Revised genetic requirements for the decatenation G2 checkpoint: the role of ATM.
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.
Bower
Jacquelyn J
JJ
Department of Pathology and Laboratory Medicine, University of North Carolina-Chapel Hill, Chapel Hill, NC, USA.
Zhou
Yingchun
Y
Zhou
Tong
T
Simpson
Dennis A
DA
Arlander
Sonnet J
SJ
Paules
Richard S
RS
Cordeiro-Stone
Marila
M
Kaufmann
William K
WK
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D064447:Checkpoint Kinase 2; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D054659:Diketopiperazines; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D016195:G2 Phase; D006657:Histones / Q000378:metabolism; D006801:Humans; D008938:Mitosis; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000378:metabolism / Q000502:physiology*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D054659:Diketopiperazines; D006657:Histones; D010879:Piperazines; D034741:RNA, Small Interfering; D059005:Topoisomerase II Inhibitors; D025521:Tumor Suppressor Proteins; C070899:4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D004250:DNA Topoisomerases, Type II