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PMID: 7759997 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Absence of autoantigen Ku in mature human neutrophils and human promyelocytic leukemia line (HL-60) cells and lymphocytes undergoing apoptosis.

The Journal of experimental medicine ·Vol. 181 ·No. 6 ·1995-06-01 ·Pages 2049-58

Ajmani AK, Satoh M, Reap E, Cohen PL, Reeves WH

Abstract

The Ku autoantigen is a heterodimer of 70- and 80-kD proteins recognized by autoantibodies from patients with systemic lupus erythematosus and related diseases that is the DNA-binding component of a DNA-dependent protein kinase. The catalytic activity of DNA-dependent protein kinase is carried by a 350-kD subunit (p350). In light of the recently described role of Ku in repairing double-strand DNA breaks, we investigated the regulation of Ku and p350 levels in neutrophils, a terminally differentiated cell type destined to undergo apoptosis. Since the appearance of double-strand DNA breaks is characteristic of apoptosis, we were interested in the possibility that Ku might oppose programmed cell death. Analysis of peripheral blood cells by flow cytometry using anti-Ku and anti-p350 monoclonal antibodies revealed that neutrophils were unstained, whereas resting (G0) lymphocytes were positive. The absence of Ku in mature neutrophils was confirmed by Western blotting and enzyme-linked immunosorbent assay for Ku antigen. In contrast, the human promyelocytic leukemia line, HL-60, which undergoes differentiation toward neutrophils after dimethylsulfoxide treatment, was positive for Ku and p350. In view of the short lifespan of neutrophils and the prolonged half-life of Ku and p350 (> 5 d), these data suggested that Ku was actively degraded during myeloid differentiation. Analysis of HL-60 cells by flow cytometry revealed that Ku staining was bimodal. Cells in G1/G0, S, or G2/M were all stained positively, whereas cells with a subdiploid DNA content characteristic of apoptosis were Ku negative. Similar results were obtained with phytohemagglutin-stimulated human lymphocytes. These data suggest that the Ku antigen is actively degraded in both myeloid cells destined to undergo apoptosis and apoptotic lymphocytes, raising the possibility that degradation of Ku may help to prevent the inappropriate repair of fragmented nuclear DNA during apoptosis.

