DOCSLIB.ORG

LKB1 Is Crucial for TRAIL-Mediated Apoptosis Induction in Osteosarcoma

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
LKB1 Is Crucial for TRAIL-Mediated Apoptosis Induction in Osteosarcoma ANTICANCER RESEARCH 27: 761-768 (2007) LKB1 is Crucial for TRAIL-mediated Apoptosis Induction in Osteosarcoma SHINTARO TAKEDA1,2, ATSUSHI IWAI2,3, MITSUKO NAKASHIMA2, DAISUKE FUJIKURA2,3, SATOKO CHIBA2,3, HONG MEI LI3, JUN UEHARA2,3, SATOSHI KAWAGUCHI1, MITSUNORI KAYA1, SATOSHI NAGOYA1, TAKURO WADA1, JUNYING YUAN4, SYDONIA RAYTER5, ALAN ASHWORTH5, JOHN C. REED6, TOSHIHIKO YAMASHITA1, TOSHIMITSU UEDE2 and TADAAKI MIYAZAKI2,3 1Department of Orthopaedic Surgery, Sapporo Medical University School of Medicine, Sapporo; 2Division of Molecular Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo; 3Department of Bioresources, Hokkaido University Research Center for Zoonosis Control, Sapporo, 0010021, Japan; 4Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, 02115, U.S.A.; 5Cancer Research UK Gene Function and Regulation Group, The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London SW3 6JB, U.K.; 6Burnham Institute for Medical Research, La Jolla, California, 92037, U.S.A. Abstract. Background: Despite improvements in Development of chemotherapy and surgery has resulted in chemotherapy and surgery in the treatment of osteosarcoma, the considerable improvement of survival rates of satisfactory results are still difficult to achieve. New therapeutic osteosarcoma patients (1), the current chemotherapeutic modalities need to be developed for the improvement of these agents still have a lot of problems to overcome, such as treatments. TRAIL (TNF-related apoptosis inducing ligand) is serious side-effects or resistance to multiple chemo- known as a selective apoptosis inducer in most tumor cells, but therapeutic agents in 20-30% of the patients (2). New not in normal cells. Therefore, TRAIL is a good candidate therapeutic modalities for these patients are driven by target for the treatment of tumors. However, sensitivity of necessity for the treatment. osteosarcoma cells to TRAIL-induced apoptosis is lower than TRAIL is a member of TNF (tumor necrosis factor) that of other types of tumor cells. Recently, DAP3 (death family proteins (3, 4). TRAIL, which selectively induces associated protein 3) was demonstrated to play a critical role in apoptosis in most tumor cells, but not in normal cells, is TRAIL-mediated apoptosis through activation of pro-caspase- expected to be a new therapeutic agent without significant 8. Here, we found that LKB1, a serine/threonine kinase, toxicities to normal tissues (5, 6). expressed in bone and soft tissue sarcoma cells, associated with Many experimental studies of the application of TRAIL DAP3. We also demonstrated that expression of DAP3 induced to osteosarcoma cells have been reported (7-15). However, apoptosis in osteosarcoma cells. Furthermore, expression of treatment of osteosarcoma cells with TRAIL did not lead LKB1 induced apoptosis and co-expression of LKB1 with to satisfactory results, since cell sensitivity to TRAIL- DAP3 strongly induced apoptosis in osteosarcoma cells. In induced apoptosis is different in various cells, and the addition, expression of LKB1 kinase dead mutant, LKB1 mechanisms for apoptosis induction remain unclear. (K78M), inhibited DAP3-induced apoptosis in these cells. Therefore, additional agents to sensitize osteosarcoma cells These results suggest that LKB1 is critical for TRAIL-induced to TRAIL-induced apoptosis have been proposed (7-11, 13- apoptosis induction, cooperating with DAP3 in osteosarcoma 15) but the problem of side-effects still remains. cells. It is predicted that LKB1 and DAP3 could be critical TRAIL receptors have been identified as TRAIL-R1 target molecules for the treatment of osteosarcomas. (DR4), TRAIL-R2 (DR5), TRAIL-R3 (DcR1), TRAIL-R4 (DcR2) and osteoprotegrin (OPG). DR4 or DR5 induces