Cross-resistance to ionizing radiation in a murine leukemic cell line resistant to cis-dichlorodiammineplatinum(II): role of Ku autoantigen.

cis-Dichlorodiammineplatinum(II) (CDDP; cisplatin) is commonly used in combination with ionizing radiation (IR) in the treatment of various malignancies. In vitro, many observations suggest that acquisition of CDDP resistance in cell lines confers cross-resistance to IR, but the molecular mechanisms involved have not been well documented yet. We report here the selection and characterization of a murine CDDP-resistant L1210 cell line (L1210/3R) that exhibits cross-resistance to IR because of an increased capacity to repair double-strand breaks compared with parental cells (L1210/P). In resistant cells, electrophoretic mobility shift assays revealed an increased DNA-end binding activity that could be ascribed, by supershifting the retardation complexes with antibodies, to the autoantigen Ku. The heterodimeric Ku protein, composed of 86-kDa (Ku80) and 70-kDa (Ku70) subunits, is the DNA-targeting component of DNA-dependent protein kinase (DNA-PK), which plays a critical role in mammalian DNA double-strand breaks repair. The increased Ku-binding activity in resistant cells was associated with an overexpression affecting specifically the Ku80 subunit. These data strongly suggest that the increase in Ku activity is responsible for the phenotype of cross-resistance to IR. In addition, these observations, along with previous results from DNA-PK- mutant cells, provide evidence in favor of a role of Ku/DNA-PK in resistance to CDDP. These results suggest that Ku activity may be an important molecular target in cancer therapy at the crossroad between cellular responses to CDDP and IR.

[1]  L. Panasci,et al.  DNA-Dependent protein kinase activity correlates with clinical and in vitro sensitivity of chronic lymphocytic leukemia lymphocytes to nitrogen mustards. , 1998, Blood.

[2]  L. Panasci,et al.  Potentiation of chlorambucil cytotoxicity in B-cell chronic lymphocytic leukemia by inhibition of DNA-dependent protein kinase activity using wortmannin. , 1998, Cancer research.

[3]  C. Muller,et al.  Human normal peripheral blood B-lymphocytes are deficient in DNA-dependent protein kinase activity due to the expression of a variant form of the Ku86 protein , 1998, Oncogene.

[4]  P. Frit,et al.  UV sensitivity and impaired nucleotide excision repair in DNA-dependent protein kinase mutant cells. , 1998, Nucleic acids research.

[5]  D. Ramsden,et al.  Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double‐strand breaks , 1998, The EMBO journal.

[6]  A. Sancar,et al.  Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions 5' to the cross-linked base and removes a 22- to 28-nucleotide-long damage-free strand , 1997, Molecular and cellular biology.

[7]  C. Muller,et al.  Regulation of DNA-dependent protein kinase activity in leukemic cells , 1997, Oncogene.

[8]  J. Turchi,et al.  Induction of apoptosis in cisplatin-sensitive and -resistant human ovarian cancer cell lines. , 1997, Cancer research.

[9]  David J. Chen,et al.  Ku70 Is Required for DNA Repair but Not for T Cell Antigen Receptor Gene Recombination In Vivo , 1997, The Journal of experimental medicine.

[10]  P. Jeggo.  DNA-PK: at the cross-roads of biochemistry and genetics. , 1997, Mutation research.

[11]  P. Frit,et al.  Deficient apoptotic process in cisplatin-resistant L1210 cells cannot account for the cellular response to various drug treatments. , 1997, Biochemical and biophysical research communications.

[12]  P. Jeggo,et al.  Molecular and biochemical characterization of xrs mutants defective in Ku80 , 1997, Molecular and cellular biology.

[13]  S. Chaney,et al.  DNA repair: enzymatic mechanisms and relevance to drug response. , 1996, Journal of the National Cancer Institute.

[14]  M. Lieber,et al.  Protein-protein and protein-DNA interaction regions within the DNA end-binding protein Ku70-Ku86 , 1996, Molecular and cellular biology.

[15]  Molly A Bogue,et al.  Ku86-Deficient Mice Exhibit Severe Combined Immunodeficiency and Defective Processing of V(D)J Recombination Intermediates , 1996, Cell.

