title: Gemcitabine sensitization by checkpoint kinase 1 inhibition correlates with inhibition of a Rad51 DNA damage response in pancreatic cancer cells.
abstract: The protein kinase checkpoint kinase 1 (Chk1) has been implicated as a key regulator of cell cycle progression and DNA repair, and inhibitors of Chk1 (e.g., UCN-01 and EXEL-9844) potentiate the cytotoxic actions of chemotherapeutic drugs in tumor cells. We have examined the ability of PD-321852, a small-molecule Chk1 inhibitor, to potentiate gemcitabine-induced clonogenic death in a panel of pancreatic cancer cell lines and evaluated the relationship between endpoints associated with Chk1 inhibition and chemosensitization. Gemcitabine chemosensitization by minimally toxic concentrations of PD-321852 ranged from minimal (<3-fold change in survival) in Panc1 cells to >30-fold in MiaPaCa2 cells. PD-321852 inhibited Chk1 in all cell lines as evidenced by stabilization of Cdc25A; in combination with gemcitabine, a synergistic loss of Chk1 protein was observed in the more sensitized cell lines. Gemcitabine chemosensitization, however, did not correlate with abrogation of the S-M or G2-M checkpoint; PD-321852 did not induce premature mitotic entry in gemcitabine-treated BxPC3 or M-Panc96 cells, which were sensitized to gemcitabine 6.2- and 4.6-fold, respectively. In the more sensitized cells lines, PD-321852 not only inhibited gemcitabine-induced Rad51 focus formation and the recovery from gemcitabine-induced replication stress, as evidenced by persistence of gamma-H2AX, but also depleted these cells of Rad51 protein. Our data suggest the inhibition of this Chk1-mediated Rad51 response to gemcitabine-induced replication stress is an important factor in determining gemcitabine chemosensitization by Chk1 inhibition in pancreatic cancer cells.
authors.affiliation: Department of Pharmacology, University of Michigan Medical School, Upjohn Center for Clinical Pharmacology, 4742E Medical Science II, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5633, USA.
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Parsels
authors.forename: Meredith A
authors.initials: MA
authors.lastname: Morgan
authors.forename: Daria M
authors.initials: DM
authors.lastname: Tanska
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Parsels
authors.forename: Brian D
authors.initials: BD
authors.lastname: Palmer
authors.forename: R John
authors.initials: RJ
authors.lastname: Booth
authors.forename: William A
authors.initials: WA
authors.lastname: Denny
authors.forename: Christine E
authors.initials: CE
authors.lastname: Canman
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Kraker
authors.forename: Theodore S
authors.initials: TS
authors.lastname: Lawrence
authors.forename: Jonathan
authors.initials: J
authors.lastname: Maybaum
mesh_terms: D055162:Biocatalysis; D002227:Carbazoles / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D010190:Pancreatic Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D051135:Rad51 Recombinase / Q000235:genetics* / Q000378:metabolism*; D000093542:Gemcitabine
chemical_list: C535157:4-(2,6-dichlorophenyl)-9-hydroxy-6-(3-methylaminopropyl)-6H-pyrrolo(3,4-c)carbazole-1,3-dione; D002227:Carbazoles; D047428:Protein Kinase Inhibitors; D003841:Deoxycytidine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D051135:Rad51 Recombinase; D000093542:Gemcitabine