apoptosis by TRAIL stimulation (16). In contrast, DcR1, DcR2 and OPG are not able to induce apoptosis, since their cytoplasmic death domains are deficient or incomplete (16, Correspondence to: Tadaaki Miyazaki, Department of Bioresources, 17, 19-21). Although the expression of DR4 or DR5 is Hokkaido University Research Center for Zoonosis Control, Sapporo, 0010021, Japan. required for TRAIL-mediated apoptosis, the expression of DcR1, DcR2 and OPG does not necessarily relate to cell Key Words: LKB1, TRAIL, apoptosis, osteosarcoma cells, DAP3. sensitivity to TRAIL-induced apoptosis (22, 23). Therefore, 0250-7005/2007 $2.00+.40 761 ANTICANCER RESEARCH 27: 761-768 (2007) the intracellular molecules located downstream of DR4 or viability assay experiments were repeated twice. Results of DR5 have been proposed to be a critical determinant of cell representative experiments are given as the mean+SD. sensitivity to apoptosis induced by TRAIL. Yeast two-hybrid screening. Human wild-type DAP3 gene was Pro-apoptotic cytoplasmic DAP3 associates with both the inserted and fused to the Gal4 DNA-binding domain in the vector intracellular death domain of TRAIL receptor and the Fas pGBKT7 (Trp) (BD Clontech, USA). After transformation into associated death domain (FADD) (23). The complex the yeast strain, AH109 with pGBKT7 (Empty vector) or pGBKT7- formation of these proteins by TRAIL receptor-stimulation DAP3, these transformants were selected on SD (Synthetic induces apoptosis through activation of pro-caspase 8 (24). Dropout) plates, in which the media lacks Trp. These selected yeast However, details of molecular mechanisms in TRAIL- or cells were transformed with a human fetal brain cDNA library DAP3-induced apoptosis are not fully understood. fused with Gal4ad in the vector pActII (Leu) (Clontech), and then selected on SD media lacking Trp and Leu. The positive clones Therefore, we screened the proteins which bind to DAP3, were selected and the confirmation of positive clones was carried using a yeast two hybrid system, in order to identify the out using ·-gal or ‚-gal assay. regulators for TRAIL-mediated apoptosis. As a result, we found that LIP1 (LKB1 interacting protein 1) associated Western blotting. Cells were harvested and the protein extracts from with DAP3. LIP1 was reported to interact with LKB1 (25). these cells were prepared as follows. The protein samples (20 Although LIP1 is not a substrate of LKB1, it was shown to Ìg/lane) were fractionated by SDS-PAGE on a 4-20% gradient anchor LKB1 to the cytoplasm (25). LKB1 was originally polyacrilamide gel (Daiichi Pure Chemicals, JAPAN). The analysed proteins were transferred to a PMDF filter with a semi- described with regard to its gene mutation causing Peutz- dry blotter. The blotted membrane filter was reacted either with Jeghers syndrome (PJS) (25-27), a rare hereditary disease, each antibody as shown in the Figure after blocking. This in which there is predisposition to develop benign and membrane was reacted with HRP-conjugated antibodies and malignant tumors of many organ systems (28, 29). LKB1 immunodetection of the proteins was performed using the ECL was demonstrated to be a tumor suppressor involved in the detection system (Amersham, Piscataway, NJ, USA). regulation of cell polarity (30-32). However, the function of 6 LKB1 in TRAIL-induced apoptosis is not clear. Therefore, Immunoprecipitation. HEK293T cells (2x10 ) in 10 cm cell culture plates were transiently transfected with 5 Ìg of each we studied the functional role of LKB1 in apoptosis expression vector plasmid (10 Ìg as a total DNA amount) induction by TRAIL stimulation. In addition, we examined together with 30 Ìl of Fugene 6 transfection reagent (Roche the function of LKB1 and DAP3 in TRAIL-mediated Diagnostic, Mannheim, Germany) and harvested 24 h later. Cells apoptosis in osteosarcoma cells. were suspended in 500 Ìl of lysis buffer containing 0.1% NP-40, 20 mM Tris-HCl at pH 7.5, 2 mM MgCl2, 1 mM EGTA, 150 mM Materials and Methods NaCl and protease inhibitors Complete Mini (Roche). Alternatively, untransfected HOS cells (5x108) were harvested Cell lines and plasmids. HOS, MNNG/HOS, MG63 (human and lysed in 1.5 ml of lysis buffer as described above. Cell osteosarcoma), HT1080, NIH3T3 (mouse fibrosarcoma) and extracts were reacted either with the indicated antibody in the HFO (primary cultured human fetal osteoblast) cells (15) were Figure for 1 h and then washed with the lysis buffer for five kindly donated by the Department of Pathology, Sapporo times. These samples were fractionated by SDS-PAGE and Medical University, Japan. HEK293T and 143B (human analyzed by Western blotting assay as described above. osteosarcoma) cells were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). These cells were Colony formation assay. HOS cells (5x105) in 35 mm cell culture cultured in Dulbeccos' modified Eagle's medium (DMEM) plates were transfected with 4 Ìg of each expression vector plasmid supplemented with 10% heat inactivated fetal bovine serum together with 10 Ìl of Lipofectamine 2000 transfection reagent (SIGMA-ALDRICH, JAPAN), 100 IU/ml penicillin and 100 (Invitrogen, CA, USA). After 24 h, these cells were cultured with Ìg/ml streptomycin (SIGMA) in a humidified atmosphere TRAIL (50 ng/ml) for 1 week. The number of colonies was determined after staining with 0.05% crystal violet solution. containing 5% CO2 at 37ÆC. Jurkat cells were cultured in the medium of RPMI-1640 supplemented with 10% heat inactivated fetal bovine serum Results (SIGMA), 100 IU/ml penicillin and 100 Ìg/ml streptomycin (SIGMA). Sensitivity of osteosarcoma cells to TRAIL-induced apoptosis. We first examined the effects of TRAIL stimulation for the Cell viability assay. Before seeding onto 96-well
Load more

Recommended publications
  • Role of Mitochondrial Ribosomal Protein S18-2 in Cancerogenesis and in Regulation of Stemness and Differentiation
    From THE DEPARTMENT OF MICROBIOLOGY TUMOR AND CELL BIOLOGY (MTC) Karolinska Institutet, Stockholm, Sweden ROLE OF MITOCHONDRIAL RIBOSOMAL PROTEIN S18-2 IN CANCEROGENESIS AND IN REGULATION OF STEMNESS AND DIFFERENTIATION Muhammad Mushtaq Stockholm 2017 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by E-Print AB 2017 © Muhammad Mushtaq, 2017 ISBN 978-91-7676-697-2 Role of Mitochondrial Ribosomal Protein S18-2 in Cancerogenesis and in Regulation of Stemness and Differentiation THESIS FOR DOCTORAL DEGREE (Ph.D.) By Muhammad Mushtaq Principal Supervisor: Faculty Opponent: Associate Professor Elena Kashuba Professor Pramod Kumar Srivastava Karolinska Institutet University of Connecticut Department of Microbiology Tumor and Cell Center for Immunotherapy of Cancer and Biology (MTC) Infectious Diseases Co-supervisor(s): Examination Board: Professor Sonia Lain Professor Ola Söderberg Karolinska Institutet Uppsala University Department of Microbiology Tumor and Cell Department of Immunology, Genetics and Biology (MTC) Pathology (IGP) Professor George Klein Professor Boris Zhivotovsky Karolinska Institutet Karolinska Institutet Department of Microbiology Tumor and Cell Institute of Environmental Medicine (IMM) Biology (MTC) Professor Lars-Gunnar Larsson Karolinska Institutet Department of Microbiology Tumor and Cell Biology (MTC) Dedicated to my parents ABSTRACT Mitochondria carry their own ribosomes (mitoribosomes) for the translation of mRNA encoded by mitochondrial DNA. The architecture of mitoribosomes is mainly composed of mitochondrial ribosomal proteins (MRPs), which are encoded by nuclear genomic DNA. Emerging experimental evidences reveal that several MRPs are multifunctional and they exhibit important extra-mitochondrial functions, such as involvement in apoptosis, protein biosynthesis and signal transduction. Dysregulations of the MRPs are associated with severe pathological conditions, including cancer.
