Early decrease in dna repair proteins, Ku70 and Ku86, and subsequent DNA fragmentation after transient focal cerebral ischemia in mice.
OBJECTIVE
Ku70 and Ku86, multifunctional DNA repair proteins, bind to broken DNA ends, including double-strand breaks, and trigger a DNA repair pathway. To investigate the involvement of these proteins in DNA fragmentation after ischemia/reperfusion, Ku protein expression was examined before and after transient focal cerebral ischemia (FCI) in mice.


METHODS
Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal suture blockade of the middle cerebral artery. Ku protein expression was studied by immunohistochemistry and Western blot analysis. DNA fragmentation was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The spatial relationship between Ku expression and DNA fragmentation was examined by double labeling with Ku and TUNEL after reperfusion.


RESULTS
Immunohistochemistry showed constitutive expression of Ku proteins in control brains. The number of Ku-expressing cells was decreased in the entire middle cerebral artery territory as early as 4 hours after reperfusion and remained reduced until 24 hours. Western blot analyses confirmed the significant reduction of these proteins (59.4% and 57.7% reduction in optical density at 4 hours of reperfusion from the normal level of Ku70 and Ku86 bands, respectively; P<0.001). DNA gel electrophoresis demonstrated DNA laddering 24 hours after reperfusion, but not at 4 hours. Double staining with Ku and TUNEL showed a concomitant loss of Ku immunoreactivity and TUNEL-positive staining.


CONCLUSIONS
These results suggest that the early reduction of Ku proteins and the loss of defense against DNA damage may underlie the mechanism of DNA fragmentation after FCI.
Kim
G W
GW
Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA.
Noshita
N
N
Sugawara
T
T
Chan
P H
PH
D000818:Animals; D034961:Antigens, Nuclear; D015153:Blotting, Western; D001921:Brain / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D002544:Cerebral Infarction / Q000378:metabolism / Q000473:pathology; D004247:DNA / Q000032:analysis / Q000378:metabolism; D053938:DNA Fragmentation; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004195:Disease Models, Animal; D004587:Electrophoresis, Agar Gel; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D002546:Ischemic Attack, Transient / Q000378:metabolism*; D000072200:Ku Autoantigen; D008297:Male; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010617:Phenanthridines / Q000032:analysis / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010617:Phenanthridines; C058428:hydroethidine; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606512:Xrcc5 protein, mouse; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen