Characteristics of gamma-H2AX foci at DNA double-strand breaks sites.

Phosphorylated H2AX (gamma-H2AX) is essential to the efficient recognition and (or) repair of DNA double strand breaks (DSBs), and many molecules, often thousands, of H2AX become rapidly phosphorylated at the site of each nascent DSB. An antibody to gamma-H2AX reveals that this highly amplified process generates nuclear foci. The phosphorylation site is a serine four residues from the C-terminus which has been evolutionarily conserved in organisms from giardia intestinalis to humans. Mice and yeast lacking the conserved serine residue demonstrate a variety of defects in DNA DSB processing. H2AX Delta/Delta mice are smaller, sensitive to ionizing radiation, defective in class switch recombination and spermatogenesis while cells from the mice demonstrate substantially increased numbers of genomic defects. gamma-H2AX foci formation is a sensitive biological dosimeter and presents new and exciting opportunities to understand important biological processes, human diseases, and individual variations in radiation sensitivity. These potentialities demonstrate the importance of understanding the parameters and functions of gamma-H2AX formation.     

Werner syndrome helicase (WRN), nuclear DNA helicase II (NDH II) and histone gammaH2AX are localized to the centrosome

Werner syndrome helicase (WRN) was found in the centrosome of human cells, both in interphase and in mitosis. Nuclear DNA helicase II (NDH II), also called RNA helicase A (RHA), an interaction partner of WRN, was also present in the centrosome. NDH II localized to the centrosome in interphase but left the centrosome with the ongoing progression of mitosis. The localization of NDH II to the centrosome was hardly affected by cytochalasin D that depolymerizes actin filaments. In contrast, treatment by the microtubules disrupting agent nocodazole strikingly detached NDH II from the centrosome, which was in contrast to WRN that remained there under this condition. Treatment of cells with the DNA damaging agent 4-nitroquinoline-1-oxide (4NQO) released NDH II, but not WRN from the centrosome. Surprisingly, the double-stranded DNA break repair-induced histone variant gammaH2AX was also found in centrosomes of interphase and mitotic cells. Following DNA damage by 4NQO, gammaH2AX left the centrosome with similar kinetics as NDH II. In vitro pull-down assays confirmed a direct physical interaction between these two proteins. Since NDH II associated with gammaH2AX after DNA damage, we suggest that complex formation between NDH II and gammaH2AX may occur in pre-assembled complexes at the centrosome, which are subsequently recruited to sites of damaged DNA for inducing the repair process.     

Elimination of radiation-induced gamma-H2AX foci in mammalian nucleus can occur by histone exchange

Double-strand breaks in mammalian DNA lead to rapid phosphorylation of C-terminal serines in histone H2AX (gamma-H2AX) and formation of large nuclear gamma-H2AX foci. After DNA repair these foci disappear, but molecular mechanism of elimination of gamma-H2AX foci remains unclear. H2AX protein can be phosphorylated and dephosphorylated in vitro in the absence of chromatin. Here, we compared global exchange of GFP-H2AX with kinetics of formation and elimination of radiation-induced gamma-H2AX foci. Maximal number of gamma-H2AX foci is observed one hour after irradiation, when approximately 20% of GFP-H2AX is exchanged suggesting that formation of the foci mostly occurs by in situ H2AX phosphorylation. However, slow elimination of gamma-H2AX foci is weakly affected by an inhibitor of protein phosphatases calyculin A which is known as an agent suppressing dephosphorylation of gamma-H2AX. This indicates that elimination of gamma-H2AX foci may be independent of dephosphorylation of H2AX which can occur after its removal from the foci by exchange.     