    [Show full text]
  • IPS-1 Is Crucial for DAP3-Mediated Anoikis Induction by Caspase-8 Activation
    Cell Death and Differentiation (2009) 16, 1615–1621 & 2009 Macmillan Publishers Limited All rights reserved 1350-9047/09 $32.00 www.nature.com/cdd IPS-1 is crucial for DAP3-mediated anoikis induction by caspase-8 activation H-M Li1, D Fujikura1, T Harada1, J Uehara1, T Kawai2,3, S Akira2,3, JC Reed4, A Iwai1 and T Miyazaki*,1 Detachment of adherent epithelial cells from the extracellular matrix induces apoptosis, a process known as anoikis. We have shown that DAP3 is critical for anoikis induction. However, the mechanism for anoikis induction mediated by DAP3 is still unclear. Here, we show that interferon-b promoter stimulator 1 (IPS-1) binds DAP3 and induces anoikis by caspase activation. Recently, IPS-1 has been shown to be critical for antiviral immune responses, although there has been no report of its function in apoptosis induction. We show that overexpression of IPS-1 induces apoptosis by activation of caspase-3, -8, and -9. In addition, IPS-1 knockout mouse embryonic fibroblasts were shown to be resistant to anoikis. Interestingly, IPS-1 expression, recruitment of caspase-8 to IPS-1, and caspase-8 activation were induced after cell detachment. Furthermore, DAP3-mediated anoikis induction was inhibited by knockdown of IPS-1 expression. Therefore, we elucidated a novel function of IPS-1 for anoikis induction by caspase-8 activation. Cell Death and Differentiation (2009) 16, 1615–1621; doi:10.1038/cdd.2009.97; published online 31 July 2009 Apoptosis, the programmed physiological cell death, is critical induces programmed cell death, a phenomenon known as for modeling tissues and maintaining homeostasis in multi- anoikis.
    [Show full text]
  • Hnoa1 Interacts with Complex I and DAP3 and Regulates
    Supplemental Material can be found at: http://www.jbc.org/cgi/content/full/M807797200/DC1 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 8, pp. 5414–5424, February 20, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. hNOA1 Interacts with Complex I and DAP3 and Regulates Mitochondrial Respiration and Apoptosis*□S Received for publication, October 9, 2008, and in revised form, December 15, 2008 Published, JBC Papers in Press, December 22, 2008, DOI 10.1074/jbc.M807797200 Tingdong Tang‡, Bin Zheng‡1, Sheng-hong Chen‡§, Anne N. Murphy¶, Krystyna Kudlicka‡, Huilin Zhou‡§, and Marilyn G. Farquhar‡2 From the Departments of ‡Cellular and Molecular Medicine and ¶Pharmacology and §Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California 92093-0651 Mitochondria are dynamic organelles that play key roles in fusion proteins such as the dynamin like large G proteins Drp1 metabolism, energy production, and apoptosis. Coordination of and Opa1, the cell’s susceptibility to apoptotic agents (3) or these processes is essential to maintain normal cellular func- ability to generate ATP (4, 5) is altered. tions. Here we characterized hNOA1, the human homologue of Apoptosis is controlled by a diverse range of cell signals, AtNOA1 (Arabidopsis thaliana nitric oxide-associated protein which may originate either extracellularly (extrinsic inducers) Downloaded from 1), a large mitochondrial GTPase. By immunofluorescence, or intracellularly (intrinsic inducers), and mitochondria play immunoelectron microscopy, and mitochondrial subfraction- central roles in both pathways (6). The apoptotic pathways ation, endogenous hNOA1 is localized within mitochondria involve a growing list of mitochondria-associated proteins, where it is peripherally associated with the inner mitochondrial such as Bad, cytochrome c, Smac, AIF, Bcl-2, and others, most membrane facing the mitochondrial matrix.
