I distribution, and hence, the activity of a trans-membrane protein called the sodium iodide symporter (NIS) responsible for iodine transport. Preliminary results indicate that the biodistribution of the tagged Q-tracker dots and ^125I co-localize very early in seemingly normal mammary glands of infected MMTV mice, while in larger palpable tumors the Q-dot signals are less apparent in comparison with the^125I signal.

  9. Insulin receptors in the mammary gland

    SciTech Connect

    Smith, D.H.

    1986-01-01

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

  10. Chemical Genetic Screen Reveals a Role for Desmosomal Adhesion in Mammary Branching Morphogenesis*

    PubMed Central

    Basham, Kaitlin J.; Kieffer, Collin; Shelton, Dawne N.; Leonard, Christopher J.; Bhonde, Vasudev R.; Vankayalapati, Hariprasad; Milash, Brett; Bearss, David J.; Looper, Ryan E.; Welm, Bryan E.

    2013-01-01

    During the process of branching morphogenesis, the mammary gland undergoes distinct phases of remodeling to form an elaborate ductal network that ultimately produces and delivers milk to newborn animals. These developmental events rely on tight regulation of critical cellular pathways, many of which are probably disrupted during initiation and progression of breast cancer. Transgenic mouse and in vitro organoid models previously identified growth factor signaling as a key regulator of mammary branching, but the functional downstream targets of these pathways remain unclear. Here, we used purified primary mammary epithelial cells stimulated with fibroblast growth factor-2 (FGF2) to model mammary branching morphogenesis in vitro. We employed a forward chemical genetic approach to identify modulators of this process and describe a potent compound, 1023, that blocks FGF2-induced branching. In primary mammary epithelial cells, we used lentivirus-mediated knockdown of the aryl hydrocarbon receptor (AHR) to demonstrate that 1023 acts through AHR to block branching. Using 1023 as a tool, we identified desmosomal adhesion as a novel target of AHR signaling and show that desmosomes are critical for AHR agonists to block branching. Our findings support a functional role for desmosomes during mammary morphogenesis and also in blocking FGF-induced invasion. PMID:23212921

  11. Form and function: how estrogen and progesterone regulate the mammary epithelial hierarchy.

    PubMed

    Arendt, Lisa M; Kuperwasser, Charlotte

    2015-06-01

    The mammary gland undergoes dramatic post-natal growth beginning at puberty, followed by full development occurring during pregnancy and lactation. Following lactation, the alveoli undergo apoptosis, and the mammary gland reverses back to resemble the nonparous gland. This process of growth and regression occurs for multiple pregnancies, suggesting the presence of a hierarchy of stem and progenitor cells that are able to regenerate specialized populations of mammary epithelial cells. Expansion of epithelial cell populations in the mammary gland is regulated by ovarian steroids, in particular estrogen acting through its receptor estrogen receptor alpha (ERα) and progesterone signaling through progesterone receptor (PR). A diverse number of stem and progenitor cells have been identified based on expression of cell surface markers and functional assays. Here we review the current understanding of how estrogen and progesterone act together and separately to regulate stem and progenitor cells within the human and mouse mammary tissues. Better understanding of the hierarchal organization of epithelial cell populations in the mammary gland and how the hormonal milieu affects its regulation may provide important insights into the origins of different subtypes of breast cancer.

  12. Expression of the whey acidic protein in transgenic pigs impairs mammary development.

    PubMed

    Shamay, A; Pursel, V G; Wilkinson, E; Wall, R J; Hennighausen, L

    1992-05-01

    The whey acidic protein has been found in milk of mice, rats, rabbits and camels, and its gene is expressed specifically in mammary tissue at late pregnancy and throughout lactation. A characteristic of whey acidic protein is the 'four-disulfide-core' signature which is also present in proteins involved in organ development. We have generated six lines of transgenic pigs which carry a mouse whey acidic protein transgene and express it at high levels in their mammary glands. Transgenic sows from three lines could not produce sufficient quantities of milk to support normal development of healthy offspring. This phenotype appears to be similar, if not identical, to the milchlos phenotype exhibited by mice expressing whey acidic protein transgenes. Mammary tissue from post-partum milchlos sows had an immature histological appearance, which was distinct from that observed during normal development or involution. Expression of the whey acidic protein transgene was found in mammary tissue from sexually immature pigs from milchlos lines, but not in sows from lines that appeared to lactate normally. We suggest that precocious synthesis of whey acidic protein impairs mammary development and function. Impaired mammary development due to inappropriate timing of whey acidic protein expression is consistent with the notion that proteins with the 'four-disulfide-core' signature participate in tissue formation. PMID:1284481

  13. Biological and genetic properties of the p53 null preneoplastic mammary epithelium

    NASA Technical Reports Server (NTRS)

    Medina, Daniel; Kittrell, Frances S.; Shepard, Anne; Stephens, L. Clifton; Jiang, Cheng; Lu, Junxuan; Allred, D. Craig; McCarthy, Maureen; Ullrich, Robert L.

    2002-01-01

    The absence of the tumor suppressor gene p53 confers an increased tumorigenic risk for mammary epithelial cells. In this report, we describe the biological and genetic properties of the p53 null preneoplastic mouse mammary epithelium in a p53 wild-type environment. Mammary epithelium from p53 null mice was transplanted serially into the cleared mammary fat pads of p53 wild-type BALB/c female to develop stable outgrowth lines. The outgrowth lines were transplanted for 10 generations. The outgrowths were ductal in morphology and progressed through ductal hyperplasia and ductal carcinoma in situ before invasive cancer. The preneoplastic outgrowth lines were immortal and exhibited activated telomerase activity. They are estrogen and progesterone receptor-positive, and aneuploid, and had various levels of tumorigenic potential. The biological and genetic properties of these lines are distinct from those found in most hyperplastic alveolar outgrowth lines, the form of mammary preneoplasia occurring in most traditional models of murine mammary tumorigenesis. These results indicate that the preneoplastic cell populations found in this genetically engineered model are similar in biological properties to a subset of precurser lesions found in human breast cancer and provide a unique model to identify secondary events critical for tumorigenicity and invasiveness.

  14. Sasa health exerts a protective effect on Her2/NeuN mammary tumorigenesis.

    PubMed

    Ren, Mingqiang; Reilly, R Todd; Sacchi, Nicoletta

    2004-01-01

    Bamboo grass leaves of different Sasa species have been widely used in food and medicine in Eastern Asia for hundreds of years. Of special interest are Kumazasa (Sasa senanensis rehder) leaves used to prepare an alkaline extract known as Sasa Health. This extract was reported to inhibit both the development and growth of mammary tumors in a mammary tumor strain of virgin SHN mice (1). We found that Sasa Health exerts a significant protective effect on spontaneous mammary tumorigenesis in another mouse model of human breast cancer, the transgenic FVB-Her2/NeuN mouse model. Two cohorts of Her2/NeuN female mice of different age (eleven-week-old and twenty-four-week-old) chronically treated with Sasa Health in drinking water showed both a delay in the development of tumors and reduced tumor multiplicity. Sasa Health also induced inhibition of mammary duct branching and side bud development in association with reduced angiogenesis. Altogether these findings indicate that Sasa Health contains phytochemicals that can effectively retard spontaneous mammary tumorigenesis.

  15. Idiopathic internal mammary artery aneurysm

    PubMed Central

    Heyn, Jens; Zimmermann, Hanna; Klose, Alexander; Luchting, Benjamin; Hinske, Christian; Sadeghi-Azandaryani, Mojtaba

    2014-01-01

    Aneurysms of the internal mammary artery are extremely rare, and their presentation and treatment are variable. Since these aneurysms often tend to rupture and cause haemothorax and life-threatening conditions, the knowledge of secure treatment options is indispensable. We here report the case of an idiopathic internal mammary aneurysm in a 46-year-old man. Open surgical resection of the aneurysm was performed in this case without any complications. The postoperative course was uneventful and the patient was in a good physical condition without any vascular or neurological abnormalities during follow-up. PMID:25452261

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

    PubMed

    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; Karantza, Vassiliki; Chen, Suzie

    2015-05-01

    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

  17. Gene expression array analyses predict increased proto-oncogene expression in MMTV induced mammary tumors.

    PubMed

    Popken-Harris, Pamela; Kirchhof, Nicole; Harrison, Ben; Harris, Lester F

    2006-08-01

    Exogenous infection by milk-borne mouse mammary tumor viruses (MMTV) typically induce mouse mammary tumors in genetically susceptible mice at a rate of 90-95% by 1 year of age. In contrast to other transforming retroviruses, MMTV acts as an insertional mutagen and under the influence of steroid hormones induces oncogenic transformation after insertion into the host genome. As these events correspond with increases in adjacent proto-oncogene transcription, we used expression array profiling to determine which commonly associated MMTV insertion site proto-oncogenes were transcriptionally active in MMTV induced mouse mammary tumors. To verify our gene expression array results we developed real-time quantitative RT-PCR assays for the common MMTV insertion site genes found in RIII/Sa mice (int-1/wnt-1, int-2/fgf-3, int-3/Notch 4, and fgf8/AIGF) as well as two genes that were consistently up regulated (CCND1, and MAT-8) and two genes that were consistently down regulated (FN1 and MAT-8) in the MMTV induced tumors as compared to normal mammary gland. Finally, each tumor was also examined histopathologically. Our expression array findings support a model whereby just one or a few common MMTV insertions into the host genome sets up a dominant cascade of events that leave a characteristic molecular signature.

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

    PubMed

    German, Tania; Barash, Itamar

    2002-05-01

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

  19. Automatic segmentation of histological structures in mammary gland tissue sections

    SciTech Connect

    Fernandez-Gonzalez, Rodrigo; Deschamps, Thomas; Idica, Adam K.; Malladi, Ravikanth; Ortiz de Solorzano, Carlos

    2004-02-17

    Real-time three-dimensional (3D) reconstruction of epithelial structures in human mammary gland tissue blocks mapped with selected markers would be an extremely helpful tool for breast cancer diagnosis and treatment planning. Besides its clear clinical application, this tool could also shed a great deal of light on the molecular basis of breast cancer initiation and progression. In this paper we present a framework for real-time segmentation of epithelial structures in two-dimensional (2D) images of sections of normal and neoplastic mammary gland tissue blocks. Complete 3D rendering of the tissue can then be done by surface rendering of the structures detected in consecutive sections of the blocks. Paraffin embedded or frozen tissue blocks are first sliced, and sections are stained with Hematoxylin and Eosin. The sections are then imaged using conventional bright field microscopy and their background is corrected using a phantom image. We then use the Fast-Marching algorithm to roughly extract the contours of the different morphological structures in the images. The result is then refined with the Level-Set method which converges to an accurate (sub-pixel) solution for the segmentation problem. Finally, our system stacks together the 2D results obtained in order to reconstruct a 3D representation of the entire tissue block under study. Our method is illustrated with results from the segmentation of human and mouse mammary gland tissue samples.

  20. Murine mammary stem/progenitor cell isolation: Different method matters?

    PubMed

    Gao, Hui; Dong, Qiaoxiang; Chen, Yuanhong; Zhang, Fuchuang; Wu, Anqi; Shi, Yuanshuo; Bandyopadhyay, Abhik; Daniel, Benjamin J; Huang, Changjiang; Sun, Lu-Zhe

    2016-01-01

    Murine mammary stem/progenitor cell isolation has been routinely used in many laboratories, yet direct comparison among different methods is lacking. In this study, we compared two frequently used digestion methods and three sets of frequently used surface markers for their efficiency in enriching mammary stem and progenitor cells in two commonly used mouse strains, C57BL/6J and FVB. Our findings revealed that the slow overnight digestion method using gentle collagenase/hyaluronidase could be easily adopted and yielded reliable and consistent results in different batches of animals. In contrast, the different fast digestion protocols, as described in published studies, yielded high percent of non-epithelial cells with very few basal epithelial cells liberated in our hands. The three sets of markers tested in our hands reveal rather equally efficiency in separating luminal and basal cells if same fluorochrome conjugations were used. However, the tendency of non-epithelial cell inclusion in the basal cell gate was highest in samples profiled by CD24/CD29 and lowest in samples profiled by CD49f/EpCAM, this is especially true in mammary cells isolated from C57BL/6J mice. This finding will have significant implication when sorted basal cells are used for subsequent gene expression analysis. PMID:26933638

  1. The Ras oncogene signals centrosome amplification in mammary epithelial cells through cyclin D1/Cdk4 and Nek2

    PubMed Central

    Zeng, X; Shaikh, FY; Harrison, MK; Adon, AM; Trimboli, AJ; Carroll, KA; Sharma, N; Timmers, C; Chodosh, LA; Leone, G; Saavedra, HI

    2010-01-01

    Centrosome amplification (CA) contributes to carcinogenesis by generating aneuploidy. Elevated frequencies of CA in most benign breast lesions and primary tumors suggest a causative role for CA in breast cancers. Clearly, identifying which and how altered signal transduction pathways contribute to CA is crucial to breast cancer control. Although a causative and cooperative role for c-Myc and Ras in mammary tumorigenesis is well documented, their ability to generate CA during mammary tumor initiation remains unexplored. To answer that question, K-RasG12D and c-Myc were induced in mouse mammary glands. Although CA was observed in mammary tumors initiated by c-Myc or K-RasG12D, it was detected only in premalignant mammary lesions expressing K-RasG12D. CA, both in vivo and in vitro, was associated with increased expression of the centrosome-regulatory proteins, cyclin D1 and Nek2. Abolishing the expression of cyclin D1, Cdk4 or Nek2 in MCF10A human mammary epithelial cells expressing H-RasG12V abrogated Ras-induced CA, whereas silencing cyclin E1 or B2 had no effect. Thus, we conclude that CA precedes mammary tumorigenesis, and interfering with centrosome-regulatory targets suppresses CA. PMID:20581865

  2. Dysfunctional telomeres promote genomic instability and metastasis in the absence of telomerase activity in oncogene induced mammary cancer.

    PubMed

    Bojovic, Bojana; Crowe, David L

    2013-02-01

    Telomerase is a ribonucleoprotein that maintains the ends of chromosomes (telomeres). In normal cells lacking telomerase activity, telomeres shorten with each cell division because of the inability to completely synthesize the lagging strand. Critically shortened telomeres elicit DNA damage responses and limit cellular division and lifespan, providing an important tumor suppressor function. Most human cancer cells express telomerase which contributes significantly to the tumor phenotype. In human breast cancer, telomerase expression is predictive of clinical outcomes such as lymph node metastasis and survival. In mouse models of mammary cancer, telomerase expression is also upregulated. Telomerase overexpression resulted in spontaneous mammary tumor development in aged female mice. Increased mammary cancer also was observed when telomerase deficient mice were crossed with p53 null mutant animals. However, the effects of telomerase and telomere length on oncogene driven mammary cancer have not been completely characterized. To address these issues we characterized neu proto-oncogene driven mammary tumor formation in G1 Terc-/- (telomerase deficient with long telomeres), G3 Terc-/- (telomerase deficient with short telomeres), and Terc+/+ mice. Telomerase deficiency reduced the number of mammary tumors and increased tumor latency regardless of telomere length. Decreased tumor formation correlated with increased apoptosis in Terc deficient tumors. Short telomeres dramatically increased lung metastasis which correlated with increased genomic instability, and specific alterations in DNA copy number and gene expression. We concluded that short telomeres promote metastasis in the absence of telomerase activity in neu oncogene driven mammary tumors.

  3. Contrast-Enhanced X-ray Detection of Microcalcifications in Radiographically Dense Mammary Tissue Using Targeted Gold Nanoparticles.

    PubMed

    Cole, Lisa E; Vargo-Gogola, Tracy; Roeder, Ryan K

    2015-09-22

    Breast density reduces the accuracy of mammography, motivating methods to improve sensitivity and specificity for detecting abnormalities within dense breast tissue, but preclinical animal models are lacking. Therefore, the objectives of this study were to investigate a murine model of radiographically dense mammary tissue and contrast-enhanced X-ray detection of microcalcifications in dense mammary tissue by targeted delivery of bisphosphonate-functionalized gold nanoparticles (BP-Au NPs). Mammary glands (MGs) in the mouse mammary tumor virus - polyomavirus middle T antigen (MMTV-PyMT or PyMT) model exhibited greater radiographic density with age and compared with strain- and age-matched wild-type (WT) controls at 6-10 weeks of age. The greater radiographic density of MGs in PyMT mice obscured radiographic detection of microcalcifications that were otherwise detectable in MGs of WT mice. However, BP-Au NPs provided enhanced contrast for the detection of microcalcifications in both radiographically dense (PyMT) and WT mammary tissues as measured by computed tomography after intramammary delivery. BP-Au NPs targeted microcalcifications to enhance X-ray contrast with surrounding mammary tissue, which resulted in improved sensitivity and specificity for detection in radiographically dense mammary tissues. PMID:26308767

  4. Ectodysplasin/NF-κB signaling in embryonic mammary gland development.

    PubMed

    Lindfors, Päivi H; Voutilainen, Maria; Mikkola, Marja L

    2013-06-01

    The ectodysplasin (Eda) signaling pathway consists of a TNF-like ligand Eda, its receptor Edar, and an adaptor protein Edaradd and its activation leads to NF-κB mediated transcription. In humans, mutations in the EDA pathway genes cause hypohidrotic ectodermal dysplasia, a disorder characterized by defective formation of hair follicles, teeth, and several exocrine glands including the breast. Embryonic mammary gland development proceeds via placode, bud, bulb and sprout stages before the onset of branching morphogenesis. Studies on mouse models have linked Eda with two aspects of embryonic mammary gland morphogenesis: placode induction and ductal growth and branching. Here we summarize the current knowledge on the role of Eda/NF-κB in mammary gland development.

  5. Mechanical strain induces involution-associated events in mammary epithelial cells

    PubMed Central

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

    2009-01-01

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

  6. Mammary Gland Specific Knockdown of the Physiological Surge in Cx26 during Lactation Retains Normal Mammary Gland Development and Function

    PubMed Central

    Stewart, Michael K. G.; Plante, Isabelle; Bechberger, John F.; Naus, Christian C.; Laird, Dale W.

    2014-01-01

    Connexin26 (Cx26) is the major Cx protein expressed in the human mammary gland and is up-regulated during pregnancy while remaining elevated throughout lactation. It is currently unknown if patients with loss-of-function Cx26 mutations that result in hearing loss and skin diseases have a greater susceptibility to impaired breast development. To investigate if Cx26 plays a critical role in mammary gland development and differentiation, a novel Cx26 conditional knockout mouse model was generated by crossing Cx26fl/fl mice with mice expressing Cre under the β-Lactoglobulin promoter. Conditional knockdown of Cx26 from the mammary gland resulted in a dramatic reduction in detectable gap junction plaques confirmed by a significant ∼65-70% reduction in Cx26 mRNA and protein throughout parturition and lactation. Interestingly, this reduction was accompanied by a decrease in mammary gland Cx30 gap junction plaques at parturition, while no change was observed for Cx32 or Cx43. Whole mount, histological and immunofluorescent assessment of breast tissue revealed comparatively normal lobuloalveolar development following pregnancy in the conditionally knockdown mice compared to control mice. In addition, glands from genetically-modified mice were capable of producing milk proteins that were evident in the lumen of alveoli and ducts at similar levels as controls, suggesting normal gland function. Together, our results suggest that low levels of Cx26 expression throughout pregnancy and lactation, and not the physiological surge in Cx26, is sufficient for normal gland development and function. PMID:24988191

  7. Influence of milk source on transplantability of histocompatible mammary tumours in mice.

    PubMed Central

    Oth, D.; Sabolovic, D.

    1977-01-01

    It is confirmed that C3H mammary tumours are much more easily transplantable in histocompatible recipients when these have been reared on C3H milk, than when they have been reared on milk from the inbred Swiss/B strain. By contrast, A.CA mammary tumours transplanted in histocompatible hosts reared on A.CA or Swiss/B milk, grow almost equally well in both sorts of recipient. Thus, rearing on Swiss/B milk has different effects on the transplantability of mammary tumours of C3H and A.CA. On the other hand, recipients which were reared on C3H or A.CA milks accept grafts of C3H mammary tumours about equally, suggesting that milks from A.CA and C3H have the same effect on the transplantability of C3H mammary tumours. The different action of Swiss/B milk on tumours of C3H and A.CA seems best attributed to differences between C3H and A.CA tumours or between mouse strain genotypes. By contrast, the transplantability of C3H mammary tumours is significantly changed when the recipients were reared on milk from the RIII strain instead of C3H. These facts suggest that the milk from RIII has an action which differs from that of both C3H and A.CA in this respect. The data are discussed on the basis of a differential tollerance-inducing action of mammary tumour viruses (MTVs) which infect C3H, A.CA and RIII, and have an important role in tumour induction. PMID:326285

  8. Mammary candidosis in lactating women.

    PubMed

    Heinig, M J; Francis, J; Pappagianis, D

    1999-12-01

    Though perceived to be a growing problem by lactation professionals, fungal infection of the breast (mammary candidosis) is largely unstudied. Candida albicans, a commensal organism encountered frequently in the vagina and gastrointestinal tract of humans, has been reported to be responsible for both superficial (cutaneous) and localized (ductal) infection of the mammary gland in lactating women, though the latter association is not universally accepted. Severe pain is considered to be characteristic of yeast infection of the breast and may be a cause of premature weaning among lactating mothers. Given that pain is often the complaint that prompts mothers to consult lactation professionals, it is important that healthcare providers working with lactating women be knowledgeable about this disease. In this article, current research regarding yeast infection of the breast is summarized, including morphology and pathology, diagnosis, risk factors, and common treatment options.

  9. 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

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

    PubMed Central

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

    2011-01-01

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

  11. Identification of genetic loci that control mammary tumor susceptibility through the host microenvironment

    SciTech Connect

    Zhang, Pengju; Lo, Alvin; Huang, Yurong; Huang, Ge; Liang, Guozhou; Mott, Joni; Karpen, Gary H.; Blakely, Eleanor A.; Bissell, Mina J.; Barcellos-Hoff, Mary Helen; Snijders, Antoine M.; Mao, Jian-Hua

    2015-03-09

    The interplay between host genetics, tumor microenvironment and environmental exposure in cancer susceptibility remains poorly understood. Here we assessed the genetic control of stromal mediation of mammary tumor susceptibility to low dose ionizing radiation (LDIR) using backcrossed F1 into BALB/c (F1Bx) between cancer susceptible (BALB/c) and resistant (SPRET/EiJ) mouse strains. Tumor formation was evaluated after transplantation of non-irradiated Trp53-/- BALB/c mammary gland fragments into cleared fat pads of F1Bx hosts. Genome-wide linkage analysis revealed 2 genetic loci that constitute the baseline susceptibility via host microenvironment. However, once challenged with LDIR, we discovered 13 additional loci that were enriched for genes involved in cytokines, including TGFβ1 signaling. Surprisingly, LDIR-treated F1Bx cohort significantly reduced incidence of mammary tumors from Trp53-/- fragments as well as prolonged tumor latency, compared to sham-treated controls. We demonstrated further that plasma levels of specific cytokines were significantly correlated with tumor latency. Using an ex vivo 3-D assay, we confirmed TGFβ1 as a strong candidate for reduced mammary invasion in SPRET/EiJ, which could explain resistance of this strain to mammary cancer risk following LDIR. Our results open possible new avenues to understand mechanisms of genes operating via the stroma that affect cancer risk from external environmental exposures.

