Caffeine Inhibits Checkpoint Responses without Inhibiting the Ataxia-Telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) Protein Kinases

Abstract

The ataxia-telangiectasia-mutated (ATM) and ATMand Rad3-related (ATR) kinases regulate cell cycle checkpoints by phosphorylating multiple substrates including the CHK1 and -2 protein kinases and p53. Caffeine has been widely used to study ATM and ATR signaling because it inhibits these kinases in vitro and overcomes cell cycle checkpoint responses in vivo. Thus, caffeine has been thought to overcome the checkpoint through its ability to prevent phosphorylation of ATM and ATR substrates. Surprisingly, I have found that multiple ATM-ATR substrates including CHK1 and -2 are hyperphosphorylated in cells treated with caffeine and genotoxic agents such as hydroxyurea or ionizing radiation. ATM autophosphorylation in cells is also increased when caffeine is used in combination with inhibitors of replication suggesting that ATM activity is not inhibited in vivo by caffeine. Furthermore, CHK1 hyperphosphorylation induced by caffeine in combination with hydroxyurea is ATR-dependent suggesting that ATR activity is stimulated by caffeine. Finally, the G 2 /M checkpoint in response to ionizing radiation or hydroxyurea is abrogated by caffeine treatment without a corresponding decrease in ATM-ATR-dependent signaling. This data suggests that although caffeine is an inhibitor of ATM-ATR kinase activity in vitro, it can block checkpoints without inhibiting ATM-ATR activation in vivo.

Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases

“…Loss of ATR in these cells promotes double-strand break formation after treatment with UV light and HU and therefore leads to ATM activation (35). Thus, we examined whether ATM was required for MCM3 phosphorylation in the absence of ATR by using siRNA in U2OS cells.…”

“…The kinase responsible for phosphorylation in response to UV light and HU is likely to be ATR. However, there were complications with this interpretation because double-strand breaks produced by treatment with UV and HU in the absence of ATR activate ATM (35), which also phosphorylates this site. Identification of MCM3 as a substrate led us to discover that MCM2 is also phosphorylated by ATR after DNA damage.…”

Poly(ADP-ribose) Polymerase Activity Prevents Signaling Pathways for Cell Cycle Arrest after DNA Methylating Agent Exposure

“…Alternatively, it may result from a passive mechanical block of DNA replication by inactive PARP-1 protein irreversibly bound to damaged DNA; replication forks are known to be slowed or arrested by specific protein-DNA complexes (26) and to initiate checkpoint activation. Caffeine has been shown to inhibit the activity of both ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) upstream checkpoint kinases and to override ATM-and ATR-dependent replication and DNA damage checkpoints (27,28). In the next experiments cells were treated for 24 h with caffeine (1 mM) in addition to MMS and 4-AN with the aim of determining whether the cell cycle arrest we observe requires active ATM-or ATR-dependent signaling.…”

Listeria monocytogenesinduces host DNA damage and delays the host cell cycle to promote infection

“…We conducted infection assays in the presence or absence of caffeine, a widely used pleiotropic inhibitor of DNA damage checkpoint responses, 24 and generated DNA histograms. Caffeine prevented the effect of Lm infection on the host cell cycle as shown by the similar cell cycle profiles of Inf and NI cells (Fig.…”