Investigational New Drugs

, Volume 29, Issue 3, pp 514–522 | Cite as

A novel Chk inhibitor, XL-844, increases human cancer cell radiosensitivity through promotion of mitotic catastrophe

  • Oliver Riesterer
  • Fumihiko Matsumoto
  • Li Wang
  • Jessica Pickett
  • David Molkentine
  • Uma Giri
  • Luka Milas
  • Uma Raju


Check point kinases (Chk) play a major role in facilitating DNA repair upon radiation exposure. We tested the potency of a novel inhibitor of Chk1 and Chk2, XL-844 (provided by Exelixis Inc., CA, USA), to radiosensitize human cancer cells grown in culture and investigated the underlying mechanisms. HT-29 cells (a human colon cancer line) were exposed to XL-844, radiation, or both, and assessed for clonogenic cell survival. Treatment-dependent effects on phosphorylated forms of Chk proteins were assessed by Western blots. Further mechanistic investigations in HT-29 cells included cell cycle analysis by flowcytometry and assessment of DNA repair kinetics by immuno-cytochemistry (ICC) for nuclear appearance of the phosphorylated form of histone 2AX protein (γ-H2AX) staining. Cells undergoing mitotic catastrophe were identified by irregular pattern of mitotic spindle markers α and γ-tubulin staining by ICC. XL-844 enhanced radiosensitivity in a dose and schedule-dependent manner and the enhancement factor was 1.42 at 0.5 survival fraction. Mechanistically XL-844 abrogated radiation-induced Chk2 phosphorylation, induced pan-nuclear γ-H2AX, and prolonged the presence of radiation-induced γ-H2AX foci, and promoted mitotic catastrophe. In conclusion, our data showed that inhibition of Chk2 activity by XL-844 enhanced cancer cell radiosensitivity that was associated with inhibition of DNA repair and induction of mitotic catastrophe.


Radiosensitivity Inhibitor of Check point kinases XL-844 Mitotic catastrophe Pan-nuclear γ-H2AX 



This work was supported partly by Exelixis Inc., CA, USA (PI: U. Raju) and partly by a UICC ACSBI fellowship supplemented by the University of Zurich (O. Riesterer). We thank Peter Lamb Ph.D. (Exelixis Incorporation, San Francisco, CA) for his involvement in designing the experiments.


