Oxidative Stress Induces Nuclear Loss of DNA Repair Proteins Ku70 and Ku80 and Apoptosis in Pancreatic Acinar AR42J Cells

Ji Yeon Song,Joo Weon Lim,Hyeyoung Kim,Tomohiro Morio,Kyung Hwan Kim

doi:10.1074/jbc.m303692200

Abstract

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.

Highlights

Cell death linked to oxidative DNA damage has been implicated in acute pancreatitis
Concentration and Time Responses of G/glucose oxidase (GO) for H2O2 Production and the Apoptotic Indices (Cell Viability, DNA Fragmentation, poly(ADP-ribose) polymerase (PARP) Cleavage, and Expression Changes of p53, Bcl-2, and Bax) in AR42J Cells—Because the susceptibility of the cells to glucose/ glucose oxidase (G/GO)-mediated damage varies among the different cell types, the cell strains, and passage numbers within the same strain (54 –56), the concentration and time responses of G/GO to the wild-type AR42J cells were determined
The number of viable cells (ϫ104 cells/ml) from the cells treated with G/GO were 7.70 Ϯ 0.23, 7.31 Ϯ 0.28, 4.11 Ϯ 0.29, and 2.41 Ϯ 0.21 at the start of the experiment and at 4, 12, and 24 h of culture, respectively (Fig. 2A)

Summary

Introduction

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. Effect of G/GO on Expression or DNA Binding Activity of Ku in AR42J Cells and the Transfected Cells (pcN-3, KuD/N, Ku70ϩ, and Ku80ϩ Cells)—In the wild-type and pcN-3 cells, 12 h of treatment with G/GO induced slight changes in the cytoplasmic Ku70 and Ku80 levels and significant decreases in the nuclear Ku70 and Ku80 levels, respectively (Fig. 6).

Results

Conclusion