Heat induces gammaH2AX foci formation in mammalian cells

H2AX is a histone variant which is present and ubiquitously distributed throughout the genome. An immunocytochemical assay using antibodies capable of recognizing histone H2AX phosphorylated at serine 139 (gammaH2AX) is very sensitive and is a specific indicator for the existence of a DNA double strand break. Although heat stress has been reported to induce the formation of gammaH2AX foci, no gammaH2AX foci formation was observed in several mammalian cell lines after heat shock. Since this was in contrast to earlier reports, the work described here was intended to verify that heat-induced gammaH2AX foci do form in mammalian cell lines other than the cell lines used in earlier reports concerning gammaH2AX foci formation. The cell lines used in this work includes cell lines with differing p53-gene status (H1299, H1299/neo, H1299/mp53 and H1299/wtp53 cells), various cancer cell lines (HeLa, HepG2, U2-OS cells), normal human cells (HEK-293 and AG1522), and cell lines established from other species (MEF normal mouse cells and CHL normal Chinese hamster cells). Exponentially growing cells were exposed to heat shock (42 degrees C for 6 h or 45.5 degrees C for 20 min) or to X-rays (3Gy). The presence of gammaH2AX was examined with immunocytochemistry and flow cytometry. Induction of gammaH2AX foci formation was observed in all of the mammalian cell lines used here after heat-treatment as well as after X-irradiation. However, the intensity of gammaH2AX was different in the different cell lines used. These results confirm that heat can induce gammaH2AX foci formation in many mammalian cell lines.     

A PP4-phosphatase complex dephosphorylates gamma-H2AX generated during DNA replication

The histone H2A variant H2AX is rapidly phosphorylated in response to DNA double-stranded breaks to produce gamma-H2AX. gamma-H2AX stabilizes cell-cycle checkpoint proteins and DNA repair factors at the break site. We previously found that the protein phosphatase PP2A is required to resolve gamma-H2AX foci and complete DNA repair after exogenous DNA damage. Here we describe a three-protein PP4 phosphatase complex in mammalian cells, containing PP4C, PP4R2, and PP4R3beta, that specifically dephosphorylates ATR-mediated gamma-H2AX generated during DNA replication. PP4 efficiently dephosphorylates gamma-H2AX within mononucleosomes in vitro and does not directly alter ATR or checkpoint kinase activity, suggesting that PP4 acts directly on gamma-H2AX in cells. When the PP4 complex is silenced, repair of DNA replication-mediated breaks is inefficient, and cells are hypersensitive to DNA replication inhibitors, but not radiomimetic drugs. Therefore, gamma-H2AX elimination at DNA damage foci is required for DNA damage repair, but accomplishing this task involves distinct phosphatases with potentially overlapping roles.     

Replication protein A and gamma-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks

Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, gamma-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and gamma-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and gamma-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and gamma-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and gamma-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with gamma-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and gamma-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with gamma-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells.     

Computational Methods for analysis of foci: validation for radiation-induced gamma-H2AX foci in human cells

Observation and counting of gamma-H2AX foci in untreated cells as well as in cells exposed to cytotoxic agents is a widely used method for documenting the presence of double-strand breaks (DSBs) in the DNA and for analysis of their repair. Similar methods are employed to analyze formation of foci by a variety of proteins implicated in the cellular responses to DNA damage. Despite the wide application of the approach, the manual counting that is frequently used is prone to inaccuracies and investigator-related biases and artifacts. To alleviate this limitation, we developed and describe here personal computer-based algorithms, operating as utilities on available software, that allow an objective and quantitative analysis of foci from confocal images. The algorithms allow focus counting as well as size definition and correct for focus coincidence due to the overlap normally occurring with an increasing number of foci per nucleus. Furthermore, the software allows measurement of the integrated optical density (IOD) of each individual focus, which enables analysis of properties of foci as a function of time. Finally, the information generated by the above analysis algorithms can be employed to evaluate colocalization between foci formed by different proteins. A validation of the software is presented for radiation-induced gamma-H2AX foci in three widely used human cell lines and colocalization tested with RAD51 and gamma-H2AX foci. The computational methods presented extend to images generated by digital cameras.     

A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage

BACKGROUND: The response of eukaryotic cells to double-strand breaks in genomic DNA includes the sequestration of many factors into nuclear foci. Recently it has been reported that a member of the histone H2A family, H2AX, becomes extensively phosphorylated within 1-3 minutes of DNA damage and forms foci at break sites. RESULTS: In this work, we examine the role of H2AX phosphorylation in focus formation by several repair-related complexes, and investigate what factors may be involved in initiating this response. Using two different methods to create DNA double-strand breaks in human cells, we found that the repair factors Rad50 and Rad51 each colocalized with phosphorylated H2AX (gamma-H2AX) foci after DNA damage. The product of the tumor suppressor gene BRCA1 also colocalized with gamma-H2AX and was recruited to these sites before Rad50 or Rad51. Exposure of cells to the fungal inhibitor wortmannin eliminated focus formation by all repair factors examined, suggesting a role for the phosphoinositide (PI)-3 family of protein kinases in mediating this response. Wortmannin treatment was effective only when it was added early enough to prevent gamma-H2AX formation, indicating that gamma-H2AX is necessary for the recruitment of other factors to the sites of DNA damage. DNA repair-deficient cells exhibit a substantially reduced ability to increase the phosphorylation of H2AX in response to ionizing radiation, consistent with a role for gamma-H2AX in DNA repair. CONCLUSIONS: The pattern of gamma-H2AX foci that is established within a few minutes of DNA damage accounts for the patterns of Rad50, Rad51, and Brca1 foci seen much later during recovery from damage. The evidence presented strongly supports a role for the gamma-H2AX and the PI-3 protein kinase family in focus formation at sites of double-strand breaks and suggests the possibility of a change in chromatin structure accompanying double-strand break repair.     

