Oxidative stress induces nuclear loss of DNA repair proteins Ku70 and Ku80 and apoptosis in pancreatic acinar AR42J cells.
Cell death linked to oxidative DNA damage has been implicated in acute pancreatitis. The severe DNA damage, which is beyond the capacity of the DNA repair proteins, triggers apoptosis. It has been hypothesized that oxidative stress may induce a decrease in the Ku70 and Ku80 levels and apoptosis in pancreatic acinar cells. In this study, it was found that oxidative stress caused by glucose oxidase (GO) acting on beta-d-glucose, glucose/glucose oxidase (G/GO), induced slight changes in cytoplasmic Ku70 and Ku80 but drastically induced a decrease in nuclear Ku70 and Ku80 both time- and concentration-dependently in AR42J cells. G/GO induced apoptosis determined by poly(ADP-ribose) polymerase cleavage, an increase in expression of p53 and Bax, and a decrease in Bcl-2 expression. G/GO-induced apoptosis was in parallel with the loss of nuclear Ku proteins in AR42J cells. Caspase-3 inhibitor prevented G/GO-induced nuclear Ku loss and cell death. G/GO did not induce apoptosis in the cells transfected with either the Ku70 or Ku80 expression gene but increased apoptosis in those transfected with the Ku dominant negative mutant. Pulse and pulse-chase results show that G/GO induced Ku70 and Ku80 syntheses, even though Ku70 and Ku80 were degraded both in cytoplasm and nucleus. G/GO-induced decrease in Ku binding to importin alpha and importin beta reflects possible modification of nuclear import of Ku proteins. The importin beta level was not changed by G/GO. These results demonstrate that nuclear decrease in Ku70 and Ku80 may result from the decrease in Ku binding to nuclear transporter importins and the degradation of Ku proteins. The nuclear loss of Ku proteins may underlie the mechanism of apoptosis in pancreatic acinar cells after oxidative stress.
Song
Ji Yeon
JY
Department of Pharmacology and Institute of Gastroenterology, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea.
Lim
Joo Weon
JW
Kim
Hyeyoung
H
Morio
Tomohiro
T
Kim
Kyung Hwan
KH
D034961:Antigens, Nuclear / Q000096:biosynthesis*; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D061945:Caspase Inhibitors; D016923:Cell Death; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D053938:DNA Fragmentation; D004265:DNA Helicases; D004260:DNA Repair; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005799:Genes, Dominant; D005949:Glucose Oxidase / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D000072200:Ku Autoantigen; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress; D010179:Pancreas / Q000166:cytology*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D028901:alpha Karyopherins / Q000737:chemistry; D051028:bcl-2-Associated X Protein; D028961:beta Karyopherins / Q000378:metabolism
D034961:Antigens, Nuclear; C494826:BAX protein, human; D061945:Caspase Inhibitors; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D028901:alpha Karyopherins; D051028:bcl-2-Associated X Protein; D028961:beta Karyopherins; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D005982:Glutathione Transferase; C505407:CASP3 protein, human; D053148:Caspase 3; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen