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Volumn 7, Issue 11, 2012, Pages

CK2-Mediated Hyperphosphorylation of Topoisomerase I Targets Serine 506, Enhances Topoisomerase I-DNA Binding, and Increases Cellular Camptothecin Sensitivity

Author keywords

[No Author keywords available]

Indexed keywords

CAMPTOTHECIN; CASEIN KINASE II; DNA TOPOISOMERASE; EPITOPE; PLASMID DNA; RECOMBINANT PROTEIN; SERINE; SERINE 506; UNCLASSIFIED DRUG;

EID: 84869803372     PISSN: None     EISSN: 19326203     Source Type: Journal    
DOI: 10.1371/journal.pone.0050427     Document Type: Article
Times cited : (23)

References (52)
  • 1
    • 0032189683 scopus 로고    scopus 로고
    • Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme
    • Pommier Y, Pourquier P, Fan Y, Strumberg D, (1998) Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme. Biochim Biophys Acta 1400: 83-105.
    • (1998) Biochim Biophys Acta , vol.1400 , pp. 83-105
    • Pommier, Y.1    Pourquier, P.2    Fan, Y.3    Strumberg, D.4
  • 2
    • 0030014783 scopus 로고    scopus 로고
    • DNA topoisomerases
    • Wang JC, (1996) DNA topoisomerases. Annu Rev Biochem 65: 635-692.
    • (1996) Annu Rev Biochem , vol.65 , pp. 635-692
    • Wang, J.C.1
  • 3
    • 0034923502 scopus 로고    scopus 로고
    • DNA topoisomerases: structure, function, and mechanism
    • Champoux JJ, (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70: 369-413.
    • (2001) Annu Rev Biochem , vol.70 , pp. 369-413
    • Champoux, J.J.1
  • 4
    • 0027139317 scopus 로고
    • Interaction between replication forks and topoisomerase I-DNA cleavable complexes: studies in a cell-free SV40 DNA replication system
    • Tsao YP, Russo A, Nyamuswa G, Silber R, Liu LF, (1993) Interaction between replication forks and topoisomerase I-DNA cleavable complexes: studies in a cell-free SV40 DNA replication system. Cancer Res 53: 5908-5914.
    • (1993) Cancer Res , vol.53 , pp. 5908-5914
    • Tsao, Y.P.1    Russo, A.2    Nyamuswa, G.3    Silber, R.4    Liu, L.F.5
  • 5
    • 0024316466 scopus 로고
    • DNA topoisomerase poisons as antitumor drugs
    • Liu LF, (1989) DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem 58: 351-375.
    • (1989) Annu Rev Biochem , vol.58 , pp. 351-375
    • Liu, L.F.1
  • 6
    • 79952081873 scopus 로고    scopus 로고
    • Protein kinase CK2 is a central regulator of topoisomerase I hyperphosphorylation and camptothecin sensitivity in cancer cell lines
    • Bandyopadhyay K, Gjerset RA, (2011) Protein kinase CK2 is a central regulator of topoisomerase I hyperphosphorylation and camptothecin sensitivity in cancer cell lines. Biochemistry 50: 704-714.
    • (2011) Biochemistry , vol.50 , pp. 704-714
    • Bandyopadhyay, K.1    Gjerset, R.A.2
  • 7
    • 37049037760 scopus 로고    scopus 로고
    • Serine phosphorylation-dependent coregulation of topoisomerase I by the p14ARF tumor suppressor
    • Bandyopadhyay K, Lee C, Haghighi A, Baneres JL, Parello J, et al. (2007) Serine phosphorylation-dependent coregulation of topoisomerase I by the p14ARF tumor suppressor. Biochemistry 46: 14325-14334.
    • (2007) Biochemistry , vol.46 , pp. 14325-14334
    • Bandyopadhyay, K.1    Lee, C.2    Haghighi, A.3    Baneres, J.L.4    Parello, J.5
  • 8
    • 0025151823 scopus 로고
    • Decreased expression of DNA topoisomerase I in camptothecin-resistant tumor cell lines as determined by a monoclonal antibody
    • Sugimoto Y, Tsukahara S, Oh-hara T, Isoe T, Tsuruo T, (1990) Decreased expression of DNA topoisomerase I in camptothecin-resistant tumor cell lines as determined by a monoclonal antibody. Cancer Res 50: 6925-6930.