    [Show full text]
  • Insight Into Glucocorticoid Receptor Signalling Through Interactome Model Analysis
    RESEARCH ARTICLE Insight into glucocorticoid receptor signalling through interactome model analysis Emyr Bakker1, Kun Tian1, Luciano Mutti1, Constantinos Demonacos2, Jean- Marc Schwartz2☯*, Marija Krstic-Demonacos1☯* 1 Biomedical Research Centre, School of Environment and Life Sciences, University of Salford, Salford, United Kingdom, 2 Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom ☯ These authors contributed equally to this work. * [email protected] (JMS); [email protected] (MKD) a1111111111 a1111111111 a1111111111 a1111111111 Abstract a1111111111 Glucocorticoid hormones (GCs) are used to treat a variety of diseases because of their potent anti-inflammatory effect and their ability to induce apoptosis in lymphoid malignan- cies through the glucocorticoid receptor (GR). Despite ongoing research, high glucocorticoid efficacy and widespread usage in medicine, resistance, disease relapse and toxicity remain OPEN ACCESS factors that need addressing. Understanding the mechanisms of glucocorticoid signalling Citation: Bakker E, Tian K, Mutti L, Demonacos C, and how resistance may arise is highly important towards improving therapy. To gain insight Schwartz J-M, Krstic-Demonacos M (2017) Insight into this we undertook a systems biology approach, aiming to generate a Boolean model of into glucocorticoid receptor signalling through interactome model analysis. PLoS Comput Biol 13 the glucocorticoid receptor protein interaction network that encapsulates functional relation- (11): e1005825. https://doi.org/10.1371/journal. ships between the GR, its target genes or genes that target GR, and the interactions pcbi.1005825 between the genes that interact with the GR. This model named GEB052 consists of 52 Editor: Andrey Rzhetsky, University of Chicago, nodes representing genes or proteins, the model input (GC) and model outputs (cell death UNITED STATES and inflammation), connected by 241 logical interactions of activation or inhibition.
    [Show full text]
  • DAP3 Antibody A
    C 0 2 - t DAP3 Antibody a e r o t S Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) 2 7 Web: [email protected] 1 www.cellsignal.com 2 # 3 Trask Lane Danvers Massachusetts 01923 USA For Research Use Only. Not For Use In Diagnostic Procedures. Applications: Reactivity: Sensitivity: MW (kDa): Source: UniProt ID: Entrez-Gene Id: WB H Endogenous 46 Rabbit P51398 7818 Product Usage Information 5. Imataka, H. et al. (1997) EMBO J 16, 817-25. 6. Levy-Strumpf, N. et al. (1997) Mol Cell Biol 17, 1615-25. Application Dilution 7. Henis-Korenblit, S. et al. (2000) Mol Cell Biol 20, 496-506. Western Blotting 1:1000 Storage Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody. Specificity / Sensitivity DAP3 Antibody detects endogenous levels of total DAP3 protein. The antibody does not cross-react with other DAP family members. Species Reactivity: Human Source / Purification Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys58 of human DAP3. Antibodies are purified by protein A and peptide affinity chromatography. Background Death associated protein 1 (DAP1) is a 15 kDa protein that functions as a positive mediator of cell death initiated by interferon-gamma (1,2). The DAP1 protein is proline rich and possesses one SH3 binding motif, as well as several consensus protein kinase phosphorylation sites (1). The protein is localized in the cytoplasm, but the detailed mechanism of its proapoptotic function is unclear.
