Akt1-mediated intracellular oxidation after UVB irradiation suppresses apoptotic cell death induced by cell detachment and serum starvation.

Apoptosis is an important cell death system that deletes damaged and mutated cells to prevent cancer. We have previously reported that a certain dose of UVB irradiation inhibited the apoptosis induced by serum starvation and cell detachment, leading to cell transformation. This antiapoptotic effect was partially inhibited by phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. UVB irradiation is known to cause the phosphorylation of Akt via the activation of PI3-kinase; however, the Akt isoform-specific relationship has not yet been clarified. Notably, the role in antiapoptotic effect of UVB has yet to be elucidated. In this study, the role of Akt1 in the UVB-induced inhibition of apoptosis was examined by Akt1 knockdown using small interfering RNA (siRNA). NIH3T3 cells showed typical apoptotic cell death by serum starvation and cell detachment, which was significantly inhibited by UVB irradiation. Akt1 knockdown decreased the antiapoptotic effect of UVB. Hydrogen peroxide-induced suppression of cell death was also decreased in Akt1 knockdown cells. An antioxidant, N-acetylcysteine, inhibited the antiapoptotic effect by UVB irradiation, whereas no inhibition was observed in Akt1 knockdown cells. Furthermore, UVB-induced intracellular peroxidation was not observed in the knockdown cells, indicating that Akt1 played an important role in mediating the intracellular redox status. Treatment with insulin had a similar antiapoptotic effect as UVB irradiation involving intracellular peroxidation, which was also attenuated in Akt1 knockdown cells. These findings suggest that appropriate intracellular oxidation after UVB irradiation prevented apoptosis, a process which might be partially regulated by the production of reactive oxygen species mediated by Akt1.

Induction of apoptosis by UV-irradiated chlorinated bisphenol A in Jurkat cells.

Chlorinated derivatives of bisphenol A (ClBPAs) have been detected in wastewater from waste paper recycling plants. We previously reported that bisphenol A (BPA) and ClBPAs [3-chlorobisphenol A, 3,3'-dichlorobisphenol A, and 3,3',5-trichlorobisphenol A] irradiated with ultraviolet (UV) B or UVC (not with UVA) induced inhibition of cell growth, and that 3-hydroxybisphenol A (3-OHBPA) was detected in the photoproducts [Mutou, Y., Ibuki, Y., Terao, Y., Kojima, S., Goto, R., 2006b. Chemical change of chlorinated bisphenol A by ultraviolet irradiation and cytotoxicity of their products on Jurkat cells. Environmental Toxicology and Pharmacology, 21, 283-289]. The formation of hydroxylated BPAs by UV irradiation might contribute to the inhibition of cell growth, but the mechanism of the growth inhibition is not clarified. In this study, we investigated whether BPA and ClBPAs exposed to UVA, UVB, or UVC, and 3-OHBPA could induce the death of Jurkat cells and whether the pattern of cell death was apoptosis. ClBPAs exposed to UVB and UVC induced significant cell death, but those exposed to UVA and BPA did not. The cell death was apoptosis because chromatin condensation and DNA fragmentation were detected. Activation of caspase-3, -8, and -9 and cytochrome c release indicated that ClBPAs exposed to UVB or UVC induced apoptosis via typical apoptotic pathways. In addition, 3-OHBPA induced apoptosis similar to UVB- or UVC-irradiated ClBPA. These results suggested that the photoproducts of ClBPAs generated by UV irradiation, containing 3-OHBPA, contributed to the induction of apoptosis.

Water soluble fraction of solar-simulated light-exposed crude oil generates phosphorylation of histone H2AX in human skin cells under UVA exposure.

