Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity.
Recent evidence indicates that arrest of mammalian cells at the G(2)/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to gamma-radiation. Prior treatment of cells with 2 mM caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC(50) of approximately 200 microM. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G(2)/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells.
Zhou
B B
BB
Department of Oncology Research, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA. Bing-Bing_S_Zhou@sbphrd.com
Chaturvedi
P
P
Spring
K
K
Scott
S P
SP
Johanson
R A
RA
Mishra
R
R
Mattern
M R
MR
Winkler
J D
JD
Khanna
K K
KK
D001260:Ataxia Telangiectasia; D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D004249:DNA Damage / Q000187:drug effects*; D004268:DNA-Binding Proteins; D016195:G2 Phase; D005720:Gamma Rays; D006801:Humans; D007700:Kinetics; D008938:Mitosis; D016297:Mutagenesis, Site-Directed; D010768:Phosphoserine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D025521:Tumor Suppressor Proteins; D020687:cdc25 Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D010768:Phosphoserine; D002110:Caffeine; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases