leupeptins and Ataxia-Telangiectasia

Ataxia Telangiectasia (A-T) is a progressive childhood disorder characterized most notably by cerebellar degeneration and predisposition to cancer. A-T is caused by mutations in the kinase ATM, a master regulator of the DNA double-strand break response. In addition to DNA-damage signaling defects, A-T cells display mitochondrial dysfunction that is thought to contribute to A-T pathogenesis. However, the molecular mechanism leading to mitochondrial dysfunction in A-T remains unclear. Here, we show that lack of ATM leads to reduced mitochondrial DNA (mtDNA) integrity and mitochondrial dysfunction, which are associated to defective mtDNA repair. While protein levels of mtDNA repair proteins are essentially normal, in the absence of ATM levels specifically of DNA ligase III (Lig3), the only DNA ligase working in mitochondria is reduced. The reduction of Lig3 is observed in different A-T patient cells, in brain and pre-B cells derived from ATM knockout mice as well as upon transient or stable knockdown of ATM. Furthermore, pharmacological inhibition of Lig3 in wild type cells phenocopies the mtDNA repair defects observed in A-T patient cells. As targeted deletion of LIG3 in the central nervous system causes debilitating ataxia in mice, reduced Lig3 protein levels and the consequent mtDNA repair defect may contribute to A-T neurodegeneration. A-T is thus the first disease characterized by diminished Lig3.

Topics: Animals; Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Biomarkers, Tumor; Cell Line; DNA Ligase ATP; DNA Ligases; DNA Repair; DNA, Mitochondrial; Gene Expression Regulation; Gene Knockdown Techniques; Humans; Leupeptins; Mice; Mice, Knockout; Mitochondria; Nervous System; Poly-ADP-Ribose Binding Proteins; Xenopus Proteins

Cells lacking an intact ATM gene are hypersensitive to ionizing radiation and show multiple defects in the cell cycle-coupled checkpoints. DNA damage usually triggers cell cycle arrest through, among other things, the activation of p53. Another DNA-damage responsive factor is NF-kappaB. It is activated by various stress situations, including oxidative stress, and by DNA-damaging compounds such as topoisomerase poisons. We found that cells from Ataxia Telangiectasia patients exhibit a defect in NF-kappaB activation in response to treatment with camptothecin, a topoisomerase I poison. In AT cells, this activation is shortened or suppressed, compared to that observed in normal cells. Ectopic expression of the ATM protein in AT cells increases the activation of NF-kappaB in response to camptothecin. MO59J glioblastoma cells that do not express the DNA-PK catalytic subunit respond normally to camptothecin. These results support the hypothesis that NF-kappaB is a DNA damage-responsive transcription factor and that its activation pathway by DNA damage shares some components with the one leading to p53 activation.

Topics: Adolescent; Age Factors; Antineoplastic Agents, Phytogenic; Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Brain Neoplasms; Camptothecin; Cell Cycle Proteins; Cells, Cultured; Child; Child, Preschool; DNA Damage; DNA-Activated Protein Kinase; DNA-Binding Proteins; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation; Glioblastoma; Humans; I-kappa B Proteins; Leupeptins; NF-kappa B; NF-KappaB Inhibitor alpha; Nuclear Proteins; Phosphoric Monoester Hydrolases; Protease Inhibitors; Protein Serine-Threonine Kinases; Proteins; Radiation Tolerance; Recombinant Fusion Proteins; Topoisomerase I Inhibitors; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53; Tumor Suppressor Proteins