Crude oil contains compounds, which have toxic and cancer-causing properties to humans. The oil spilled in environments is usually exposed to sunlight; however, the toxicity of sunlight-exposed oil is poorly understood. In this study, we found that the water soluble fraction (WSF) of crude oil irradiated with solar-simulated light (SSL) generated phosphorylation of histone H2AX (gamma-H2AX) in human skin cells under UVA irradiation, which was due to the formation of DNA double strand breaks (DSBs). Crude oil was exposed to SSL for approximately 7 days. The WSF obtained from unexposed crude oil showed no toxicity, whereas the WSF obtained from crude oil pre-exposed to SSL induced acute cell death on exposure to UVA irradiation (induction of phototoxicity), which was more remarkable in human skin fibroblasts than human skin keratinocytes. gamma-H2AX was detected in both cell lines immediately after treatment with the WSF plus UVA. Interestingly, gamma-H2AX was detectable even at low SSL- and UVA-doses, which induced no cytotoxicity. The WSF of crude oil irradiated with SSL, generated DSBs under UVA irradiation, which were detected by biased sinusoidal field gel electrophoresis. This was confirmed using xrs-5 cells isolated from CHO-K1 cells, which are deficient in a repair enzyme for DSBs; the WSF plus UVA induced a more dramatic decrease in survival in xrs-5 cells than CHO-K1 cells. These findings demonstrate that exposure of crude oil to sunlight makes the WSF phototoxic, generating DSBs accompanying the appearance of gamma-H2AX in human skin cells.

Coexposure to benzo[a]pyrene and UVA induces DNA damage: first proof of double-strand breaks in a cell-free system.

DNA damage induced by solar ultraviolet (UV) radiation plays an important role in the induction of skin cancer. Although UVA constitutes the majority of solar UV radiation, it is less damaging to DNA than UVB. The DNA damage produced by UVA radiation, however, can be augmented in the presence of a photosensitizer. We previously used benzo[a]pyrene (BaP), an environmental carcinogenic polycyclic aromatic hydrocarbon, as an exogenous photosensitizer, and demonstrated that combined exposure to BaP and UVA resulted in DNA double-strand breaks (DSBs) in cultured Chinese hamster ovary (CHO-K1) cells. In this study, we investigated whether coexposure to BaP and UVA induces DSBs in a cell-free system and whether reactive oxygen species (ROS) were involved in the generation of the DSBs. DSBs were induced by the coexposure both in the cell-free system (in vitro) and in CHO-K1 cells (in vivo), but not by treatment with BaP or UVA alone. DSB induction in vitro required higher doses of UVA and BaP than were required in vivo, suggesting that the mechanism of DSB induction differed. A similar difference in efficiency also was observed in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) by coexposure to BaP and UVA in vitro and in vivo. A singlet oxygen ((1)O2) scavenger (NaN3) effectively inhibited the production of DSBs and 8-oxodG, suggesting that (1)O2 is a principal ROS generated by BaP and UVA both in vitro and in vivo. Furthermore, repair-deficient xrs-5 cells were more sensitive to coexposure with BaP and UVA than were CHO-K1 cells, but the two cell lines were equally sensitive to the combined treatment in the presence of NaN3. This result suggested that the cell death produced by coexposure to BaP and UVA was at least partly due to the DSBs generated by (1)O2. Our findings indicate that coexposure to BaP and UVA effectively induced DNA damage, especially DSBs, which results in phototoxicity and possibly photocarcinogenesis.

Chemical change of chlorinated bisphenol A by ultraviolet irradiation and cytotoxicity of their products on Jurkat cells.

Chlorinated derivatives of bisphenol A (ClBPAs) have been detected in wastewater from waste paper recycling plants. BPA and ClBPAs are always exposed to ultraviolet (UV) radiation in the environment and consequently various photoproducts might be produced. Acute cytotoxicity of photoproducts of BPA and ClBPAs are not known. In this study, we investigated the cytotoxicity and chemical structure of photoproducts of BPA and ClBPAs (3-chlorobisphenol A (3-ClBPA), 3,3'-dichlorobisphenol A (3,3'-diClBPA) and 3,3',5-trichlorobisphenol A (3,3',5-triClBPA)) after UV irradiation (UVA, UVB and UVC). The toxicities of photoproducts on Jurkat cells were determined by Alamar Blue assay, and the chemical structures of the photoproducts were identified using GC/MS. The cytotoxicities of 3-ClBPA and 3,3'-diClBPA were higher than that of BPA and 3,3',5-triClBPA. In addition, the toxicities of ClBPAs were increased by the irradiation of UVB and UVC at 100J/cm(2) and decreased at 1000J/cm(2) in comparison with those at 100J/cm(2), indicating that their structures had changed. 3-Hydroxybisphenol A (3-OHBPA) was detected in the photoproducts of 3-ClBPA irradiated with UVB and UVC at 100J/cm(2), and 3-OHBPA and 3-chloro-3'-hydroxybisphenol A (3-Cl-3'-OHBPA) were detected in those of 3,3'-diClBPA. However, these hydroxylated BPAs were not detected in the photoproducts exposed to 1000J/cm(2). The cytotoxicity of 3-OHBPA was the almost same as ClBPAs after UVB and UVC irradiation. These results indicate that the formation of hydroxylated BPAs might contribute to the increase in toxicity caused by UV irradiation.