    • (1990) Cancer Res , vol.50 , pp. 6925-6930
    • Sugimoto, Y.1    Tsukahara, S.2    Oh-hara, T.3    Isoe, T.4    Tsuruo, T.5
  • 10
    • 0032189251 scopus 로고    scopus 로고
    • Cellular resistance to topoisomerase-targeted drugs: from drug uptake to cell death
    • Larsen AK, Skladanowski A, (1998) Cellular resistance to topoisomerase-targeted drugs: from drug uptake to cell death. Biochim Biophys Acta 1400: 257-274.
    • (1998) Biochim Biophys Acta , vol.1400 , pp. 257-274
    • Larsen, A.K.1    Skladanowski, A.2
  • 11
    • 0025183609 scopus 로고
    • Phosphorylation sites for type N II protein kinase in DNA-topoisomerase I from calf thymus
    • Coderoni S, Paparelli M, Gianfranceschi GL, (1990) Phosphorylation sites for type N II protein kinase in DNA-topoisomerase I from calf thymus. Int J Biochem 22: 737-746.
    • (1990) Int J Biochem , vol.22 , pp. 737-746
    • Coderoni, S.1    Paparelli, M.2    Gianfranceschi, G.L.3
  • 12
    • 0025297468 scopus 로고
    • Phosphorylation of mammalian DNA topoisomerase I and activation by protein kinase C
    • Pommier Y, Kerrigan D, Hartman KD, Glazer RI, (1990) Phosphorylation of mammalian DNA topoisomerase I and activation by protein kinase C. J Biol Chem. 265: 9418-9422.
    • (1990) J Biol Chem , vol.265 , pp. 9418-9422
    • Pommier, Y.1    Kerrigan, D.2    Hartman, K.D.3    Glazer, R.I.4
  • 13
    • 0027437792 scopus 로고
    • A casein kinase type II (CKII)-like nuclear protein kinase associates with, phosphorylates, and activates topoisomerase I
    • Turman MA, Douvas A, (1993) A casein kinase type II (CKII)-like nuclear protein kinase associates with, phosphorylates, and activates topoisomerase I. Biochem Med Metab Biol. 50: 210-225.
    • (1993) Biochem Med Metab Biol , vol.50 , pp. 210-225
    • Turman, M.A.1    Douvas, A.2
  • 14
    • 47749138222 scopus 로고    scopus 로고
    • Mitotic phosphorylation stimulates DNA relaxation activity of human topoisomerase I
    • Hackbarth JS, Galvez-Peralta M, Dai NT, Loegering DA, Peterson KL, et al. (2008) Mitotic phosphorylation stimulates DNA relaxation activity of human topoisomerase I. J Biol Chem. 283: 16711-16722.
    • (2008) J Biol Chem , vol.283 , pp. 16711-16722
    • Hackbarth, J.S.1    Galvez-Peralta, M.2    Dai, N.T.3    Loegering, D.A.4    Peterson, K.L.5
  • 15
    • 0025377431 scopus 로고
    • Role of calf thymus DNA-topoisomerase I phosphorylation on relaxation activity expression and on DNA-protein interaction
    • Role of DNA-topoisomerase I phosphorylation
    • Coderoni S, Paparelli M, Gianfranceschi GL, (1990) Role of calf thymus DNA-topoisomerase I phosphorylation on relaxation activity expression and on DNA-protein interaction. Role of DNA-topoisomerase I phosphorylation. Mol Biol Rep 14: 35-39.
    • (1990) Mol Biol Rep , vol.14 , pp. 35-39
    • Coderoni, S.1    Paparelli, M.2    Gianfranceschi, G.L.3
  • 16
    • 70349320107 scopus 로고    scopus 로고
    • Protein kinase CK2 in health and disease: CK2: a key player in cancer biology
    • Trembley JH, Wang G, Unger G, Slaton J, Ahmed K, (2009) Protein kinase CK2 in health and disease: CK2: a key player in cancer biology. Cell Mol Life Sci 66: 1858-1867.