  12. Identification of genetic loci that control mammary tumor susceptibility through the host microenvironment

    DOE PAGES

    Zhang, Pengju; Lo, Alvin; Huang, Yurong; Huang, Ge; Liang, Guozhou; Mott, Joni; Karpen, Gary H.; Blakely, Eleanor A.; Bissell, Mina J.; Barcellos-Hoff, Mary Helen; et al

    2015-03-09

    The interplay between host genetics, tumor microenvironment and environmental exposure in cancer susceptibility remains poorly understood. Here we assessed the genetic control of stromal mediation of mammary tumor susceptibility to low dose ionizing radiation (LDIR) using backcrossed F1 into BALB/c (F1Bx) between cancer susceptible (BALB/c) and resistant (SPRET/EiJ) mouse strains. Tumor formation was evaluated after transplantation of non-irradiated Trp53-/- BALB/c mammary gland fragments into cleared fat pads of F1Bx hosts. Genome-wide linkage analysis revealed 2 genetic loci that constitute the baseline susceptibility via host microenvironment. However, once challenged with LDIR, we discovered 13 additional loci that were enriched for genesmore » involved in cytokines, including TGFβ1 signaling. Surprisingly, LDIR-treated F1Bx cohort significantly reduced incidence of mammary tumors from Trp53-/- fragments as well as prolonged tumor latency, compared to sham-treated controls. We demonstrated further that plasma levels of specific cytokines were significantly correlated with tumor latency. Using an ex vivo 3-D assay, we confirmed TGFβ1 as a strong candidate for reduced mammary invasion in SPRET/EiJ, which could explain resistance of this strain to mammary cancer risk following LDIR. Our results open possible new avenues to understand mechanisms of genes operating via the stroma that affect cancer risk from external environmental exposures.« less

  13. A Multifaceted Role for Myd88-Dependent Signaling in Progression of Murine Mammary Carcinoma

    PubMed Central

    Higgins, Mary J.; Serrano, Antonio; Boateng, Kofi Y.; Parsons, Victoria A.; Phuong, Tiffany; Seifert, Alyssa; Ricca, Jacob M.; Tucker, Kyle C.; Eidelman, Alec S.; Carey, Maureen A.; Kurt, Robert A.

    2016-01-01

    Previous data obtained in our laboratory suggested that there may be constitutive signaling through the myeloid differentiation primary response gene 88 (Myd88)-dependent signaling cascade in murine mammary carcinoma. Here, we extended these findings by showing that, in the absence of an added Toll-like receptor (TLR) agonist, the myddosome complex was preformed in 4T1 tumor cells, and that Myd88 influenced cytoplasmic extracellular signal–regulated kinase (Erk)1/Erk2 levels, nuclear levels of nuclear factor-kappaB (NFκB) and signal transducer and activator of transcription 5 (STAT5), tumor-derived chemokine (C–C motif) ligand 2 (CCL2) expression, and in vitro and in vivo tumor growth. In addition, RNA-sequencing revealed that Myd88-dependent signaling enhanced the expression of genes that could contribute to breast cancer progression and genes previously associated with poor outcome for patients with breast cancer, in addition to suppressing the expression of genes capable of inhibiting breast cancer progression. Yet, Myd88-dependent signaling in tumor cells also suppressed expression of genes that could contribute to tumor progression. Collectively, these data revealed a multifaceted role for Myd88-dependent signaling in murine mammary carcinoma. PMID:27812285

  14. Mammary tumor growth and metastasis are reduced in c-Kit mutant Sash mice.

    PubMed

    He, Licai; Zhu, Zhenfeng; Chen, Shang; Wang, Yongping; Gu, Haihua

    2016-06-01

    Besides its well-known function in allergic response, mast cell, one of the key immune cells present in tumor microenvironment, plays important roles in cancer progression. However, the functional role of mast cells in breast cancer development and metastasis is not well understood. To test the involvement of mast cells in breast cancer, we examined the effects of loss of mast cells on mammary tumor development by crossing the well-known mast cell deficient mouse strain sash (Kit(W-sh/W-sh) ) with the mammary tumor transgenic mouse strain MMTV-Polyoma Middle T antigen (PyMT). Although mammary tumor onset was not affected in the absence of mast cells, mammary growth and metastasis were reduced in PyMT/Kit(W-sh/W-sh) mice compared with PyMT/wild-type mice (WT). Histological and immunofluorescent analyses showed that tumors from PyMT/Kit(W-sh/W-sh) mice showed largely differentiated morphology with reduced angiogenesis compared with MMTV-PyMT/WT mice. Our results suggest that mast cells may promote breast cancer growth and metastasis. Agents that can block mast cells growth are potential new therapies to treat metastatic breast cancer. PMID:26992445

  15. 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

    2016-01-01

    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.

  16. Telomerase and estrogen-sensing activities are essential for continued mammary growth in vivo but dispensable for “reprogramming” neural stem cells

    PubMed Central

    George, Andrea L.; Boulanger, Corinne A.; Smith, Gilbert H.

    2016-01-01

    It has been proposed that the erosion of telomere length is a limiting factor in replicative capacity and important in cell senescence. To determine if this activity was essential in the mouse mammary gland in vivo, we serially transplanted mammary fragments from wild type (TER+/+), heterozygous (TER+/−), and homozygous (TER−/−) mammary tissues into the cleared mammary fat pads of immune-compromised nude mice. Individual implants from both homozygous and heterozygous TER null outgrowths showed growth senescence beginning at transplant generation two, earlier than implants from TER+/+ mammary glands which continued to show growth. This result suggests that either mammary epithelial stem cells maintain their telomere length in order to self renew, or that the absence or reduction of telomerase template results in more frequent death/extinction of stem cells during symmetric divisions. A third possibility is the inability of signaling cells in the niche to replicate resulting in reduction of the maintenance signals necessary for stem cell renewal. Consistent with this, examination of senescent outgrowths revealed the absence of estrogen receptor alpha (ERα+) epithelium although progesterone receptor (PR+) cells were abundant. Despite their inability to establish mammary growth in vivo, TER+/− cells were able to direct neural stem cells to mammary cell fates. PMID:27347776

  17. Telomerase and estrogen-sensing activities are essential for continued mammary growth in vivo but dispensable for "reprogramming" neural stem cells.

    PubMed

    George, Andrea L; Boulanger, Corinne A; Smith, Gilbert H

    2016-07-01

    It has been proposed that the erosion of telomere length is a limiting factor in replicative capacity and important in cell senescence. To determine if this activity was essential in the mouse mammary gland in vivo, we serially transplanted mammary fragments from wild type (TER+/+), heterozygous (TER+/-), and homozygous (TER-/-) mammary tissues into the cleared mammary fat pads of immune-compromised nude mice. Individual implants from both homozygous and heterozygous TER null outgrowths showed growth senescence beginning at transplant generation two, earlier than implants from TER+/+ mammary glands which continued to show growth. This result suggests that either mammary epithelial stem cells maintain their telomere length in order to self renew, or that the absence or reduction of telomerase template results in more frequent death/extinction of stem cells during symmetric divisions. A third possibility is the inability of signaling cells in the niche to replicate resulting in reduction of the maintenance signals necessary for stem cell renewal. Consistent with this, examination of senescent outgrowths revealed the absence of estrogen receptor alpha (ERα+) epithelium although progesterone receptor (PR+) cells were abundant. Despite their inability to establish mammary growth in vivo, TER+/- cells were able to direct neural stem cells to mammary cell fates. PMID:27347776

  18. Evo-devo of the mammary gland.

    PubMed

    Oftedal, Olav T; Dhouailly, Danielle

    2013-06-01

    We propose a new scenario for mammary evolution based on comparative review of early mammary development among mammals. Mammary development proceeds through homologous phases across taxa, but evolutionary modifications in early development produce different final morphologies. In monotremes, the mammary placode spreads out to form a plate-like mammary bulb from which more than 100 primary sprouts descend into mesenchyme. At their distal ends, secondary sprouts develop, including pilosebaceous anlagen, resulting in a mature structure in which mammary lobules and sebaceous glands empty into the infundibula of hair follicles; these structural triads (mammolobular-pilo-sebaceous units or MPSUs) represent an ancestral condition. In marsupials a flask-like mammary bulb elongates as a sprout, but then hollows out; its secondary sprouts include hair and sebaceous anlagen (MPSUs), but the hairs are shed during nipple formation. In some eutherians (cat, horse, human) MPSUs form at the distal ends of primary sprouts; pilosebaceous components either regress or develop into mature structures. We propose that a preexisting structural triad (the apocrine-pilo-sebaceous unit) was incorporated into the evolving mammary structure, and coupled to additional developmental processes that form the mammary line, placode, bulb and primary sprout. In this scenario only mammary ductal trees and secretory tissue derive from ancestral apocrine-like glands. The mammary gland appears to have coopted signaling pathways and genes for secretory products from even earlier integumentary structures, such as odontode (tooth-like) or odontode-derived structures. We speculate that modifications in signal use (such as PTHrP and BMP4) may contribute to taxonomic differences in MPSU development. PMID:23681303

  19. The calcineurin/NFAT pathway is activated in diagnostic breast cancer cases and is essential to survival and metastasis of mammary cancer cells.

    PubMed

    Quang, C Tran; Leboucher, S; Passaro, D; Fuhrmann, L; Nourieh, M; Vincent-Salomon, A; Ghysdael, J

    2015-02-26

    Nuclear factor of activated T cells 1 (NFAT1) expression has been associated with increased migratory/invasive properties of mammary tumor-derived cell lines in vitro. It is unknown, however, if NFAT activation actually occurs in breast cancer cases and whether the calcineurin/NFAT pathway is important to mammary tumorigenesis. Using a cohort of 321 diagnostic cases of the major subgroup of breast cancer, we found Cn/NFAT pathway activated in ER(-)PR(-)HER2(-) triple-negative breast cancer subtype, whereas its prevalence is less in other subgroups. Using a small hairpin RNA-based gene expression silencing approach in murine mammary tumor cell line (4T1), we show that not only NFAT1 but also NFAT2 and their upstream activator Cn are essential to the migratory and invasive properties of mammary tumor cells. We also demonstrate that Cn, NFAT1 and NFAT2 are essential to the tumorigenic and metastatic properties of these cells in mice, a phenotype which coincides with increased apoptosis in vivo. Finally, global gene expression analyses identified several NFAT-deregulated genes, many of them being previously associated with mammary tumorigenesis. In particular, we identified the gene encoding a disintegrin and metalloproteinase with thrombonspondin motifs 1, as being a potential direct target of NFAT1. Thus, our results show that the Cn/NFAT pathway is activated in diagnostic cases of breast cancers and is essential to the tumorigenic and metastatic potential of mammary tumor cell line. These results suggest that pharmacological inhibition of the Cn/NFAT pathway at different levels could be of therapeutical interest for breast cancer patients.

  20. Mammary hypertrophy in an ovariohysterectomized cat.

    PubMed

    Pukay, B P; Stevenson, D A

    1983-05-01

    A four year old ovariohysterectomized domestic short-haired cat under treatment for behavioral urine spraying and idiopathic alopecia developed mammary gland hypertrophy following treatment with megestrol acetate. Withdrawal of the progestin and treatment with androgen failed to cause regression of the hypertrophy. The affected mammary gland was surgically excised and recovery was uneventful.

  1. Cytokine networks that mediate epithelial cell-macrophage crosstalk in the mammary gland: implications for development and cancer.

    PubMed

    Sun, Xuan; Ingman, Wendy V

    2014-07-01

    Dynamic interactions between the hormone responsive mammary gland epithelium and surrounding stromal macrophage populations are critical for normal development and function of the mammary gland. Macrophages are versatile cells capable of diverse roles in mammary gland development and maintenance of homeostasis, and their function is highly dependent on signals within the local cytokine microenvironment. The mammary epithelium secretes a number of cytokines, including colony stimulating factor 1 (CSF1), transforming growth factor beta 1 (TGFB1), and chemokine ligand 2 (CCL2) that affect the abundance, phenotype and function of macrophages. However, aberrations in these interactions have been found to increase the risk of tumour formation, and utilisation of stromal macrophage support by tumours can increase the invasive and metastatic potential of the cancer. Studies utilising genetically modified mouse models have shed light on the significance of epithelial cell-macrophage crosstalk, and the cytokines that mediate this communication, in mammary gland development and tumourigenesis. This article reviews the current status of our understanding of the roles of epithelial cell-derived cytokines in mammary gland development and cancer, with a focus on the crosstalk between epithelial cells and the local macrophage population.

  2. Scribble Modulates the MAPK/Fra1 Pathway to Disrupt Luminal and Ductal Integrity and Suppress Tumour Formation in the Mammary Gland

    PubMed Central

    Godde, Nathan J.; Sheridan, Julie M.; Smith, Lorey K.; Pearson, Helen B.; Britt, Kara L.; Galea, Ryan C.; Yates, Laura L.; Visvader, Jane E.; Humbert, Patrick O.

    2014-01-01

    Polarity coordinates cell movement, differentiation, proliferation and apoptosis to build and maintain complex epithelial tissues such as the mammary gland. Loss of polarity and the deregulation of these processes are critical events in malignant progression but precisely how and at which stage polarity loss impacts on mammary development and tumourigenesis is unclear. Scrib is a core polarity regulator and tumour suppressor gene however to date our understanding of Scrib function in the mammary gland has been limited to cell culture and transplantation studies of cell lines. Utilizing a conditional mouse model of Scrib loss we report for the first time that Scrib is essential for mammary duct morphogenesis, mammary progenitor cell fate and maintenance, and we demonstrate a critical and specific role for Scribble in the control of the early steps of breast cancer progression. In particular, Scrib-deficiency significantly induced Fra1 expression and basal progenitor clonogenicity, which resulted in fully penetrant ductal hyperplasia characterized by high cell turnover, MAPK hyperactivity, frank polarity loss with mixing of apical and basolateral membrane constituents and expansion of atypical luminal cells. We also show for the first time a role for Scribble in mammalian spindle orientation with the onset of mammary hyperplasia being associated with aberrant luminal cell spindle orientation and a failure to apoptose during the final stage of duct tubulogenesis. Restoring MAPK/Fra1 to baseline levels prevented Scrib-hyperplasia, whereas persistent Scrib deficiency induced alveolar hyperplasia and increased the incidence, onset and grade of mammary tumours. These findings, based on a definitive genetic mouse model provide fundamental insights into mammary duct maturation and homeostasis and reveal that Scrib loss activates a MAPK/Fra1 pathway that alters mammary progenitor activity to drive premalignancy and accelerate tumour progression. PMID:24852022

  3. 9 CFR 310.17 - Inspection of mammary glands.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Inspection of mammary glands. 310.17... INSPECTION AND CERTIFICATION POST-MORTEM INSPECTION § 310.17 Inspection of mammary glands. (a) Lactating mammary glands and diseased mammary glands of cattle, sheep, swine, and goats shall be removed...

  4. Transcriptional repressor Tbx3 is required for the hormone-sensing cell lineage in mammary epithelium.

    PubMed

    Kunasegaran, Kamini; Ho, Victor; Chang, Ted H-T; De Silva, Duvini; Bakker, Martijn L; Christoffels, Vincent M; Pietersen, Alexandra M

    2014-01-01

    The transcriptional repressor Tbx3 is involved in lineage specification in several tissues during embryonic development. Germ-line mutations in the Tbx3 gene give rise to Ulnar-Mammary Syndrome (comprising reduced breast development) and Tbx3 is required for mammary epithelial cell identity in the embryo. Notably Tbx3 has been implicated in breast cancer, which develops in adult mammary epithelium, but the role of Tbx3 in distinct cell types of the adult mammary gland has not yet been characterized. Using a fluorescent reporter knock-in mouse, we show that in adult virgin mice Tbx3 is highly expressed in luminal cells that express hormone receptors, and not in luminal cells of the alveolar lineage (cells primed for milk production). Flow cytometry identified Tbx3 expression already in progenitor cells of the hormone-sensing lineage and co-immunofluorescence confirmed a strict correlation between estrogen receptor (ER) and Tbx3 expression in situ. Using in vivo reconstitution assays we demonstrate that Tbx3 is functionally relevant for this lineage because knockdown of Tbx3 in primary mammary epithelial cells prevented the formation of ER+ cells, but not luminal ER- or basal cells. Interestingly, genes that are repressed by Tbx3 in other cell types, such as E-cadherin, are not repressed in hormone-sensing cells, highlighting that transcriptional targets of Tbx3 are cell type specific. In summary, we provide the first analysis of Tbx3 expression in the adult mammary gland at a single cell level and show that Tbx3 is important for the generation of hormone-sensing cells.

  5. Transcriptional Repressor Tbx3 Is Required for the Hormone-Sensing Cell Lineage in Mammary Epithelium

    PubMed Central

    Kunasegaran, Kamini; Ho, Victor; Chang, Ted H-. T.; De Silva, Duvini; Bakker, Martijn L.; Christoffels, Vincent M.; Pietersen, Alexandra M.

    2014-01-01

    The transcriptional repressor Tbx3 is involved in lineage specification in several tissues during embryonic development. Germ-line mutations in the Tbx3 gene give rise to Ulnar-Mammary Syndrome (comprising reduced breast development) and Tbx3 is required for mammary epithelial cell identity in the embryo. Notably Tbx3 has been implicated in breast cancer, which develops in adult mammary epithelium, but the role of Tbx3 in distinct cell types of the adult mammary gland has not yet been characterized. Using a fluorescent reporter knock-in mouse, we show that in adult virgin mice Tbx3 is highly expressed in luminal cells that express hormone receptors, and not in luminal cells of the alveolar lineage (cells primed for milk production). Flow cytometry identified Tbx3 expression already in progenitor cells of the hormone-sensing lineage and co-immunofluorescence confirmed a strict correlation between estrogen receptor (ER) and Tbx3 expression in situ. Using in vivo reconstitution assays we demonstrate that Tbx3 is functionally relevant for this lineage because knockdown of Tbx3 in primary mammary epithelial cells prevented the formation of ER+ cells, but not luminal ER- or basal cells. Interestingly, genes that are repressed by Tbx3 in other cell types, such as E-cadherin, are not repressed in hormone-sensing cells, highlighting that transcriptional targets of Tbx3 are cell type specific. In summary, we provide the first analysis of Tbx3 expression in the adult mammary gland at a single cell level and show that Tbx3 is important for the generation of hormone-sensing cells. PMID:25343378

  6. Expression of class II beta-tubulin by proliferative myoepithelial cells in canine mammary mixed tumors.

    PubMed

    Arai, K; Nakano, H; Shibutani, M; Naoi, M; Matsuda, H

    2003-11-01

    Benign mammary mixed tumors in dogs resemble human salivary pleomorphic adenomas with regard to their histogenesis, including the occurrence of cartilaginous or bony metaplasia as well as the expression pattern of cytoskeletal proteins in proliferative myoepithelial cells. Recently, a monoclonal antibody specific for class II beta-tubulin has been developed. The epitope it recognizes was determined to be the heptapeptide Glu-Glu-Glu-Glu-Gly-Glu-Asp, which is the common sequence found among the canine, rat, mouse, and human class II beta-tubulin-specific regions. We carried out immunohistochemical studies on mammary mixed tumors obtained from three female dogs using this the monoclonal antibody. The antibody to class II beta-tubulin reacted intensely with proliferative myoepithelial cells in canine mammary mixed tumors, whereas staining was barely detectable in normal myoepithelial cells surrounding alveoli and alveolar ducts within the tumor and adjacent normal tissue. Proliferative myoepithelial cells also expressed vimentin, but alpha-smooth muscle actin (alphaSMA) staining was barely detectable. Immunoblot analysis showed that class II beta-tubulin and vimentin were expressed in myoepithelial cell lines prepared from the three mammary mixed tumors. On the other hand, only one cell line, which was negative for alphaSMA, produced cartilage-specific type II collagen. These results suggest that class II beta-tubulin could be a new molecular marker of proliferating myoepithelial cells in canine mammary mixed tumors and that differential expression of cytoskeletal components is associated with cartilaginous metaplasia of proliferative myoepithelial cells in mixed mammary tumors.

  7. TFAP2C Governs the Luminal Epithelial Phenotype in Mammary Development and Carcinogenesis

    PubMed Central

    Cyr, Anthony R; Kulak, Mikhail V.; Park, Jung M.; Bogachek, Maria V.; Spanheimer, Philip M.; Woodfield, George W.; White-Baer, Lola S.; O’Malley, Yunxia Q.; Sugg, Sonia L.; Olivier, Alicia K.; Zhang, Weizhou; Domann, Frederick E.; Weigel, Ronald J.

    2014-01-01

    Molecular subtypes of breast cancer are characterized by distinct patterns of gene expression that are predictive of outcome and response to therapy. The luminal breast cancer subtypes are defined by the expression of ER-alpha (ERα)-associated genes, many of which are directly responsive to the Transcription Factor Activator Protein 2C (TFAP2C). TFAP2C participates in a gene regulatory network controlling cell growth and differentiation during ectodermal development and regulating ESR1/ERα and other luminal cell-associated genes in breast cancer. TFAP2C has been established as a prognostic factor in human breast cancer, however, its role in the establishment and maintenance of the luminal cell phenotype during carcinogenesis and mammary gland development have remained elusive. Herein, we demonstrate a critical role for TFAP2C in maintaining the luminal phenotype in human breast cancer and in influencing the luminal cell phenotype during normal mammary development. Knockdown of TFAP2C in luminal breast carcinoma cells induced EMT with morphological and phenotypic changes characterized by a loss of luminal-associated gene expression and a concomitant gain of basal-associated gene expression. Conditional knockout of the mouse homolog of TFAP2C, Tcfap2c, in mouse mammary epithelium driven by MMTV-Cre promoted aberrant growth of the mammary tree leading to a reduction in the CD24hi/CD49fmid luminal cell population and concomitant gain of the CD24mid/CD49fhi basal cell population at maturity. Our results establish TFAP2C as a key transcriptional regulator for maintaining the luminal phenotype in human breast carcinoma. Furthermore, Tcfap2c influences development of the luminal cell type during mammary development. The data suggest that TFAP2C plays an important role in regulated luminal specific genes and may be a viable therapeutic target in breast cancer. PMID:24469049

  8. Remodeling of Endogenous Mammary Epithelium by Breast Cancer Stem Cells

    PubMed Central

    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.

    2014-01-01

    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. PMID:22899386

  9. 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. PMID:22899386

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

    SciTech Connect

    Rauner, Gat; Barash, Itamar

    2014-10-15

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

  11. Mammary and extramammary Paget's disease.

    PubMed

    Lopes Filho, Lauro Lourival; Lopes, Ione Maria Ribeiro Soares; Lopes, Lauro Rodolpho Soares; Enokihara, Milvia M S S; Michalany, Alexandre Osores; Matsunaga, Nobuo

    2015-01-01

    Paget's disease, described by Sir James Paget in 1874, is classified as mammary and extramammary. The mammary type is rare and often associated with intraductal cancer (93-100% of cases). It is more prevalent in postmenopausal women and it appears as an eczematoid, erythematous, moist or crusted lesion, with or without fine scaling, infiltration and inversion of the nipple. It must be distinguished from erosive adenomatosis of the nipple, cutaneous extension of breast carcinoma, psoriasis, atopic dermatitis, contact dermatitis, chronic eczema, lactiferous ducts ectasia, Bowen's disease, basal cell carcinoma, melanoma and intraductal papilloma. Diagnosis is histological and prognosis and treatment depend on the type of underlying breast cancer. Extramammary Paget's disease is considered an adenocarcinoma originating from the skin or skin appendages in areas with apocrine glands. The primary location is the vulvar area, followed by the perianal region, scrotum, penis and axillae. It starts as an erythematous plaque of indolent growth, with well-defined edges, fine scaling, excoriations, exulcerations and lichenification. In most cases it is not associated with cancer, although there are publications linking it to tumors of the vulva, vagina, cervix and corpus uteri, bladder, ovary, gallbladder, liver, breast, colon and rectum. Differential diagnoses are candidiasis, psoriasis and chronic lichen simplex. Histopathology confirms the diagnosis. Before treatment begins, associated malignancies should be investigated. Surgical excision and micrographic surgery are the best treatment options, although recurrences are frequent. PMID:25830993

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

    PubMed

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

    2005-05-24

    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.

