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 chec kpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216, Data obtained with a phospho-s pecific antibody against serine 216 suggest that activation of the DNA dama ge checkpoint is accompanied by an increase in serine 216 phosphorylated Cd c25C in the nucleus after exposure of cells to gamma-radiation. Prior treat ment of cells with 2 mM caffeine inhibits such a change and markedly reduce s radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucl eus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not i nhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Ch k2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk 2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC50 of approximately 200 mu M. Using a phospho-spe cific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensit ive. 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. Inhi bition of ATM provides a molecular explanation for the increased radiosensi tivity of caffeine-treated cells.