[16]  M. Nussenzweig,et al.  Requirement for Ku80 in growth and immunoglobulin V(D)J recombination , 1996, Nature.

[17]  B. Fertil,et al.  A new model describing the curves for repair of both DNA double-strand breaks and chromosome damage. , 1996, Radiation research.

[18]  J. Um,et al.  Isolation of mammalian cell mutants that are X-ray sensitive, impaired in DNA double-strand break repair and defective for V(D)J recombination. , 1995, Mutation research.

[19]  M. Oshimura,et al.  Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[20]  M. Satoh,et al.  Role of free p70 (Ku) subunit in posttranslational stabilization of newly synthesized p80 during DNA-dependent protein kinase assembly. , 1995, European journal of cell biology.

[21]  P. Jeggo,et al.  DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Lieber,et al.  Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. , 1994, Science.

[23]  F. Alt,et al.  A DNA repair defect in Chinese hamster ovary cells affects V(D)J recombination similarly to the murine scid mutation. , 1994, The Journal of biological chemistry.

[24]  H. Sugiyama,et al.  The scid mutation in mice causes defects in the repair system for both double-strand DNA breaks and DNA cross-links. , 1993, Mutation research.

[25]  M. Falzon,et al.  EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA. , 1993, The Journal of biological chemistry.

[26]  A. Griffith,et al.  Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks. , 1993, The Journal of biological chemistry.

[27]  J. Barret,et al.  DNA excision-repair synthesis is enhanced in a murine leukemia L1210 cell line resistant to cisplatin. , 1993, European journal of biochemistry.

[28]  S. Jackson,et al.  The DNA-dependent protein kinase: Requirement for DNA ends and association with Ku antigen , 1993, Cell.

[29]  W. Zhang,et al.  On the mechanisms of Ku protein binding to DNA. , 1992, Biochemical and biophysical research communications.

[30]  J. Quinn,et al.  The Ku complex is modulated in response to viral infection and other cellular changes. , 1992, Biochimica et biophysica acta.

[31]  P. Jeggo,et al.  Cross-sensitivity of γ-ray-sensitive hamster mutants to cross-linking agents , 1991 .

[32]  P. Scalliet,et al.  Resistance patterns between cis-diamminedichloroplatinum(II) and ionizing radiation. , 1991, Cancer research.

[33]  D. Ettinger,et al.  Ifosfamide in the treatment of non-small cell lung cancer. , 1989, Seminars in oncology.

[34]  H. Schuitemaker,et al.  cis-diamminedichloroplatinum(II)-resistant sublines derived from two human ovarian tumor cell lines. , 1988, Cancer research.

[35]  R. Wood,et al.  Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts , 1988, Cell.

[36]  K. Wallner,et al.  Effect of cisplatin resistance on cellular radiation response. , 1987, International journal of radiation oncology, biology, physics.

[37]  E. Tan,et al.  Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity. , 1986, Journal of immunology.

[38]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[39]  C. Badie,et al.  Hypersensitivity of ataxia telangiectasia fibroblasts to ionizing radiation is associated with a repair deficiency of DNA double-strand breaks. , 1997, International journal of radiation biology.

[40]  K. Sakaguchi,et al.  The human DNA-activated protein kinase, DNA-PK: Substrate specificity , 1995 .

[41]  P. Jeggo,et al.  Cross-sensitivity of gamma-ray-sensitive hamster mutants to cross-linking agents. , 1991, Mutation research.

[42]  B. Hill.  INTERACTION BETWEEN ANTI TUMOR AGENT AND RADIATION AND EXPRESSION OF RESISTANCE , 1991 .

[43]  C. Rogers,et al.  Genotoxicity of 1-nitronaphthalene in Chinese hamster V79 cells. , 1991, Mutation research.

[44]  C. Coughlin,et al.  Biologic and clinical developments of cisplatin combined with radiation: concepts, utility, projections for new trials, and the emergence of carboplatin. , 1989, Seminars in oncology.

[45]  P. Sharp,et al.  In vitro transcription: whole-cell extract. , 1983, Methods in enzymology.

[46]  R. Geran,et al.  PROTOCOLS FOR SCREENING CHEMICAL AGENTS AND NATURAL PRODUCTS AGAINST ANIMAL TUMORS AND OTHER BIOLOGICAL SYSTEMS , 1972 .