  13. Distinct Luminal-Type Mammary Carcinomas Arise from Orthotopic Trp53-Null Mammary Transplantation of Juvenile versus Adult Mice

    SciTech Connect

    Nguyen, David H.; Ouyang, Haoxu; Mao, Jian-Hua; Hlatky, Lynn; Barcellos-Hoff, M. H.

    2014-12-01

    Age and physiologic status, such as menopause, are risk factors for breast cancer. Less clear is what factors influence the diversity of breast cancer. In this study, we investigated the effect of host age on the distribution of tumor subtypes in mouse mammary chimera consisting of wild-type hosts and Trp53 nullizygous epithelium, which undergoes a high rate of neoplastic transformation. Wild-type mammary glands cleared of endogenous epithelium at 3 weeks of age were subsequently transplanted during puberty (5 weeks) or at maturation (10 weeks) with syngeneic Trp53-null mammary tissue fragments and monitored for one year. Tumors arose sooner from adult hosts (AH) compared with juvenile hosts (JH). However, compared with AH tumors, JH tumors grew several times faster, were more perfused, exhibited a two-fold higher mitotic index, and were more highly positive for insulin-like growth factor receptor phosphorylation. Most tumors in each setting were estrogen receptor (ER)-positive (80% JH vs. 70% AH), but JH tumors were significantly more ER-immunoreactive (P = 0.0001) than AH tumors. A differential expression signature (JvA) of juvenile versus adult tumors revealed a luminal transcriptional program. Centroids of the human homologs of JvA genes showed that JH tumors were more like luminal A tumors and AH tumors were more like luminal B tumors. Hierarchical clustering with the JvA human ortholog gene list segregated luminal A and luminal B breast cancers across datasets. Lastly, these data support the notion that age-associated host physiology greatly influences the intrinsic subtype of breast cancer.

  14. Distinct Luminal-Type Mammary Carcinomas Arise from Orthotopic Trp53-Null Mammary Transplantation of Juvenile versus Adult Mice

    DOE PAGES

    Nguyen, David H.; Ouyang, Haoxu; Mao, Jian-Hua; Hlatky, Lynn; Barcellos-Hoff, M. H.

    2014-12-01

    Age and physiologic status, such as menopause, are risk factors for breast cancer. Less clear is what factors influence the diversity of breast cancer. In this study, we investigated the effect of host age on the distribution of tumor subtypes in mouse mammary chimera consisting of wild-type hosts and Trp53 nullizygous epithelium, which undergoes a high rate of neoplastic transformation. Wild-type mammary glands cleared of endogenous epithelium at 3 weeks of age were subsequently transplanted during puberty (5 weeks) or at maturation (10 weeks) with syngeneic Trp53-null mammary tissue fragments and monitored for one year. Tumors arose sooner from adultmore » hosts (AH) compared with juvenile hosts (JH). However, compared with AH tumors, JH tumors grew several times faster, were more perfused, exhibited a two-fold higher mitotic index, and were more highly positive for insulin-like growth factor receptor phosphorylation. Most tumors in each setting were estrogen receptor (ER)-positive (80% JH vs. 70% AH), but JH tumors were significantly more ER-immunoreactive (P = 0.0001) than AH tumors. A differential expression signature (JvA) of juvenile versus adult tumors revealed a luminal transcriptional program. Centroids of the human homologs of JvA genes showed that JH tumors were more like luminal A tumors and AH tumors were more like luminal B tumors. Hierarchical clustering with the JvA human ortholog gene list segregated luminal A and luminal B breast cancers across datasets. Lastly, these data support the notion that age-associated host physiology greatly influences the intrinsic subtype of breast cancer.« less

  15. ER and PR signaling nodes during mammary gland development

    PubMed Central

    2012-01-01

    The ovarian hormones estrogen and progesterone orchestrate postnatal mammary gland development and are implicated in breast cancer. Most of our understanding of the molecular mechanisms of estrogen receptor (ER) and progesterone receptor (PR) signaling stems from in vitro studies with hormone receptor-positive cell lines. They have shown that ER and PR regulate gene transcription either by binding to DNA response elements directly or via other transcription factors and recruiting co-regulators. In addition they cross-talk with other signaling pathways through nongenomic mechanisms. Mouse genetics combined with tissue recombination techniques have provided insights about the action of these two hormones in vivo. It has emerged that hormones act on a subset of mammary epithelial cells and relegate biological functions to paracrine factors. With regards to hormonal signaling in breast carcinomas, global gene expression analyses have led to the identification of gene expression signatures that are characteristic of ERα-positive tumors that have stipulated functional studies of hitherto poorly understood transcription factors. Here, we highlight what has been learned about ER and PR signaling nodes in these different systems and attempt to lay out in which way the insights may converge. PMID:22809143

  16. Transcriptome analysis of mammary epithelial subpopulations identifies novel determinants of lineage commitment and cell fate

    PubMed Central

    Kendrick, Howard; Regan, Joseph L; Magnay, Fiona-Ann; Grigoriadis, Anita; Mitsopoulos, Costas; Zvelebil, Marketa; Smalley, Matthew J

    2008-01-01

    Background Understanding the molecular control of cell lineages and fate determination in complex tissues is key to not only understanding the developmental biology and cellular homeostasis of such tissues but also for our understanding and interpretation of the molecular pathology of diseases such as cancer. The prerequisite for such an understanding is detailed knowledge of the cell types that make up such tissues, including their comprehensive molecular characterisation. In the mammary epithelium, the bulk of the tissue is composed of three cell lineages, namely the basal/myoepithelial, luminal epithelial estrogen receptor positive and luminal epithelial estrogen receptor negative cells. However, a detailed molecular characterisation of the transcriptomic differences between these three populations has not been carried out. Results A whole transcriptome analysis of basal/myoepithelial cells, luminal estrogen receptor negative cells and luminal estrogen receptor positive cells isolated from the virgin mouse mammary epithelium identified 861, 326 and 488 genes as highly differentially expressed in the three cell types, respectively. Network analysis of the transcriptomic data identified a subpopulation of luminal estrogen receptor negative cells with a novel potential role as non-professional immune cells. Analysis of the data for potential paracrine interacting factors showed that the basal/myoepithelial cells, remarkably, expressed over twice as many ligands and cell surface receptors as the other two populations combined. A number of transcriptional regulators were also identified that were differentially expressed between the cell lineages. One of these, Sox6, was specifically expressed in luminal estrogen receptor negative cells and functional assays confirmed that it maintained mammary epithelial cells in a differentiated luminal cell lineage. Conclusion The mouse mammary epithelium is composed of three main cell types with distinct gene expression patterns

  17. Dmp1α Inhibits HER2/neu-Induced Mammary Tumorigenesis

    PubMed Central

    Fry, Elizabeth A.; Taneja, Pankaj; Maglic, Dejan; Zhu, Sinan; Sui, Guangchao; Inoue, Kazushi

    2013-01-01

    Our recent study shows a pivotal role of Dmp1 in quenching hyperproliferative signals from HER2 to the Arf-p53 pathway as a safety mechanism to prevent breast carcinogenesis. To directly demonstrate the role of Dmp1 in preventing HER2/neu-driven oncogenic transformation, we established Flag-Dmp1α transgenic mice (MDTG) under the control of the mouse mammary tumor virus (MMTV) promoter. The mice were viable but exhibited poorly developed mammary glands with markedly reduced milk production; thus more than half of parous females were unable to support the lives of new born pups. The mammary glands of the MDTG mice had very low Ki-67 expression but high levels of Arf, Ink4a, p53, and p21Cip1, markers of senescence and accelerated aging. In all strains of generated MDTG;neu mice, tumor development was significantly delayed with decreased tumor weight. Tumors from MDTG;neu mice expressed Flag-Dmp1α and Ki-67 in a mutually exclusive fashion indicating that transgenic Dmp1α prevented tumor growth in vivo. Genomic DNA analyses showed that the Dmp1α transgene was partially lost in half of the MDTG;neu tumors, and Western blot analyses showed Dmp1α protein downregulation in 80% of the cases. Our data demonstrate critical roles of Dmp1 in preventing mammary tumorigenesis and raise the possibility of treating breast cancer by restoring Dmp1α expression. PMID:24205004

  18. The Construction and Expression of Lysine-Rich Gene in the Mammary Gland of Transgenic Mice

    PubMed Central

    Ma, Xin; Zhang, Peng; Song, Guangqi; Chen, Yue; Wang, Zhongwei; Yin, Yupeng; Kong, Delong; Zhang, Sheng; Zhao, Zhihui; Ouyang, Hongsheng

    2012-01-01

    Lysine is the limiting amino acid in cereal grains, which represent a major source of human food and animal feed worldwide, and is considered the most important of the essential amino acids. In this study, β-casein, αS2-casein, and lactotransferrin cDNA clone fragments encoding lysine-rich peptides were fused together to generate a lysine-rich (LR) gene and the mammary gland-specific expression vector pBC1-LR-NEOr was constructed. Transgenic mice were generated by pronuclear microinjection of the linearized expression vectors harboring the LR transgene. The transgenic mice and their offspring were examined using multiplex polymerase chain reaction (PCR), Southern blotting, reverse transcriptase–PCR, in situ hybridization, and Western blotting techniques. Our results showed that the LR gene was successfully integrated into the mouse genome and was transmitted stably. The specific LR gene expression was restricted to the mammary gland, active alveoli of the transgenic female mice during lactation. The lysine level of the two transgenic lines was significantly higher than that of nontransgenic controls (p<0.05). In addition, the growth performance of transgenic pups was enhanced by directly feeding them the LR protein-enriched transgenic milk. Our results demonstrated that lysine-rich gene was successfully constructed and expressed in mammary gland of transgenic mice. This study will provide a better understanding of how mammary gland expression systems that increase the lysine content of milk can be applied to other mammals, such as cows. PMID:22577831

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

    PubMed

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

    2015-02-01

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

  20. RANKL/RANK control Brca1 mutation-driven mammary tumors.

    PubMed

    Sigl, Verena; Owusu-Boaitey, Kwadwo; Joshi, Purna A; Kavirayani, Anoop; Wirnsberger, Gerald; Novatchkova, Maria; Kozieradzki, Ivona; Schramek, Daniel; Edokobi, Nnamdi; Hersl, Jerome; Sampson, Aishia; Odai-Afotey, Ashley; Lazaro, Conxi; Gonzalez-Suarez, Eva; Pujana, Miguel A; Cimba, For; Heyn, Holger; Vidal, Enrique; Cruickshank, Jennifer; Berman, Hal; Sarao, Renu; Ticevic, Melita; Uribesalgo, Iris; Tortola, Luigi; Rao, Shuan; Tan, Yen; Pfeiler, Georg; Lee, Eva Yhp; Bago-Horvath, Zsuzsanna; Kenner, Lukas; Popper, Helmuth; Singer, Christian; Khokha, Rama; Jones, Laundette P; Penninger, Josef M

    2016-07-01

    Breast cancer is the most common female cancer, affecting approximately one in eight women during their life-time. Besides environmental triggers and hormones, inherited mutations in the breast cancer 1 (BRCA1) or BRCA2 genes markedly increase the risk for the development of breast cancer. Here, using two different mouse models, we show that genetic inactivation of the key osteoclast differentiation factor RANK in the mammary epithelium markedly delayed onset, reduced incidence, and attenuated progression of Brca1;p53 mutation-driven mammary cancer. Long-term pharmacological inhibition of the RANK ligand RANKL in mice abolished the occurrence of Brca1 mutation-driven pre-neoplastic lesions. Mechanistically, genetic inactivation of Rank or RANKL/RANK blockade impaired proliferation and expansion of both murine Brca1;p53 mutant mammary stem cells and mammary progenitors from human BRCA1 mutation carriers. In addition, genome variations within the RANK locus were significantly associated with risk of developing breast cancer in women with BRCA1 mutations. Thus, RANKL/RANK control progenitor cell expansion and tumorigenesis in inherited breast cancer. These results present a viable strategy for the possible prevention of breast cancer in BRCA1 mutant patients. PMID:27241552

  1. Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein Id-1

    SciTech Connect

    Desprez, P.; Hara, E.; Bissell, M.J.

    1995-06-01

    Cell proliferation and differentiation are precisely coordinated during the development and maturation of the mammary gland, and this balance invariably is disrupted during carcinogenesis. Little is known about the cell-specific transcription factors that regulate these processes in the mammary gland. The mouse mammary epithelial cell line SCp2 grows well under standard culture conditions but arrests growth, forms alveolus-like structures, and expresses {beta}-casein, a differentiation marker, 4 to 5 days after exposure to basement membrane and lactogenic hormones (differentiation signals). The authors show that this differentiation entails a marked decline in the expression of Id-1, a helix-loop-helix (HLH) protein that inactivates basic HLH transcription factors in other cell types. SCp2 cells stably transfected with an Id-1 expression vector grew more rapidly than control cells under standard conditions, but in response to differentiation signals, they lost three-dimensional organization, invaded the basement membrane, and then resumed growth. SCp2 cells expressing an Id-1 antisense vector grew more slowly than controls; in response to differentiation signals, they remained stably growth arrested and fully differentiated, as did control cells. The authors suggest that Id-1 renders cells refractory to differentiation signals and receptive to growth signals by inactivating one or more basic HLH proteins that coordinate growth and differentiation in the mammary epithelium. 53 refs., 6 figs.

  2. Breast cancer protein PS2 synthesis in mammary gland of transgenic mice and secretion into milk.

    PubMed

    Tomasetto, C; Wolf, C; Rio, M C; Mehtali, M; LeMeur, M; Gerlinger, P; Chambon, P; Lathe, R

    1989-10-01

    PS2, a small estrogen-inducible secretory polypeptide with structural analogies to a growth factor, is produced by approximately 50% of human breast tumors. The function of PS2 is, however, unknown. To determine whether PS2 may play an autocrine role in the development of mammary tumors we constructed transgenic mice bearing fusion constructs designed to direct the expression of human PS2 in the lactating mammary gland under the control of the whey acidic protein (WAP) promoter. Mouse lines bearing the genomic PS2 gene under the control of the WAP promoter region (WAP-PS2-2) failed to express the transgene. However, mice harboring the fusion construct WAP-PS2-1, in which the PS2 coding sequence is inserted into the 5' untranslated region of the complete WAP gene, were observed to express the transgene. Expression was restricted to the secretory epithelium of the mammary gland during lactation, and PS2 protein was secreted into the milk. Nevertheless, no mammary gland dysplasia was observed, and PS2 expression had no discernable effect upon the physiology and/or development of the suckling young or the transgenic mother. PMID:2481815

  3. RANKL/RANK control Brca1 mutation-driven mammary tumors.

    PubMed

    Sigl, Verena; Owusu-Boaitey, Kwadwo; Joshi, Purna A; Kavirayani, Anoop; Wirnsberger, Gerald; Novatchkova, Maria; Kozieradzki, Ivona; Schramek, Daniel; Edokobi, Nnamdi; Hersl, Jerome; Sampson, Aishia; Odai-Afotey, Ashley; Lazaro, Conxi; Gonzalez-Suarez, Eva; Pujana, Miguel A; Cimba, For; Heyn, Holger; Vidal, Enrique; Cruickshank, Jennifer; Berman, Hal; Sarao, Renu; Ticevic, Melita; Uribesalgo, Iris; Tortola, Luigi; Rao, Shuan; Tan, Yen; Pfeiler, Georg; Lee, Eva Yhp; Bago-Horvath, Zsuzsanna; Kenner, Lukas; Popper, Helmuth; Singer, Christian; Khokha, Rama; Jones, Laundette P; Penninger, Josef M

    2016-07-01

    Breast cancer is the most common female cancer, affecting approximately one in eight women during their life-time. Besides environmental triggers and hormones, inherited mutations in the breast cancer 1 (BRCA1) or BRCA2 genes markedly increase the risk for the development of breast cancer. Here, using two different mouse models, we show that genetic inactivation of the key osteoclast differentiation factor RANK in the mammary epithelium markedly delayed onset, reduced incidence, and attenuated progression of Brca1;p53 mutation-driven mammary cancer. Long-term pharmacological inhibition of the RANK ligand RANKL in mice abolished the occurrence of Brca1 mutation-driven pre-neoplastic lesions. Mechanistically, genetic inactivation of Rank or RANKL/RANK blockade impaired proliferation and expansion of both murine Brca1;p53 mutant mammary stem cells and mammary progenitors from human BRCA1 mutation carriers. In addition, genome variations within the RANK locus were significantly associated with risk of developing breast cancer in women with BRCA1 mutations. Thus, RANKL/RANK control progenitor cell expansion and tumorigenesis in inherited breast cancer. These results present a viable strategy for the possible prevention of breast cancer in BRCA1 mutant patients.

  4. Oncogenic AKT1(E17K) mutation induces mammary hyperplasia but prevents HER2-driven tumorigenesis

    PubMed Central

    Mancini, Maria L.; Lien, Evan C.; Toker, Alex

    2016-01-01

    One of the most frequently deregulated signaling pathways in breast cancer is the PI 3-K/Akt cascade. Genetic lesions are commonly found in PIK3CA, PTEN, and AKT, which lead to excessive and constitutive activation of Akt and downstream signaling that results in uncontrolled proliferation and increased cellular survival. One such genetic lesion is the somatic AKT1(E17K) mutation, which has been identified in 4-8% of breast cancer patients. To determine how this mutation contributes to mammary tumorigenesis, we constructed a genetically engineered mouse model that conditionally expresses human AKT1(E17K) in the mammary epithelium. Although AKT1(E17K) is only weakly constitutively active and does not promote proliferation in vitro, it is capable of escaping negative feedback inhibition to exhibit sustained signaling dynamics in vitro. Consistently, both virgin and multiparous AKT1(E17K) mice develop mammary gland hyperplasia that do not progress to carcinoma. This hyperplasia is accompanied by increased estrogen receptor expression, although exposure of the mice to estrogen does not promote tumor development. Moreover, AKT1(E17K) prevents HER2-driven mammary tumor formation, in part through negative feedback inhibition of RTK signaling. Analysis of TCGA breast cancer data revealed that the mRNA expression, total protein levels, and phosphorylation of various RTKs are decreased in human tumors harboring AKT1(E17K). PMID:27004402

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

    PubMed

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

    2007-06-01

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

  6. Inactivation of RARβ inhibits Wnt1-induced mammary tumorigenesis by suppressing epithelial-mesenchymal transition

    PubMed Central

    Liu, Xingxing; Giguère, Vincent

    2014-01-01

    Retinoic acid receptor β (RARβ) has been proposed to act as a tumor suppressor in breast cancer. In contrast, recent data have shown that RARβ promotes ERBB2-induced mammary gland tumorigenesis through remodeling of the stromal compartment and activation of cancer-associated fibroblasts. However, it is currently unknown whether RARβ oncogenic activity is specific to ERBB2-induced tumors, or whether it influences the initiation and progression of other breast cancer subtypes. Accordingly, we set out to investigate the involvement of RARβ in basal-like breast cancer using mouse mammary tumor virus (MMTV)-wingless-related integration site 1 (Wnt1)-induced mammary gland tumorigenesis as a model system. We found that compared with wild type mice, inactivation of Rarb resulted in a lengthy delay in Wnt1-induced mammary gland tumorigenesis and in a significantly slower tumor growth rate. Ablation of Rarb altered the composition of the stroma, repressed the activation of cancer-associated fibroblasts, and reduced the recruitment of inflammatory cells and angiogenesis. Reduced expression of IGF-1 and activity of its downstream signaling pathway contribute to attenuate EMT in the Rarb-null tumors. Our results show that, in the absence of retinoid signaling via RARβ, reduced IGF-1 signaling results in suppression of epithelial-mesenchymal transition and delays tumorigenesis induced by the Wnt1 oncogene. Accordingly, our work reinforces the concept that antagonizing RARβ-dependent retinoid signaling could provide a therapeutic avenue to treat poor outcome breast cancers. PMID:25422594

  7. The mammary cellular hierarchy and breast cancer.

    PubMed

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

    2014-11-01

    Advances in the study of hematopoietic cell maturation have paved the way to a deeper understanding the stem and progenitor cellular hierarchy in the mammary gland. The mammary epithelium, unlike the hematopoietic cellular hierarchy, sits in a complex niche where communication between epithelial cells and signals from the systemic hormonal milieu, as well as from extra-cellular matrix, influence cell fate decisions and contribute to tissue homeostasis. We review the discovery, definition and regulation of the mammary cellular hierarchy and we describe the development of the concepts that have guided our investigations. We outline recent advances in in vivo lineage tracing that is now challenging many of our assumptions regarding the behavior of mammary stem cells, and we show how understanding these cellular lineages has altered our view of breast cancer.

  8. Gordon Research Conference on Mammary Gland Biology

    SciTech Connect

    Not Available

    1989-01-01

    The 1989 conference was the tenth in the series of biennial Gordon Research Conferences on Mammary Gland Biology. Traditionally this conference brings together scientists from diverse backgrounds and experience but with a common interest in the biology of the mammary gland. Investigators from agricultural and medical schools, biochemists, cell and molecular biologists, endocrinologists, immunologists, and representatives from the emerging biotechnology industries met to discuss current concepts and results on the function and regulation of the normal and neoplastic mammary gland in a variety of species. Of the participants, approximately three-fourths were engaged in studying the normal mammary gland function, whereas the other quarter were engaged in studying the neoplastic gland. The interactions between scientists, clinicians, veterinarians examining both normal and neoplastic cell function serves to foster the multi-disciplinary goals of the conference and has stimulated many cooperative projects among participants in previous years.

  9. Mammary gland: From embryogenesis to adult life.

    PubMed

    Musumeci, Giuseppe; Castrogiovanni, Paola; Szychlinska, Marta Anna; Aiello, Flavia Concetta; Vecchio, Giada Maria; Salvatorelli, Lucia; Magro, Gaetano; Imbesi, Rosa

    2015-01-01

    The aim of this review is to focus on the molecular factors that ensure the optimal development and maintenance of the mammary gland thanks to their integration and coordination. The development of the mammary gland is supported, not only by endocrine signals, but also by regulatory molecules, which are able to integrate signals from the surrounding microenvironment. A major role is certainly played by homeotic genes, but their incorrect expression during the spatiotemporal regulation of proliferative, functional and differentiation cycles of the mammary gland, may result in the onset of neoplastic processes. Attention is directed also to the endocrine aspects and sexual dimorphism of mammary gland development, as well as the role played by ovarian steroids and their receptors in adult life.

  10. Genetic component in rat mammary carcinogenesis

    SciTech Connect

    Vogel, H.J. Jr.; Turner, J.E.

    1982-02-01

    Five genetically defined strains of female rats were exposed to whole-body radiation with a single dose of 50 rad of fission neutrons. After 1 year 56% of the Long-Evans and Sprague-Dawely strains showed at least one mammary tumor; 25-29% of the Buffalo and Fischer-344 strains and only 5% of the Wistar-Lewis strain and palpable mammary tumors. Only one tumor was found among 73 unirradiated controls during the 1-year observation period. Approximately two-thirds of 50 mammary analyzed pathologically were adenofibromas and fibroadenomas; one-third were adenocarcinomas. The Kaplan-Meier method of life table analysis was used to deal with the problem of intercurrent mortality. A genetic factor seems evident in rat mammary tumorigenesis following exposure to fission neutrons.

  11. Mammary Adipose Tissue-Derived Lysophospholipids Promote Estrogen Receptor-Negative Mammary Epithelial Cell Proliferation.

