Revised genetic requirements for the decatenation G2 checkpoint: The role of ATM
| Autor: | Yingchun Zhou, Dennis A. Simpson, Sonnet J.H. Arlander, Marila Cordeiro-Stone, Tong Zhou, Jacquelyn J. Bower, William K. Kaufmann, Richard S. Paules |
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| Rok vydání: | 2010 |
| Předmět: |
G2 Phase
Mitotic index Cell cycle checkpoint Mitosis Cell Cycle Proteins Ataxia Telangiectasia Mutated Proteins Diketopiperazines Protein Serine-Threonine Kinases Biology Piperazines Article Cell Line Histones medicine Humans Topoisomerase II Inhibitors CHEK1 Phosphorylation RNA Small Interfering Molecular Biology Checkpoint Kinase 2 Tumor Suppressor Proteins Cell Biology Fibroblasts G2-M DNA damage checkpoint medicine.disease Molecular biology Cell biology DNA-Binding Proteins DNA Topoisomerases Type II Mitotic exit Ataxia-telangiectasia RNA Interference biological phenomena cell phenomena and immunity Developmental Biology |
| Zdroj: | Cell Cycle. 9:1617-1628 |
| ISSN: | 1551-4005 1538-4101 |
| DOI: | 10.4161/cc.9.8.11470 |
| Popis: | The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in Chk2, but no appreciable phosphorylation of Ser139-H2AX or Ser345-Chk1. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage. |
| Databáze: | OpenAIRE |
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