Ataxia telangiectasia-mutated gene product inhibits DNA damage-induced apoptosis via ceramide synthase

DNA double-stranded breaks (dsb) activate surveillance systems that identif y DNA damage and either initiate repair or signal cell death. Failure of ce lls to undergo appropriate death in response to DNA damage leads to misrepa ir, mutations, and neoplastic transformation. Pathways linking DNA dsb to r eproductive or apoptotic death are virtually unknown. Here we report that m etabolic incorporation of I-125-labeled 5-iodo-2'deoxyuridine, which produc es DNA dsb, signaled de novo ceramide synthesis by post-translational activ ation of ceramide synthase (CS) and apoptosis. CS activation was obligatory , since fumonisin B1, a fungal pathogen that acts as a specific CS inhibito r, abrogated DNA damage-induced death. X-irradiation yielded similar result s. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activat ion, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, isa member of the phosphati dylinositol 3-kinase family that constitutes the DNA damage surveillance/re pair system. Epstein-Barr virus-immortalized B cell lines from six ataxia t elangiectasia patients with different mutations exhibited radiation-induced CS activation, ceramide generation, and apoptosis, whereas three lines fro m normal patients failed to manifest these responses. Stable transfection o f wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred t he ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of C S, thereby regulating DNA damage-induced apoptosis.