Gemcitabine sensitization by checkpoint kinase 1 inhibition correlates with inhibition of a Rad51 DNA damage response in pancreatic cancer cells.
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.
Parsels
Leslie A
LA
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.
Morgan
Meredith A
MA
Tanska
Daria M
DM
Parsels
Joshua D
JD
Palmer
Brian D
BD
Booth
R John
RJ
Denny
William A
WA
Canman
Christine E
CE
Kraker
Alan J
AJ
Lawrence
Theodore S
TS
Maybaum
Jonathan
J
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
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