    [Show full text]
  • DAP3 Mouse Monoclonal Antibody [Clone ID: OTI2D4] Product Data
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for TA802818 DAP3 Mouse Monoclonal Antibody [Clone ID: OTI2D4] Product data: Product Type: Primary Antibodies Clone Name: OTI2D4 Applications: WB Recommended Dilution: WB 1:2000 Reactivity: Human Host: Mouse Isotype: IgG1 Clonality: Monoclonal Immunogen: Human recombinant protein fragment corresponding to amino acids 161-398 of human DAP3 (NP_387506) produced in E.coli. Formulation: PBS (PH 7.3) containing 1% BSA, 50% glycerol and 0.02% sodium azide. Concentration: 1 mg/ml Purification: Purified from mouse ascites fluids or tissue culture supernatant by affinity chromatography (protein A/G) Conjugation: Unconjugated Storage: Store at -20°C as received. Stability: Stable for 12 months from date of receipt. Predicted Protein Size: 45.4 kDa Gene Name: death associated protein 3 Database Link: NP_387506 Entrez Gene 7818 Human P51398 This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 2 DAP3 Mouse Monoclonal Antibody [Clone ID: OTI2D4] – TA802818 Background: Mammalian mitochondrial ribosomal proteins are encoded by nuclear genes and help in protein synthesis within the mitochondrion. Mitochondrial ribosomes (mitoribosomes) consist of a small 28S subunit and a large 39S subunit. They have an estimated 75% protein to rRNA composition compared to prokaryotic ribosomes, where this ratio is reversed. Another difference between mammalian mitoribosomes and prokaryotic ribosomes is that the latter contain a 5S rRNA.
    [Show full text]
  • Recent Insights Into the Mechanism of Glucocorticosteroid- Induced Apoptosis
    Cell Death and Differentiation (2002) 9, 6±19 ã 2002 Nature Publishing Group All rights reserved 1350-9047/02 $25.00 www.nature.com/cdd Review Recent insights into the mechanism of glucocorticosteroid- induced apoptosis CW Distelhorst*,1 apoptosis.1 But progress in understanding corticosteroid- induced apoptosis has lagged behind remarkable advances in 1 Division of Hematology/Oncology and Comprehensive Cancer Center, understanding other forms of apoptosis, such as that induced Departments of Medicine and Pharmacology, Case Western Reserve by the ligands of death receptors (e.g., Fas/CD95, tumor University School of Medicine and University Hospitals of Cleveland, necrosis factor). In part, this is because corticosteroid-induced Cleveland, Ohio, USA apoptosis is initiated by a steroid receptor-mediated change in * Corresponding author: CW Distelhorst, Division of Hematology/Oncology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio, gene expression, but specific genes that mediate cell death in OH 44106-4937, USA. Tel: 216-368-1175; Fax: 216-368-1166; response to corticosteroid treatment have not been identified. E-mail: [email protected] Recent findings have shed light on events in the cell death pathway downstream from gene regulation, including the Received 20.7.01; revised 7.9.01; accepted 3.10.01 caspases responsible for the execution phase of cell death, Edited by G Nunez the unexpected involvement of the multicatalytic proteasome in the death process, the suppression of prosurvival transcrip- tion factors (e.g., AP-1, c-myc,NF-kB), and crosstalk between Abstract T cell receptor and cytokine signaling pathways. Moreover, Glucocorticosteroid hormones induce apoptosis in lympho- evidence that mitochondrial dysfunction lies downstream of cytes.
    [Show full text]
  • Glucocorticoid Receptor Changes Its Cellular Location with Breast Cancer Development
    Histol Histopathol (2008) 23: 77-85 Histology and http://www.hh.um.es Histopathology Cellular and Molecular Biology Glucocorticoid receptor changes its cellular location with breast cancer development Isabel Conde1, Ricardo Paniagua1, Benito Fraile1, Javier Lucio2 and Maria I. Arenas1 1Department of Cell Biology and Genetics, and 2Department of Physiology. University of Alcalá, Alcalá de Henares, Madrid. Spain Summary. Glucocorticoids play a major role in Introduction attenuation of the inflammatory response and they are useful in the primary combination chemotherapy of The hormonal regulation of breast cancer cell breast cancer, since in vitro studies have demonstrated an growth and survival is multifaceted and very complex antiproliferative effect in human breast cancer cells. In (Nass and Davidson, 1999) due to the potential for contrast, it was recently shown that glucocorticoids interaction or cross talk between the different classes of protect against apoptotic signals evoked by cytokines, hormones, especially the ability of certain hormones to cAMP, tumour suppressors, and death genes in regulate the abundance of the receptors for other mammary gland epithelia. Their actions are mediated by hormones (Traynor, 1995). Activated steroid receptors intracellular receptor (GR) that functions as a hormone- affect many genes that are involved in cellular dependent transcription factor; however, no previous metabolism and often affect the transcription of other studies have been focused on GR expression in different steroid receptors (Dickson and Lippman, 1988). One of pathologies of the human breast, and the possible these receptors is the glucocorticoid receptor (GR) relationship with that of mineralocorticoid receptor which operates on the mechanisms of cellular (MR) and COX-2.