Induction and repair of DNA double-strand breaks assessed by gamma-H2AX foci after irradiation with pulsed or continuous proton beams

In particle tumor therapy including beam scanning at accelerators, the dose per voxel is delivered within about 100 ms. In contrast, the new technology of laser plasma acceleration will produce ultimately shorter particle packages that deliver the dose within a nanosecond. Here, possible differences for relative biological effectiveness in creating DNA double-strand breaks in pulsed or continuous irradiation mode are studied. HeLa cells were irradiated with 1 or 5 Gy of 20-MeV protons at the Munich tandem accelerator, either at continuous mode (100 ms), or applying a single pulse of 1-ns duration. Cells were fixed 1 h after 1-Gy irradiation and 24 h after 5-Gy irradiation, respectively. A dose–effect curve based on five doses of X-rays was taken as reference. The total number of phosphorylated histone H2AX (gamma-H2AX) foci per cell was determined using a custom-made software macro for gamma-H2AX foci counting. For 1 h after 1-Gy 20-MeV proton exposures, values for the relative biological effectiveness (RBE) of 0.97 ± 0.19 for pulsed and 1.13 ± 0.21 for continuous irradiations were obtained in the first experiment 1.13 ± 0.09 and 1.16 ± 0.09 in the second experiment. After 5 Gy and 24 h, RBE values of 0.99 ± 0.29 and 0.91 ± 0.23 were calculated, respectively. Based on the gamma-H2AX foci numbers obtained, no significant differences in RBE between pulsed and continuous proton irradiation in HeLa cells were detected. These results are well in line with our data on micronucleus induction in HeLa cells.     

NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain

DNA double-strand breaks represent the most potentially serious damage to a genome; hence, many repair proteins are recruited to nuclear damage sites by as yet poorly characterized sensor mechanisms. Here, we show that NBS1, the gene product defective in Nijmegen breakage syndrome (NBS), physically interacts with histone, rather than damaged DNA, by direct binding to gamma-H2AX. We also demonstrate that NBS1 binding can occur in the absence of interaction with hMRE11 or BRCA1. Furthermore, this NBS1 physical interaction was reduced when anti-gamma-H2AX antibody was introduced into normal cells and was also delayed in AT cells, which lack the kinase activity for phosphorylation of H2AX. NBS1 has no DNA binding region but carries a combination of the fork-head associated (FHA) and the BRCA1 C-terminal domains (BRCT). We show that the FHA/BRCT domain of NBS1 is essential for this physical interaction, since NBS1 lacking this domain failed to bind to gamma-H2AX in cells, and a recombinant FHA/BRCT domain alone can bind to recombinant gamma-H2AX. Consequently, the FHA/BRCT domain is likely to have a crucial role for both binding to histone and for relocalization of hMRE11/hRAD50 nuclease complex to the vicinity of DNA damage.     

DNA double-strand breaks and gamma-H2AX signaling in the testis

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms gamma-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These gamma-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, gamma-H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit gamma-H2AX foci but show homogeneous nuclear gamma-H2AX staining, whereas in pachytene spermatocytes gamma-H2AX is only present in the sex vesicle. In response to ionizing radiation, gamma-H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, gamma-H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear gamma-H2AX staining in leptotene spermatocytes demonstrates a function for gamma-H2AX during meiosis. gamma-H2AX staining in intermediate and B spermatogonia, preleptotene spermatocytes, and sex vesicles and round spermatids, however, indicates that the function of H2AX phosphorylation during spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA double-strand breaks.     

