title: A novel Chk inhibitor, XL-844, increases human cancer cell radiosensitivity through promotion of mitotic catastrophe.
abstract: 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.
authors.affiliation: Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Unit 66, 1515 Holcombe Blvd., Houston, TX 77030, USA.
authors.forename: Oliver
authors.initials: O
authors.lastname: Riesterer
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Matsumoto
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Jessica
authors.initials: J
authors.lastname: Pickett
authors.forename: David
authors.initials: D
authors.lastname: Molkentine
authors.forename: Uma
authors.initials: U
authors.lastname: Giri
authors.forename: Luka
authors.initials: L
authors.lastname: Milas
authors.forename: Uma
authors.initials: U
authors.lastname: Raju
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D006657:Histones / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008938:Mitosis / Q000187:drug effects*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011836:Radiation Tolerance / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D014404:Tubulin / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; C468783:H2AX protein, human; D006657:Histones; D047428:Protein Kinase Inhibitors; D014404:Tubulin; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases