ABSTRACT
BACKGROUND: Artemis has a defined role in V(D)J recombination and has been implicated in the repair of radiation induced double-strand breaks. However the exact function(s) of Artemis in DNA repair and its preferred substrate(s) in vivo remain undefined. Our previous work suggests that Artemis is important for the repair of complex DNA damage like that inflicted by high Linear Energy Transfer (LET) radiation. To establish the contribution of Artemis in repairing DNA damage caused by various radiation qualities, we evaluated the effect of over-expressing Artemis on cell survival, DNA repair, and cell cycle arrest after exposure to high and low LET radiation. RESULTS: Our data reveal that Artemis over-expression confers marked radioprotection against both types of radiation, although the radioprotective effect was greater following high LET radiation. Inhibitor studies reveal that the radioprotection imparted by Artemis is primarily dependent on DNA-PK activity, and to a lesser extent on ATM kinase activity. Together, these data suggest a DNA-PK dependent role for Artemis in the repair of complex DNA damage. CONCLUSIONS: These findings indicate that Artemis levels significantly influence radiation toxicity in human cells and suggest that Artemis inhibition could be a practical target for adjuvant cancer therapies.
Subject(s)
Cell Cycle/radiation effects , Cell Survival/radiation effects , DNA Breaks, Double-Stranded/radiation effects , DNA Repair , Linear Energy Transfer , Nuclear Proteins/physiology , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/metabolism , Cell Proliferation , DNA-Binding Proteins/metabolism , Endonucleases , Flow Cytometry , HEK293 Cells , Humans , Kinetics , Nuclear Proteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Time Factors , Tumor Suppressor Proteins/metabolismABSTRACT
The multifunctional structural protein 4.1R is required for assembly and maintenance of functional nuclei but its nuclear roles are unidentified. 4.1R localizes within nuclei, at the nuclear envelope, and in cytoplasm. Here we show that 4.1R, the nuclear envelope protein emerin and the intermediate filament protein lamin A/C co-immunoprecipitate, and that 4.1R-specific depletion in human cells by RNA interference produces nuclear dysmorphology and selective mislocalization of proteins from several nuclear subcompartments. Such 4.1R-deficiency causes emerin to partially redistribute into the cytoplasm, whereas lamin A/C is disorganized at nuclear rims and displaced from nucleoplasmic foci. The nuclear envelope protein MAN1, nuclear pore proteins Tpr and Nup62, and nucleoplasmic proteins NuMA and LAP2α also have aberrant distributions, but lamin B and LAP2ß have normal localizations. 4.1R-deficient mouse embryonic fibroblasts show a similar phenotype. We determined the functional effects of 4.1R-deficiency that reflect disruption of the association of 4.1R with emerin and A-type lamin: increased nucleus-centrosome distances, increased ß-catenin signaling, and relocalization of ß-catenin from the plasma membrane to the nucleus. Furthermore, emerin- and lamin-A/C-null cells have decreased nuclear 4.1R. Our data provide evidence that 4.1R has important functional interactions with emerin and A-type lamin that impact upon nuclear architecture, centrosome-nuclear envelope association and the regulation of ß-catenin transcriptional co-activator activity that is dependent on ß-catenin nuclear export.
