Early decrease of XRCC1, a DNA base excision repair protein, may contribute to DNA fragmentation after transient focal cerebral ischemia in mice.
OBJECTIVE
DNA damage and the DNA repair mechanism are known to be involved in ischemia/reperfusion injury in the brain. The x-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in the DNA base excision repair pathway by interacting with DNA ligase III and DNA polymerase beta. The present study examined the protein expression of XRCC1 and DNA fragmentation before and after transient focal cerebral ischemia (FCI).


METHODS
Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal blockade of the middle cerebral artery. XRCC1 protein expression was analyzed by immunohistochemistry and Western blot analysis. DNA damage was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL). The spatial relationship between XRCC1 expression and DNA damage was examined by double staining with XRCC1 and TUNEL after FCI.


RESULTS
Immunohistochemistry showed the nuclear expression of XRCC1 in all regions of the control brains and that it was predominant in the hippocampus. The XRCC1 level was markedly reduced in the caudate putamen at 10 minutes, further decreased in the entire middle cerebral artery territory at 1 hour, and remained reduced until 4 and 24 hours after FCI. Western blot analysis of the normal control brain showed a characteristic band of 70 kDa, which decreased after FCI. A significant amount of DNA fragmentation was detected by DNA gel electrophoresis 24 hours but not 4 hours after FCI. Double staining showed that the neurons that lost XRCC1 immunoreactivity became TUNEL positive.


CONCLUSIONS
These results suggest that the early decrease of XRCC1 and the failure of the DNA repair mechanism may contribute, at least in part, to DNA fragmentation after FCI.
Fujimura
M
M
Departments of Neurosurgery, Neurology and Neurological Sciences, and Program in Neurosciences, Stanford University School of Medicine, Palo Alto, CA 94304, USA.
Morita-Fujimura
Y
Y
Sugawara
T
T
Chan
P H
PH
D000818:Animals; D015153:Blotting, Western; D004396:Coloring Agents; D004249:DNA Damage / Q000235:genetics; D053938:DNA Fragmentation / Q000235:genetics*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics; D019951:DNA Polymerase beta / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D005786:Gene Expression Regulation; D006624:Hippocampus / Q000378:metabolism; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D020244:Infarction, Middle Cerebral Artery / Q000235:genetics; D002546:Ischemic Attack, Transient / Q000235:genetics*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D020768:Middle Cerebral Artery / Q000503:physiopathology; D000075223:Poly-ADP-Ribose Binding Proteins; D011699:Putamen / Q000378:metabolism; D015427:Reperfusion Injury / Q000235:genetics; D000076105:X-ray Repair Cross Complementing Protein 1; D029867:Xenopus Proteins
D004396:Coloring Agents; D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; D029867:Xenopus Proteins; C000619352:Xrcc1 protein, mouse; D019951:DNA Polymerase beta; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618818:Lig3 protein, mouse