    [Show full text]
  • Effects of the Knockdown of Death-Associated Protein 3 Expression on Cell Adhesion, Growth and Migration in Breast Cancer Cells
    ONCOLOGY REPORTS 33: 2575-2582, 2015 Effects of the knockdown of death-associated protein 3 expression on cell adhesion, growth and migration in breast cancer cells UMAR WAZIR1,2, ANDREW J. SANDERS3, AHMAD M.A. WAZIR4, LIN YE3, WEN G. JIANG3, IRINA C. STER2, ANUP K. SHARMA2 and KEFAH MOKBEL1,2 1The London Breast Institute, Princess Grace Hospital; 2Department of Breast Surgery, St. George's Hospital and Medical School, University of London, London; 3Cardiff University-Peking University Cancer Institute (CUPUCI), Cardiff University School of Medicine, Cardiff University, Cardiff, Wales, UK; 4Peshawar Medical College, Peshawar, Pakistan Received December 30, 2014; Accepted February 4, 2015 DOI: 10.3892/or.2015.3825 Abstract. The death-associated protein 3 (DAP3) is a compared to the controls. Furthermore, invasion and migra- highly conserved phosphoprotein involved in the regula- tion were significantly increased in the MDA-MB-231DAP3kd tion of autophagy. A previous clinical study by our group cells vs. the controls. The expression of caspase 9 and IPS1, suggested an association between low DAP3 expression known components of the apoptosis pathway, were signifi- and clinicopathological parameters of human breast cancer. cantly reduced in the MCF7DAP3kd cells (p=0.05 and p=0.003, In the present study, we intended to determine the role of respectively). We conclude that DAP3 silencing contributes to DAP3 in cancer cell behaviour in the context of human breast breast carcinogenesis by increasing cell adhesion, migration cancer. We developed knockdown sub-lines of MCF7 and and invasion. It is possible that this may be due to the activity MDA-MB-231, and performed growth, adhesion, invasion of focal adhesion kinase further downstream of the anoikis assays and electric cell-substrate impedance sensing (ECIS) pathway.
    [Show full text]
  • And Fas-Induced Cell Death
    The EMBO Journal Vol.18 No.2 pp.353–362, 1999 Structure–function analysis of an evolutionary conserved protein, DAP3, which mediates TNF-α- and Fas-induced cell death Joseph L.Kissil, Ofer Cohen, Tal Raveh and have been studied extensively: ced-3, ced-4 and ced- Adi Kimchi1 9 (reviewed in Hengartner and Horvitz, 1994). Their mammalian homologs were identified as the caspases Department of Molecular Genetics, The Weizmann Institute of (cysteine proteases), Apaf-1 and members of the Bcl-2 Science, Rehovot 76100, Israel family, respectively. The fact that these protein families 1Corresponding author constitute the core of the apoptotic machinery in mamma- e-mail: [email protected] lian cell-death systems was subsequently established (reviewed in Peter et al., 1997). Another approach con- A novel approach to the isolation of positive mediators sisted of the isolation of proteins which are recruited to of programmed cell death, based on random inactiva- the intracellular domains of cytokine receptors belonging tion of genes by expression of anti sense RNAs, was to the tumor necrosis factor (TNF) family. The yeast two- employed to identify mediators of interferon-γ-induced hybrid system served as a major tool for the isolation of apoptosis. One of the several genes identified is DAP3, these cytoplasmic proteins that bind to the intracellular which codes for a 46 kDa protein with a potential part of TNF-α receptor (p55 TNF-R1), the Fas receptor, nucleotide-binding motif. Structure–function studies of and to each other (Yuan, 1997; Ashkenazi and Dixit, 1998; the protein indicate that the intact full-length protein Wallach et al., 1998).