Slow elimination of phosphorylated histone gamma-H2AX from DNA of terminally differentiated mouse heart cells in situ

Phosphorylation of replacement histone H2AX occurs in megabase chromatin domains around double-strand DNA breaks (DSBs) and this modification (called gamma-H2AX) may serve as a useful marker of genome damage and repair in terminally differentiated cells. Here using immunohistochemistry we studied kinetics of gamma-H2AX formation and elimination in the X-irradiated mouse heart and renal epithelial tissues in situ. Unirradiated tissues have 3-5% gamma-H2AX-positive cells and in tissues fixed 1h after X-irradiation gamma-H2AX-positive nuclei are induced in a dose-dependent manner approaching 20-30% after 3 Gy of IR. Analysis of mouse tissues at different times after 3 Gy of IR showed that maximal induction of gamma-H2AX in heart is observed 20 min after IR and then is decreased slowly with about half remaining 23 h later. In renal epithelium maximum of the gamma-H2AX-positive cells is observed 40 min after IR and then decreases to control values in 23 h. This indicates that there are significant variations between non-proliferating mammalian tissues in the initial H2AX phosphorylation rate as well as in the rate of gamma-H2AX elimination after X-irradiation, which should be taken into account in the analysis of radiation responses.     

Human immunodeficiency virus type 1 Vpr-mediated G2 arrest requires Rad17 and Hus1 and induces nuclear BRCA1 and gamma-H2AX focus formation

Eukaryotic cells have evolved a complex mechanism for sensing DNA damage during genome replication. Activation of this pathway prevents entry into mitosis to allow for either DNA repair or, in the event of irreparable damage, commitment to apoptosis. Under conditions of replication stress, the damage signal is initiated by the ataxia-telangiectasia-mutated and Rad3-related kinase ATR. We recently demonstrated that the human immunodeficiency virus type 1 (HIV-1) gene product viral protein R (Vpr) arrests infected cells in the G(2) phase via the activation of ATR. In the present study, we show that the activation of ATR by Vpr is analogous to activation by certain genotoxic agents, both mechanistically and in its downstream consequences. Specifically, we show a requirement for Rad17 and Hus1 to induce G(2) arrest as well as Vpr-induced phosphorylation of histone 2A variant X (H2AX) and formation of nuclear foci containing H2AX and breast cancer susceptibility protein 1. These results demonstrate that G(2) arrest mediated by the HIV-1 gene product Vpr utilizes the cellular signaling pathway whose physiological function is to recognize replication stress. These findings should contribute to a greater understanding of how HIV-1 manipulates the CD4(+)-lymphocyte cell cycle and apoptosis induction in the progressive CD4(+)-lymphocyte depletion characteristic of HIV-1 pathogenesis.     

DNA damage-induced RPA focalization is independent of gamma-H2AX and RPA hyper-phosphorylation

Replication protein A (RPA) is the major eukaryotic single stranded DNA binding protein that plays a central role in DNA replication, repair and recombination. Like many DNA repair proteins RPA is heavily phosphorylated (specifically on its 32 kDa subunit) in response to DNA damage. Phosphorylation of many repair proteins has been shown to be important for their recruitment to DNA damage-induced intra-nuclear foci. Further, phosphorylation of H2AX (gamma-H2AX) has been shown to be important for either the recruitment or stable retention of DNA repair proteins to these intra-nuclear foci. We address here the relationship between DNA damage-induced hyper-phosphorylation of RPA and its intra-nuclear focalization, and whether gamma-H2AX is required for RPA's presence at these foci. Using GFP-conjugated RPA, we demonstrate the formation of extraction-resistant RPA foci induced by DNA damage or stalled replication forks. The strong DNA damage-induced RPA foci appear after phosphorylated histone H2AX and Chk1, but earlier than the appearance of hyper-phosphorylated RPA. We demonstrate that while the functions of phosphoinositol-3-kinase-related protein kinases are essential for DNA damage-induced H2AX phosphorylation and RPA hyper-phosphorylation, they are dispensable for the induction of extraction-resistant RPA and RPA foci. Furthermore, in mouse cells genetically devoid of H2AX, DNA damage-induced extraction-resistant RPA appears with the same kinetics as in normal mouse cells. These results demonstrate that neither RPA hyper-phosphorylation nor H2AX are required for the formation in RPA intra-nuclear foci in response to DNA damage/replicational stress and are consistent with a role for RPA as a DNA damage sensor involved in the initial recognition of damaged DNA or blocked replication forks.     

Biochemical kinetics model of DSB repair and induction of gamma-H2AX foci by non-homologous end joining

We developed a biochemical kinetics approach to describe the repair of double-strand breaks (DSBs) produced by low-LET radiation by modeling molecular events associated with non-homologous end joining (NHEJ). A system of coupled nonlinear ordinary differential equations describes the induction of DSBs and activation pathways for major NHEJ components including Ku70/80, DNA-PKcs, and the ligase IV-XRCC4 heterodimer. The autophosphorylation of DNA-PKcs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of regulation of repair by DNA-PKcs was developed with an initial step allowing access of other NHEJ components to breaks and a second step limiting access to ligase IV-XRCC4. Our model assumes that the transition from the first to the second step depends on DSB complexity, with a much slower rate for complex DSBs. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field gel electrophoresis (PFGE) at 10 min postirradiation or longer and quantification of the induction of gamma-H2AX foci. A process that is independent of DNA-PKcs is required for the model to reproduce experimental data for rejoining before 10 min postirradiation. Predictions are made for the behaviors of NHEJ components at low doses and dose rates, and a steady state is found at dose rates of 0.1 Gy/h or lower.     

Dephosphorylation of histone gamma-H2AX during repair of DNA double-strand breaks in mammalian cells and its inhibition by calyculin A

The induction of DNA double-strand breaks (DSBs) by ionizing radiation in mammalian chromosomes leads to the phosphorylation of Ser-139 in the replacement histone H2AX, but the molecular mechanism(s) of the elimination of phosphorylated H2AX (called gamma-H2AX) from chromatin in the course of DSB repair remains unknown. We showed earlier that gamma-H2AX cannot be replaced by exchange with free H2AX, suggesting the direct dephosphorylation of H2AX in chromatin by a protein phosphatase. Here we studied the dynamics of dephosphorylation of gamma-H2AX in vivo and found that more than 50% was dephosphorylated in 3 h, but a significant amount of gamma-H2AX could be detected even 6 h after the induction of DSBs. At this time, a significant fraction of the gamma-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites. However, gamma-H2AX could be detected in some cells treated with methyl methanesulfonate which accumulated RAD18 protein at stalled replication sites. We also found that calyculin A inhibited early elimination of gamma-H2AX and DSB rejoining in vivo and that protein phosphatase 1 was able to remove phosphate groups from gamma-H2AX-containing chromatin in vitro. Our results confirm the tight association between DSBs and gamma-H2AX and the coupling of its in situ dephosphorylation to DSB repair.     

The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks

The maintenance of genome stability requires efficient DNA double-stranded break (DSB) repair mediated by the phosphorylation of multiple histone H2AX molecules near the break sites. The phosphorylated H2AX (γ-H2AX) molecules form foci covering many megabases of chromatin. The formation of g-H2AX foci is critical for efficient DNA damage response (DDR) and for the maintenance of genome stability, however, the mechanisms of protein organization in foci is largely unknown. To investigate the nature of γ-H2AX foci formation, we analyzed the distribution of γ-H2AX and other DDR proteins at DSB sites using a variety of techniques to visualize, expand and partially disrupt chromatin. We report here that γ-H2AX foci change composition during the cell cycle, with proteins 53BP1, NBS1 and MRE11 dissociating from foci in G2 and mitosis to return at the beginning of the following G1. In contrast, MDC1 remained colocalized with g-H2AX during mitosis. In addition, while γ-H2AX was found to span large domains flanking DSB sites, 53BP1 and NBS1 were more localized and MDC1 colocalized in doublets in foci. H2AX and MDC1 were found to be involved in chromatin relaxation after DSB formation. Our data demonstrates that the DSB repair focus is a heterogeneous and dynamic structure containing internal complexity.     

Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (gamma-H2AX) demonstrates that gamma-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of (125)I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of (125)IdU and processed immunocytochemically to determine the number of gamma-H2AX foci. The numbers of (125)IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of (125)I decays and the number of foci per cell, consistent with the assumptions that each (125)I decay yields a DNA DSB and each DNA DSB yields a visible gamma-H2AX focus. Based on these findings, we conclude that gamma-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.     

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.