    PubMed

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

    2016-05-01

    Lysophosphatidic acid (LPA), acting in an autocrine or paracrine fashion through G protein-coupled receptors, has been implicated in many physiologic and pathologic processes, including cancer. LPA is converted from lysophosphatidylcholine (LPC) by the secreted phospholipase autotaxin (ATX). Although various cell types can produce ATX, adipocyte-derived ATX is believed to be the major source of circulating ATX and also to be the major regulator of plasma LPA levels. In addition to ATX, adipocytes secrete numerous other factors (adipokines); although several adipokines have been implicated in breast cancer biology, the contribution of mammary adipose tissue-derived LPC/ATX/LPA (LPA axis) signaling to breast cancer is poorly understood. Using murine mammary fat-conditioned medium, we investigated the contribution of LPA signaling to mammary epithelial cancer cell biology and identified LPA signaling as a significant contributor to the oncogenic effects of the mammary adipose tissue secretome. To interrogate the role of mammary fat in the LPA axis during breast cancer progression, we exposed mammary adipose tissue to secreted factors from estrogen receptor-negative mammary epithelial cell lines and monitored changes in the mammary fat pad LPA axis. Our data indicate that bidirectional interactions between mammary cancer cells and mammary adipocytes alter the local LPA axis and increase ATX expression in the mammary fat pad during breast cancer progression. Thus, the LPC/ATX/LPA axis may be a useful target for prevention in patients at risk of ER-negative breast cancer. Cancer Prev Res; 9(5); 367-78. ©2016 AACR. PMID:26862086

  12. Kinase-independent role of cyclin D1 in chromosomal instability and mammary tumorigenesis.

    PubMed

    Casimiro, Mathew C; Di Sante, Gabriele; Crosariol, Marco; Loro, Emanuele; Dampier, William; Ertel, Adam; Yu, Zuoren; Saria, Elizabeth A; Papanikolaou, Alexandros; Li, Zhiping; Wang, Chenguang; Addya, Sankar; Lisanti, Michael P; Fortina, Paolo; Cardiff, Robert D; Tozeren, Aydin; Knudsen, Erik S; Arnold, Andrew; Pestell, Richard G

    2015-04-20

    Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1(-/-) mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1(WT) or cyclin D1(KE) in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis. PMID:25940700

  13. Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss.

    PubMed

    Arun, Gayatri; Diermeier, Sarah; Akerman, Martin; Chang, Kung-Chi; Wilkinson, J Erby; Hearn, Stephen; Kim, Youngsoo; MacLeod, A Robert; Krainer, Adrian R; Norton, Larry; Brogi, Edi; Egeblad, Mikala; Spector, David L

    2016-01-01

    Genome-wide analyses have identified thousands of long noncoding RNAs (lncRNAs). Malat1 (metastasis-associated lung adenocarcinoma transcript 1) is among the most abundant lncRNAs whose expression is altered in numerous cancers. Here we report that genetic loss or systemic knockdown of Malat1 using antisense oligonucleotides (ASOs) in the MMTV (mouse mammary tumor virus)-PyMT mouse mammary carcinoma model results in slower tumor growth accompanied by significant differentiation into cystic tumors and a reduction in metastasis. Furthermore, Malat1 loss results in a reduction of branching morphogenesis in MMTV-PyMT- and Her2/neu-amplified tumor organoids, increased cell adhesion, and loss of migration. At the molecular level, Malat1 knockdown results in alterations in gene expression and changes in splicing patterns of genes involved in differentiation and protumorigenic signaling pathways. Together, these data demonstrate for the first time a functional role of Malat1 in regulating critical processes in mammary cancer pathogenesis. Thus, Malat1 represents an exciting therapeutic target, and Malat1 ASOs represent a potential therapy for inhibiting breast cancer progression.

  14. Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss

    PubMed Central

    Arun, Gayatri; Diermeier, Sarah; Akerman, Martin; Chang, Kung-Chi; Wilkinson, J. Erby; Hearn, Stephen; Kim, Youngsoo; MacLeod, A. Robert; Krainer, Adrian R.; Norton, Larry; Brogi, Edi; Egeblad, Mikala; Spector, David L.

    2016-01-01

    Genome-wide analyses have identified thousands of long noncoding RNAs (lncRNAs). Malat1 (metastasis-associated lung adenocarcinoma transcript 1) is among the most abundant lncRNAs whose expression is altered in numerous cancers. Here we report that genetic loss or systemic knockdown of Malat1 using antisense oligonucleotides (ASOs) in the MMTV (mouse mammary tumor virus)-PyMT mouse mammary carcinoma model results in slower tumor growth accompanied by significant differentiation into cystic tumors and a reduction in metastasis. Furthermore, Malat1 loss results in a reduction of branching morphogenesis in MMTV-PyMT- and Her2/neu-amplified tumor organoids, increased cell adhesion, and loss of migration. At the molecular level, Malat1 knockdown results in alterations in gene expression and changes in splicing patterns of genes involved in differentiation and protumorigenic signaling pathways. Together, these data demonstrate for the first time a functional role of Malat1 in regulating critical processes in mammary cancer pathogenesis. Thus, Malat1 represents an exciting therapeutic target, and Malat1 ASOs represent a potential therapy for inhibiting breast cancer progression. PMID:26701265

  15. Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss.

    PubMed

    Arun, Gayatri; Diermeier, Sarah; Akerman, Martin; Chang, Kung-Chi; Wilkinson, J Erby; Hearn, Stephen; Kim, Youngsoo; MacLeod, A Robert; Krainer, Adrian R; Norton, Larry; Brogi, Edi; Egeblad, Mikala; Spector, David L

    2016-01-01

    Genome-wide analyses have identified thousands of long noncoding RNAs (lncRNAs). Malat1 (metastasis-associated lung adenocarcinoma transcript 1) is among the most abundant lncRNAs whose expression is altered in numerous cancers. Here we report that genetic loss or systemic knockdown of Malat1 using antisense oligonucleotides (ASOs) in the MMTV (mouse mammary tumor virus)-PyMT mouse mammary carcinoma model results in slower tumor growth accompanied by significant differentiation into cystic tumors and a reduction in metastasis. Furthermore, Malat1 loss results in a reduction of branching morphogenesis in MMTV-PyMT- and Her2/neu-amplified tumor organoids, increased cell adhesion, and loss of migration. At the molecular level, Malat1 knockdown results in alterations in gene expression and changes in splicing patterns of genes involved in differentiation and protumorigenic signaling pathways. Together, these data demonstrate for the first time a functional role of Malat1 in regulating critical processes in mammary cancer pathogenesis. Thus, Malat1 represents an exciting therapeutic target, and Malat1 ASOs represent a potential therapy for inhibiting breast cancer progression. PMID:26701265

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. 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.

  18. Mammary Fat Can Adjust Prolactin Effect on Mammary Epithelial Cells via Leptin and Estrogen.

    PubMed

    Feuermann, Yonatan; Mabjeesh, Sameer J; Shamay, Avi

    2009-01-01

    Leptin, like estrogen, is one of the endo/paracrine factors, which are synthesized in and secreted from mature adipocytes. The roles of the mammary fat pad and mammary adipocytes in the initiation of lactation are not clear. In this study, we showed that combination of prolactin, leptin and estrogen elevated the expression of the milk protein beta-lactoglobulin. We also showed that after prolactin stimulate the secretion of leptin from the mammary fat, leptin upregulated the expression of estrogen receptor alpha in the mammary epithelial cells. Also, prolactin affected aromatase mRNA expression in the bovine mammary fat and we demonstrated that leptin and prolactin can affect cholesterol secretion from explants in culture to the medium. Therefore, we suggest that prolactin initiates estrogen expression (as represented by aromatase mRNA) in the mammary fat pad, whereas leptin stimulates estrogen receptor alpha expression in the mammary epithelial cells. We hypothesize that leptin and estrogen, secreted from the mammary fat regulate lactation after stimulation of prolactin. PMID:20049155

  19. A role for interleukin-1 alpha in the 1,25 dihydroxyvitamin D3 response in mammary epithelial cells.

    PubMed

    Maund, Sophia L; Shi, Lihong; Cramer, Scott D

    2013-01-01

    Breast cancer is the most common non-cutaneous malignancy in American women, and better preventative strategies are needed. Epidemiological and laboratory studies point to vitamin D3 as a promising chemopreventative agent for breast cancer. Vitamin D3 metabolites induce anti-proliferative effects in breast cancer cells in vitro and in vivo, but few studies have investigated their effects in normal mammary epithelial cells. We hypothesized that 1,25(OH)2D3, the metabolically active form of vitamin D3, is growth suppressive in normal mouse mammary epithelial cells. In addition, we have previously established a role for the cytokine interleukin-1 alpha (IL1α) in the anti-proliferative effects of 1,25(OH)2D3 in normal prostate cells, and so we hypothesized that IL1α is involved in the 1,25(OH)2D3 response in mammary cells. Evaluation of cell viability, clonogenicity, senescence, and induction of cell cycle regulators p21 and p27 supported an anti-proliferative role for 1,25(OH)2D3 in mammary epithelial cells. Furthermore, 1,25(OH)2D3 increased the intracellular expression of IL1α, which was necessary for the anti-proliferative effects of 1,25(OH)2D3 in mammary cells. Together, these findings support the chemopreventative potential of vitamin D3 in the mammary gland and present a role for IL1α in regulation of mammary cell proliferation by 1,25(OH)2D3.

  20. A Role for Interleukin-1 Alpha in the 1,25 Dihydroxyvitamin D3 Response in Mammary Epithelial Cells

    PubMed Central

    Maund, Sophia L.; Shi, Lihong; Cramer, Scott D.

    2013-01-01

    Breast cancer is the most common non-cutaneous malignancy in American women, and better preventative strategies are needed. Epidemiological and laboratory studies point to vitamin D3 as a promising chemopreventative agent for breast cancer. Vitamin D3 metabolites induce anti-proliferative effects in breast cancer cells in vitro and in vivo, but few studies have investigated their effects in normal mammary epithelial cells. We hypothesized that 1,25(OH)2D3, the metabolically active form of vitamin D3, is growth suppressive in normal mouse mammary epithelial cells. In addition, we have previously established a role for the cytokine interleukin-1 alpha (IL1α) in the anti-proliferative effects of 1,25(OH)2D3 in normal prostate cells, and so we hypothesized that IL1α is involved in the 1,25(OH)2D3 response in mammary cells. Evaluation of cell viability, clonogenicity, senescence, and induction of cell cycle regulators p21 and p27 supported an anti-proliferative role for 1,25(OH)2D3 in mammary epithelial cells. Furthermore, 1,25(OH)2D3 increased the intracellular expression of IL1α, which was necessary for the anti-proliferative effects of 1,25(OH)2D3 in mammary cells. Together, these findings support the chemopreventative potential of vitamin D3 in the mammary gland and present a role for IL1α in regulation of mammary cell proliferation by 1,25(OH)2D3. PMID:24244740

  1. ZnT2 is a critical mediator of lysosomal-mediated cell death during early mammary gland involution

    PubMed Central

    Hennigar, Stephen R.; Seo, Young Ah; Sharma, Supriya; Soybel, David I.; Kelleher, Shannon L.

    2015-01-01

    Mammary gland involution is the most dramatic example of physiological cell death. It occurs through an initial phase of lysosomal-mediated cell death (LCD) followed by mitochondrial-mediated apoptosis. Zinc (Zn) activates both LCD and apoptosis in vitro. The Zn transporter ZnT2 imports Zn into vesicles and mitochondria and ZnT2-overexpression activates cell death in mammary epithelial cells (MECs). We tested the hypothesis that ZnT2-mediated Zn transport is critical for mammary gland involution in mice. Following weaning, ZnT2 abundance increased in lysosomes and mitochondria, which paralleled Zn accumulation in each of these organelles. Adenoviral expression of ZnT2 in lactating mouse mammary glands in vivo increased Zn in lysosomes and mitochondria and activated LCD and apoptosis, promoting a profound reduction in MECs and alveoli. Injection of TNFα, a potent activator of early involution, into the mammary gland fat pads of lactating mice increased ZnT2 and Zn in lysosomes and activated premature involution. Exposure of cultured MECs to TNFα redistributed ZnT2 to lysosomes and increased lysosomal Zn, which activated lysosomal swelling, cathepsin B release, and LCD. Our data implicate ZnT2 as a critical mediator of cell death during involution and importantly, that as an initial involution signal, TNFα redistributes ZnT2 to lysosomes to activate LCD. PMID:25620235

  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. Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk

    PubMed Central

    Wagner, Stefan; Thresher, Rosemary; Bland, Ross; Laible, Götz

    2015-01-01

    Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland. PMID:26463440

  4. Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk.

    PubMed

    Wagner, Stefan; Thresher, Rosemary; Bland, Ross; Laible, Götz

    2015-01-01

    Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland. PMID:26463440

  5. Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk.

    PubMed

    Wagner, Stefan; Thresher, Rosemary; Bland, Ross; Laible, Götz

    2015-10-14

    Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland.

  6. The nipple: a simple intersection of mammary gland and integument, but focal point of organ function.

    PubMed

    Koyama, Sachiko; Wu, Hsin-Jung; Easwaran, Teresa; Thopady, Sunil; Foley, John

    2013-06-01

    Having glands that secrete milk to nourish neonatal offspring characterizes all mammals. We provide a brief overview of the development and anatomy of nipples and mammary glands in monotremes, marsupials, and marine mammals, and focus on the nipples and mammary glands in terrestrial eutherian species. We first classify eutherians into three groups: the altricial, precocial, and arboreal types based on their rearing system. We then summarize the physiology of lactation and the cell biology of nipples with specific focus on comparing these in the mouse, cow, and human, which represent the three different groups. Finally we propose that the nipple is an example of specialized epidermis. As specialized epidermis, it is dependent the underlying stroma for development and maintenance in adult life. The development of the nipple and signaling pathways that regulate its formation are described. PMID:23674217

  7. The nipple: a simple intersection of mammary gland and integument, but focal point of organ function.

    PubMed

    Koyama, Sachiko; Wu, Hsin-Jung; Easwaran, Teresa; Thopady, Sunil; Foley, John

    2013-06-01

    Having glands that secrete milk to nourish neonatal offspring characterizes all mammals. We provide a brief overview of the development and anatomy of nipples and mammary glands in monotremes, marsupials, and marine mammals, and focus on the nipples and mammary glands in terrestrial eutherian species. We first classify eutherians into three groups: the altricial, precocial, and arboreal types based on their rearing system. We then summarize the physiology of lactation and the cell biology of nipples with specific focus on comparing these in the mouse, cow, and human, which represent the three different groups. Finally we propose that the nipple is an example of specialized epidermis. As specialized epidermis, it is dependent the underlying stroma for development and maintenance in adult life. The development of the nipple and signaling pathways that regulate its formation are described.

  8. Extracellular matrix proteins regulate epithelial-mesenchymal transition in mammary epithelial cells

    PubMed Central

    Chen, Qike K.; Lee, KangAe; Radisky, Derek C.; Nelson, Celeste M.

    2013-01-01

    Mouse mammary epithelial cells undergo transdifferentiation via epithelial-mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland. PMID:23660532

  9. Prevalence of Glomerulopathies in Canine Mammary Carcinoma

    PubMed Central

    2016-01-01

    The incidence and prevalence of paraneoplastic glomerulopathy, especially associated with carcinoma, are a matter of debate and the causal link between cancer and glomerular diseases remains unclear. The aim of this study was to evaluate renal biopsies of selected bitches with spontaneous mammary gland carcinoma. We hypothesized that dogs with mammary carcinomas would show histologic evidence of glomerular pathology. A prospective study was performed in dogs with naturally occurring mammary carcinoma that were undergoing tumor resection and ovariohysterectomy. We evaluated renal biopsies of 32 bitches with spontaneous mammary gland carcinoma and 11 control dogs without mammary gland neoplasia. Samples were obtained from the left kidney and the biopsy material was divided for light microscopy (LM), immunofluorescence (IF) and transmission electron microscopy (TEM). Light microscopy abnormalities were identified in 78.1% of dogs with mammary carcinoma (n = 25) and in none of the dogs in the control group. Focal glomerular mesangial matrix expansion was the most common alteration (n = 15, 60.0%), but mesangial cell proliferation (n = 9, 36.0%) and focal segmental glomerulosclerosis (n = 9, 36.0%), synechiae (n = 7, 28.0%), and globally sclerotic glomeruli (n = 6, 24.0%) were also frequent in dogs with malignancy. Immunofluorescence microscopy revealed strong IgM staining was demonstrated in 64.3% (n = 18) of carcinoma dogs. Transmission electron microscopy from dogs with carcinoma revealed slight changes, the most frequent of which was faint sub-endothelial and mesangial deposits of electron-dense material (78%). Mesangial cell interpositioning and segmental effacement of podocyte foot processes were identified in some specimens (45%). Changes in the glomerulus and proteinuria are common in dogs with naturally occurring mammary carcinoma and this condition appears to provide an excellent large animal model for cancer-associated glomerulopathy in humans. PMID:27764139

  10. Lipopolysaccharide disrupts the milk-blood barrier by modulating claudins in mammary alveolar tight junctions.

    PubMed

    Kobayashi, Ken; Oyama, Shoko; Numata, Atsushi; Rahman, Md Morshedur; Kumura, Haruto

    2013-01-01

    Mastitis, inflammation of the mammary gland, is the most costly common disease in the dairy industry, and is caused by mammary pathogenic bacteria, including Escherichia coli. The bacteria invade the mammary alveolar lumen and disrupt the blood-milk barrier. In normal mammary gland, alveolar epithelial tight junctions (TJs) contribute the blood-milk barrier of alveolar epithelium by blocking the leakage of milk components from the luminal side into the blood serum. In this study, we focused on claudin subtypes that participate in the alveolar epithelial TJs, because the composition of claudins is an important factor that affects TJ permeability. In normal mouse lactating mammary glands, alveolar TJs consist of claudin-3 without claudin-1, -4, and -7. In lipopolysaccharide (LPS)-induced mastitis, alveolar TJs showed 2-staged compositional changes in claudins. First, a qualitative change in claudin-3, presumably caused by phosphorylation and participation of claudin-7 in alveolar TJs, was recognized in parallel with the leakage of fluorescein isothiocyanate-conjugated albumin (FITC-albumin) via the alveolar epithelium. Second, claudin-4 participated in alveolar TJs with claudin-3 and claudin-7 12 h after LPS injection. The partial localization of claudin-1 was also observed by immunostaining. Coinciding with the second change of alveolar TJs, the severe disruption of the blood-milk barrier was recognized by ectopic localization of β-casein and much leakage of FITC-albumin. Furthermore, the localization of toll-like receptor 4 (TLR4) on the luminal side and NFκB activation by LPS was observed in the alveolar epithelial cells. We suggest that the weakening and disruption of the blood-milk barrier are caused by compositional changes of claudins in alveolar epithelial TJs through LPS/TLR4 signaling.

  11. Does protein intake alter the precursors for synthesis of lactose and non-essential amino acids by the mammary glands of lactating mice?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aims were to: 1) develop a [U-13C]glucose tracer approach to establish the pathways of and substrates used for milk lactose and casein synthesis in the mouse mammary gland and 2) determine the influence of protein intake on this partition and use for milk synthesis. In Study 1, we determined th...

  12. 9 CFR 310.17 - Inspection of mammary glands.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... mammary glands and diseased mammary glands of cattle, sheep, swine, and goats shall be removed without..., swine, and goats shall not be saved for edible purposes. (d) The udders from cows officially...

  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. Cloning the laboratory mouse.

    PubMed

    Wakayama, T; Yanagimachi, R

    1999-06-01

    A brief account is given of early attempts to clone mammals (mice) by transferring cells (nuclei) of preimplantation embryos into enucleated oocytes, zygotes or blastomeres of two-cell embryos. This is followed by a brief review of recent successes using adult somatic cells: mammary gland cells for sheep, muscle cells for cattle and cumulus cells for mice. We have developed a technique for cloning the laboratory mouse by transferring cumulus cell nuclei into enucleated oocytes. With this technique, we have produced a population of over 80 cloned animals, and have carried the process over four generations. Development and fertility of these appear normal. However, the yield is very low; only approximately 1% of injected oocytes are carried to term. The challenge is now to understand the reason for this high loss. Is it a problem of technique, genomic reprogramming, somatic mutation, imprinting or incompatible cell cycle phases?

  15. Prolactin affects leptin action in the bovine mammary gland via the mammary fat pad.

    PubMed

    Feuermann, Y; Mabjeesh, S J; Niv-Spector, L; Levin, D; Shamay, A

    2006-11-01

    One of the roles of the endocrine system is to synchronize mammary function. Hormones, such as estrogen, progesterone, and prolactin act directly on the mammary gland. Metabolic hormones, such as GH, glucocorticoids, insulin, and leptin are responsible for coordinating the body's response to metabolic homeostasis. Leptin has been shown to be an important factor in regulating the metabolic adaptation of nutrient partitioning during the energy-consuming processes of lactation. In the present study, we show that leptin is secreted from the mammary fat, and is regulated by prolactin. The expression of alpha-casein in a co-culture of epithelial cells and fat explants was enhanced by prolactin compared with that in epithelial cells cultured alone. Leptin antagonist abolished the effect of leptin on alpha-casein expression in mammary gland explants when exogenous leptin was not present in the medium. This finding supports our hypothesis that the antagonist abolishes the action of endogenous leptin secreted by the mammary adipocytes. These results lead us to the hypothesis that prolactin and leptin act in the bovine mammary gland, via mammary fat pad/adipocytes. PMID:17088410

  16. The Cleared Mammary Fat Pad Transplantation Assay for Mammary Epithelial Organogenesis.

    PubMed

    Lawson, Devon A; Werb, Zena; Zong, Yang; Goldstein, Andrew S

    2015-12-01

    Cleared mammary fat pad (MFP) transplantation has been a standard technique for studies of mammary development and cancer for several decades. The mammary gland is comprised of several fundamental components: The epithelial compartment contains basal/myoepithelial cells and luminal cells, and the stromal compartment (called the MFP) contains adipocytes, smooth muscle cells, fibroblasts, and immune cells. In 3- to 4-wk-old female mice, the mammary epithelium is concentrated very close to the nipple and has not yet grown beyond the mammary lymph node to penetrate the bulk of the MFP. This developmental feature provides an anatomical fixed point, and enables one to cut away the portion of the MFP from the nipple to the lymph node, leaving behind the majority of the MFP free of epithelium. The "cleared" MFP can serve as a supportive native microenvironment fully sufficient for the organogenesis of injected donor epithelium. Normal mammary epithelial donor cells will produce histologically and functionally normal mammary ductal epithelium several weeks posttransplant, with the exception that the ducts will not be connected to the nipple. The assay described here provides a powerful platform for assessing the developmental and tumorigenic potential of engineered cells of interest. PMID:26631119

  17. The Cleared Mammary Fat Pad Transplantation Assay for Mammary Epithelial Organogenesis.

    PubMed

    Lawson, Devon A; Werb, Zena; Zong, Yang; Goldstein, Andrew S

    2015-12-02

    Cleared mammary fat pad (MFP) transplantation has been a standard technique for studies of mammary development and cancer for several decades. The mammary gland is comprised of several fundamental components: The epithelial compartment contains basal/myoepithelial cells and luminal cells, and the stromal compartment (called the MFP) contains adipocytes, smooth muscle cells, fibroblasts, and immune cells. In 3- to 4-wk-old female mice, the mammary epithelium is concentrated very close to the nipple and has not yet grown beyond the mammary lymph node to penetrate the bulk of the MFP. This developmental feature provides an anatomical fixed point, and enables one to cut away the portion of the MFP from the nipple to the lymph node, leaving behind the majority of the MFP free of epithelium. The "cleared" MFP can serve as a supportive native microenvironment fully sufficient for the organogenesis of injected donor epithelium. Normal mammary epithelial donor cells will produce histologically and functionally normal mammary ductal epithelium several weeks posttransplant, with the exception that the ducts will not be connected to the nipple. The assay described here provides a powerful platform for assessing the developmental and tumorigenic potential of engineered cells of interest.

  18. 9 CFR 310.17 - Inspection of mammary glands.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... mammary glands and diseased mammary glands of cattle, sheep, swine, and goats shall be removed without... accordance with the provisions of part 311 of this subchapter. (c) Lactating mammary glands of cattle, sheep... as “Brucellosis reactors” or as “Mastitis elimination cows” shall be condemned....

