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Induction of a pro-apoptotic ATM-NF-kappaB pathway and its repression by ATR in response to replication stress.

ABSTRACT: The transcription factor NF-kappaB has critical functions in biologic responses to genotoxic stimuli. Activation of NF-kappaB in response to DNA double strand break (DSB) inducers can be mediated by ATM (ataxia telangiectasia mutated)-dependent phosphorylation of NEMO (NF-kappaB essential modulator). Here, we show that the replication stress inducers hydroxyurea (HU) and aphidicolin also activate this ATM-dependent signalling pathway. We further show that ATR (ATM- and Rad3-related) interacts with NEMO but surprisingly does not cause NEMO phosphorylation. Consequently, ATR represses NF-kappaB activation induced by replication stress. Reduction or increase of ATR expression by RNA interference or overexpression increased or reduced ATM-NEMO association and NF-kappaB activation induced by HU. Apoptosis gene expression and chromatin immunoprecipitation analyses indicated that HU and the DSB inducer etoposide caused complex patterns of NF-kappaB-dependent pro- and antiapoptotic gene expression with the overall outcome for the former being pro-apoptotic, whereas the latter antiapoptotic. Thus, replication stress and DSB inducers activate NF-kappaB through a conserved pathway with opposite biologic outcomes, and ATR antagonizes ATM function at least in part by competing for NEMO association.

SUBMITTER: Wu ZH

PROVIDER: S-EPMC2486281 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The ability of the transcription factor NF-kappaB to upregulate anti-apoptotic proteins has been linked to the chemoresistance of solid tumors to standard chemotherapy. In contrast, recent studies have proposed that, in response to doxorubicin, NF-kappaB can be pro-apoptotic through repression of anti-apoptotic target genes. However, there is little evidence analyzing the outcome of NF-kappaB inhibition on the cytotoxicity of doxorubicin in studies describing pro-apoptotic NF-kappaB activity. In this study, we further characterize the activation of NF-kappaB in response to doxorubicin and evaluate its role in chemotherapy-induced cell death in sarcoma cells where NF-kappaB is reported to be pro-apoptotic. Doxorubicin treatment in U2OS cells induced canonical NF-kappaB activity as evidenced by increased nuclear accumulation of phosphorylated p65 at serine 536 and increased DNA-binding activity. Co-treatment with a small molecule IKKbeta inhibitor, Compound A, abrogated this response. RT-PCR evaluation of anti-apoptotic gene expression revealed that doxorubicin-induced transcription of cIAP2 was inhibited by Compound A, while doxorubicin-induced repression of other anti-apoptotic genes was unaffected by Compound A or siRNA to p65. Furthermore, the combination of doxorubicin and canonical NF-kappaB inhibition with Compound A or siRNA to p65 resulted in decreased cell viability measured by trypan blue staining and MTS assay and increased apoptosis measured by cleaved poly (ADP-ribose) polymerase and cleaved caspase 3 when compared to doxorubicin alone. Our results demonstrate that doxorubicin-induced canonical NF-kappaB activity associated with phosphorylated p65 is anti-apoptotic in its function and that doxorubicin-induced repression of anti-apoptotic genes occurs independent of p65. Therefore, combination therapies incorporating NF-kappaB inhibitors together with standard chemotherapies remains a viable method to improve the clinical outcomes in patients with advanced stage malignancies.

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Induction of a pro-apoptotic ATM-NF-kappaB pathway and its repression by ATR in response to replication stress.

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