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(PP283) Ionizing radiation-induced G2-checkpoint control by ATR and Chk1 in p53 deficient prostate cancer cells.
Ray, Subrata2, Gupta, Damodar1, 2, Macklis, Roger 1, Almasan, Alex*,1, 2, 2 Cancer Biology/Lerner Research Institute, Cleveland, OH1 Radiation Oncology/Taussig Cancer Center, Cleveland, OH
ABSTRACT- G2-checkpoints are activated by DNA damaging agents, such as ionizing radiation, to cause cell cycle arrest and provide time for repair of the DNA and thus maintain the genomic integrity of mammalian cells. The p53 tumor suppressor regulates the G2-cherckpoint(s) in response to DNA damage. To identify the mechanisms by which the G2-checkpoint is regulated by p53 following ionizing radiation, we have used C4-2 human prostate cancer cells and its p53-mutant (dominant negative) derivatives. Ionizing radiation induced G2-arrest in both cell lines, with enhanced G2-arrest in p53-mutant cells. In p53 wild type C4-2 cells, G2-checkpoint occured through the p53-dependent down-regulation of Cyclin B1, Cdk1 (Cdc2), of their association and kinase activities and upregulation of p21 within Cyclin B1-Cdk1 multicomplexes in the nucleus. Prolonged and enhanced activation of ATM might also add to this checkpoint activation by activating and phosphorylating p53 on Ser15. However, in the absence of p53, the G2-checkpoint switched to a p53-independent ATR-Chk1-dependent pathway. This involved induction of ATR, a prolonged and enhanced Chk1 activation, and inactivation of Cdc25C, causing the release of (p)Ser216Cdc25C into the cytoplasm. There was also significantly increased accumulation of inhibitory (p)Tyr15-Cdk1 in the nucleus, which resulted in the inhibition of Cyclin B1-Cdk1 complexes and a transient decrease in their kinase activities. Moreover, inhibition of atr and chk1 by siRNAs abrogated stably the G2-arrest and sensitized p53-mutant C4-2 cells to ionizing radiation. In contrast, inhibition of either chk1 and chk2, alone or in combination, or atm using siRNAs did not overcome the G2-arrest in p53 wild type C4-2 cells. These data indicate that p53 but not ATM, Chk1 or Chk2 plays an important role in G2-checkpoint activation of p53 wild type C4-2 cells following ionizing radiation.
Key words: checkpoint activation, ATM/ATR, p53, Cell cycle control
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