    • (2009) Cell Mol Life Sci , vol.66 , pp. 1858-1867
    • Trembley, J.H.1    Wang, G.2    Unger, G.3    Slaton, J.4    Ahmed, K.5
  • 17
    • 0030003562 scopus 로고    scopus 로고
    • Phosphorylation of topoisomerase I in L5178Y-S cells is associated with poly(ADP-ribose) metabolism
    • Staron K, Kowalska-Loth B, Nieznanski K, Szumiel I, (1996) Phosphorylation of topoisomerase I in L5178Y-S cells is associated with poly(ADP-ribose) metabolism. Carcinogenesis 17: 383-387.
    • (1996) Carcinogenesis , vol.17 , pp. 383-387
    • Staron, K.1    Kowalska-Loth, B.2    Nieznanski, K.3    Szumiel, I.4
  • 18
  • 19
    • 0035805563 scopus 로고    scopus 로고
    • Constitutive ERK1/2 activation in esophagogastric rib bone marrow micrometastatic cells is MEK-independent
    • Barry OP, Mullan B, Sheehan D, Kazanietz MG, Shanahan F, et al. (2001) Constitutive ERK1/2 activation in esophagogastric rib bone marrow micrometastatic cells is MEK-independent. J Biol Chem 276: 15537-15546.
    • (2001) J Biol Chem , vol.276 , pp. 15537-15546
    • Barry, O.P.1    Mullan, B.2    Sheehan, D.3    Kazanietz, M.G.4    Shanahan, F.5
  • 20
    • 0028963195 scopus 로고
    • Functionally distinct human marrow stromal cell lines immortalized by transduction with the human papilloma virus E6/E7 genes
    • Roecklein BA, Torok-Storb B, (1995) Functionally distinct human marrow stromal cell lines immortalized by transduction with the human papilloma virus E6/E7 genes. Blood 85: 997-1005.
    • (1995) Blood , vol.85 , pp. 997-1005
    • Roecklein, B.A.1    Torok-Storb, B.2
  • 21
    • 0029927505 scopus 로고    scopus 로고
    • Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels
    • Shevchenko A, Wilm M, Vorm O, Mann M, (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68: 850-858.
    • (1996) Anal Chem , vol.68 , pp. 850-858
    • Shevchenko, A.1    Wilm, M.2    Vorm, O.3    Mann, M.4
  • 23
    • 0028134964 scopus 로고
    • Isolation of nuclear extracts from fragile cells: a simplified procedure applied to thymocytes
    • Olnes MI, Kurl RN, (1994) Isolation of nuclear extracts from fragile cells: a simplified procedure applied to thymocytes. Biotechniques 17: 828-829.
    • (1994) Biotechniques , vol.17 , pp. 828-829
    • Olnes, M.I.1    Kurl, R.N.2
  • 24
    • 27544444006 scopus 로고    scopus 로고
    • DNA damage disrupts the p14ARF-B23(nucleophosmin) interaction and triggers a transient subnuclear redistribution of p14ARF
    • Lee C, Smith BA, Bandyopadhyay K, Gjerset RA, (2005) DNA damage disrupts the p14ARF-B23(nucleophosmin) interaction and triggers a transient subnuclear redistribution of p14ARF. Cancer Res 65: 9834-9842.
    • (2005) Cancer Res , vol.65 , pp. 9834-9842
    • Lee, C.1    Smith, B.A.2    Bandyopadhyay, K.3    Gjerset, R.A.4
  • 25
    • 0019889733 scopus 로고
    • The effect of salt on the binding of the eucaryotic DNA nicking-closing enzyme to DNA and chromatin
    • McConaughy BL, Young LS, Champoux JJ, (1981) The effect of salt on the binding of the eucaryotic DNA nicking-closing enzyme to DNA and chromatin. Biochim Biophys Acta 655: 1-8.