MeSH Terms
Antibodies, Monoclonal Antigens, Nuclear Apoptosis Autoantibodies/immunology Autoantigens/analysis Blotting, Western Cell Line DNA/analysis DNA Helicases DNA-Activated Protein Kinase DNA-Binding Proteins/analysis,metabolism Humans Kinetics Ku Autoantigen Leukemia, Promyelocytic, Acute Lupus Erythematosus, Systemic/immunology Lymphocytes/enzymology,immunology,physiology Macromolecular Substances Neutrophils/enzymology,immunology,physiology Nuclear Proteins/analysis,metabolism Protein Serine-Threonine Kinases/metabolism Tumor Cells, Cultured
Chemicals
Antibodies, Monoclonal Antigens, Nuclear Autoantibodies Autoantigens DNA-Binding Proteins Macromolecular Substances Nuclear Proteins DNA DNA-Activated Protein Kinase PRKDC protein, human Protein Serine-Threonine Kinases DNA Helicases XRCC5 protein, human Xrcc6 protein, human Ku Autoantigen
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Ajmani A K
Department of Medicine, Thurston Arthritis Research Center, Chapel Hill, North Carolina, USA.
Satoh M
Reap E
Cohen P L
Reeves W H
References (49)
49 references, click to expand
  1. The development of neutrophilic polymorphonuclear leukocytes in human bone marrow.
    J Exp Med. 1971 Oct 1;134(4):907-34 PMID: 4106490
  2. Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA.
    Science. 1994 Oct 14;266(5183):288-91 PMID: 7939667
  3. Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers.
    J Cell Sci. 1994 Nov;107 ( Pt 11):3223-33 PMID: 7699019
  4. Role of free p70 (Ku) subunit in posttranslational stabilization of newly synthesized p80 during DNA-dependent protein kinase assembly.
    Eur J Cell Biol. 1995 Feb;66(2):127-35 PMID: 7774599
  5. Induction of activation-driven death (apoptosis) in activated but not resting peripheral blood T cells.
    J Immunol. 1993 May 15;150(10):4338-45 PMID: 8482839
  6. Normal functional characteristics of cultured human promyelocytic leukemia cells (HL-60) after induction of differentiation by dimethylsulfoxide.
    J Exp Med. 1979 Apr 1;149(4):969-74 PMID: 219131
  7. Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease.
    Proc Natl Acad Sci U S A. 1981 May;78(5):2737-41 PMID: 6789322
  8. Characterization of a high molecular weight acidic nuclear protein recognized by autoantibodies in sera from patients with polymyositis-scleroderma overlap.
    J Clin Invest. 1981 Sep;68(3):611-20 PMID: 7276162
  9. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor.
    Cell. 1983 Dec;35(3 Pt 2):603-10 PMID: 6606489
  10. Use of monoclonal antibodies for the characterization of novel DNA-binding proteins recognized by human autoimmune sera.
    J Exp Med. 1985 Jan 1;161(1):18-39 PMID: 2578541
  11. Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity.
    J Immunol. 1986 Mar 1;136(5):1648-53 PMID: 2419410
  12. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis.
    Science. 1986 Oct 17;234(4774):364-8 PMID: 2876518
  13. Levels of synthesis of primate-specific nuclear proteins differ between growth-arrested and proliferating cells.
    Exp Cell Res. 1987 Feb;168(2):389-401 PMID: 3542540
  14. Antinuclear antibodies as probes to explore the structural organization of the genome.
    J Rheumatol Suppl. 1987 Jun;14 Suppl 13:97-105 PMID: 3497271
  15. The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression.
    Blood. 1987 Nov;70(5):1233-44 PMID: 3311197
  16. Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites.
    J Cell Biol. 1987 Oct;105(4):1549-54 PMID: 2889739
  17. Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages.
    J Clin Invest. 1989 Mar;83(3):865-75 PMID: 2921324
  18. Flow cytometric correlation of the c-myc oncoprotein and cell cycle kinetics of HL60 leukaemia during induced maturation with cytosine arabinoside and dimethylsulphoxide.
    Cell Tissue Kinet. 1989 Jan;22(1):1-11 PMID: 2676187
  19. Cell cycle-related proteins and flow cytometry.
    Haematologica. 1990 May-Jun;75(3):252-64 PMID: 2146199
  20. A DNA-activated protein kinase from HeLa cell nuclei.
    Mol Cell Biol. 1990 Dec;10(12):6460-71 PMID: 2247066
  21. Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen.
    Mol Cell Biol. 1990 Dec;10(12):6472-81 PMID: 2247067
  22. Degradation of nuclear oncoproteins by the ubiquitin system in vitro.
    Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):139-43 PMID: 1846034
  23. Identification of nuclear factor IV/Ku autoantigen in a human 2D-gel protein database. Modification of the large subunit depends on cellular proliferation.
    FEBS Lett. 1991 Apr 22;282(1):189-92 PMID: 2026259
  24. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.
    J Immunol Methods. 1991 Jun 3;139(2):271-9 PMID: 1710634
  25. Expression of the Ku protein during cell proliferation.
    Biochim Biophys Acta. 1991 Oct 8;1090(2):181-7 PMID: 1932109
  26. Dependence of cell survival on DNA repair in human mononuclear phagocytes.
    J Immunol. 1991 Dec 15;147(12):4302-6 PMID: 1753100
  27. bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells.
    Blood. 1992 Mar 1;79(5):1291-8 PMID: 1536952
  28. Induction of apoptosis in fibroblasts by c-myc protein.
    Cell. 1992 Apr 3;69(1):119-28 PMID: 1555236
  29. Apoptosis and programmed cell death in immunity.
    Annu Rev Immunol. 1992;10:267-93 PMID: 1590988
  30. Antibodies to snRNPs in systemic lupus erythematosus.
    Rheum Dis Clin North Am. 1992 May;18(2):311-35 PMID: 1626072
  31. Apoptotic cell death induced by c-myc is inhibited by bcl-2.
    Nature. 1992 Oct 8;359(6395):552-4 PMID: 1406975
  32. Regulation of p26-bcl-2 protein levels in human peripheral blood lymphocytes.
    Lab Invest. 1992 Oct;67(4):443-9 PMID: 1434528
  33. Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II.
    Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11920-4 PMID: 1465419
  34. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen.
    Cell. 1993 Jan 15;72(1):131-42 PMID: 8422676
  35. HL-60 cells induced to differentiate towards neutrophils subsequently die via apoptosis.
    Clin Exp Immunol. 1990 Mar;79(3):448-53 PMID: 2317949
  36. Purification and characterization of proximal sequence element-binding protein 1, a transcription activating protein related to Ku and TREF that binds the proximal sequence element of the human U1 promoter.
    J Biol Chem. 1990 Oct 15;265(29):17911-20 PMID: 2211668
  37. Site-specific proteolytic cleavage of Ku protein bound to DNA.
    Proteins. 1993 Mar;15(3):330-7 PMID: 8456100
  38. Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens.
    Mol Biol Rep. 1993 Jun;18(1):15-28 PMID: 7694076
  39. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme.
    Cell. 1993 Nov 19;75(4):641-52 PMID: 8242740
  40. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3.
    Cell. 1993 Nov 19;75(4):653-60 PMID: 8242741
  41. DNA damage and the DNA-activated protein kinase.
    Trends Biochem Sci. 1993 Nov;18(11):433-7 PMID: 8291090
  42. bcl-2 inhibits apoptosis of neutrophils but not their engulfment by macrophages.
    J Exp Med. 1994 Mar 1;179(3):1047-52 PMID: 8113673
  43. Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes.
    J Exp Med. 1994 Apr 1;179(4):1317-30 PMID: 7511686
  44. Bcl2 inhibits apoptosis associated with terminal differentiation of HL-60 myeloid leukemia cells.
    Blood. 1994 Apr 15;83(8):2261-7 PMID: 8161792
  45. Calpain activation in apoptosis.
    J Cell Physiol. 1994 May;159(2):229-37 PMID: 8163563
  46. Decreased membrane phospholipid packing and decreased cell size precede DNA cleavage in mature mouse B cell apoptosis.
    J Immunol. 1994 May 15;152(10):4832-42 PMID: 8176206
  47. Involvement of the Ku autoantigen in the cellular response to DNA double-strand breaks.
    Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7623-7 PMID: 8052631
  48. Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination.
    Science. 1994 Sep 2;265(5177):1442-5 PMID: 8073286
  49. Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen.
    Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):890-4 PMID: 7846073
Article Info
Journal
The Journal of experimental medicine
Abbr.
J Exp Med
ISSN
0022-1007
DOI
10.1084/jem.181.6.2049
Published
1995-06-01
Pages
2049-58
Language
English
Region
United States
NLM ID
2985109R
PMCID
PMC2192057
Subset
IM
Grants
NIAMS NIH HHS · P50-AR42573 · United States
NIAMS NIH HHS · P60-AR30701 · United States
NIAMS NIH HHS · R01-AR40391 · United States
External Links
PubMed source DOI: 10.1084/jem.181.6.2049 PMC Full Text View journal details
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