Role of checkpoint kinase 1 in preventing premature mitosis in response to gemcitabine.
The deoxycytidine analogue 2',2'-difluoro-2'-deoxycytidine (gemcitabine) is a potent radiation sensitizer in a variety of solid tumors and tumor cell lines. Previous studies have shown that radiosensitization by gemcitabine is accompanied by simultaneous depletion of dATP pools (through ribonucleotide reductase inhibition) and accumulation in the S-phase of the cell cycle. Because of the importance of cell cycle redistribution in gemcitabine-mediated radiosensitization, we investigated the role of checkpoint kinase (Chk) 1 and Chk2 in gemcitabine-induced cell cycle arrest. We hypothesized that gemcitabine might induce Chk1 or Chk2 signal transduction pathways that mediate S-phase arrest. We found that radiosensitizing concentrations of gemcitabine induced accumulation of phosphorylated Chk1 and Chk2 and down-regulation of Cdc25A in BxPC-3 (10 nmol/L), Panc-1 (100 nmol/L), A549 (30 nmol/L), RKO (30 nmol/L), and SW620 (30 nmol/L) cells. Depletion of Chk1 from Panc-1 cells prevented the down-regulation of Cdc25A in response to gemcitabine. Furthermore, Chk1 depletion permitted Panc-1 and SW620 cells treated with gemcitabine to enter mitosis despite incomplete DNA synthesis. However, depletion of neither Chk1 nor Chk2 abrogated the inhibition of DNA synthesis in response to gemcitabine. These results provide evidence that Chk1 negatively regulates entry into mitosis in response to gemcitabine. Furthermore, these data imply that Chk1 acts to coordinate the cell cycle with DNA synthesis, thus preventing premature mitotic entry in gemcitabine-treated cells.
Morgan
Meredith A
MA
Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, Michigan 49109-0010, USA.
Parsels
Leslie A
LA
Parsels
Joshua D
JD
Mesiwala
Alefiyah K
AK
Maybaum
Jonathan
J
Lawrence
Theodore S
TS
D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004273:DNA, Neoplasm / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008938:Mitosis / Q000187:drug effects* / Q000502:physiology; D011494:Protein Kinases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D014162:Transfection; D020687:cdc25 Phosphatases / Q000378:metabolism; D000093542:Gemcitabine
D004273:DNA, Neoplasm; D034741:RNA, Small Interfering; D011838:Radiation-Sensitizing Agents; D003841:Deoxycytidine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases; D000093542:Gemcitabine