    • (1981) Biochim Biophys Acta , vol.655 , pp. 1-8
    • McConaughy, B.L.1    Young, L.S.2    Champoux, J.J.3
  • 26
    • 0035419786 scopus 로고    scopus 로고
    • A human topoisomerase I cleavage complex is recognized by an additional human topisomerase I molecule in vitro
    • Soe K, Dianov G, Nasheuer HP, Bohr VA, Grosse F, et al. (2001) A human topoisomerase I cleavage complex is recognized by an additional human topisomerase I molecule in vitro. Nucleic Acids Res 29: 3195-3203.
    • (2001) Nucleic Acids Res , vol.29 , pp. 3195-3203
    • Soe, K.1    Dianov, G.2    Nasheuer, H.P.3    Bohr, V.A.4    Grosse, F.5
  • 27
    • 0024411478 scopus 로고
    • Interactions between eukaryotic DNA topoisomerase I and a specific binding sequence
    • Stevnsner T, Mortensen UH, Westergaard O, Bonven BJ, (1989) Interactions between eukaryotic DNA topoisomerase I and a specific binding sequence. J Biol Chem 264: 10110-10113.
    • (1989) J Biol Chem , vol.264 , pp. 10110-10113
    • Stevnsner, T.1    Mortensen, U.H.2    Westergaard, O.3    Bonven, B.J.4
  • 29
    • 0032489634 scopus 로고    scopus 로고
    • Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA
    • Redinbo MR, Stewart L, Kuhn P, Champoux JJ, Hol WG, (1998) Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. Science 279: 1504-1513.
    • (1998) Science , vol.279 , pp. 1504-1513
    • Redinbo, M.R.1    Stewart, L.2    Kuhn, P.3    Champoux, J.J.4    Hol, W.G.5
  • 30
    • 0344668840 scopus 로고    scopus 로고
    • p53 stimulates human topoisomerase I activity by modulating its DNA binding
    • Soe K, Grosse F, (2003) p53 stimulates human topoisomerase I activity by modulating its DNA binding. Nucleic Acids Res 31: 6585-6592.
    • (2003) Nucleic Acids Res , vol.31 , pp. 6585-6592
    • Soe, K.1    Grosse, F.2
  • 31
    • 0032861343 scopus 로고    scopus 로고
    • Megabase chromatin domains involved in DNA double-strand breaks in vivo
    • Rogakou EP, Boon C, Redon C, Bonner WM, (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146: 905-916.
    • (1999) J Cell Biol , vol.146 , pp. 905-916
    • Rogakou, E.P.1    Boon, C.2    Redon, C.3    Bonner, W.M.4
  • 32
    • 0034746224 scopus 로고    scopus 로고
    • Irinotecan (CPT-11): recent developments and future directions-colorectal cancer and beyond
    • Rothenberg ML, (2001) Irinotecan (CPT-11): recent developments and future directions-colorectal cancer and beyond. Oncologist 6: 66-80.
    • (2001) Oncologist , vol.6 , pp. 66-80
    • Rothenberg, M.L.1
  • 33
    • 84871097466 scopus 로고    scopus 로고
    • National Comprehensive Cancer Network website. Available: www.NCCN.org. Accessed 2012 Oct 25.
  • 34
    • 1642579676 scopus 로고    scopus 로고
    • The role of topotecan in the treatment of brain metastases
    • Wong ET, Berkenblit A, (2004) The role of topotecan in the treatment of brain metastases. Oncologist 9: 68-79.
    • (2004) Oncologist , vol.9 , pp. 68-79
    • Wong, E.T.1    Berkenblit, A.2
  • 35
    • 0035215752 scopus 로고    scopus 로고
    • The development of camptothecin analogs in childhood cancers
    • Bomgaars L, Berg SL, Blaney SM, (2001) The development of camptothecin analogs in childhood cancers. Oncologist 6: 506-516.
    • (2001) Oncologist , vol.6 , pp. 506-516
    • Bomgaars, L.1    Berg, S.L.2    Blaney, S.M.3
  • 36
    • 0028226829 scopus 로고
    • Activation of a tumor-associated protein kinase (p40TAK) and casein kinase 2 in human squamous cell carcinomas and adenocarcinomas of the lung
    • Daya-Makin M, Sanghera JS, Mogentale TL, Lipp M, Parchomchuk J, et al. (1994) Activation of a tumor-associated protein kinase (p40TAK) and casein kinase 2 in human squamous cell carcinomas and adenocarcinomas of the lung. Cancer Res 54: 2262-2268.
