Double strand break repair by homologous recombination is regulated by cell cycle-independent signaling via ATM in human glioma cells.
To investigate double strand break (DSB) repair and signaling in human glioma cells, we stably transfected human U87 (ATM(+), p53(+)) glioma cells with a plasmid having a single I-SceI site within an inactive green fluorescent protein (GFP) expression cassette, allowing for the detection of homologous recombination repair (HRR) by GFP ... expression. HRR and nonhomologous end joining (NHEJ) were also determined by PCR. DSB repair was first detected at 12 h postinfection with an adenovirus expressing I-SceI with repair reaching plateau levels between 24 and 48 h. Within this time frame, NHEJ predominated over HRR in the range of 3-50-fold. To assess the involvement of ATM in DSB repair, we first examined whether ATM was associated with the DSB. Chromatin immunoprecipitation showed that ATM was present at the site of the DSB as early as 18 h postinfection. In cells treated with caffeine, an inhibitor of ATM, HRR was reduced, whereas NHEJ was not. In support of this finding, GFP flow cytometry demonstrated that caffeine reduced HRR by 90% under conditions when ATM kinase activity was inhibited. Dominant-negative ATM expressed from adenovirus inhibited HRR by 45%, also having little to no effect on NHEJ. Furthermore, HRR was inhibited by caffeine in serum-starved cells arrested in G(0)/G(1), suggesting that ATM is also important for HRR outside of the S and G(2) cell cycle phases. Altogether, these results demonstrate that HRR contributes substantially to DSB repair in human glioma cells, and, importantly, ATM plays a critical role in regulating HRR but not NHEJ throughout the cell cycle.
Mesh Terms:
Adenoviridae, Ataxia Telangiectasia Mutated Proteins, Blotting, Western, Caffeine, Cell Cycle, Cell Cycle Proteins, Cell Line, Tumor, Central Nervous System Stimulants, Chromatin, Culture Media, Serum-Free, DNA, DNA Damage, DNA Repair, DNA-Binding Proteins, G2 Phase, Genes, Dominant, Glioma, Green Fluorescent Proteins, Humans, Luminescent Proteins, Precipitin Tests, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, Recombination, Genetic, S Phase, Signal Transduction, Time Factors, Transfection, Tumor Suppressor Proteins
Adenoviridae, Ataxia Telangiectasia Mutated Proteins, Blotting, Western, Caffeine, Cell Cycle, Cell Cycle Proteins, Cell Line, Tumor, Central Nervous System Stimulants, Chromatin, Culture Media, Serum-Free, DNA, DNA Damage, DNA Repair, DNA-Binding Proteins, G2 Phase, Genes, Dominant, Glioma, Green Fluorescent Proteins, Humans, Luminescent Proteins, Precipitin Tests, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, Recombination, Genetic, S Phase, Signal Transduction, Time Factors, Transfection, Tumor Suppressor Proteins
J. Biol. Chem.
Date: Apr. 09, 2004
PubMed ID: 14744854
View in: Pubmed Google Scholar
Download Curated Data For This Publication
199937
Switch View:
- Interactions 1