    [Show full text]
  • DAP3 Antibody
    Product Datasheet DAP3 Antibody Catalog No: #31066 Package Size: #31066-1 50ul #31066-2 100ul Orders: [email protected] Support: [email protected] Description Product Name DAP3 Antibody Host Species Rabbit Clonality Polyclonal Applications ELISA WB IHC Species Reactivity Hu Specificity The antibody detects endogenous level of total DAP3 protein. Immunogen Type Recombinant protein Immunogen Description Fusion protein corresponding to a C terminal 300 amino acids of human death associated protein 3 Target Name DAP3 Other Names death associated protein 3, DAP-3, MRPS29, MRP-S29, bMRP-10 Accession No. Swiss-Prot:Q16828Gene ID:1848 Formulation Rabbit IgG in pH7.4 PBS, 0.05% NaN3, 40% Glycerol. Storage Store at -20°C/1 year Application Details Predicted MW: 46kd ELISA: 1:1000-1:5000 Western blotting: 1:200-1:1000 Immunohistochemistry: 1:5-1:20 Images Gel: 12%SDS-PAGE Lane1: 293T cell lysate Lane2: Jurkat cell lysate Lane3: Human Lymphoma tissue lysate Lysates: 30ug per lane Primary antibody: 1/300 dilution Secondary antibody: Goat anti Rabbit IgG - H&L (HRP) at 1/10000 dilution Exposure time: 20 seconds Address: 8400 Baltimore Ave., Suite 302, College Park, MD 20740, USA http://www.sabbiotech.com 1 The image on the left is immunohistochemistry of paraffin-embedded Human colon cancer tissue using 31066 (DAP3 Antibody) at dilution 1/10, on the right is treated with the fusion protein. Background This gene encodes a 28S subunit protein that also participates in apoptotic pathways which are initiated by tumor necrosis factor-alpha, Fas ligand, and gamma interferon. This protein potentially binds ATP/GTP and might be a functional partner of the mitoribosomal protein S27.
    [Show full text]
  • Novel Function of DAP3 As a Tumor Suppressor Gene Tadaaki Miyazaki
    A SHORT RESEARCH REVIEW ARTICLE Novel Function of DAP3 as A Tumor Suppressor Gene Tadaaki Miyazaki Department of Probiotics Immunology, Institute for Genetic Medicine, Hokkaido University, North 15, West 7, Kita-Ku, Sapporo, 0600815, Japan ABSTRACT DAP3 is a cell death mediator of the signaling pathways induced by interferon-γ, Fas ligand, tumor necrosis factor (TNF)-α, and TNF-related apoptosis-inducing ligand (TRAIL). DAP3 is also critical for the mitochondrial homeostasis during cell death and the anoikis induction. Anoikis is induced by detachment of adherent epithelial cells from the extracellular matrix. The deregulation of anoikis induction leads to the survival of cancer cells to promote the metastasis formation. Since the metastasis causes 90% of human cancer deaths, DAP3 might be a critical target for the effective cancer therapy. In fact, in the case of human breast cancer patients, lower DAP3 expression levels were significantly associated with local recurrence, high tumor grade, distant metastasis, and high mortality. Therefore, DAP3 could be used as a prognostic marker of the cancer patients and represents a potential therapeutic target as a novel tumor suppressor gene for the cancer treatment. Osteosarcoma is the primary bone tumor, and the sensitivity of cancer cells to TRAIL-induced apoptosis is lower than that of other type tumor cells. We found that DAP3 was interacted with LKB1, a serine/threonine kinase, expressed in bone and soft tissue sarcoma cells, and DAP3 and LKB1 cooperatively induce apoptosis by TRAIL stimulation. Therefore, new therapeutic modalities targeting these molecules are required to be developed for the improvement of the therapy. Furthermore, we identified a novel DAP3-binding protein termed death ligand signal enhancer (DELE).
    [Show full text]