    • (1994) Cancer Res , vol.54 , pp. 2262-2268
    • Daya-Makin, M.1    Sanghera, J.S.2    Mogentale, T.L.3    Lipp, M.4    Parchomchuk, J.5
  • 37
    • 0030021720 scopus 로고    scopus 로고
    • Elevated protein kinase CK2 activity in chromatin of head and neck tumors: association with malignant transformation
    • Faust RA, Gapany M, Tristani P, Davis A, Adams GL, et al. (1996) Elevated protein kinase CK2 activity in chromatin of head and neck tumors: association with malignant transformation. Cancer Lett 101: 31-35.
    • (1996) Cancer Lett , vol.101 , pp. 31-35
    • Faust, R.A.1    Gapany, M.2    Tristani, P.3    Davis, A.4    Adams, G.L.5
  • 39
    • 79951983174 scopus 로고    scopus 로고
    • Overexpression of nuclear protein kinase CK2 alpha catalytic subunit (CK2alpha) as a poor prognosticator in human colorectal cancer
    • Lin KY, Tai C, Hsu JC, Li CF, Fang CL, et al. (2011) Overexpression of nuclear protein kinase CK2 alpha catalytic subunit (CK2alpha) as a poor prognosticator in human colorectal cancer. PLoS One 6: e17193.
    • (2011) PLoS One , vol.6
    • Lin, K.Y.1    Tai, C.2    Hsu, J.C.3    Li, C.F.4    Fang, C.L.5
  • 41
    • 0028115657 scopus 로고
    • Nuclear casein kinase 2 (CK-2) activity in human normal, benign hyperplastic, and cancerous prostate
    • Yenice S, Davis AT, Goueli SA, Akdas A, Limas C, et al. (1994) Nuclear casein kinase 2 (CK-2) activity in human normal, benign hyperplastic, and cancerous prostate. Prostate 24: 11-16.
    • (1994) Prostate , vol.24 , pp. 11-16
    • Yenice, S.1    Davis, A.T.2    Goueli, S.A.3    Akdas, A.4    Limas, C.5
  • 42
    • 0029360679 scopus 로고
    • Association of elevated protein kinase CK2 activity with aggressive behavior of squamous cell carcinoma of the head and neck
    • Gapany M, Faust RA, Tawfic S, Davis A, Adams GL, et al. (1995) Association of elevated protein kinase CK2 activity with aggressive behavior of squamous cell carcinoma of the head and neck. Mol Med 1: 659-666.
    • (1995) Mol Med , vol.1 , pp. 659-666
    • Gapany, M.1    Faust, R.A.2    Tawfic, S.3    Davis, A.4    Adams, G.L.5
  • 43
    • 33847655629 scopus 로고    scopus 로고
    • Nuclear localization of protein kinase CK2 catalytic subunit (CK2alpha) is associated with poor prognostic factors in human prostate cancer
    • Laramas M, Pasquier D, Filhol O, Ringeisen F, Descotes JL, et al. (2007) Nuclear localization of protein kinase CK2 catalytic subunit (CK2alpha) is associated with poor prognostic factors in human prostate cancer. Eur J Cancer 43: 928-934.
    • (2007) Eur J Cancer , vol.43 , pp. 928-934
    • Laramas, M.1    Pasquier, D.2    Filhol, O.3    Ringeisen, F.4    Descotes, J.L.5
  • 44
    • 4444298254 scopus 로고    scopus 로고
    • Casein kinase II alpha subunit and C1-inhibitor are independent predictors of outcome in patients with squamous cell carcinoma of the lung
    • O-Charoenrat P, Rusch V, Talbot SG, Sarkaria I, Viale A, et al. (2004) Casein kinase II alpha subunit and C1-inhibitor are independent predictors of outcome in patients with squamous cell carcinoma of the lung. Clin Cancer Res 10: 5792-5803.
