title: Ubiquitination capabilities in response to neocarzinostatin and H(2)O(2) stress in cell lines from patients with ataxia-telangiectasia.
abstract: The human genetic disorder ataxia-telangiectasia (A-T) is due to lack of functional ATM, a protein kinase which is involved in cellular responses to DNA double strand breaks (DSBs) and possibly other oxidative stresses, as well as in regulation of several fundamental cellular functions. Studies regarding responses in A-T cells to the induction of DSBs utilize ionizing radiation or radiomimetic chemicals, such as neocarzinostatin (NCS), which induce DNA DSBs. This critical DNA lesion activates many defense systems, such as the cell cycle checkpoints. The cell cycle is also regulated through a timed and coordinated degradation of regulatory proteins via the ubiquitin pathway. Our recent studies indicate that the ubiquitin pathway is influenced by the cellular redox status and that it is the major cellular pathway for removal of oxidized proteins. Accordingly, we hypothesized that the absence of a functional ATM protein might involve perturbations to the ubiquitin pathway as well. We show here that upon treatment with NCS, there was a transient 50-70% increase in endogenous ubiquitin conjugates in A-T and wt lymphoblastoid cells. Ubiquitin conjugation capabilities per se and levels of substrates for conjugation were also similarly enhanced in wt and A-T cells upon NCS treatment. We also compared the ubiquitination response in A-T and wt cells using H(2)O(2) as the stress, in view of preexisting evidence of the effects of H(2)O(2) on ubiquitination capabilities in other types of cells. As with NCS treatment, there was an approximately 45% increase in endogenous ubiquitin conjugates by 2-4 h after exposure to H(2)O(2). Both cell types showed a rapid 50-150% increase in de novo formed 125I-ubiquitin conjugates. As compared with wt cells, unexposed A-T cells had higher endogenous levels of conjugates and enhanced conjugation capability. However, A-T cells mounted a more muted ubiquitination response to the stress. The enhanced ubiquitin conjugation in unstressed A-T cells and attenuated ability of these cells to respond to stress are consistent with the A-T cells being under oxidative stress and with their having an 'aged' phenotype. The indication that ubiquitin conjugate levels and ubiquitin conjugation capabilities are enhanced upon oxidative stress without significant changes in GSSG/GSH ratios indicates that assays of ubiquitination provide a sensitive measure of cellular stress. The data also add support to the impression that potentiated ubiquitination response to mild oxidative stress is a generalizable phenomenon.
authors.affiliation: JM USDA Human Nutrition Research Center on Aging at Tufts University, Laboratory for Nutrition and Vision Research, 711 Washington Street, Boston, Massachusetts 02111, USA. ataylor@hnrc.tufts.edu
authors.forename: Allen
authors.initials: A
authors.lastname: Taylor
authors.forename: Fu
authors.initials: F
authors.lastname: Shang
authors.forename: Thomas
authors.initials: T
authors.lastname: Nowell
authors.forename: Yaron
authors.initials: Y
authors.lastname: Galanty
authors.forename: Yosef
authors.initials: Y
authors.lastname: Shiloh
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001260:Ataxia Telangiectasia / Q000378:metabolism* / Q000473:pathology; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D004249:DNA Damage / Q000187:drug effects; D004268:DNA-Binding Proteins; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008025:Ligases / Q000378:metabolism*; D008214:Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D009353:Zinostatin / Q000494:pharmacology*
chemical_list: D000903:Antibiotics, Antineoplastic; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D016877:Oxidants; D025521:Tumor Suppressor Proteins; D014452:Ubiquitins; D009353:Zinostatin; D006861:Hydrogen Peroxide; D044767:Ubiquitin-Protein Ligases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D008025:Ligases; D005978:Glutathione