Caffeine Abolishes the Mammalian G2/M DNA Damage Checkpoint by Inhibiting Ataxia-Telangiectasia-mutated Kinase Activity

Abstract

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 ␥-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 M. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeinesensitive. 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.

Caffeine and human DNA metabolism: the magic and the mystery

“…Previous studies had indicated that caffeine inhibited ATM and ATR signaling in checkpoints acting in G1, S and G2 [2,41,51]. We were not surprised therefore to find that caffeine fully abrogated ATM-and ATR-dependent S and G2 checkpoint responses in IRand UVC-treated human fibroblasts.…”

“…Inactivation of either enzyme attenuates the G2 checkpoint response by about 50%. The full ablation by caffeine of G2 checkpoint response has been attributed to its inhibition of ATM [41]. A recent publication disputes the interpretation that ATM and ATR are directly inhibited by caffeine in cultured cells [42].…”

Ceramide-induced G2 arrest in rhabdomyosarcoma (RMS) cells requires p21Cip1/Waf1 induction and is prevented by MDM2 overexpression

“…34 Caffeine is capable of abrogating cell cycle check points, thereby releasing cells from DNAdamaged evoked G 2 -arrest. 40 Although caffeine reversed IR-induced G 2 /M arrest in RD cells, it did not prevent ceramide-mediated G 2 -arrest. Given the lack of phosphorylation of components of the DNA damage pathway (ATM, Chk1 and Chk2; Supplementary Figure 1) and no evidence of double-strand breaks (a-Histone H2A.X phosphorylation, Ser319), our data precludes ceramide-induced G 2 -arrest and stabilization of p21 Cip1/Waf1 protein expression to be a result of DNA damage.…”

Low frequency of CHEK2 1100delC allele in Australian multiple-case breast cancer families: functional analysis in heterozygous individuals

“…In response to ionising radiation (IR), threonine 68 of CHK2 is rapidly phosphorylated by ataxia telangiectasia mutated (ATM) (Matsuoka et al, 2000;Zhou et al, 2000), allowing oligomerisation and transautophosphorylation of CHK2 (Ahn et al, 2002;Xu et al, 2002). Activated CHK2 is involved in maintaining the G1/S and G2/M checkpoints by phosphorylation of CDC25A, CDC25C and p53 (Chaturvedi et al, 1999;Shieh et al, 2000;Falck et al, 2001), and repair of double-strand DNA breaks via homologous recombination (HR) through phosphorylation of BRCA1 (Lee et al, 2000).…”