    • (2004) Clin Cancer Res , vol.10 , pp. 5792-5803
    • O-Charoenrat, P.1    Rusch, V.2    Talbot, S.G.3    Sarkaria, I.4    Viale, A.5
  • 45
    • 0032871379 scopus 로고    scopus 로고
    • Subcellular immunolocalization of protein kinase CK2 in normal and carcinoma cells
    • Faust RA, Niehans G, Gapany M, Hoistad D, Knapp D, et al. (1999) Subcellular immunolocalization of protein kinase CK2 in normal and carcinoma cells. Int J Biochem Cell Biol 31: 941-949.
    • (1999) Int J Biochem Cell Biol , vol.31 , pp. 941-949
    • Faust, R.A.1    Niehans, G.2    Gapany, M.3    Hoistad, D.4    Knapp, D.5
  • 46
    • 0343069462 scopus 로고
    • Vaccinia DNA topoisomerase I promotes illegitimate recombination in Escherichia coli
    • Shuman S, (1989) Vaccinia DNA topoisomerase I promotes illegitimate recombination in Escherichia coli. Proc Natl Acad Sci U S A 86: 3489-3493.
    • (1989) Proc Natl Acad Sci U S A , vol.86 , pp. 3489-3493
    • Shuman, S.1
  • 47
    • 0026657545 scopus 로고
    • DNA strand transfer reactions catalyzed by vaccinia topoisomerase I
    • Shuman S, (1992) DNA strand transfer reactions catalyzed by vaccinia topoisomerase I. J Biol Chem. 267: 8620-8627.
    • (1992) J Biol Chem , vol.267 , pp. 8620-8627
    • Shuman, S.1
  • 48
    • 0029960067 scopus 로고    scopus 로고
    • Topoisomerase I involvement in illegitimate recombination in Saccharomyces cerevisiae
    • Zhu J, Schiestl RH, (1996) Topoisomerase I involvement in illegitimate recombination in Saccharomyces cerevisiae. Mol Cell Biol 16: 1805-1812.
    • (1996) Mol Cell Biol , vol.16 , pp. 1805-1812
    • Zhu, J.1    Schiestl, R.H.2
  • 49
    • 77952581077 scopus 로고    scopus 로고
    • Inhibition of topoisomerase I prevents chromosome breakage at common fragile sites
    • Arlt MF, Glover TW, (2010) Inhibition of topoisomerase I prevents chromosome breakage at common fragile sites. DNA Repair (Amst) 9: 678-689.
    • (2010) DNA Repair (Amst) , vol.9 , pp. 678-689
    • Arlt, M.F.1    Glover, T.W.2
  • 50
    • 70449522304 scopus 로고    scopus 로고
    • Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription
    • Tuduri S, Crabbe L, Conti C, Tourriere H, Holtgreve-Grez H, et al. (2009) Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription. Nat Cell Biol 11: 1315-1324.
    • (2009) Nat Cell Biol , vol.11 , pp. 1315-1324
    • Tuduri, S.1    Crabbe, L.2    Conti, C.3    Tourriere, H.4    Holtgreve-Grez, H.5
  • 51
    • 0037417128 scopus 로고    scopus 로고
    • Delineation of the domains required for physical and functional interaction of p14ARF with human topoisomerase I
    • Ayrault O, Karayan L, Riou JF, Larsen CJ, Seite P, (2003) Delineation of the domains required for physical and functional interaction of p14ARF with human topoisomerase I. Oncogene. 22: 1945-1954.
    • (2003) Oncogene , vol.22 , pp. 1945-1954
    • Ayrault, O.1    Karayan, L.2    Riou, J.F.3    Larsen, C.J.4    Seite, P.5
  • 52
    • 0035864758 scopus 로고    scopus 로고
    • Human ARF protein interacts with topoisomerase I and stimulates its activity
    • Karayan L, Riou JF, Seite P, Migeon J, Cantereau A, et al. (2001) Human ARF protein interacts with topoisomerase I and stimulates its activity. Oncogene 20: 836-848.
    • (2001) Oncogene , vol.20 , pp. 836-848
    • Karayan, L.1    Riou, J.F.2    Seite, P.3    Migeon, J.4    Cantereau, A.5


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