  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. 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

  1. Stromal Effects on Mammary Gland Development and Breast Cancer

    NASA Astrophysics Data System (ADS)

    Wiseman, Bryony S.; Werb, Zena

    2002-05-01

    Breast cancer manifests itself in the mammary epithelium, yet there is a growing recognition that mammary stromal cells also play an important role in tumorigenesis. During its developmental cycle, the mammary gland displays many of the properties associated with breast cancer, and many of the stromal factors necessary for mammary development also promote or protect against breast cancer. Here we review our present knowledge of the specific factors and cell types that contribute to epithelial-stromal crosstalk during mammary development. To find cures for diseases like breast cancer that rely on epithelial-stromal crosstalk, we must understand how these different cell types communicate with each other.

  2. 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.

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

    SciTech Connect

    Sharma, Rohit B.; Wang, Qingde; Khillan, Jaspal S.

    2013-07-12

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

  4. Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

    PubMed Central

    Jhappan, C; Geiser, A G; Kordon, E C; Bagheri, D; Hennighausen, L; Roberts, A B; Smith, G H; Merlino, G

    1993-01-01

    Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland. Images PMID:8491177

  5. CLINICOPATHOLOGIC FEATURES OF MAMMARY MASSES IN CAPTIVE LIONS (PANTHERA LEO).

    PubMed

    Sadler, Ryan A; Craig, Linden E; Ramsay, Edward C; Helmick, Kelly; Collins, Darin; Garner, Michael M

    2016-03-01

    A multi-institutional retrospective analysis of 330 pathology accessions from 285 different lions found 15 captive, female African lions (Panthera leo) with confirmed mammary masses. Aside from the presence of a mammary mass, the most common initial clinical sign was inappetence. Histologic diagnoses were predominantly adenocarcinoma (n = 12), though two benign masses (mammary hyperplasia and a mammary cyst) and one squamous cell carcinoma were identified. Nine of 13 malignant tumors had metastasized to lymph nodes or viscera at the time of necropsy. Six lions with adenocarcinoma and two lions with benign mammary masses had received hormonal contraception, though little evidence of mammary lobular hyperplasia was seen in association with the adenocarcinomas. The most common concurrent disease processes found at necropsy were chronic urinary tract disease and other malignancies. These cases demonstrate that mammary malignancies occur in captive lions and frequently metastasize.

  6. CLINICOPATHOLOGIC FEATURES OF MAMMARY MASSES IN CAPTIVE LIONS (PANTHERA LEO).

    PubMed

    Sadler, Ryan A; Craig, Linden E; Ramsay, Edward C; Helmick, Kelly; Collins, Darin; Garner, Michael M

    2016-03-01

    A multi-institutional retrospective analysis of 330 pathology accessions from 285 different lions found 15 captive, female African lions (Panthera leo) with confirmed mammary masses. Aside from the presence of a mammary mass, the most common initial clinical sign was inappetence. Histologic diagnoses were predominantly adenocarcinoma (n = 12), though two benign masses (mammary hyperplasia and a mammary cyst) and one squamous cell carcinoma were identified. Nine of 13 malignant tumors had metastasized to lymph nodes or viscera at the time of necropsy. Six lions with adenocarcinoma and two lions with benign mammary masses had received hormonal contraception, though little evidence of mammary lobular hyperplasia was seen in association with the adenocarcinomas. The most common concurrent disease processes found at necropsy were chronic urinary tract disease and other malignancies. These cases demonstrate that mammary malignancies occur in captive lions and frequently metastasize. PMID:27010273

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

    PubMed

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

    2016-07-01

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

  8. Effects of endotoxin on the lactating mouse

    SciTech Connect

    Carr, J.K.

    1985-01-01

    The regulation of endogenous mouse mammary tumor virus (MMTV) sequences in trans by a host gene, the Lps locus on mouse chromosome 4, was suspected from a genetic linkage analysis. The Lps locus mediates the mouse's response to the injection of lipopolysaccharide (LPS) in the responder mouse while mice with the deficient allele are incapable of responding. Others have found that endotoxin exposure reduces milk production in lactating animals. This observation was confirmed in mice and extended by examining /sup 125/I-prolactin binding to liver membranes of lactating mice. Endotoxin treatment of responder mice increases liver prolactin binding within 15 minutes, followed by a decline over 6 hours. Scatchard analysis shows that the immediate increase comes from both increased affinity and abundance of the prolactin receptor. No such change in prolactin binding is seen in the non-responder following endotoxin treatment nor in /sup 125/I-insulin binding in responders.

  9. Mammary Stem Cells: A Clinician's View.

    PubMed

    Schneider, José

    2015-01-01

    Mammary stem cells were identified and isolated more than a decade ago and, although much remains to be learned, a lot has been revealed about their properties and behavior. Yet there is a gap between the newly acquired knowledge and its successful clinical application. This chapter presented a critical view from the perspective of a clinician. PMID:26040694

  10. Dietary genistein stimulates mammary development in gilts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The possible role of the phytoestrogen, genistein, on prepubertal development of mammary glands, hormonal status and bone resorption was investigated in gilts. Forty-five gilts were fed a control diet containing soya (CTLS, n = 15), a control diet without soya (CTL0, n = 15) or the CTLS diet supplem...

  11. Stromal regulation of embryonic and postnatal mammary epithelial development and differentiation.

    PubMed

    Howard, Beatrice A; Lu, Pengfei

    2014-01-01

    The stroma, which is composed of supporting cells and connective tissue, comprises a large component of the local microenvironment of many epithelial cell types, and influences several fundamental aspects of cell behaviour through both tissue interactions and niche regulation. The significance of the stroma in development and disease has been increasingly recognised. Whereas normal stroma is essential for various developmental processes during vertebrate organogenesis, it can be deregulated and become abnormal, which in turn can initiate or promote a disease process, including cancer. The mouse mammary gland has emerged in recent years as an excellent model system for understanding stromal function in both developmental and cancer biology. Here, we take a systematic approach and focus on the dynamic interactions that the stroma engages with the epithelium during mammary specification, cell differentiation, and branching morphogenesis of both the embryonic and postnatal development of the mammary gland. Similar stromal-epithelial interactions underlie the aetiology of breast cancer, making targeting the cancer stroma an increasingly important and promising therapeutic strategy to pursue for breast cancer treatment.

  12. Epimorphin mediates mammary luminal morphogenesis through control of C/EBPbeta

    SciTech Connect

    Hirai, Yohei; Radisky, Derek; Boudreau, Rosanne; Simian, Marina; Stevens, Mary E.; Oka, Yumiko; Takebe, Kyoko; Niwa, Shinichiro; Bissell, Mina J.

    2002-03-22

    We have previously shown that epimorphin, a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which epimorphin mediates luminal morphogenesis. Treatment of cells with epimorphin to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, as constitutive expression of LIP was sufficient to produce lumen formation, while constitutive expression of LAP blocked epimorphin-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which epimorphin expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPbeta, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for epimorphin in luminal morphogenesis through control of C/EBPbeta expression.

  13. 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.

  14. Identification of a stable molecular signature in mammary tumor endothelial cells that persists in vitro

    PubMed Central

    Xiao, Lin; Harrell, J. Chuck; Perou, Charles M.; Dudley, Andrew C.

    2013-01-01

    Long-term, in vitro propagation of tumor-specific endothelial cells (TEC) allows for functional studies and genome-wide expression profiling of clonally-derived, well-characterized subpopulations. Using a genetically engineered mouse model (GEMM) of mammary adenocarcinoma, we have optimized an isolation procedure and defined growth conditions for long-term propagation of mammary TEC. The isolated TEC maintain their endothelial specification and phenotype in culture. Furthermore, gene expression profiling of multiple TEC subpopulations revealed striking, persistent overexpression of several candidate genes including Irx2 and Zfp503 (transcription factors), Alcam and Cd133 (cell surface markers), Ccl4 and neurotensin (Nts) (angiocrine factors), and Gpr182 and Cnr2 (G protein-coupled receptors, GPCRs). Taken together, we have developed an effective method for isolating and culture-expanding mammary TEC, and uncovered several new TEC-selective genes whose overexpression persists even after long-term in vitro culture. These results suggest that the tumor microenvironment may induce changes in vascular endothelium in vivo that are stably transmittable in vitro. PMID:24257808

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

    PubMed

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

    2016-01-01

    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

  16. Runx2 is a novel regulator of mammary epithelial cell fate in development and breast cancer

    PubMed Central

    Best, Sarah; Ledger, Anita; Mooney, Anne-Marie; Ferguson, Alison; Shore, Paul; Swarbrick, Alexander; Ormandy, Christopher J; Simpson, Peter T; Carroll, Jason S; Visvader, Jane; Naylor, Matthew J

    2014-01-01

    Regulators of differentiated cell fate can offer targets for managing cancer development and progression. Here we identify Runx2 as a new regulator of epithelial cell fate in mammary gland development and breast cancer. Runx2 is expressed in the epithelium of pregnant mice in a strict temporally and hormonally-regulated manner. During pregnancy, Runx2 genetic deletion impaired alveolar differentiation in a manner that disrupted alveolar progenitor cell populations. Conversely, exogenous transgenic expression of Runx2 in mammary epithelial cells blocked milk production, suggesting that the decrease in endogenous Runx2 observed late in pregnancy is necessary for full differentiation. In addition, overexpression of Runx2 drove EMT-like changes in normal mammary epithelial cells, while Runx2 deletion in basal breast cancer cells inhibited cellular phenotypes associated with tumorigenesis. Notably, loss of Runx2 expression increased tumor latency and enhanced overall survival in a mouse model of breast cancer, with Runx2-deficient tumors exhibiting reduced cell proliferation. Together, our results establish a novel function for Runx2 in breast cancer that may offer a novel generalized route for therapeutic interventions. PMID:25056120

  17. 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. PMID:27681436

  18. 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

  19. Expression of constitutively activated Akt in the mammary gland leads to excess lipid synthesis during pregnancy and lactation.

    PubMed

    Schwertfeger, Kathryn L; McManaman, James L; Palmer, Carol A; Neville, Margaret C; Anderson, Steven M

    2003-06-01

    Expression of constitutively activated Akt in the mammary glands of transgenic mice results in a delay in post-lactational involution. We now report precocious lipid accumulation in the alveolar epithelium of mouse mammary tumor virus-myr-Akt transgenic mice accompanied by a lactation defect that results in a 50% decrease in litter weight over the first 9 days of lactation. Although ductal structures and alveolar units develop normally during pregnancy, cytoplasmic lipid droplets appeared precociously in mammary epithelial cells in early pregnancy and were accompanied by increased expression of adipophilin, which is associated with lipid droplets. By late pregnancy the lipid droplets had become significantly larger than in nontransgenic mice, and they persisted into lactation. The fat content of milk from lactating myr-Akt transgenic mice was 65-70% by volume compared to 25-30% in wild-type mice. The diminished growth of pups nursed by transgenic mothers could result from the high viscosity of the milk and the inability of the pups to remove sufficient quantities of milk by suckling. Transduction of the CIT3 mammary epithelial cell line with a recombinant human adenovirus encoding myr-Akt resulted in an increase in glucose transport and lipid biosynthesis, suggesting that Akt plays an important role in regulation of lipid metabolism. PMID:12700340

  20. The Chromatin Remodeling Component Arid1a Is a Suppressor of Spontaneous Mammary Tumors in Mice.

    PubMed

    Kartha, Nithya; Shen, Lishuang; Maskin, Carolyn; Wallace, Marsha; Schimenti, John C

    2016-08-01

    Human cancer genome studies have identified the SWI/SNF chromatin remodeling complex member ARID1A as one of the most frequently altered genes in several tumor types. Its role as an ovarian tumor suppressor has been supported in compound knockout mice. Here, we provide genetic and functional evidence that Arid1a is a bona fide mammary tumor suppressor, using the Chromosome aberrations occurring spontaneously 3 (Chaos3) mouse model of sporadic breast cancer. About 70% of mammary tumors that formed in these mice contained a spontaneous deletion removing all or part of one Arid1a allele. Restoration of Arid1a expression in a Chaos3 mammary tumor line with low Arid1a levels greatly impaired its ability to form tumors following injection into cleared mammary glands, indicating that ARID1A insufficiency is crucial for maintenance of these Trp53-proficient tumors. Transcriptome analysis of tumor cells before and after reintroduction of Arid1a expression revealed alterations in growth signaling and cell-cycle checkpoint pathways, in particular the activation of the TRP53 pathway. Consistent with the latter, Arid1a reexpression in tumor cells led to increased p21 (Cdkn1a) expression and dramatic accumulation of cells in G2 phase of the cell cycle. These results not only provide in vivo evidence for a tumor suppressive and/or maintenance role in breast cancer, but also indicate a potential opportunity for therapeutic intervention in ARID1A-deficient human breast cancer subtypes that retain one intact copy of the gene and also maintain wild-type TRP53 activity. PMID:27280691

  1. c-Myc Transforms Human Mammary Epithelial Cells through Repression of the Wnt Inhibitors DKK1 and SFRP1▿ †

    PubMed Central

    Cowling, Victoria H.; D'Cruz, Celina M.; Chodosh, Lewis A.; Cole, Michael D.

    2007-01-01

    c-myc is frequently amplified in breast cancer; however, the mechanism of myc-induced mammary epithelial cell transformation has not been defined. We show that c-Myc induces a profound morphological transformation in human mammary epithelial cells and anchorage-independent growth. c-Myc suppresses the Wnt inhibitors DKK1 and SFRP1, and derepression of DKK1 or SFRP1 reduces Myc-dependent transforming activity. Myc-dependent repression of DKK1 and SFRP1 is accompanied by Wnt target gene activation and endogenous T-cell factor activity. Myc-induced mouse mammary tumors have repressed SFRP1 and increased expression of Wnt target genes. DKK1 and SFRP1 inhibit the transformed phenotype of breast cancer cell lines, and DKK1 inhibits tumor formation. We propose a positive feedback loop for activation of the c-myc and Wnt pathways in breast cancer. PMID:17485441

  2. Mammary gland specific expression of Brk/PTK6 promotes delayed involution and tumor formation associated with activation of p38 MAPK

    PubMed Central

    2011-01-01

    Introduction Protein tyrosine kinases (PTKs) are frequently overexpressed and/or activated in human malignancies, and regulate cancer cell proliferation, cellular survival, and migration. As such, they have become promising molecular targets for new therapies. The non-receptor PTK termed breast tumor kinase (Brk/PTK6) is overexpressed in approximately 86% of human breast tumors. The role of Brk in breast pathology is unclear. Methods We expressed a WAP-driven Brk/PTK6 transgene in FVB/n mice, and analyzed mammary glands from wild-type (wt) and transgenic mice after forced weaning. Western blotting and immunohistochemistry (IHC) studies were conducted to visualize markers of mammary gland involution, cell proliferation and apoptosis, as well as Brk, STAT3, and activated p38 mitogen-activated protein kinase (MAPK) in mammary tissues and tumors from WAP-Brk mice. Human (HMEC) or mouse (HC11) mammary epithelial cells were stably or transiently transfected with Brk cDNA to assay p38 MAPK signaling and cell survival in suspension or in response to chemotherapeutic agents. Results Brk-transgenic dams exhibited delayed mammary gland involution and aged mice developed infrequent tumors with reduced latency relative to wt mice. Consistent with delayed involution, mammary glands of transgenic animals displayed decreased STAT3 phosphorylation, a marker of early-stage involution. Notably, p38 MAPK, a pro-survival signaling mediator downstream of Brk, was activated in mammary glands of Brk transgenic relative to wt mice. Brk-dependent signaling to p38 MAPK was recapitulated by Brk overexpression in the HC11 murine mammary epithelial cell (MEC) line and human MEC, while Brk knock-down in breast cancer cells blocked EGF-stimulated p38 signaling. Additionally, human or mouse MECs expressing Brk exhibited increased anchorage-independent survival and resistance to doxorubicin. Finally, breast tumor biopsies were subjected to IHC analysis for co-expression of Brk and phospho-p38 MAPK

  3. Disparate impact of butyroyloxymethyl diethylphosphate (AN-7), a histone deacetylase inhibitor, and doxorubicin in mice bearing a mammary tumor.

    PubMed

    Tarasenko, Nataly; Cutts, Suzanne M; Phillips, Don R; Inbal, Aida; Nudelman, Abraham; Kessler-Icekson, Gania; Rephaeli, Ada

    2012-01-01

    The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) synergizes the cytotoxic effect of doxorubicin (Dox) and anti-HER2 on mammary carcinoma cells while protecting normal cells against their insults. This study investigated the concomitant changes occurring in heart tissue and tumors of mice bearing a subcutaneous 4T1 mammary tumor following treatment with AN-7, Dox, or their combination. Dox or AN-7 alone led to inhibition of both tumor growth and lung metastases, whereas their combination significantly increased their anticancer efficacy and attenuated Dox- toxicity. Molecular analysis revealed that treatment with Dox, AN-7, and to a greater degree, AN-7 together with Dox increased tumor levels of γH2AX, the marker for DNA double-strand breaks and decreased the expression of Rad51, a protein needed for DNA repair. These events culminated in increased apoptosis, manifested by the appearance of cytochrome-c in the cytosol. In the myocardium, Dox-induced cardiomyopathy was associated with an increase in γH2AX expression and a reduction in Rad51 and MRE11 expression and increased apoptosis. The addition of AN-7 to the Dox treatment protected the heart from Dox insults as was manifested by a decrease in γH2AX levels, an increase in Rad51 and MRE11 expression, and a diminution of cytochrome-c release. Tumor fibrosis was high in untreated mice but diminished in Dox- and AN-7-treated mice and was almost abrogated in AN-7+Dox-treated mice. By contrast, in the myocardium, Dox alone induced a dramatic increase in fibrosis, and AN7+Dox attenuated it. The high expression levels of c-Kit, Ki-67, c-Myc, lo-FGF, and VEGF in 4T1 tumors were significantly reduced by Dox or AN-7 and further attenuated by AN-7+Dox. In the myocardium, Dox suppressed these markers, whereas AN-7+Dox restored their expression. In conclusion, the combination of AN-7 and Dox results in two beneficial effects, improved anticancer efficacy and cardioprotection.

  4. GH-producing mammary tumors in two dogs with acromegaly.

    PubMed

    Murai, Atsuko; Nishii, Naohito; Morita, Takehito; Yuki, Masashi

    2012-06-01

    Two intact female dogs were admitted for growing mammary tumors. They had symptoms of acromegaly including weight gain, enlargement of the head, excessive skin folds, and inspiratory stridor. Serum concentrations of growth hormone (GH), insulin-like growth factor-I (IGF-I), and insulin were elevated in the two cases. From these findings, both dogs were diagnosed with acromegaly. In case 1, the GH, IGF-I, and insulin levels subsided after removal of the focal benign mammary tumors and ovariohysterectomy. In case 2, those levels subsided after removal of only focal mammary carcinoma. In both cases, immunohistochemical investigations for GH were positive in the mammary tumor cells but not in the normal mammary glands. We concluded that GH-producing mammary tumors caused the present acromegaly.

  5. Cox-2 levels in canine mammary tumors, including inflammatory mammary carcinoma: clinicopathological features and prognostic significance.

    PubMed

    Queiroga, Felisbina Luisa; Perez-Alenza, Maria Dolores; Silvan, Gema; Peña, Laura; Lopes, Carlos; Illera, Juan Carlos

    2005-01-01

    Cyclo-oxygenase (Cox-2) plays an important role in mammary carcinogenesis, nevertheless, its role in canine mammary tumors, and particularly in inflammatory mammary carcinoma (IMC), is unknown. Tumor Cox-2 levels were analyzed by enzyme immunoassay, in post-surgical tumor homogenates of 129 mammary tumors (62 dysplasias and benign tumors, 57 malignant non-IMC and 10 IMC) from 57 female dogs. The highest Cox-2 values were detected in the IMC group. In non-IMC malignant tumors, high values of Cox-2 were related to skin ulceration (p < 0.001) and tumor size (p < 0.001). The follow-up study revealed that high Cox-2 levels were related with recurrence (p = 0.002), metastases (p < 0.001), disease-free survival (p < 0.001) and overall survival (p < 0.001). This study demonstrates an association between intra-tumor Cox-2 levels and poor prognosis. The high levels found in IMC cases could indicate a special role of Cox-2 in the inflammatory phenotype and open the possibility of additional new therapeutic approaches in this special type of mammary cancer in humans and dogs.

  6. Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction

    SciTech Connect

    Roskelley, C.D.; Desprez, P.Y.; Bissell, M.J. )

    1994-12-20

    Extracellular matrix (ECM) profoundly influences the growth and differentiation of the mammary gland epithelium, both in culture and in vivo. Utilizing a clonal population of mouse mammary epithelial cells that absolutely requires an exogenous ECM for function, we developed a rapid assay to study signal transduction by ECM. Two components of the cellular response to a basement membrane overlay that result in the expression of the milk protein [beta]-casein were defined. The first component of this response involves a rounding and clustering of the cells that can be physically mimicked by plating the cells on a nonadhesive substratum. The second component is biochemical in nature, and it is associated with [beta][sub 1] integrin clustering and increased tyrosine phosphorylation. The second component is initiated in a morphology-independent manner, but the proper translation of this biochemical signal into a functional response requires cell rounding and cell clustering. Thus, physical and biochemical signal transduction events contribute to the ECM-dependent regulation of tissue-specific gene expression in mouse mammary epithelial cells. 44 refs., 6 figs.

  7. Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization

    PubMed Central

    Resemann, Henrike K.; Ramos-Montoya, Antonio; Skepper, Jeremy; Watson, Christine J.

    2014-01-01

    We have previously demonstrated that Stat3 regulates lysosomal mediated-programmed cell death (LM-PCD) during mouse mammary gland involution in vivo. However, the mechanism that controls the release of lysosomal cathepsins to initiate cell death in this context has not been elucidated. We show here that Stat3 regulates the formation of large lysosomal vacuoles that contain triglyceride. Furthermore, we demonstrate that milk fat globules (MFGs) are toxic to epithelial cells and that, when applied to purified lysosomes, the MFG hydrolysate oleic acid potently induces lysosomal leakiness. Additionally, uptake of secreted MFGs coated in butyrophilin 1A1 is diminished in Stat3 ablated mammary glands while loss of the phagocytosis bridging molecule MFG-E8 results in reduced leakage of cathepsins in vivo. We propose that Stat3 regulates LM-PCD in mouse mammary gland by switching cellular function from secretion to uptake of MFGs. Thereafter, perturbation of lysosomal vesicle membranes by high levels of free fatty acids results in controlled leakage of cathepsins culminating in cell death. PMID:25283994

  8. Quantitative proteomic analyses of mammary organoids reveals distinct signatures after exposure to environmental chemicals.

    PubMed

    Williams, Katherine E; Lemieux, George A; Hassis, Maria E; Olshen, Adam B; Fisher, Susan J; Werb, Zena

    2016-03-01

    Common environmental contaminants such as bisphenols and phthalates and persistent contaminants such as polychlorinated biphenyls are thought to influence tissue homeostasis and carcinogenesis by acting as disrupters of endocrine function. In this study we investigated the direct effects of exposure to bisphenol A (BPA), mono-n-butyl phthalate (Pht), and polychlorinated biphenyl 153 (PCB153) on the proteome of primary organotypic cultures of the mouse mammary gland. At low-nanomolar doses each of these agents induced distinct effects on the proteomes of these cultures. Although BPA treatment produced effects that were similar to those induced by estradiol, there were some notable differences, including a reduction in the abundance of retinoblastoma-associated protein and increases in the Rho GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle protein CDC42. Both Pht and PCB153 induced changes that were distinct from those induced by estrogen, including decreased levels of the transcriptional corepressor C-terminal binding protein 1. Interestingly, the three chemicals appeared to alter the abundance of distinct splice forms of many proteins as well as the abundance of several proteins that regulate RNA splicing. Our combined results indicate that the three classes of chemical have distinct effects on the proteome of normal mouse mammary cultures, some estrogen-like but most estrogen independent, that influence diverse biological processes including apoptosis, cell adhesion, and proliferation.

  9. Quantitative proteomic analyses of mammary organoids reveals distinct signatures after exposure to environmental chemicals

    PubMed Central

    Williams, Katherine E.; Lemieux, George A.; Hassis, Maria E.; Olshen, Adam B.; Fisher, Susan J.; Werb, Zena

    2016-01-01

    Common environmental contaminants such as bisphenols and phthalates and persistent contaminants such as polychlorinated biphenyls are thought to influence tissue homeostasis and carcinogenesis by acting as disrupters of endocrine function. In this study we investigated the direct effects of exposure to bisphenol A (BPA), mono-n-butyl phthalate (Pht), and polychlorinated biphenyl 153 (PCB153) on the proteome of primary organotypic cultures of the mouse mammary gland. At low-nanomolar doses each of these agents induced distinct effects on the proteomes of these cultures. Although BPA treatment produced effects that were similar to those induced by estradiol, there were some notable differences, including a reduction in the abundance of retinoblastoma-associated protein and increases in the Rho GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle protein CDC42. Both Pht and PCB153 induced changes that were distinct from those induced by estrogen, including decreased levels of the transcriptional corepressor C-terminal binding protein 1. Interestingly, the three chemicals appeared to alter the abundance of distinct splice forms of many proteins as well as the abundance of several proteins that regulate RNA splicing. Our combined results indicate that the three classes of chemical have distinct effects on the proteome of normal mouse mammary cultures, some estrogen-like but most estrogen independent, that influence diverse biological processes including apoptosis, cell adhesion, and proliferation. PMID:26903627

  10. Quantitative proteomic analyses of mammary organoids reveals distinct signatures after exposure to environmental chemicals.

    PubMed

    Williams, Katherine E; Lemieux, George A; Hassis, Maria E; Olshen, Adam B; Fisher, Susan J; Werb, Zena

    2016-03-01

    Common environmental contaminants such as bisphenols and phthalates and persistent contaminants such as polychlorinated biphenyls are thought to influence tissue homeostasis and carcinogenesis by acting as disrupters of endocrine function. In this study we investigated the direct effects of exposure to bisphenol A (BPA), mono-n-butyl phthalate (Pht), and polychlorinated biphenyl 153 (PCB153) on the proteome of primary organotypic cultures of the mouse mammary gland. At low-nanomolar doses each of these agents induced distinct effects on the proteomes of these cultures. Although BPA treatment produced effects that were similar to those induced by estradiol, there were some notable differences, including a reduction in the abundance of retinoblastoma-associated protein and increases in the Rho GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle protein CDC42. Both Pht and PCB153 induced changes that were distinct from those induced by estrogen, including decreased levels of the transcriptional corepressor C-terminal binding protein 1. Interestingly, the three chemicals appeared to alter the abundance of distinct splice forms of many proteins as well as the abundance of several proteins that regulate RNA splicing. Our combined results indicate that the three classes of chemical have distinct effects on the proteome of normal mouse mammary cultures, some estrogen-like but most estrogen independent, that influence diverse biological processes including apoptosis, cell adhesion, and proliferation. PMID:26903627

  11. A mammary adenocarcinoma murine model suitable for the study of cancer immunoediting

    PubMed Central

    2014-01-01

    Background Cancer immunoediting is a dynamic process composed of three phases: elimination (EL), equilibrium (EQ) and escape (ES) that encompasses the potential host-protective and tumor-sculpting functions of the immune system throughout tumor development. Animal models are useful tools for studying diseases such as cancer. The present study was designed to characterize the interaction between mammary adenocarcinoma M-406 and CBi, CBi− and CBi/L inbred mice lines. Results The mammary adenocarcinoma M-406 developed spontaneously in a CBi mouse. CBi/L and CBi− mice were artificially selected for body conformation from CBi. When CBi mice are s.c. challenged with M-406, tumor growths exponentially in 100% of animals, while in CBi− the tumor growths briefly and then begins a rejection process in 100% of the animals. In CBi/L the growth of the tumor shows the three phases: 51.6% in ES, 18.5% in EQ and 29.8% in EL. Conclusions The results obtained support the conclusion that the system M-406 plus the inbred mouse lines CBi, CBi− and CBi/L, is a good murine model to study the process of tumor immunoediting. PMID:24885995

  12. Targeting Serglycin Prevents Metastasis in Murine Mammary Carcinoma

    PubMed Central

    Roy, Ananya; Femel, Julia; Huijbers, Elisabeth J. M.; Spillmann, Dorothe; Larsson, Erik; Ringvall, Maria; Olsson, Anna-Karin; Åbrink, Magnus

    2016-01-01

    In hematopoietic cells, serglycin proteoglycans mainly contribute to proper storage and secretion of inflammatory mediators via their negatively charged glycosaminoglycans. Serglycin proteoglycans are also expressed in cancer cells where increased expression has been linked to poor prognosis. However, the serglycin-dependent mediators promoting cancer progression remain to be determined. In the present study we report that genetic ablation of serglycin proteoglycan completely blocks lung metastasis in the MMTV-PyMT-driven mouse breast cancer model, while serglycin-deficiency did not affect primary tumour growth or number of mammary tumours. Although E-cadherin expression was higher in the serglycin-deficient primary tumour tissue, indicating reduced invasiveness, serglycin-deficient tumour cells were still detected in the circulation. These data suggest that serglycin proteoglycans play a role in extravasation as well as colonization and growth of metastatic cells. A microarray expression analysis and functional annotation of differentially expressed genes identified several biological pathways where serglycin may be important. Our results suggest that serglycin and serglycin-dependent mediators are potential drug targets to prevent metastatic disease/dissemination of cancer. PMID:27223472

  13. 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.

  14. Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes.

    PubMed

    Veltmaat, Jacqueline M; Relaix, Frédéric; Le, Lendy T; Kratochwil, Klaus; Sala, Frédéric G; van Veelen, Wendy; Rice, Ritva; Spencer-Dene, Bradley; Mailleux, Arnaud A; Rice, David P; Thiery, Jean Paul; Bellusci, Saverio

    2006-06-01

    Little is known about the regulation of cell fate decisions that lead to the formation of five pairs of mammary placodes in the surface ectoderm of the mouse embryo. We have previously shown that fibroblast growth factor 10 (FGF10) is required for the formation of mammary placodes 1, 2, 3 and 5. Here, we have found that Fgf10 is expressed only in the somites underlying placodes 2 and 3, in gradients across and within these somites. To test whether somitic FGF10 is required for the formation of these two placodes, we analyzed a number of mutants with different perturbations of somitic Fgf10 gradients for the presence of WNT signals and ectodermal multilayering, markers for mammary line and placode formation. The mammary line is displaced dorsally, and formation of placode 3 is impaired in Pax3ILZ/ILZ mutants, which do not form ventral somitic buds. Mammary line formation is impaired and placode 3 is absent in Gli3Xt-J/Xt-J and hypomorphic Fgf10 mutants, in which the somitic Fgf10 gradient is shortened dorsally and less overall Fgf10 is expressed, respectively. Recombinant FGF10 rescued mammogenesis in Fgf10(-/-) and Gli3Xt-J/Xt-J flanks. We correlate increasing levels of somitic FGF10 with progressive maturation of the surface ectoderm, and show that full expression of somitic Fgf10, co-regulated by GLI3, is required for the anteroposterior pattern in which the flank ectoderm acquires a mammary epithelial identity. We propose that the intra-somitic Fgf10 gradient, together with ventral elongation of the somites, determines the correct dorsoventral position of mammary epithelium along the flank.

  15. Ceramide Kinase Promotes Tumor Cell Survival and Mammary Tumor Recurrence

    PubMed Central

    Payne, Ania W.; Pant, Dhruv K.; Pan, Tien-chi; Chodosh, Lewis A.

    2014-01-01

    Recurrent breast cancer is typically an incurable disease and, as such, is disproportionately responsible for deaths from this disease. Recurrent breast cancers arise from the pool of disseminated tumor cells (DTCs) that survive adjuvant or neoadjuvant therapy, and patients with detectable DTCs following therapy are at substantially increased risk for recurrence. Consequently, the identification of pathways that contribute to the survival of breast cancer cells following therapy could aid in the development of more effective therapies that decrease the burden of residual disease and thereby reduce the risk of breast cancer recurrence. We now report that Ceramide Kinase (Cerk) is required for mammary tumor recurrence following HER2/neu pathway inhibition and is spontaneously up-regulated during tumor recurrence in multiple genetically engineered mouse models for breast cancer. We find that Cerk is rapidly up-regulated in tumor cells following HER2/neu down-regulation or treatment with Adriamycin and that Cerk is required for tumor cell survival following HER2/neu down-regulation. Consistent with our observations in mouse models, analysis of gene expression profiles from over 2,200 patients revealed that elevated CERK expression is associated with an increased risk of recurrence in women with breast cancer. Additionally, although CERK expression is associated with aggressive subtypes of breast cancer, including those that are ER–, HER2+, basal-like, or high grade, its association with poor clinical outcome is independent of these clinicopathological variables. Together, our findings identify a functional role for Cerk in breast cancer recurrence and suggest the clinical utility of agents targeted against this pro-survival pathway. PMID:25164007

  16. Adipose and mammary epithelial tissue engineering.

    PubMed

    Zhu, Wenting; Nelson, Celeste M

    2013-01-01

    Breast reconstruction is a type of surgery for women who have had a mastectomy, and involves using autologous tissue or prosthetic material to construct a natural-looking breast. Adipose tissue is the major contributor to the volume of the breast, whereas epithelial cells comprise the functional unit of the mammary gland. Adipose-derived stem cells (ASCs) can differentiate into both adipocytes and epithelial cells and can be acquired from autologous sources. ASCs are therefore an attractive candidate for clinical applications to repair or regenerate the breast. Here we review the current state of adipose tissue engineering methods, including the biomaterials used for adipose tissue engineering and the application of these techniques for mammary epithelial tissue engineering. Adipose tissue engineering combined with microfabrication approaches to engineer the epithelium represents a promising avenue to replicate the native structure of the breast.

  17. Cytokine signalling in mammary gland development.

    PubMed

    Watson, Christine J; Oliver, Carrie H; Khaled, Walid T

    2011-03-01

    Mammary gland development occurs in three distinct stages during the lifetime of the female mammal: in embryonic, pubertal and reproductive life. At each of these developmental stages, different signalling molecules induce changes in both the epithelium and the surrounding stroma. However, it is during pregnancy that the most dramatic changes occur, resulting in a massive increase in the number of epithelial cells and in their function. Pregnancy initiates the development of a new epithelial lineage, the alveolar cells, which form the milk-producing lobuloalveolar structures. These cells become redundant at the end of lactation and are removed in an exquisitely controlled process of tissue remodelling coupled with extensive cell death. All of these events require not only steroid hormones but also sequential signalling by cytokines. A recent surprising discovery was that the signalling pathways and cytokines that regulate lineage determination in T helper cells are also involved in mammary gland development during pregnancy.

  18. Adipose and mammary epithelial tissue engineering

    PubMed Central

    Zhu, Wenting; Nelson, Celeste M.

    2013-01-01

    Breast reconstruction is a type of surgery for women who have had a mastectomy, and involves using autologous tissue or prosthetic material to construct a natural-looking breast. Adipose tissue is the major contributor to the volume of the breast, whereas epithelial cells comprise the functional unit of the mammary gland. Adipose-derived stem cells (ASCs) can differentiate into both adipocytes and epithelial cells and can be acquired from autologous sources. ASCs are therefore an attractive candidate for clinical applications to repair or regenerate the breast. Here we review the current state of adipose tissue engineering methods, including the biomaterials used for adipose tissue engineering and the application of these techniques for mammary epithelial tissue engineering. Adipose tissue engineering combined with microfabrication approaches to engineer the epithelium represents a promising avenue to replicate the native structure of the breast. PMID:23628872

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

    PubMed

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

    2014-01-01

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

  20. Epigenetic regulation of LSD1 during mammary carcinogenesis

    PubMed Central

    Wu, Yadi; Zhou, Binhua P

    2014-01-01

    Inheritable epigenetic regulation is integral to the dynamic control of gene expression under different stimuli for cellular homeostasis and disease progression. Histone methylation is a common and important type of chromatin modification. LSD1, the first known histone lysine-specific demethylase, operates as a key component of several corepressor complexes during development and in disease states. In this review, we focus on the regulation of LSD1 in mammary carcinogenesis. LSD1 plays a role in promoting mammary tumor metastasis and proliferation and in maintaining mammary cancer stem cells. Therefore, LSD1 represents a viable therapeutic target for effective treatment of mammary carcinogenesis. PMID:27308339

  1. Absence of canine papillomavirus sequences in canine mammary tumours.

    PubMed

    Sardon, D; Blundell, R; Burrai, G P; Alberti, A; Tore, G; Passino, E Sanna; Antuofermo, E

    2015-01-01

    Human papillomaviruses (PVs) are found in human breast cancer tissue; however, it remains controversial as to whether these viruses play a role in the aetiology of this tumour. There has been minimal study of whether PVs are found in normal or abnormal mammary glands of animals. The present study investigated whether a PV sequence could be found in the mammary glands of 33 female dogs by rolling circle amplification and polymerase chain reaction. No PV DNA was found in normal or neoplastic canine mammary tissues, suggesting that canine PVs are probably not involved in the pathogenesis of canine mammary neoplasia. PMID:25435511

  2. 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.

  3. The severity of mammary gland developmental defects is linked to the overall functional status of Cx43 as revealed by genetically modified mice

    PubMed Central

    Stewart, Michael K. G.; Gong, Xiang-Qun; Barr, Kevin J.; Bai, Donglin; Fishman, Glenn I.; Laird, Dale W.

    2012-01-01

    Genetically modified mice mimicking ODDD (oculodentodigital dysplasia), a disease characterized by reduced Cx43 (connexin 43)-mediated gap junctional intercellular communication, represent an in vivo model to assess the role of Cx43 in mammary gland development and function. We previously reported that severely compromised Cx43 function delayed mammary gland development and impaired milk ejection in mice that harboured a G60S Cx43 mutant, yet there are no reports of lactation defects in ODDD patients. To address this further, we obtained a second mouse model of ODDD expressing an I130T Cx43 mutant to assess whether a mutant with partial gap junction channel activity would be sufficient to retain mammary gland development and function. The results of the present study show that virgin Cx43I130T/+ mice exhibited a temporary delay in ductal elongation at 4 weeks. In addition, Cx43I130T/+ mice develop smaller mammary glands at parturition due to reduced cell proliferation despite similar overall gland architecture. Distinct from Cx43G60S/+ mice, Cx43I130T/+ mice adequately produce and deliver milk to pups, suggesting that milk ejection is unaffected. Thus the present study suggests that a loss-of-function mutant of Cx43 with partial gap junction channel coupling conductance results in a less severe mammary gland phenotype, which may partially explain the lack of reported lactation defects associated with ODDD patients. PMID:23075222

  4. Over-expression of an endogenous milk protein gene in transgenic mice is associated with impaired mammary alveolar development and a milchlos phenotype.

    PubMed

    Burdon, T; Wall, R J; Shamay, A; Smith, G H; Hennighausen, L

    1991-12-01

    The whey acidic protein (WAP) gene is expressed in mammary epithelial cells at late pregnancy and throughout lactation. We have generated transgenic mice in which a mouse WAP transgene is expressed precociously in pregnancy. From 13 founder mice bearing WAP transgenes, two female founders and the daughters from a male founder failed to lactate and nurture their offspring. We named this phenotype milchlos. Mammary tissue from postpartum milchlos mice was underdeveloped, contained too few alveoli and resembled the glands of non-transgenic mid-pregnant mice. The hypothesis that alveolar development in milchlos mice was functionally arrested in a prelactational state is consistent with low levels of alpha-lactalbumin mRNA, and an unidentified keratin RNA in mammary tissue from postpartum mice. Defects in alveolar function in milchlos mice were detected at mid-pregnancy; in non-transgenic mice, WAP was secreted into the alveolar lumen but remained preferentially in the cytoplasm of the alveolar epithelial cells in the milchlos mice. Since deregulated WAP expression resulted in impaired mammary development, it is possible that WAP plays a regulatory role in the terminal differentiation and development of mammary alveolar cells. PMID:1782141

  5. Mammary stem cells: expansion and animal productivity

    PubMed Central

    2014-01-01

    Identification and characterization of mammary stem cells and progenitor cells from dairy animals is important in the understanding of mammogenesis, tissue turnover, lactation persistency and regenerative therapy. It has been realized by many investigators that altered lactation, long dry periods (non-milking period between two consecutive lactation cycles), abrupt cessation of lactation (common in water buffaloes) and disease conditions like mastitis, greatly reduce milk yield thus render huge financial losses within the dairy sector. Cellular manipulation of specialized cell types within the mammary gland, called mammary stem cells (MaSCs)/progenitor cells, might provide potential solutions to these problems and may improve milk production. In addition, MaSCs/progenitor cells could be used in regenerative therapy against tissue damage caused by mastitis. This review discusses methods of MaSC/progenitor cell manipulation and their mechanisms in bovine and caprine animals. Author believes that intervention of MaSCs/progenitor cells could lessen the huge financial losses to the dairy industry globally. PMID:25057352

  6. PKA signaling drives mammary tumorigenesis through Src.

    PubMed

    Beristain, A G; Molyneux, S D; Joshi, P A; Pomroy, N C; Di Grappa, M A; Chang, M C; Kirschner, L S; Privé, G G; Pujana, M A; Khokha, R

    2015-02-26

    Protein kinase A (PKA) hyperactivation causes hereditary endocrine neoplasias; however, its role in sporadic epithelial cancers is unknown. Here, we show that heightened PKA activity in the mammary epithelium generates tumors. Mammary-restricted biallelic ablation of Prkar1a, which encodes for the critical type-I PKA regulatory subunit, induced spontaneous breast tumors characterized by enhanced type-II PKA activity. Downstream of this, Src phosphorylation occurs at residues serine-17 and tyrosine-416 and mammary cell transformation is driven through a mechanism involving Src signaling. The phenotypic consequences of these alterations consisted of increased cell proliferation and, accordingly, expansion of both luminal and basal epithelial cell populations. In human breast cancer, low PRKAR1A/high SRC expression defines basal-like and HER2 breast tumors associated with poor clinical outcome. Together, the results of this study define a novel molecular mechanism altered in breast carcinogenesis and highlight the potential strategy of inhibiting SRC signaling in treating this cancer subtype in humans. PMID:24662820

  7. Oxytocin binding sites in bovine mammary tissue

    SciTech Connect

    Zhao, Xin.

    1989-01-01

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

  8. Mammary stem cells: expansion and animal productivity.

    PubMed

    Choudhary, Ratan K

    2014-01-01

    Identification and characterization of mammary stem cells and progenitor cells from dairy animals is important in the understanding of mammogenesis, tissue turnover, lactation persistency and regenerative therapy. It has been realized by many investigators that altered lactation, long dry periods (non-milking period between two consecutive lactation cycles), abrupt cessation of lactation (common in water buffaloes) and disease conditions like mastitis, greatly reduce milk yield thus render huge financial losses within the dairy sector. Cellular manipulation of specialized cell types within the mammary gland, called mammary stem cells (MaSCs)/progenitor cells, might provide potential solutions to these problems and may improve milk production. In addition, MaSCs/progenitor cells could be used in regenerative therapy against tissue damage caused by mastitis. This review discusses methods of MaSC/progenitor cell manipulation and their mechanisms in bovine and caprine animals. Author believes that intervention of MaSCs/progenitor cells could lessen the huge financial losses to the dairy industry globally.

  9. Secretory pathway Ca2+/Mn2+-ATPase isoform 2 and lactation: specific localization of plasmalemmal and secretory pathway Ca2+ pump isoforms in the mammary gland

    SciTech Connect

    Faddy, Helen M.; Smart, Chanel E.; Xu, Ren; Lee, Genee Y.; Kenny, Paraic A.; Feng, Mingye; Rao, Rajini; Brown, Melissa A.; Bissell, Mina J.; Roberts-Thomson, Sarah J.; Monteith, Gregory R.

    2008-04-09

    The supply of calcium to the developing neonate via milk is an important physiological process. Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely mediated via the secretory pathway. However, recent studies suggest that a specific isoform of the plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during different stages of development. SPCA2 levels increased over 35 fold during lactation, while SPCA1 increased only a modest two fold. The potential importance of SPCA2 in lactation was also highlighted by its localization to luminal secretory cells of the mammary gland during lactation, while SPCA1 was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1 during lactation. Using the SCp2 mouse mammary epithelial cell 3D culture model, differences in the sub-cellular distribution of PMCA2 and PMCA1 were clear. These studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation, and link the recently characterized SPCA2 calcium pump to the supply of calcium into milk and the regulation of Golgi resident enzymes important in lactation. They also indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.

  10. 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. PMID:26678338

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

    PubMed Central

    Zhao, Wang-Sheng; Hu, Shi-Liang; Yu, Kang; Wang, Hui; Wang, Wei; Loor, Juan; Luo, Jun

    2014-01-01

    Lipoprotein lipase (LPL) serves as a central factor in hydrolysis of triacylglycerol and uptake of free fatty acids from the plasma. However, there are limited data concerning the action of LPL on the regulation of milk fat synthesis in goat mammary gland. In this investigation, we describe the cloning and sequencing of the LPL gene from Xinong Saanen dairy goat mammary gland, along with a study of its phylogenetic relationships. Sequence analysis showed that goat LPL shares similarities with other species including sheep, bovine, human and mouse. LPL mRNA expression in various tissues determined by RT-qPCR revealed the highest expression in white adipose tissue, with lower expression in heart, lung, spleen, rumen, small intestine, mammary gland, and kidney. Expression was almost undetectable in liver and muscle. The expression profiles of LPL gene in mammary gland at early, peak, mid, late lactation, and the dry period were also measured. Compared with the dry period, LPL mRNA expression was markedly greater at early lactation. However, compared with early lactation, the expression was lower at peak lactation and mid lactation. Despite those differences, LPL mRNA expression was still greater at peak, mid, and late lactation compared with the dry period. Using goat mammary epithelial cells (GMEC), the in vitro knockdown of LPL via shRNA or with Orlistat resulted in a similar degree of down-regulation of LPL (respectively). Furthermore, knockdown of LPL was associated with reduced mRNA expression of SREBF1, FASN, LIPE and PPARG but greater expression of FFAR3. There was no effect on ACACA expression. Orlistat decreased expression of LIPE, FASN, ACACA, and PPARG, and increased FFAR3 and SREBF1 expression. The pattern of LPL expression was similar to the changes in milk fat percentage in lactating goats. Taken together, results suggest that LPL may play a crucial role in fatty acid synthesis. PMID:25501331

  12. p63 Sustains self-renewal of mammary cancer stem cells through regulation of Sonic Hedgehog signaling.

