DNA-PK: the major target for wortmannin-mediated radiosensitization by the inhibition of DSB repair via NHEJ pathway.

The effect of wortmannin posttreatment was studied in cells derived from different species (hamster, mouse, chicken, and human) with normal and defective DNA-dependent protein kinase (DNA-PK) activity, cells with and without the ataxia telangiectasia (ATM) gene, and cells lacking other regulatory proteins involved in the DNA double-strand break (DSB) repair pathways. Clonogenic assays were used to obtain all results. Wortmannin radiosensitization was observed in Chinese hamster cells (V79-B310H , CHO-K1), mouse mammary carcinoma cells (SR-1), transformed human fibroblast (N2KYSV), chicken B lymphocyte wild-type cells (DT40), and chicken Rad54 knockout cells (Rad54-/-). However, mouse mammary carcinoma cells (SX9) with defects in the DNA-PK and chicken DNA-PK catalytic subunit (DNA-PKcs) knockout cells (DNA-PKcs-/-/-) failed to exhibit wortmannin radiosensitization. On the other hand, SCID mouse cells (SC3VA2) exposed to wortmannin exhibited significant increases in radiosensitivity, possibly because of some residual function of DNA-PKcs. Moreover, the transformed human cells derived from AT patients (AT2KYSV) and chicken ATM knockout cells (ATM-/-) showed pronounced wortmannin radiosensitization. These studies demonstrate confirm that the mechanism underlying wortmannin radiosensitization is the inhibition of DNA-PK, but not of ATM, thereby resulting in the inhibition of DSB repair via nonhomologous endjoining (NHEJ).

[1]  T. Honjo,et al.  DNA Double-Strand Breaks , 2002, The Journal of experimental medicine.

[2]  Y. Hosoi,et al.  Roles of DNA-dependent protein kinase and ATM in cell-cycle-dependent radiation sensitivity in human cells , 2002, International journal of radiation biology.

[3]  P. Dhar,et al.  Genetic Analysis of the DNA-dependent Protein Kinase Reveals an Inhibitory Role of Ku in Late S–G2 Phase DNA Double-strand Break Repair* , 2001, The Journal of Biological Chemistry.

[4]  H. Utsumi,et al.  Requirement for Repair of DNA Double-Strand Breaks by Homologous Recombination in Split-Dose Recovery , 2001, Radiation research.

[5]  Y. Matsumoto,et al.  Sensitization by wortmannin of heat- or X-ray induced cell death in cultured Chinese hamster V79 cells. , 2000, Journal of radiation research.

[6]  T Hyslop,et al.  DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus. , 2000, Cancer research.

[7]  A. Shinohara,et al.  The controlling role of ATM in homologous recombinational repair of DNA damage , 2000, The EMBO journal.

[8]  R. Wells,et al.  Wortmannin sensitizes mammalian cells to radiation by inhibiting the DNA-dependent protein kinase-mediated rejoining of double-strand breaks. , 1999, Radiation research.

[9]  S. Okada,et al.  The phosphatidylinositol 3-kinase inhibitor wortmannin sensitizes quiescent but not proliferating MG-63 human osteosarcoma cells to radiation. , 1998, Cancer letters.

[10]  J. Sarkaria,et al.  Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. , 1998, Cancer research.

[11]  Y. Yamaguchi-Iwai,et al.  Homologous recombination and non‐homologous end‐joining pathways of DNA double‐strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells , 1998, The EMBO journal.

[12]  R. Fukumura,et al.  Murine Cell Line SX9 Bearing a Mutation in the dna-pkcsGene Exhibits Aberrant V(D)J Recombination Not Only in the Coding Joint but Also in the Signal Joint* , 1998, The Journal of Biological Chemistry.

[13]  R. Okayasu,et al.  Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells. , 1998, Radiation research.

[14]  Akira Shinohara,et al.  Rad51‐deficient vertebrate cells accumulate chromosomal breaks prior to cell death , 1998, The EMBO journal.

