Annual Report HS02243-01 Maintenance of genome integrity is essential for survival of all organisms, from bacteria to human. In order to maintain genome integrity, a cell must be able to sense DNA damage, repair the DNA damage and at the same time, halt cell cycle at one of several checkpoints to prevent cell division before DNA damage is repaired. Checkpoints maintain the order and fidelity of the eukaryotic cell cycle and defects in checkpoints contribute to genetic instability and cancer. Much of our current understanding of checkpoints comes from genetic studies conducted in yeast. In the fission yeast Schizosaccharomyces pombe, SpRad3 is an essential component of both the DNA damage and DNA replication checkpoints. The SpChk1 and SpCds1 protein kinases function downstream of SpRad3. SpChk1 is an effector of the DNA damage checkpoint and, in the absence of SpCds1, serves an essential function in the DNA replication checkpoint. SpCds1 functions in the DNA replication checkpoint and in the S-phase DNA damage checkpoint. Human homologs of both SpRad3 and SpChk1 but not SpCds1 have been identified. We have identified a human cDNA encoding a protein (designated HuCds1) that shares sequence, structural and functional similarity to SpCds1. The human homolog of Cds1, Chk2, was modified by phosphorylation and activated in response to ionizing radiation. It was also modified in response to hydroxyurea (HU) treatment. Functional ATM protein was required for HuCds1 modification after ionizing radiation but not after HU treatment. Like its fission yeast counterpart, Chk2 phosphorylated Cdc25C. Over the past year, we explored the possibility that Chk2 may be functionally linke to the product of the PML gene. The promyelocytic leukemia gene (PML), which is translocated in most acute promyelocytic leukemias, encodes a tumor suppressor. PML-/- mice are sensitive to papillomas, carcinomas, and T and B cell lymphomas supporting the tumor suppressor role of PML. Although the molecular mechanism remains largely unknown, PML is involved in multiple apoptotic pathways. PML -/- mice and PML -/- cells are resistant to the lethal effects of ionizing irradiation suggesting that PML plays an important role in DNA damage-induced apoptosis. However, the signaling cascade of the PML-mediated apoptosis after DNA damage is not understood. Chk2, a cell-cycle checkpoint kinase which is activated in response to DNA damage is necessary for ionizing irradiation-induced apoptosis. The mechanism by which Chk2 induces ionizing irradiation-induced apoptosis is poorly understood, but is thought to involve stabilization of p53. However, ionizing irradiation-induced apoptosis also occurs via p53-independent mechanisms. We found that hCds1/Chk2 mediates ionizing irradiation-induced apoptosis in a p53-independent manner by an ATM-Chk2-PML pathway. The apoptotic function of PML is regulated by phosphorylation of serine 117 by hCds1/Chk2 after g irradiation. Our results provide the first evidence for a functional relationship between PML and a checkpoint kinase in ionizing irradiation-induced apoptosis.
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