Abstract
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.
Highlights
Chromosomal double strand breaks (DSBs)1 are considered the most toxic of DNA lesions [1, 2]
We show that homologous recombination repair (HRR) is an important DSB repair pathway in these cells and that ATM binds to the DSB, and using pharmacologic and genetic inhibitors, we demonstrate that ATM regulates HRR but not nonhomologous end joining (NHEJ)
ATM Is Expressed in Human Glioma Cell Lines—To demonstrate that ATM is expressed in the U87 cells and other glioma cells, ATM was immunoprecipitated from U87 cells and transferred onto a membrane for Western blot analysis
Summary
Reagents—Caffeine, propidium iodide, and other chemicals were obtained from Sigma, and endonuclease I-SceI and restriction enzyme BcgI were obtained from New England Biolabs (Beverly, MA). Due to low protein expression, cells lysates were immunoprecipitated with antibody prior to Western blot analysis. Upon infection with Ad-SceI-NG, a single site-specific DSB is generated with a number of different scenarios possible; the break can religate without any evidence of DNA cleavage, or the ends can be joined by NHEJ and at the same time destroy the I-SceI site, or HRR may use the 3Ј internal copy of GFP DNA (iGFP) as a homologous template for repair, resulting in GFPϩ cells. NHEJ and HRR/SSA events can be detected by a PCR-based assay using GFP primers directed to sites flanking the I-SceI restriction site. Cells were harvested at different times after I-SceI virus infection and proteins cross-linked to DNA by treating with 1% formaldehyde for 10 min followed by quenching with 2 mM glycine. PCR was carried out with ChIP primers directed to the 5Ј DNA sequence-flanking site of the I-SceI site
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