    PubMed

    Memmi, Elisa Maria; Sanarico, Anna Giulia; Giacobbe, Arianna; Peschiaroli, Angelo; Frezza, Valentina; Cicalese, Angelo; Pisati, Federica; Tosoni, Daniela; Zhou, Huiqing; Tonon, Giovanni; Antonov, Alexey; Melino, Gerry; Pelicci, Pier Giuseppe; Bernassola, Francesca

    2015-03-17

    The predominant p63 isoform, ΔNp63, is a master regulator of normal epithelial stem cell (SC) maintenance. However, in vivo evidence of the regulation of cancer stem cell (CSC) properties by p63 is still limited. Here, we exploit the transgenic MMTV-ErbB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2) mouse model of carcinogenesis to dissect the role of p63 in the regulation of mammary CSC self-renewal and breast tumorigenesis. ErbB2 tumor cells enriched for SC-like properties display increased levels of ΔNp63 expression compared with normal mammary progenitors. Down-regulation of p63 in ErbB2 mammospheres markedly restricts self-renewal and expansion of CSCs, and this action is fully independent of p53. Furthermore, transplantation of ErbB2 progenitors expressing shRNAs against p63 into the mammary fat pads of syngeneic mice delays tumor growth in vivo. p63 knockdown in ErbB2 progenitors diminishes the expression of genes encoding components of the Sonic Hedgehog (Hh) signaling pathway, a driver of mammary SC self-renewal. Remarkably, p63 regulates the expression of Sonic Hedgehog (Shh), GLI family zinc finger 2 (Gli2), and Patched1 (Ptch1) genes by directly binding to their gene regulatory regions, and eventually contributes to pathway activation. Collectively, these studies highlight the importance of p63 in maintaining the self-renewal potential of mammary CSCs via a positive modulation of the Hh signaling pathway. PMID:25739959

  13. Elevated GH/IGF-I promotes mammary tumors in high-fat, but not low-fat, fed mice.

    PubMed

    Gahete, Manuel D; Córdoba-Chacón, José; Lantvit, Daniel D; Ortega-Salas, Rosa; Sanchez-Sanchez, Rafael; Pérez-Jiménez, Francisco; López-Miranda, José; Swanson, Steven M; Castaño, Justo P; Luque, Raúl M; Kineman, Rhonda D

    2014-11-01

    Growth hormone (GH) and/or insulin-like growth factor I (IGF-I) are thought to promote breast cancer based on reports showing circulating IGF-I levels correlate, in epidemiological studies, with breast cancer risk. Also, mouse models with developmental GH/IGF-I deficiency/resistance are less susceptible to genetic- or chemical-induced mammary tumorigenesis. However, given the metabolic properties of GH, medical strategies have been considered to raise GH to improve body composition and metabolic function in elderly and obese patients. Since hyperlipidemia, inflammation, insulin resistance and obesity increase breast cancer risk, elevating GH may serve to exacerbate cancer progression. To better understand the role GH/IGF-I plays in tumor formation, this study used unique mouse models to determine if reducing GH/IGF-I in adults protects against 7,12-dimethylbenz[α]anthracene (DMBA)-induced mammary tumor development, and if moderate elevations in endogenous GH/IGF-I alter DMBA-induced tumorigenesis in mice fed a standard-chow diet or in mice with altered metabolic function due to high-fat feeding. We observed that adult-onset isolated GH-deficient mice, which also have reduced IGF-I levels, were less susceptible to DMBA-treatment. Specifically, fewer adult-onset isolated GH-deficient mice developed mammary tumors compared with GH-replete controls. In contrast, chow-fed mice with elevated endogenous GH/IGF-I (HiGH mice) were not more susceptible to DMBA-treatment. However, high-fat-fed, HiGH mice showed reduced tumor latency and increased tumor incidence compared with diet-matched controls. These results further support a role of GH/IGF-I in regulating mammary tumorigenesis but suggest the ultimate consequences of GH/IGF-I on breast tumor development are dependent on the diet and/or metabolic status.

  14. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

    SciTech Connect

    Lund, Leif R; Romer, John; Thomasset, Nicole; Solberg, Helene; Pyke, Charles; Bissell, Mina J; Dano, Keld; Werb, Zena

    1996-01-01

    Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin

  15. An autoregulatory enhancer controls mammary-specific STAT5 functions

    PubMed Central

    Metser, Gil; Shin, Ha Youn; Wang, Chaochen; Yoo, Kyung Hyun; Oh, Sumin; Villarino, Alejandro V.; O'Shea, John J.; Kang, Keunsoo; Hennighausen, Lothar

    2016-01-01

    Signal Transducers and Activators of Transcription (STATs) are principal transcription factors downstream of cytokine receptors. Although STAT5A is expressed in most tissues it remains to be understood why its premier, non-redundant functions are restricted to prolactin-induced mammary gland development and function. We report that the ubiquitously expressed Stat5a/b locus is subject to additional lineage-specific transcriptional control in mammary epithelium. Genome-wide surveys of epigenetic status and transcription factor occupancy uncovered a putative mammary-specific enhancer within the intergenic sequences separating the two Stat5 genes. This region exhibited several hallmarks of genomic enhancers, including DNaseI hypersensitivity, H3K27 acetylation and binding by GR, NFIB, ELF5 and MED1. Mammary-specific STAT5 binding was obtained at two canonical STAT5 binding motifs. CRISPR/Cas9-mediated genome editing was used to delete these sites in mice and determine their biological function. Mutant animals exhibited an 80% reduction of Stat5 levels in mammary epithelium and a concomitant reduction of STAT5-dependent gene expression. Transcriptome analysis identified a class of mammary-restricted genes that was particularly dependent on high STAT5 levels as a result of the intergenic enhancer. Taken together, the mammary-specific enhancer enables a positive feedback circuit that contributes to the remarkable abundance of STAT5 and, in turn, to the efficacy of STAT5-dependent mammary physiology. PMID:26446995

  16. An autoregulatory enhancer controls mammary-specific STAT5 functions.

    PubMed

    Metser, Gil; Shin, Ha Youn; Wang, Chaochen; Yoo, Kyung Hyun; Oh, Sumin; Villarino, Alejandro V; O'Shea, John J; Kang, Keunsoo; Hennighausen, Lothar

    2016-02-18

    Signal Transducers and Activators of Transcription (STATs) are principal transcription factors downstream of cytokine receptors. Although STAT5A is expressed in most tissues it remains to be understood why its premier, non-redundant functions are restricted to prolactin-induced mammary gland development and function. We report that the ubiquitously expressed Stat5a/b locus is subject to additional lineage-specific transcriptional control in mammary epithelium. Genome-wide surveys of epigenetic status and transcription factor occupancy uncovered a putative mammary-specific enhancer within the intergenic sequences separating the two Stat5 genes. This region exhibited several hallmarks of genomic enhancers, including DNaseI hypersensitivity, H3K27 acetylation and binding by GR, NFIB, ELF5 and MED1. Mammary-specific STAT5 binding was obtained at two canonical STAT5 binding motifs. CRISPR/Cas9-mediated genome editing was used to delete these sites in mice and determine their biological function. Mutant animals exhibited an 80% reduction of Stat5 levels in mammary epithelium and a concomitant reduction of STAT5-dependent gene expression. Transcriptome analysis identified a class of mammary-restricted genes that was particularly dependent on high STAT5 levels as a result of the intergenic enhancer. Taken together, the mammary-specific enhancer enables a positive feedback circuit that contributes to the remarkable abundance of STAT5 and, in turn, to the efficacy of STAT5-dependent mammary physiology.

  17. 9 CFR 310.17 - Inspection of mammary glands.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... mammary glands and diseased mammary glands of cattle, sheep, swine, and goats shall be removed without opening the milk ducts or sinuses. If pus or other objectionable material is permitted to come in contact..., swine, and goats shall not be saved for edible purposes. (d) The udders from cows officially...

  18. 9 CFR 310.17 - Inspection of mammary glands.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... mammary glands and diseased mammary glands of cattle, sheep, swine, and goats shall be removed without opening the milk ducts or sinuses. If pus or other objectionable material is permitted to come in contact..., swine, and goats shall not be saved for edible purposes. (d) The udders from cows officially...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. Mammary Development and Breast Cancer: A Wnt Perspective

    PubMed Central

    Yu, Qing Cissy; Verheyen, Esther M.; Zeng, Yi Arial

    2016-01-01

    The Wnt pathway has emerged as a key signaling cascade participating in mammary organogenesis and breast oncogenesis. In this review, we will summarize the current knowledge of how the pathway regulates stem cells and normal development of the mammary gland, and discuss how its various components contribute to breast carcinoma pathology. PMID:27420097

  1. Mammary Development and Breast Cancer: A Wnt Perspective.

    PubMed

    Yu, Qing Cissy; Verheyen, Esther M; Zeng, Yi Arial

    2016-01-01

    The Wnt pathway has emerged as a key signaling cascade participating in mammary organogenesis and breast oncogenesis. In this review, we will summarize the current knowledge of how the pathway regulates stem cells and normal development of the mammary gland, and discuss how its various components contribute to breast carcinoma pathology. PMID:27420097

  2. P-cadherin expression in feline mammary tissues.

    PubMed

    Figueira, Ana Catarina; Teodósio, Ana Sofia; Carvalheira, Júlio; Lacerda, Manuela; de Matos, Augusto; Gärtner, Fátima

    2012-01-01

    The search for molecular markers in the feline mammary gland, namely, the adhesion molecules belonging to the cadherin family, is useful in the understanding of the development of mammary carcinomas in felines and humans. To study P-cadherin expression in the feline mammary gland, 61 samples of normal (n = 4), hyperplastic (n = 12), and neoplastic (n = 45) feline mammary tissues were examined. In both normal and hyperplastic mammary tissues as well as in benign tumours, P-cadherin immunolabelling was restricted to myoepithelial cells. In malignant tumours, however, there was an aberrant epithelial P-cadherin immunoexpression in 64.1% (n = 25) of cases, with a membranous and/or cytoplasmic pattern of distribution. A statistically significant relationship was seen between epithelial P-cadherin expression and malignant mammary lesions (P = 0.0001). In malignant mammary tumours, there was likewise a statistically significant relationship between aberrant P-cadherin immunoexpression and histological grade (P = 0.0132). Aberrant epithelial P-cadherin expression seems to be related to malignancy in the feline mammary gland. To confirm the results of this investigation, further studies with larger samples and follow-up studies are warranted.

  3. Sustained trophism of the mammary gland is sufficient to accelerate and synchronize development of ErbB2/Neu-induced tumors

    PubMed Central

    Landis, MD; Seachrist, DD; Abdul-Karim, FW; Keri, RA

    2006-01-01

    Epidemiological studies indicate that parity enhances HER2/ErbB2/Neu-induced breast tumorigenesis. Furthermore, recent studies using multiparous, ErbB2/Neu-overexpressing mouse mammary tumor virus (MMTV-Neu) mice have shown that parity induces a population of cells that are targeted for ErbB2/Neu-induced transformation. Although parity accelerates mammary tumorigenesis, the pattern of tumor development in multiparous MMTV-Neu mice remains stochastic, suggesting that additional events are required for ErbB2/Neu to cause mammary tumors. Whether such events are genetic in nature or reflective of the dynamic hormonal control of the gland that occurs with pregnancy remains unclear. We postulated that young age at pregnancy initiation or chronic trophic maintenance of mammary epithelial cells might provide a cellular environment that significantly increases susceptibility to ErbB2/Neu-induced tumorigenesis. MMTV-Neu mice that were maintained pregnant or lactating beginning at 3 weeks of age demonstrated accelerated tumorigenesis, but this process was still stochastic, indicating that early pregnancy does not provide the requisite events of tumorigenesis. However, bitransgenic mice that were generated by breeding MMTV-Neu mice with a luteinizing hormone-overexpressing mouse model of ovarian hyperstimulation developed multifocal mammary tumors in an accelerated, synchronous manner compared to virgin MMTV-Neu animals. This synchrony of tumor development in the bitransgenic mice suggests that trophic maintenance of the mammary gland provides the additional events required for tumor formation and maintains the population of cells that are targeted by ErbB2/Neu for transformation. Both the synchrony of tumor appearance and the ability to characterize a window of commitment by ovariectomy/palpation studies permitted microarray analysis to evaluate changes in gene expression over a defined timeline that spans the progression from normal to preneoplastic mammary tissue. These

  4. Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis

    PubMed Central

    Karantza-Wadsworth, Vassiliki; Patel, Shyam; Kravchuk, Olga; Chen, Guanghua; Mathew, Robin; Jin, Shengkan; White, Eileen

    2007-01-01

    Autophagy is a catabolic process involving self-digestion of cellular organelles during starvation as a means of cell survival; however, if it proceeds to completion, autophagy can lead to cell death. Autophagy is also a haploinsufficient tumor suppressor mechanism for mammary tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in breast carcinomas. However, the mechanism by which autophagy suppresses breast cancer remains elusive. Here we show that allelic loss of beclin1 and defective autophagy sensitized mammary epithelial cells to metabolic stress and accelerated lumen formation in mammary acini. Autophagy defects also activated the DNA damage response in vitro and in mammary tumors in vivo, promoted gene amplification, and synergized with defective apoptosis to promote mammary tumorigenesis. Therefore, we propose that autophagy limits metabolic stress to protect the genome, and that defective autophagy increases DNA damage and genomic instability that ultimately facilitate breast cancer progression. PMID:17606641

  5. Prenatal TCDD Exposure Predisposes for Mammary Cancer in Rats

    PubMed Central

    Jenkins, Sarah; Rowell, Craig; Wang, Jun; Lamartiniere, Coral A.

    2007-01-01

    Epidemiological data are conflicting in the link between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure and breast cancer causation. We have hypothesized that timing of exposure to endocrine disruptors, such as TCDD, will alter breast cancer susceptibility. Using a carcinogen induced rat mammary cancer model, we have shown that prenatal exposure to TCDD alters mammary gland differentiation and increases susceptibility for mammary cancer. Investigations into imprinting via DNA methylation mechanisms showed that there were no changes in protein expression in DNA methyltransferases, ER-alpha, ER-beta, GST-pi, or MDGI. Using 2-D gels and mass spectrometry, we have found seven proteins to be differentially regulated, including a decrease in superoxide dismutase 1 (SOD1). Down-regulation of SOD1 could provide an environment ill equipped to deal with subsequent free radical exposure. We conclude that prenatal TCDD can predispose for mammary cancer susceptibility in the adult offspring by altering the mammary proteome. PMID:17157473

  6. 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.

  7. 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

  8. Genistein-mediated inhibition of mammary stromal adipocyte differentiation limits expansion of mammary stem/progenitor cells by paracrine signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mammary adiposity may contribute to breast cancer development and progression by releasing cytokines and other inflammatory mediators that promote mammary epithelial proliferation. We evaluated the effects of soy isoflavone genistein (GEN) on the adipogenic differentiation of a SV40-immortalized mou...

  9. Annexin A8 Identifies a Subpopulation of Transiently Quiescent c-Kit Positive Luminal Progenitor Cells of the Ductal Mammary Epithelium

    PubMed Central

    Iglesias, Juan Manuel; Cairney, Claire J.; Ferrier, Roderick K.; McDonald, Laura; Soady, Kelly; Kendrick, Howard; Pringle, Marie-Anne; Morgan, Reginald O.; Martin, Finian; Smalley, Matthew J.; Blyth, Karen; Stein, Torsten

    2015-01-01

    We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G0/G1 arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin. PMID:25803307

  10. Effectiveness of photodynamic therapy for mammary and extra-mammary Paget's disease: a state of the science review

    PubMed Central

    2011-01-01

    Background Paget's disease is a rare skin disorder occurring in the breast (mammary) or in the groin, genital, peri-anal and axillary regions (extra-mammary). Typical treatment involves surgical excision, which in the case of extra-mammary Paget's disease, can lead to significant morbidity. Photodynamic therapy (PDT) which uses a topical or intravenous photosensitizing agent that is activated by a light source to ablate abnormal tissue, offers a minimally invasive alternative. The purpose of this study was to assess the effectiveness of photodynamic therapy in the treatment of Paget's disease. Methods Following Cochrane guidelines, a comprehensive systematic review of all clinical studies and reports examining the use of PDT for mammary and extra-mammary Paget's disease was conducted. Study quality was assessed using the Oxford Levels of Evidence Scale. Results 21 retrospective and 2 prospective non-comparative studies were identified and included in the review: 9 case reports with 1-2 patients and 14 case series with 1-16 patients. These reports totalled 99 patients with 133 extra-mammary Paget's lesions and 3 patients (with 3 lesions) with mammary Paget's disease. Follow-up periods were typically one year or less, with 77/133 extra-mammary lesions exhibiting complete response to PDT. One recurrent mammary skin lesion and two mammary lesions treated concomitantly with surgery also exhibited complete responses. Conclusions Evidence of the effectiveness of PDT for Paget's disease is promising, but limited. This may, in part, be explained by the rarity of the condition, making controlled comparative clinical trials challenging. PMID:21676258

  11. Surface scanning: an application to mammary surgery

    NASA Astrophysics Data System (ADS)

    Rigotti, Camilla; Ferrigno, Giancarlo; Aliverti, Andrea; Pedotti, Antonio

    1998-04-01

    The possibility of mathematically describing the body surface represents a useful tool for several medical sectors, such as prosthetics or plastic surgery, and could improve diagnosis and objective evaluation of deformities and the follow-up of progressive diseases. The approach presented is based on the acquisition of a surface scanned by a laser beam. The 3D coordinates of the spot generated on the surface by the laser beam are computed by an automatic image analyzer. Using at least two different views of the subject, the 3D coordinates are obtained by stereophotogrammetry. A software package for graphic representation and extraction of linear superficial and volumetric features from the acquired surface has been developed and some preliminary results with mammary reconstruction are presented. A good mammary reconstruction after mastectomy must achieve two results. First, the reconstruction should follow the patients' wishes and second, the reconstructed breast should be as similar as possible to the contralateral one. To achieve these goals, a knowledge of breast volume, area, and shape features are essential for the surgeon. In such a context, this system could be a valuable tool in improving breast reconstructive surgery.

  12. Gene expression pattern in canine mammary osteosarcoma.

    PubMed

    Pawłowski, K M; Majewska, A; Szyszko, K; Dolka, I; Motyl, T; Król, M

    2011-01-01

    Canine mammary sarcomas are usually very aggressive and easily metastasize. Unfortunately the biology of this type of tumor is not well known because they are a very rare type of tumors. The aim of this study was to find differences in gene expression patterns in canine mammary osteosarcomas (malignant) versus osteomas (benign) using DNA microarrays. Our microarray experiment showed that 11 genes were up-regulated in osteosarcoma in comparison to osteoma whereas 36 genes were down-regulated. Among the up-regulated genes were: PDK1, EXT1, and EIF4H which are involved in AKT/PI3K and GLI/Hedgehog pathways. These genes play an important role in cell biology (cancer cell proliferation) and may be essential in osteosarcoma formation and development. Analyzing the down-regulated genes, the most interesting seemed to be HSPB8 and SEPP1. HSPB8 is a small heat shock protein that plays an important role in cell cycle regulation, apoptosis, and breast carcinogenesis. Also SEPP1 may play a role in carcinogenesis, as its down-regulation may induce oxidative stress possibly resulting in carcinogenesis. The preliminary results of the present study indicate that the up-regulation of three genes EXT1, EIF4H, and PDK1 may play an essential role in osteosarcoma formation, development and proliferation. In our opinion the cross-talk between GLI/Hedgehog and PI3K/AKT pathways may be a key factor to increase tumor proliferation and malignancy. PMID:21528706

  13. Mammary expression of new genes to combat mastitis.

    PubMed

    Kerr, D E; Wellnitz, O

    2003-01-01

    Continual advances in the ability to produce transgenic animals make it likely that such animals will become important components of animal agriculture. The full benefit of the technology, and justification of its initial cost outlay, will be dependent on the establishment within these animals of new traits not easily achievable by other means. Potential applications include enhanced nutrient digestibility with reduced fecal losses, significantly altered milk composition with superior nutritional properties, and enhanced disease resistance. Our goal is to enhance mastitis resistance of dairy cows by enabling the cells of the mammary gland to secrete additional antibacterial proteins. Proof of concept has been obtained through experimentation with a transgenic mouse model. Three lines of mice were developed that produce varying levels of lysostaphin in their milk. This protein has potent anti-staphylococcal activity and its secretion into milk confers substantial resistance to infection caused by intramammary challenge with Staphylococcus aureus, a major mastitis pathogen. Additional antibacterial proteins are being sought that will complement lysostaphin. A potential benefit of transgenic application of antibacterial proteins is the concomitant sparing in the agricultural use of antibiotics currently used as human therapeutics. Antibacterial proteins, such as lysostaphin, are not typically used as injectable or oral therapeutics because of immune-mediated or digestive destruction of their activity. In contrast, the immune system of transgenic animals will not consider the transgenic protein as being foreign. In addition we are exploring the potential of involution or mastitis responsive promoter elements for use in subsequent transgenic experiments designed to restrict lysostaphin production to these important time points. It is anticipated that genomics will play a role in unveiling candidate genes whose promoter elements will enable desired temporal expression

  14. Fibronectin Expression Modulates Mammary Epithelial Cell Proliferation during Acinar Differentiation

    PubMed Central

    Williams, Courtney M.; Engler, Adam J.; Slone, R. Daniel; Galante, Leontine L.; Schwarzbauer, Jean E.

    2009-01-01

    The mammary gland consists of a polarized epithelium surrounded by a basement membrane matrix that forms a series of branching ducts ending in hollow, sphere-like acini. Essential roles for the epithelial basement membrane during acinar differentiation, in particular laminin and its integrin receptors, have been identified using mammary epithelial cells cultured on a reconstituted basement membrane. Contributions from fibronectin, which is abundant in the mammary gland during development and tumorigenesis, have not been fully examined. Here, we show that fibronectin expression by mammary epithelial cells is dynamically regulated during the morphogenic process. Experiments with synthetic polyacrylamide gel substrates implicate both specific extracellular matrix components, including fibronectin itself, and matrix rigidity in this regulation. Alterations in fibronectin levels perturbed acinar organization. During acinar development, increased fibronectin levels resulted in overproliferation of mammary epithelial cells and increased acinar size. Addition of fibronectin to differentiated acini stimulated proliferation and reversed growth arrest of mammary epithelial cells negatively affecting maintenance of proper acinar morphology. These results show that expression of fibronectin creates a permissive environment for cell growth that antagonizes the differentiation signals from the basement membrane. These effects suggest a link between fibronectin expression and epithelial cell growth during development and oncogenesis in the mammary gland. PMID:18451144

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

    PubMed

    Haricharan, S; Li, Y

    2014-01-25

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

  16. The mammary gland and its origin during synapsid evolution.

    PubMed

    Oftedal, Olav T

    2002-07-01

    Lactation appears to be an ancient reproductive trait that predates the origin of mammals. The synapsid branch of the amniote tree that separated from other taxa in the Pennsylvanian (>310 million years ago) evolved a glandular rather than scaled integument. Repeated radiations of synapsids produced a gradual accrual of mammalian features. The mammary gland apparently derives from an ancestral apocrine-like gland that was associated with hair follicles. This association is retained by monotreme mammary glands and is evident as vestigial mammary hair during early ontogenetic development of marsupials. The dense cluster of mammo-pilo-sebaceous units that open onto a nipple-less mammary patch in monotremes may reflect a structure that evolved to provide moisture and other constituents to permeable eggs. Mammary patch secretions were coopted to provide nutrients to hatchlings, but some constituents including lactose may have been secreted by ancestral apocrine-like glands in early synapsids. Advanced Triassic therapsids, such as cynodonts, almost certainly secreted complex, nutrient-rich milk, allowing a progressive decline in egg size and an increasingly altricial state of the young at hatching. This is indicated by the very small body size, presence of epipubic bones, and limited tooth replacement in advanced cynodonts and early mammaliaforms. Nipples that arose from the mammary patch rendered mammary hairs obsolete, while placental structures have allowed lactation to be truncated in living eutherians. PMID:12751889

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

    PubMed Central

    2013-01-01

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

  18. Mammary Epithelial Cell Hierarchy in the Dairy Cow Throughout Lactation.

    PubMed

    Perruchot, Marie-Hélène; Arévalo-Turrubiarte, Magdalena; Dufreneix, Florence; Finot, Laurence; Lollivier, Vanessa; Chanat, Eric; Mayeur, Frédérique; Dessauge, Frédéric

    2016-10-01

    The plasticity of the mammary gland relies on adult mammary stem cells (MaSCs) and their progenitors, which give rise to various populations of mammary epithelial cells (MECs). To face global challenges, an in-depth characterization of milk-producing animal mammary gland plasticity is required, to select more sustainable and robust dairy cows. The identification and characterization of MaSC and their progenitors will also provide innovative tools in veterinary/human medicine regarding mammary tissue damage (carcinogenesis, bacterial infections). This study aimed to determine the dynamics of mammary cell populations throughout a lactation cycle. Using mammary biopsies from primiparous lactating dairy cows at 30, 90, 150, and 250 days of lactation, we phenotyped cell populations by flow cytometry. To investigate cell lineages, we used specific cell-surface markers, including CD49f, CD24, EpCAM (epithelial cell adhesion molecule), and CD10. Two cell populations linked to milk production were identified: CD49f(+)/EpCAM(-) (y = 0.88x + 4.42, R(2) = 0.36, P < 0.05) and CD49f(-)/EpCAM(-) (y = -1.15x + 92.44, R(2) = 0.51, P < 0.05) cells. Combining immunostaining analysis, flow cytometry, daily milk production data, and statistical approaches, we defined a stem cell population (CD24(+)/CD49f(+)) and four progenitor cell populations that include bipotent luminal progenitors (CD24(-)/CD49f(+)), lumino-alveolar progenitors (CD24(-)/EpCAM(+)), myoepithelial progenitors (CD24(+)/CD10(-)), and lumino-ductal progenitors (CD49f(-)/EpCAM(+)). Interestingly, we found that the bipotent luminal progenitors (CD24(-)/CD49f(+)) decreased significantly (P < 0.05) during lactation. This study provides the first results of mammary cell lineage, allowing insight into mammary cell plasticity during lactation.