  1. 1.
    Zhou BB, Elledge SJ (2000) The DNA damage response: putting checkpoints in perspective. Nature 408(6811):433–439PubMedCrossRefGoogle Scholar
  2. 2.
    Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Chessa L, Smorodinsky NI, Prives C, Reiss Y, Shiloh Y, Ziv Y (1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281(5383):1674–1677PubMedCrossRefGoogle Scholar
  3. 3.
    Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguchi K, Appella E, Kastan MB, Siliciano JD (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281(5383):1677–1679PubMedCrossRefGoogle Scholar
  4. 4.
    Elledge SJ (1996) Cell cycle checkpoints: preventing an identity crisis. Science 274(5293):1664–1672PubMedCrossRefGoogle Scholar
  5. 5.
    Painter RB, Young BR (1980) Radiosensitivity in ataxia-telangiectasia: a new explanation. Proc Natl Acad Sci USA 77(12):7315–7317PubMedCrossRefGoogle Scholar
  6. 6.
    Samuel T, Weber HO, Funk JO (2002) Linking DNA damage to cell cycle checkpoints. Cell Cycle 1(3):162–168PubMedCrossRefGoogle Scholar
  7. 7.
    Chaturvedi P, Eng WK, Zhu Y, Mattern MR, Mishra R, Hurle MR, Zhang X, Annan RS, Lu Q, Faucette LF, Scott GF, Li X, Carr SA, Johnson RK, Winkler JD, Zhou BB (1999) Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Oncogene 18(28):4047–4054PubMedCrossRefGoogle Scholar
  8. 8.
    Pommier Y, Sordet O, Rao VA, Zhang H, Kohn KW (2005) Targeting chk2 kinase: molecular interaction maps and therapeutic rationale. Curr Pharm Des 11(22):2855–2872PubMedCrossRefGoogle Scholar
  9. 9.
    Bartek J, Falck J, Lukas J (2001) CHK2 kinase—a busy messenger. Nat Rev Mol Cell Biol 2(12):877–886PubMedCrossRefGoogle Scholar
  10. 10.
    Taylor WR, Stark GR (2001) Regulation of the G2/M transition by p53. Oncogene 20(15):1803–1815PubMedCrossRefGoogle Scholar
  11. 11.
    Choudhury A, Cuddihy A, Bristow RG (2006) Radiation and new molecular agents part I: targeting ATM-ATR checkpoints, DNA repair, and the proteasome. Semin Radiat Oncol 16(1):51–58PubMedCrossRefGoogle Scholar
  12. 12.
    Xu B, Kim ST, Lim DS, Kastan MB (2002) Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation. Mol Cell Biol 22(4):1049–1059PubMedCrossRefGoogle Scholar
  13. 13.
    Matthews DJ, Yakes FM, Chen J, Tadano M, Bornheim L, Clary DO, Tai A, Wagner JM, Miller N, Kim YD, Robertson S, Murray L, Karnitz LM (2007) Pharmacological abrogation of S-phase checkpoint enhances the anti-tumor activity of gemcitabine in vivo. Cell Cycle 6(1):104–110PubMedCrossRefGoogle Scholar
  14. 14.
    Busby EC, Leistritz DF, Abraham RT, Karnitz LM, Sarkaria JN (2000) The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1. Cancer Res 60(8):2108–2112PubMedGoogle Scholar
  15. 15.
    Jobson AG, Lountos GT, Lorenzi PL, Llamas J, Connelly J, Cerna D, Tropea JE, Onda A, Zoppoli G, Kondapaka S, Zhang G, Caplen NJ, Cardellina JH, Yoo SS, Monks A, Self C, Waugh DS, Shoemaker RH, Pommier Y (2009) Cellular inhibition of Chk2 kinase and potentiation of camptothecins and radiation by the novel Chk2 inhibitor PV1019. J Pharmacol Exp Ther Epub ahead of printGoogle Scholar
  16. 16.
    Ashwell S, Janetka JW, Zabludoff S (2008) Keeping checkpoint kinases in line: new selective inhibitors in clinical trials. Expert Opin Investig Drugs 17(9):1331–1340PubMedCrossRefGoogle Scholar
  17. 17.
    Fertil B, Malaise EP (1981) Inherent cellular radiosensitivity as a basic concept for human tumor radiotherapy. Int J Radiat Oncol Biol Phys 7(5):621–629PubMedCrossRefGoogle Scholar
  18. 18.
    Antoni L, Sodha N, Collins I, Garrett MD (2007) CHK2 kinase: cancer susceptibility and cancer therapy—two sides of the same coin? Nat Rev Cancer 7(12):925–936PubMedCrossRefGoogle Scholar
  19. 19.
    Matsuoka S, Huang M, Elledge SJ (1998) Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 282(5395):1893–1897PubMedCrossRefGoogle Scholar
  20. 20.
    Ahn JY, Schwarz JK, Piwnica-Worms H, Canman CE (2000) Threonine 68 phosphorylation by ataxia telangiectasia mutated is required for efficient activation of Chk2 in response to ionizing radiation. Cancer Res 60(21):5934–5936PubMedGoogle Scholar
  21. 21.
    Tse AN, Rendahl KG, Sheikh T, Cheema H, Aardalen K, Embry M, Ma S, Moler EJ, Ni ZJ, Lopes de Menezes DE, Hibner B, Gesner TG, Schwartz GK (2007) CHIR-124, a novel potent inhibitor of Chk1, potentiates the cytotoxicity of topoisomerase I poisons in vitro and in vivo. Clin Cancer Res 13(2 Pt 1):591–602PubMedCrossRefGoogle Scholar
  22. 22.
    Xiao Z, Xue J, Sowin TJ, Rosenberg SH, Zhang H (2005) A novel mechanism of checkpoint abrogation conferred by Chk1 downregulation. Oncogene 24(8):1403–1411PubMedCrossRefGoogle Scholar
  23. 23.
    Ganzinelli M, Carrassa L, Crippa F, Tavecchio M, Broggini M, Damia G (2008) Checkpoint Kinase 1 down-regulation by an inducible small interfering RNA expression system sensitized in vivo tumors to treatment with 5-fluorouracil. Clin Cancer Res 14(16):5131–5141PubMedCrossRefGoogle Scholar
