Radiosensitization of p53 mutant cells by PD0166285, a novel G(2) checkpoint abrogator.
The lack of functional p53 in many cancer cells offers a therapeutic target for treatment. Cells lacking p53 would not be anticipated to demonstrate a G(1) checkpoint and would depend on the G(2) checkpoint to permit DNA repair prior to undergoing mitosis. We hypothesized that the G(2) checkpoint abrogator could preferentially kill p53-inactive cancer cells by removing the only checkpoint that protects these cells from premature mitosis in response to DNA damage. Because Wee1 kinase is crucial in maintaining G(2) arrest through its inhibitory phosphorylation of Cdc2, we developed a high-throughput mass screening assay and used it to screen chemical library for Wee1 inhibitors. A pyridopyrimidine class of molecule, PD0166285 was identified that inhibited Wee1 at a nanomolar concentration. At the cellular level, 0.5 microM PD0166285 dramatically inhibits irradiation-induced Cdc2 phosphorylation at the Tyr-15 and Thr-14 in seven of seven cancer cell lines tested. PD0166285 abrogates irradiation-induced G(2) arrest as shown by both biochemical markers and fluorescence-activated cell sorter analysis and significantly increases mitotic cell populations. Biologically, PD0166285 acts as a radiosensitizer to sensitize cells to radiation-induced cell death with a sensitivity enhancement ratio of 1.23 as shown by standard clonogenic assay. This radiosensitizing activity is p53 dependent with a higher efficacy in p53-inactive cells. Thus, G(2) checkpoint abrogators represent a novel class of anticancer drugs that enhance cell killing of conventional cancer therapy through the induction of premature mitosis.
Wang
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Department of Cancer Molecular Sciences, Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA. Yuli.Wang@Pfizer.com
Li
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J
Booher
R N
RN
Kraker
A
A
Lawrence
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Leopold
W R
WR
Sun
Y
Y
D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D018797:Cell Cycle Proteins; D016923:Cell Death / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016195:G2 Phase / Q000187:drug effects* / Q000502:physiology; D019073:HT29 Cells / Q000187:drug effects / Q000528:radiation effects; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011743:Pyrimidines / Q000494:pharmacology*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology*
D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; C440869:PD 0166285; D011743:Pyrimidines; D011838:Radiation-Sensitizing Agents; D016159:Tumor Suppressor Protein p53; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; C499348:Wee1 protein, mouse; D016203:CDC2 Protein Kinase