  19. Mammary Epithelial Cell Hierarchy in the Dairy Cow Throughout Lactation.

    PubMed

    Perruchot, Marie-Hélène; Arévalo-Turrubiarte, Magdalena; Dufreneix, Florence; Finot, Laurence; Lollivier, Vanessa; Chanat, Eric; Mayeur, Frédérique; Dessauge, Frédéric

    2016-10-01

    The plasticity of the mammary gland relies on adult mammary stem cells (MaSCs) and their progenitors, which give rise to various populations of mammary epithelial cells (MECs). To face global challenges, an in-depth characterization of milk-producing animal mammary gland plasticity is required, to select more sustainable and robust dairy cows. The identification and characterization of MaSC and their progenitors will also provide innovative tools in veterinary/human medicine regarding mammary tissue damage (carcinogenesis, bacterial infections). This study aimed to determine the dynamics of mammary cell populations throughout a lactation cycle. Using mammary biopsies from primiparous lactating dairy cows at 30, 90, 150, and 250 days of lactation, we phenotyped cell populations by flow cytometry. To investigate cell lineages, we used specific cell-surface markers, including CD49f, CD24, EpCAM (epithelial cell adhesion molecule), and CD10. Two cell populations linked to milk production were identified: CD49f(+)/EpCAM(-) (y = 0.88x + 4.42, R(2) = 0.36, P < 0.05) and CD49f(-)/EpCAM(-) (y = -1.15x + 92.44, R(2) = 0.51, P < 0.05) cells. Combining immunostaining analysis, flow cytometry, daily milk production data, and statistical approaches, we defined a stem cell population (CD24(+)/CD49f(+)) and four progenitor cell populations that include bipotent luminal progenitors (CD24(-)/CD49f(+)), lumino-alveolar progenitors (CD24(-)/EpCAM(+)), myoepithelial progenitors (CD24(+)/CD10(-)), and lumino-ductal progenitors (CD49f(-)/EpCAM(+)). Interestingly, we found that the bipotent luminal progenitors (CD24(-)/CD49f(+)) decreased significantly (P < 0.05) during lactation. This study provides the first results of mammary cell lineage, allowing insight into mammary cell plasticity during lactation. PMID:27520504

  20. RNA synthesis in isolated nuclei of lactating mammary cells in presence of unmodified and mercury-labeled CTP.

    PubMed Central

    Ganguly, R; Banerjee, M R

    1978-01-01

    Isolated nuclei of lactating mouse mammary gland were capable of supporting DNA-dependent RNA synthesis in vitro in presence of unmodified and mercurated CTP (Hg-CTP) at high ionic condition at 25 degrees C. In presence of unmodified CTP, [3H]UMP incorporation into RNA increased linearly upto 180 min. The kinetic pattern of the reaction and the rate of RNA synthesis were essentially similar when CTP was replaced by Hg-CTP. Both in unmodified and Hg-CTP containing reactions, 70-80% of RNA synthesis was inhibited by alpha-amanitin. Presence of poly(A) in a small portion of the in vitro synthesized messenger-like RNA was detectable by oligo(dT) cellulose chromatography. Both poly(A)+ and poly(A)- RNAs sedimented with a clear peak around 15S region in a formamide-sucrose denaturing gradient. The Hg-RNA after separation from endogenous nuclear RNA by SH-agarose affinity column chromatography also sedimented around 15S region in a formamide-sucrose gradient. The Hg-RNA synthesized in the isolated mammary cell nuclei in vitro should now permit monitoring hormonal regulation of specific gene (casein) transcription in the mammary cells by molecular hybridization of the Hg-RNA with cDNA to casein mRNA. PMID:724523

  1. Miz1 Deficiency in the Mammary Gland Causes a Lactation Defect by Attenuated Stat5 Expression and Phosphorylation

    PubMed Central

    Sanz-Moreno, Adrián; Fuhrmann, David; Wolf, Elmar; von Eyss, Björn; Eilers, Martin; Elsässer, Hans-Peter

    2014-01-01

    Miz1 is a zinc finger transcription factor with an N-terminal POZ domain. Complexes with Myc, Bcl-6 or Gfi-1 repress expression of genes like Cdkn2b (p15Ink4) or Cdkn1a (p21Cip1). The role of Miz1 in normal mammary gland development has not been addressed so far. Conditional knockout of the Miz1 POZ domain in luminal cells during pregnancy caused a lactation defect with a transient reduction of glandular tissue, reduced proliferation and attenuated differentiation. This was recapitulated in vitro using mouse mammary gland derived HC11 cells. Further analysis revealed decreased Stat5 activity in Miz1ΔPOZ mammary glands and an attenuated expression of Stat5 targets. Gene expression of the Prolactin receptor (PrlR) and ErbB4, both critical for Stat5 phosphorylation (pStat5) or pStat5 nuclear translocation, was decreased in Miz1ΔPOZ females. Microarray, ChIP-Seq and gene set enrichment analysis revealed a down-regulation of Miz1 target genes being involved in vesicular transport processes. Our data suggest that deranged intracellular transport and localization of PrlR and ErbB4 disrupt the Stat5 signalling pathway in mutant glands and cause the observed lactation phenotype. PMID:24586582

  2. Genetic suppression reveals DNA repair-independent antagonism between BRCA1 and COBRA1 in mammary gland development.

    PubMed

    Nair, Sreejith J; Zhang, Xiaowen; Chiang, Huai-Chin; Jahid, Md Jamiul; Wang, Yao; Garza, Paula; April, Craig; Salathia, Neeraj; Banerjee, Tapahsama; Alenazi, Fahad S; Ruan, Jianhua; Fan, Jian-Bing; Parvin, Jeffrey D; Jin, Victor X; Hu, Yanfen; Li, Rong

    2016-01-01

    The breast cancer susceptibility gene BRCA1 is well known for its function in double-strand break (DSB) DNA repair. While BRCA1 is also implicated in transcriptional regulation, the physiological significance remains unclear. COBRA1 (also known as NELF-B) is a BRCA1-binding protein that regulates RNA polymerase II (RNAPII) pausing and transcription elongation. Here we interrogate functional interaction between BRCA1 and COBRA1 during mouse mammary gland development. Tissue-specific deletion of Cobra1 reduces mammary epithelial compartments and blocks ductal morphogenesis, alveologenesis and lactogenesis, demonstrating a pivotal role of COBRA1 in adult tissue development. Remarkably, these developmental deficiencies due to Cobra1 knockout are largely rescued by additional loss of full-length Brca1. Furthermore, Brca1/Cobra1 double knockout restores developmental transcription at puberty, alters luminal epithelial homoeostasis, yet remains deficient in homologous recombination-based DSB repair. Thus our genetic suppression analysis uncovers a previously unappreciated, DNA repair-independent function of BRCA1 in antagonizing COBRA1-dependent transcription programme during mammary gland development. PMID:26941120

  3. Truncating Prolactin Receptor Mutations Promote Tumor Growth in Murine Estrogen Receptor-Alpha Mammary Carcinomas.

    PubMed

    Griffith, Obi L; Chan, Szeman Ruby; Griffith, Malachi; Krysiak, Kilannin; Skidmore, Zachary L; Hundal, Jasreet; Allen, Julie A; Arthur, Cora D; Runci, Daniele; Bugatti, Mattia; Miceli, Alexander P; Schmidt, Heather; Trani, Lee; Kanchi, Krishna-Latha; Miller, Christopher A; Larson, David E; Fulton, Robert S; Vermi, William; Wilson, Richard K; Schreiber, Robert D; Mardis, Elaine R

    2016-09-27

    Estrogen receptor alpha-positive (ERα+) luminal tumors are the most frequent subtype of breast cancer. Stat1(-/-) mice develop mammary tumors that closely recapitulate the biological characteristics of this cancer subtype. To identify transforming events that contribute to tumorigenesis, we performed whole genome sequencing of Stat1(-/-) primary mammary tumors and matched normal tissues. This investigation identified somatic truncating mutations affecting the prolactin receptor (PRLR) in all tumor and no normal samples. Targeted sequencing confirmed the presence of these mutations in precancerous lesions, indicating that this is an early event in tumorigenesis. Functional evaluation of these heterozygous mutations in Stat1(-/-) mouse embryonic fibroblasts showed that co-expression of truncated and wild-type PRLR led to aberrant STAT3 and STAT5 activation downstream of the receptor, cellular transformation in vitro, and tumor formation in vivo. In conclusion, truncating mutations of PRLR promote tumor growth in a model of human ERα+ breast cancer and warrant further investigation. PMID:27681435

  4. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

    SciTech Connect

    Robinson, Claire; Kolb, Andreas F.

    2009-02-01

    The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A {beta}-galactosidase reporter gene was inserted in place of the {beta}-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the {beta}-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal {beta}-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the {beta}-casein gene.

  5. Isolation and sequence characterization of mammary derived growth inhibitor gene of riverine buffalo (Bubalus bubalis).

    PubMed

    Mukesh, M; Mishra, B P; Kataria, R S; Ahlawat, S P S; Sobti, R C

    2007-01-01

    In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5' UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3' UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI/fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.

  6. Extra-mammary findings on breast MRI: a pictorial review.

    PubMed

    Karp, Norna L; Price, Elissa R; Wisner, Dorota J; Chang, C Belinda; Hylton, Nola M; Joe, Bonnie N

    2015-01-01

    Recent improvements in breast coil performance have made detection of extra-mammary findings increasingly common. Some of these findings have important clinical implications. The radiologist should be aware of the spectrum of extra-mammary pathologies found on breast magnetic resonance imaging (MRI) and be able to distinguish clinically significant findings from those that are inconsequential. The purpose of this essay is to demonstrate various common and uncommon extra-mammary findings encountered while interpreting breast MRI and to detail appropriate management recommendations.

  7. Bone sialoprotein mediates the tumor cell-targeted prometastatic activity of TGF-β in a mouse model of breast cancer.

    PubMed Central

    Nam, Jeong-Seok; Suchar, Adam M.; Kang, Mi-Jin; Stuelten, Christina H.; Tang, Binwu; Michalowska, Aleksandra M.; Fisher, Larry W.; Fedarko, Neal S.; Jain, Alka; Pinkas, Jan; Lonning, Scott; Wakefield, Lalage M.

    2006-01-01

    Transforming growth factor-βs (TGF-βs) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as pro-metastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-β antagonists can suppress metastasis without the predicted toxicities (Yang et al., J. Clin. Invest. (2002) 109:1607-1615). To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer. Treatment of mice with a monoclonal anti-TGF-β antibody (1D11) significantly suppressed metastasis of 4T1 cells to the lungs. When metastatic 4T1 cells were recovered from lungs of 1D11-treated and control mice, the most differentially expressed gene was found to be bone sialoprotein (Bsp). Immunostaining confirmed the loss of Bsp protein in 1D11-treated lung metastases, and TGF-β was shown to regulate and correlate with Bsp expression in vitro. Functionally, knockdown of Bsp in 4T1 cells reduced the ability of TGF-β to induce local collagen degradation and invasion in vitro, and treatment with recombinant BSP protected 4T1 cells from complement-mediated lysis. Finally, suppression of Bsp in 4T1 cells reduced metastasis in vivo. We conclude that Bsp is a plausible mediator of at least some of the tumor cell-targeted prometastatic activity of TGF-β in this model, and that Bsp expression in metastases can be successfully suppressed by systemic treatment with anti-TGF-β antibodies. PMID:16778210

  8. Peroxisome proliferator-activated receptor δ (PPARδ) induces estrogen receptor-positive mammary neoplasia through an inflammatory and metabolic phenotype linked to mTor activation

    PubMed Central

    Yuan, Hongyan; Lu, Jin; Xiao, Junfeng; Upadhyay, Geeta; Umans, Rachel; Kallakury, Bhaskar; Yin, Yuhzi; Fant, Michael E.; Kopelovich, Levy; Glazer, Robert I.

    2013-01-01

    The peroxisome proliferator-activated receptor-δ (PPARδ) regulates a multitude of physiological processes associated with glucose and lipid metabolism, inflammation and proliferation. One or more of these processes are potential risk factors for the ability of PPARδ agonists to promote tumorigenesis in the mammary gland. In the present study, we describe a new transgenic mouse model in which activation of PPARδ in the mammary epithelium by endogenous or synthetic ligands resulted in progressive histopathological changes that culminated in the appearance of estrogen receptor- and progesterone receptor-positive and ErbB2-negative infiltrating ductal carcinomas. Multiparous mice presented with mammary carcinomas after a latency of 12 months, and administration of the PPARδ ligand GW501516 reduced tumor latency to five months. Histopathological changes occurred concurrently with an increase in an inflammatory, invasive, metabolic and proliferative gene signature, including expression of the trophoblast gene, Plac1, beginning one week after GW501516 treatment, and remained elevated throughout tumorigenesis. The appearance of malignant changes correlated with a pronounced increase in phosphatidylcholine and lysophosphatidic acid metabolites, which coincided with activation of Akt and mTor signaling that were attenuated by treatment with the mTor inhibitor everolimus. Our findings are the first to demonstrate a direct role of PPARδ in the pathogenesis of mammary tumorigenesis, and suggest a rationale for therapeutic approaches to prevent and treat this disease. PMID:23811944

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

    PubMed

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

    2016-11-15

    The regulation of the tammar wallaby (Macropus eugenii) early lactation protein (ELP) gene is complex. ELP is responsive to the lactogenic hormones; insulin (I), hydrocortisone (HC) and prolactin (PRL) in mammary gland explants but could not be induced with lactogenic hormones in tammar primary mammary gland cells, nor in KIM-2 conditionally immortalised murine mammary epithelial cells. Similarly, ELP promoter constructs transiently-transfected into human embryonic kidney (HEK293T) cells constitutively expressing the prolactin receptor (PRLR) and Signal Transducer and Activator of Transcription (STAT)5A were unresponsive to prolactin, unlike the rat and mouse β-casein (CSN2) promoter constructs. Identification of the minimal promoter required for the hormone-independent transcription of tammar ELP in HEK293Ts and comparative analysis of the proximal promoters of marsupial ELP and the orthologous eutherian colostrum trypsin inhibitor (CTI) gene suggests that mammary cell-activating factor (MAF), an E26 transformation-specific (ETS) factor, may bind to an AGGAAG motif and activate tammar ELP. PMID:27452799

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

    PubMed

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

    2016-11-15

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

  11. Mammary Stem Cells and Tumor-Initiating Cells Are More Resistant to Apoptosis and Exhibit Increased DNA Repair Activity in Response to DNA Damage

    PubMed Central

    Chang, Chi-Hsuan; Zhang, Mei; Rajapakshe, Kimal; Coarfa, Cristian; Edwards, Dean; Huang, Shixia; Rosen, Jeffrey M.

    2015-01-01

    Summary Adult stem cells and tumor-initiating cells (TICs) often employ different mechanisms of DNA damage response (DDR) as compared to other tissue cell types. However, little is known about how mammary stem cells (MaSCs) and mammary TICs respond to DNA damage. Using the mouse mammary gland and syngeneic p53-null tumors as models, we investigated the molecular and physiological consequences of DNA damage in wild-type MaSCs, p53-null MaSCs, and p53-null TICs. We showed that wild-type MaSCs and basal cells are more resistant to apoptosis and exhibit increased non-homologous end joining (NHEJ) activity. Loss of p53 in mammary epithelium affected both cell-cycle regulation and DNA repair efficiency. In p53-null tumors, we showed that TICs are more resistant to ionizing radiation (IR) due to decreased apoptosis, elevated NHEJ activity, and more-rapid DNA repair. These results have important implications for understanding DDR mechanisms involved in both tumorigenesis and therapy resistance. PMID:26300228

  12. The use of alternative polyadenylation sites renders integrin β1 (Itgb1) mRNA isoforms with differential stability during mammary gland development.

    PubMed

    Naipauer, Julian; Gattelli, Albana; Degese, Maria Sol; Slomiansky, Victoria; Wertheimer, Eva; LaMarre, Jonathan; Castilla, Lucio; Abba, Martin; Kordon, Edith C; Coso, Omar A

    2013-09-01

    Integrins are heterodimeric cell-surface adhesion receptors that play a critical role in tissue development. Characterization of the full-length mRNA encoding the β1 subunit (Itgb1) revealed an alternative functional cleavage and polyadenylation site that yields a new Itgb1 mRNA isoform 578 bp shorter than that previously reported. Using a variety of experimental and bioinformatic approaches, we found that the two Itgb1 isoforms are expressed at different levels in a variety of mouse tissues, including the mammary gland, where they are differentially regulated at successive developmental stages. The longer mRNA species is prevelant during lactation, whereas the shorter is induced after weaning. In 3D cultures, where expression of integrin β1 protein is required for normal formation of acini, experimental blockade of the longer isoform induced enhanced expression of the shorter species which allowed normal morphological mammary differentiation. The short isoform lacks AU-rich motifs and miRNA target sequences that are potentially implicated in the regulation of mRNA stability and translation efficiency. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. In summary, the results of the present study identify a new regulatory instance involved in the fine-tuning of Itgb1 expression during mammary gland development and function.

  13. Erbb4 Signaling in the Mammary Gland Is Required for Lobuloalveolar Development and Stat5 Activation during Lactation

    PubMed Central

    Jones, Frank E.; Welte, Thomas; Fu, Xin-Yuan; Stern, David F.

    1999-01-01

    Signaling by members of the epidermal growth factor receptor family plays an important role in breast development and breast cancer. Earlier work suggested that one of these receptors, ErbB4, is coupled to unique responses in this tissue. To determine the function of ErbB4 signaling in the normal mouse mammary gland, we inactivated ErbB4 signaling by expressing a COOH terminally deleted dominant-negative allele of ErbB4 (ErbB4ΔIC) as a transgene in the mammary gland. Despite the expression of ErbB4ΔIC from puberty through later stages of mammary development, an ErbB4ΔIC-specific phenotype was not observed until mid-lactation. At 12-d postpartum, lobuloalveoli expressing ErbB4ΔIC protein were condensed and lacked normal lumenal lactation products. In these lobuloalveoli, β-casein mRNA, detected by in situ hybridization, was normal. However, whey acidic protein mRNA was reduced, and α-lactalbumin mRNA was undetectable. Stat5 expression was detected by immunohistochemistry in ErbB4ΔIC-expressing tissue. However, Stat5 was not phosphorylated at Y694 and was, therefore, probably inactive. When expressed transiently in 293T cells, ErbB4 induced phosphorylation of Stat5. This phosphorylation required an intact Stat5 SH2 domain. In summary, our results demonstrate that ErbB4 signaling is necessary for mammary terminal differentiation and Stat5 activation at mid-lactation. PMID:10508857

  14. IL-17A Is an Important Effector of the Immune Response of the Mammary Gland to Escherichia coli Infection.

    PubMed

    Porcherie, Adeline; Gilbert, Florence B; Germon, Pierre; Cunha, Patricia; Trotereau, Angélina; Rossignol, Christelle; Winter, Nathalie; Berthon, Patricia; Rainard, Pascal

    2016-01-15

    The cytokine IL-17A has been shown to play critical roles in host defense against bacterial and fungal infections at different epithelial sites, but its role in the defense of the mammary gland (MG) has seldom been investigated, although infections of the MG constitute the main pathology afflicting dairy cows. In this study, we showed that IL-17A contributes to the defense of the MG against Escherichia coli infection by using a mouse mastitis model. After inoculation of the MG with a mastitis-causing E. coli strain, the bacterial load increased rapidly, triggering an intense influx of leukocytes into mammary tissue and increased concentrations of IL-6, IL-22, TNF-α, and IL-10. Neutrophils were the first cells that migrated intensely to the mammary tissue, in line with an early production of CXCL2. Depletion of neutrophils induced an increased mammary bacterial load. There was a significant increase of IL-17-containing CD4(+) αβ T lymphocyte numbers in infected glands. Depletion of IL-17A correlated with an increased bacterial colonization and IL-10 production. Intramammary infusion of IL-17A at the onset of infection was associated with markedly decreased bacterial numbers, decreased IL-10 production, and increased neutrophil recruitment. Depletion of CD25(+) regulatory T cells correlated with a decreased production of IL-10 and a reduced bacterial load. These results indicate that IL-17A is an important effector of MG immunity to E. coli and suggest that an early increased local production of IL-17A would improve the outcome of infection. These findings point to a new lead to the development of vaccines against mastitis. PMID:26685206

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

    PubMed Central

    Gurzov, Esteban N; Nabha, Sanaa M; Yamamoto, Hamilto; Meng, Hong; Scharovsky, O Graciela; Bonfil, R Daniel

    2007-01-01

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

  16. Modeling Prolactin Actions in Breast Cancer in vivo: Insights from the NRL-PRL Mouse

    PubMed Central

    O'Leary, Kathleen A.; Shea, Michael P.; Schuler, Linda A.

    2016-01-01

    Elevated exposure to prolactin is epidemiologically associated with an increased risk of aggressive ER+ breast cancer. To understand the underlying mechanisms and crosstalk with other oncogenic factors, we developed the NRL-PRL mouse. In this model, mammary expression of a rat prolactin transgene raises local exposure to prolactin without altering estrous cycling. Nulliparous females develop metastatic, histotypically diverse mammary carcinomas independent from ovarian steroids, and most are ER+. These characteristics resemble the human clinical disease, facilitating study of tumorigenesis, and identification of novel preventive and therapeutic approaches. PMID:25472540

  17. Developmental biology: cell fate in the mammary gland

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. The biology of progesterone receptor in the normal mammary gland and in breast cancer.

    PubMed

    Obr, Alison E; Edwards, Dean P

    2012-06-24

    This paper reviews work on progesterone and the progesterone receptor (PR) in the mouse mammary gland that has been used extensively as an experimental model. Studies have led to the concept that progesterone controls proliferation and morphogenesis of the luminal epithelium in a tightly orchestrated manner at distinct stages of development by paracrine signaling pathways, including receptor activator of nuclear factor κB ligand (RANKL) as a major paracrine factor. Progesterone also drives expansion of stem cells by paracrine signals to generate progenitors required for alveologenesis. During mid-to-late pregnancy, progesterone has another role to suppress secretory activation until parturition mediated in part by crosstalk between PR and prolactin/Stat5 signaling to inhibit induction of