  24. 24.
    Koniaras K, Cuddihy AR, Christopoulos H, Hogg A, O’Connell MJ (2001) Inhibition of Chk1-dependent G2 DNA damage checkpoint radiosensitizes p53 mutant human cells. Oncogene 20(51):7453–7463PubMedCrossRefGoogle Scholar
  25. 25.
    Zhao H, Watkins JL, Piwnica-Worms H (2002) Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints. Proc Natl Acad Sci USA 99(23):14795–14800PubMedCrossRefGoogle Scholar
  26. 26.
    Yu Q, La Rose J, Zhang H, Takemura H, Kohn KW, Pommier Y (2002) UCN-01 inhibits p53 up-regulation and abrogates gamma-radiation-induced G(2)-M checkpoint independently of p53 by targeting both of the checkpoint kinases, Chk2 and Chk1. Cancer Res 62(20):5743–5748PubMedGoogle Scholar
  27. 27.
    Atherton-Fessler S, Parker LL, Geahlen RL, Piwnica-Worms H (1993) Mechanisms of p34cdc2 regulation. Mol Cell Biol 13(3):1675–1685PubMedGoogle Scholar
  28. 28.
    Galaktionov K, Lee AK, Eckstein J, Draetta G, Meckler J, Loda M, Beach D (1995) CDC25 phosphatases as potential human oncogenes. Science 269(5230):1575–1577PubMedCrossRefGoogle Scholar
  29. 29.
    Norbury C, Nurse P (1992) Animal cell cycles and their control. Annu Rev Biochem 61:441–470PubMedCrossRefGoogle Scholar
  30. 30.
    Watanabe N, Broome M, Hunter T (1995) Regulation of the human WEE1Hu CDK tyrosine 15-kinase during the cell cycle. Embo J 14(9):1878–1891PubMedGoogle Scholar
  31. 31.
    Hunter T (1995) Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80(2):225–236PubMedCrossRefGoogle Scholar
  32. 32.
    Kastan MB, Lim DS, Kim ST, Yang D (2001) ATM—a key determinant of multiple cellular responses to irradiation. Acta Oncol 40(6):686–688PubMedCrossRefGoogle Scholar
  33. 33.
    Raju U, Ariga H, Dittmann K, Nakata E, Ang KK, Milas L (2005) Inhibition of DNA repair as a mechanism of enhanced radioresponse of head and neck carcinoma cells by a selective cyclooxygenase-2 inhibitor, celecoxib. Int J Radiat Oncol Biol Phys 63(2):520–528PubMedCrossRefGoogle Scholar
  34. 34.
    Syljuasen RG, Sorensen CS, Hansen LT, Fugger K, Lundin C, Johansson F, Helleday T, Sehested M, Lukas J, Bartek J (2005) Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. Mol Cell Biol 25(9):3553–3562PubMedCrossRefGoogle Scholar
  35. 35.
    Belmont LD, Hyman AA, Sawin KE, Mitchison TJ (1990) Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 62(3):579–589PubMedCrossRefGoogle Scholar
  36. 36.
    Eriksson D, Lofroth PO, Johansson L, Riklund KA, Stigbrand T (2007) Cell cycle disturbances and mitotic catastrophes in HeLa Hep2 cells following 2.5 to 10 Gy of ionizing radiation. Clin Cancer Res 13(18 Pt 2):5501s–5508sPubMedCrossRefGoogle Scholar
  37. 37.
    McIntosh JR (1984) Cell biology.Microtubule catastrophe. Nature 312(5991):196–197PubMedCrossRefGoogle Scholar
  38. 38.
    Robinson HM, Black EJ, Brown R, Gillespie DA (2007) DNA mismatch repair and Chk1-dependent centrosome amplification in response to DNA alkylation damage. Cell Cycle 6(8):982–992PubMedCrossRefGoogle Scholar
  39. 39.
    Castedo M, Perfettini JL, Roumier T, Yakushijin K, Horne D, Medema R, Kroemer G (2004) The cell cycle checkpoint kinase Chk2 is a negative regulator of mitotic catastrophe. Oncogene 23(25):4353–4361PubMedCrossRefGoogle Scholar
  40. 40.
    Roninson IB, Broude EV, Chang BD (2001) If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells. Drug Resist Updat 4(5):303–313PubMedCrossRefGoogle Scholar
  41. 41.
    Cenciarelli C, Tanzarella C, Vitale I, Pisano C, Crateri P, Meschini S, Arancia G, Antoccia A (2008) The tubulin-depolymerising agent combretastatin-4 induces ectopic aster assembly and mitotic catastrophe in lung cancer cells H460. Apoptosis 13(5):659–669PubMedCrossRefGoogle Scholar
  42. 42.
    Brown JM, Attardi LD (2005) The role of apoptosis in cancer development and treatment response. Nat Rev Cancer 5(3):231–237PubMedCrossRefGoogle Scholar
  43. 43.
    Niida H, Tsuge S, Katsuno Y, Konishi A, Takeda N, Nakanishi M (2005) Depletion of Chk1 leads to premature activation of Cdc2-cyclin B and mitotic catastrophe. J Biol Chem 280(47):39246–39252PubMedCrossRefGoogle Scholar
  44. 44.
    Mitchison T, Kirschner M (1984) Dynamic instability of microtubule growth. Nature 312(5991):237–242PubMedCrossRefGoogle Scholar
  45. 45.
    Oakley CE, Oakley BR (1989) Identification of gamma-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans. Nature 338(6217):662–664PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Oliver Riesterer
    • 1
    • 2
  • Fumihiko Matsumoto
    • 1
  • Li Wang
    • 1
  • Jessica Pickett
    • 1
  • David Molkentine
    • 1
  • Uma Giri
    • 1
  • Luka Milas
    • 1
  • Uma Raju
    • 1
  1. 1.Department of Experimental Radiation OncologyThe University of Texas M. D. Anderson Cancer CenterHoustonUSA
  2. 2.Department of Radiation OncologyUniversity Hospital ZurichZurichSwitzerland

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