Role of ATM and ATR kinases in regulating gene expression and cellular survival after genotoxic stress

ATM (Ataxia-telangiectasia mutated) and ATR (ATM and Rad-3 related) are structurally homologous, functionally non-redundant large molecular weight nuclear protein kinases that belong to the PIKK gene superfamily. ATM/ATR have been implicated as critical regulators of cellular responses to genotoxic stress including the coordination of cell cycle checkpoints, DNA repair, gene expression and induction of apoptosis. Although ATM/ATR-dependent pathways that regulate activation of cell cycle checkpoints are well understood, the identities of the relevant factors that mediate ATM/ATR-dependent changes in gene expression and cell survival remain largely undetermined. We delineated a novel stress-induced signal transduction pathway directly linking ATM to the Ca2+/cAMP response element-binding protein, CREB, a transcription factor that regulates cell growth, homeostasis, and survival. ATM phosphorylated CREB in vitro and in vivo in response to ionizing radiation (IR) and oxidative stress. IR-induced phosphorylation of CREB correlated with a decrease in CREB transactivation potential and reduced interaction between CREB and its transcriptional co-activator. CBP. To establish the CREB target genes that may be regulated by the ATM-CREB pathway, we performed a comparative gene expression microarray analysis. This analysis identified homeobox transcription factor HOXB13, as a potential downstream target repressed by the ATM-CREB pathway. Given that HOXB13 is implicated as an anti-proliferative and proapoptotic factor in cells, we propose that ATM-CREB pathway functions in prosurvival capacity by altering the expression of HOXB13. Parallely, we studied the role of ATR in replication stress-induced apoptosis in hydroxyurea (HU)hypersensitive ML-1 cells. HU is a competitive inhibitor of the enzyme ribonucleotide reducatse that is required for DNA synthesis. Exposure of ML-1 cells to HU caused rapid cell death that correlated well with ATR-dependent accumulation of transcription factor p53 and pro-apoptotic p53 target gene PUMA. In addition, microtubule inhibitor nocodazole suppressed HU-induced p53 accumulation in ML-1 cells suggesting that a microtubule-dependent event is also required for apoptosis in ML-1 cells. Our findings outline a HU-induced cell death pathway and suggest that activation of the ATR is necessary, but not sufficient, for stabilization of p53 in response to DNA replication stress.