[15]  Shengfang Jin,et al.  Double‐strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions , 1997, The EMBO journal.

[16]  J. Nickoloff,et al.  Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells , 1997, Molecular and cellular biology.

[17]  K. Rosenzweig,et al.  Radiosensitization of human tumor cells by the phosphatidylinositol3-kinase inhibitors wortmannin and LY294002 correlates with inhibition of DNA-dependent protein kinase and prolonged G2-M delay. , 1997, Clinical cancer research : an official journal of the American Association for Cancer Research.

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

[19]  L. Mi,et al.  The p53 status of Chinese hamster V79 cells frequently used for studies on DNA damage and DNA repair. , 1997, Nucleic acids research.

[20]  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.

[21]  S. Boulton,et al.  Wortmannin is a potent inhibitor of DNA double strand break but not single strand break repair in Chinese hamster ovary cells. , 1996, Carcinogenesis.

[22]  B. Price,et al.  The phosphatidylinositol 3-kinase inhibitor wortmannin sensitizes murine fibroblasts and human tumor cells to radiation and blocks induction of p53 following DNA damage. , 1996, Cancer research.

[23]  D. Weaver,et al.  Regulation and repair of double-strand DNA breaks. , 1996, Critical reviews in eukaryotic gene expression.

[24]  M. Meyn,et al.  Ataxia-telangiectasia and cellular responses to DNA damage. , 1995, Cancer research.

[25]  S. Schreiber,et al.  PIK-Related Kinases: DNA Repair, Recombination, and Cell Cycle Checkpoints , 1995, Science.

[26]  M. Connelly,et al.  DNA-dependent protein kinase catalytic subunit: A relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product , 1995, Cell.

[27]  K. Khanna,et al.  Nature of G1/S cell cycle checkpoint defect in ataxia-telangiectasia. , 1995, Oncogene.

[28]  M. Lovett,et al.  A single ataxia telangiectasia gene with a product similar to PI-3 kinase. , 1995, Science.

[29]  F. Alt,et al.  Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation , 1995, Cell.

[30]  M. Oettinger,et al.  DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect , 1995, Science.

[31]  J. Ward,et al.  The complexity of DNA damage: relevance to biological consequences. , 1994, International journal of radiation biology.

[32]  J. Dodge,et al.  Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase. , 1994, Cancer research.

[33]  T. Okada,et al.  Blockage of chemotactic peptide-induced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase. , 1994, The Journal of biological chemistry.

[34]  A. Arcaro,et al.  Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. , 1993, The Biochemical journal.

[35]  N. Niikawa,et al.  Functional complementation in mouse-human radiation hybrids assigns the putative murine scid gene to the pericentric region of human chromosome 8. , 1993, Human molecular genetics.

[36]  B. Vogelstein,et al.  A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia , 1992, Cell.

[37]  G. Iliakis,et al.  Level of DNA double-strand break rejoining in Chinese hamster xrs-5 cells is dose-dependent: implications for the mechanism of radiosensitivity. , 1992, International journal of radiation biology.

[38]  D. Schatz,et al.  A link between double-strand break-related repair and V(D)J recombination: the scid mutation. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[39]  A. Giaccia,et al.  scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[40]  A. Giaccia,et al.  Human chromosome 5 complements the DNA double-strand break-repair deficiency and gamma-ray sensitivity of the XR-1 hamster variant. , 1990, American journal of human genetics.

[41]  Naomi Rosenberg,et al.  The defect in murine severe combined immune deficiency: Joining of signal sequences but not coding segments in V(D)J recombination , 1988, Cell.

[42]  M. M. Elkind,et al.  Two forms of potentially lethal damage have similar repair kinetics in plateau- and in log-phase cells. , 1985, International journal of radiation biology and related studies in physics, chemistry, and medicine.

[43]  P. Jeggo,et al.  X-ray sensitive mutants of Chinese hamster ovary cells defective in double-strand break rejoining. , 1984, Mutation research.