Dysregulation of apoptosis by benzene metabolites and their relationships with carcinogenesis.

Benzene is a widely recognized human carcinogen, the effect of which is attributed to the production of reactive oxygen species (ROS) from its metabolites. Although there have been many reports on the relationship between DNA damage induced by benzene metabolites and carcinogenesis, only a report approached the subject by examining the benzene-induced dysregulation of apoptosis. Inhibition of apoptosis, aberrantly prolonging cell survival, may contribute to cancer by facilitating the insurgence of mutations and by creating a permissive environment for genetic instability. In this study, we examined the mechanism of antiapoptotic effects by benzene metabolites, p-benzoquinone (BQ) and hydroquinone (HQ), and their relationships with carcinogenesis. BQ and HQ inhibited the apoptotic death of NIH3T3 cells induced by both serum starvation and lack of an extracellular matrix (ECM). An antioxidant agent, N-acetylcysteine, significantly inhibited the antiapoptotic effects induced by benzene metabolites, indicating that the effects were mainly due to the production of ROS. Furthermore, BQ and HQ inhibited the in vitro caspase-3 activation, suggesting that the inhibition of caspase-3 activation due to ROS produced by BQ- and HQ-treatment was related to the suppression of apoptosis. The cells that escaped apoptosis could survive with the addition of serum and attachment to the ECM. Levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine were higher in the cells which survived after BQ- and HQ-treatment than in the normal cells. Furthermore, the cells treated with BQ and HQ showed greater proliferation than normal cells under low-serum conditions and anchorage-independent growth in soft agar. These findings suggested that benzene metabolites induced dysregulation of apoptosis due to caspase-3 inhibition, which contributes to carcinogenesis.

gamma-Irradiation-induced DNA damage enhances NO production via NF-kappaB activation in RAW264.7 cells.

We investigated the mechanism of augmentation of nitric oxide (NO) production in the murine macrophage cell line RAW264.7 after gamma-irradiation. The cells treated with interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) showed enhanced NO production by gamma-irradiation in a dose-dependent manner, accompanying the induction of inducible nitric oxide synthase (iNOS) expression. Nuclear factor kappa B (NF-kappaB) activation was induced 1 h after gamma-irradiation dose-dependently, which was detected by the degradation of I-kappaB. Inhibitors of I-kappaB degradation, MG132 and N(alpha)-p-tosyl-L-lysine chloromethyl ketone (TLCK), suppressed the further increase by gamma-irradiation in IFN-gamma-induced NO production, showing that gamma-irradiation induced NO production via NF-kappaB activation. Although NF-kappaB is known to be a redox-sensitive transcription factor, the antioxidant agents N-acetyl-cysteine (NAC) and 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (trolox) showed no suppression and treatment with H(2)O(2) showed only slight enhancement of IFN-gamma-induced NO production. The DNA damaging agents camptothecin and etoposide enhanced IFN-gamma-induced NO production and showed I-kappaB degradation, indicating that the increase in NO production was due to direct DNA damage. Furthermore, 3-aminobenzamide (3AB) and benzamide, inhibitors of poly (ADP-ribose) polymerase (PARP) that are activated upon recognition of DNA strand breaks, suppressed the further increase by gamma-irradiation in IFN-gamma-induced NO production and the I-kappaB degradation by gamma-irradiation. We concluded that (1) the increase in NO production was due to direct DNA damage by gamma-irradiation, and that (2) PARP activation through DNA damage induced NF-kappaB activation, leading to iNOS expression and NO production.

Proteome analysis of UV-B-induced anti-apoptotic regulatory factors.

Ultraviolet (UV) irradiation is well known to induce apoptosis, a hallmark event of which is the occurrence of sunburn cells in the epidermis. Keratinocytes in which DNA damaged by UV irradiation is not repaired undergo apoptosis as sunburn cells. However, we have previously reported that low-dose UV-B irradiation (approximately 0.1 J/cm2) suppressed the apoptosis induced by cell detachment and serum depletion. Dysregulation of apoptosis is important in tumor progression and malignancy and in promoting resistance to cancer therapy. To develop a better understanding of the antiapoptotic effect of UV irradiation, and to design the effective induction of apoptosis, we tried the proteome analysis of the molecules regulating apoptosis in low-dose UV-B-irradiated NIH3T3 cells, using two-dimensional difference gel electrophoresis (DIGE). Of a total of 3811 protein spots detected, 42 were found to be different between the cells undergoing apoptosis and cells after the irradiation. Of the spots selected, 25 were identified using MALDI-TOF/TOF-MS, some as structural proteins. Although typical apoptosis-related molecules were not detected, possibly because proteins with low molecular weights were difficult to identify in the gel conditions used in this study, some of the proteins were considered to be involved in apoptosis. The DIGE system used in this experiment has advantages (including a high level of statistical confidence) for discovering new functional proteins related to the regulation of apoptosis.

Antiapoptotic effects induced by different wavelengths of ultraviolet light.

Cells receive signals for survival as well as for death, and the balance between the two ultimately determines the fate of the cells. UV-triggered apoptotic signaling has been well documented, whereas UV-induced survival effects have received little attention. We have reported previously that UVB irradiation prevented apoptosis, which is partly dependent on activation of the phosphatidylinositol 3-kinase (PI3-kinase)-Akt pathway (Ibuki Y. and Goto, R. [2000] Biochem. Biophys. Res. Commun. 279, 872-878). In this study, antiapoptotic effects and survival signals of UV with different wavelength ranges, UVA, UVB and UVC, were examined. NIH3T3 cells showed apoptotic cell death by detachment from the extracellular matrix under serum-free conditions, which was prevented by all wavelengths. However, the effect of UVA was less than those of UVB and UVC, as determined by metabolism of fluoresceine diacetate and the appearance of chromatin-condensed cells. Furthermore, the effects of three wavelengths of UV on the apoptotic pathway upstream of the nuclear signals were examined. Reduction of mitochondrial transmembrane potential (delta psi) and activation of caspase-9 and -3 were suppressed by all three wavelengths of UV, showing wavelength-dependent effects as mentioned previously. Shorter wavelengths showed stronger inhibitory effects on caspase-8 activity. The P13-kinase inhibitor wortmannin partially inhibited the UVB- and UVC-induced suppression of apoptosis but not the inhibitory effect of UVA. Furthermore, normal delta psi maintained by UVA was not changed in the presence of wortmannin, but those by UVB and UVC were reduced. Akt was clearly phosphorylated by all three wavelengths. The phosphorylation by UVB and UVC was completely inhibited by addition of wortmannin, but that by UVA was not, in agreement with the results of survival and of delta psi. These results suggested the existence of two different survival pathways leading to suppression of apoptosis, one for UVA that is independent of the PI3-kinase-Akt pathway and the other for UVB and UVC that is dependent on this pathway.

The antiapoptotic effect of low-dose UVB irradiation in NIH3T3 cells involves caspase inhibitions.

UVB irradiation is a well-known apoptosis induction factor. However, we have previously found that low doses of UVB irradiation inhibited apoptosis induced by both serum starvation and lack of extracellular matrix, involving a significant inhibition of caspase-3/7 activation. In this study, we report on the relationship between the UVB-induced anti-apoptotic effect and caspase-3/7 inhibition by reactive oxygen species (ROS). The UVB-induced antiapoptotic effect was partially prevented by an antioxidant agent, N-acetylcysteine. A ROS-generating agent, menadione and a pro-oxidant agent, H2O2 also showed an effect that was similar to the UVB-induced antiapoptotic effect, indicating that ROS contributed to the antiapoptotic effect. UVB irradiation significantly suppressed caspase-3/7 activation, which was caused by the inhibition of proteolysis and not by the inhibition of enzymatic activity itself. The prevention of proteolysis was also confirmed by both the following results: one is the inhibition of in vitro caspase-3/7 and -9 activation in cell lysates exposed to UVB in the presence of cytochrome c and dATP, which was caused by the production of ROS, and the other is the inhibition of in vitro caspase-3/7 activation in the presence of active caspase-9. These results showed that the inhibition of the caspase cascade downstream mitochondria by ROS production, leading to a significant inhibition of caspase-3/7 activation, was one of the causes of the antiapoptotic effect by small doses of UVB irradiation.

Degradation of nonylphenol polyethoxylates by ultraviolet B irradiation and effects of their products on mammalian cultured cells.

Nonylphenol polyethoxylates (NPEOs) are widely used as non-ionic surfactants and their biodegradation products such as 4-n-nonylphenol are stable and have been demonstrated to be cytotoxic. In the aquatic environment, these compounds are usually exposed to sunlight, and while the correlation between the biodegradation of NPEOs and changes in cytotoxicity has been reported, the relationship between the photodegradation of NPEOs and cytotoxicity has not. In this study, we investigated the degradation of NPEO by ultraviolet (UV) irradiation, especially UVB irradiation, and the effects on mammalian cell lines. NPEO with a smaller number of ethylene oxide (EO) units showed greater cytotoxicity. Although NPEO (10) completely inhibited the proliferation of the cells, NPEO (70) showed no toxicity. UVB irradiation significantly induced a shortening of the side chain, which was due to the production of ROS. The EO side chain of NPEO (10), was gradually degradated, but that of NPEO (70) was degradated near the benzene ring. Furthermore, the degradation of the benzene ring was more effective in NPEO (70) than NPEO (10). The toxicity of NPEO (10) in cultured cells decreased following UVB irradiation, whereas that of NPEO (70) was induced after UVB irradiation at 500 J/cm2 and disappeared at 1000 J/cm2. This might be due to the production of NPEO with a short side chain and 4-n-nonylphenol by the degradation of EO and due to the degradation of the benzene ring at higher doses of UVB irradiation. This study shows the significance of UV exposure to the degradation of alkylphenol polyethoxylates in the environment.

Phototoxicity of benzo[a]pyrene by ultraviolet A irradiation: induction of apoptosis in Jurkat cells.

The toxicology of benzo[a]pyrene (BaP) has been mainly studied with regard to the carcinogenicity of its metabolites, but its phototoxicity is not well understood. Although some studies have indicated the lethal phototoxicity of BaP, there have been no reports regarding the pattern of cell death induced by this agent. In this study, we investigated the pattern and mechanism of cell death induced by coexposure to BaP plus ultraviolet A (UVA) in Jurkat cells. Coexposure to BaP plus UVA showed dose-dependent cytotoxicity. The pattern of cell death was apoptotic as determined by cell shrinkage, chromatin condensation, appearance of subdiploid apoptotic nuclei and translocation of phosphatidylserine to the outer membrane leaflet. Coexposure also strongly increased caspase-3/7 activity and slightly elevated those of caspase-8/6 and -9. The pan caspase inhibitor Z-VAD-CH(2)-DCB partially inhibited the phototoxicity of BaP. Cytochrome c release was observed 6 h after coexposure, but not after 1 h. Furthermore, the phototoxicity was inhibited by NaN(3) (quencher of singlet oxygen), but not by mannitol (quencher of hydroxy radicals). Chromatin condensation and translocation of phosphatidylserine were also inhibited by NaN(3), suggesting that the induction of apoptosis by coexposure to BaP plus UVA was due to singlet oxygen production.

Coexposure to benzo[a]pyrene plus ultraviolet A induces 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in human skin fibroblasts: preventive effects of anti-oxidant agents.

The influence of environmental benzo[a]pyrene (BaP) contamination under ultraviolet A (UVA) on normal human skin fibroblasts was examined. Treatment of human skin fibroblasts with UVA in the presence of BaP induced cytotoxicity in a UVA- and BaP-dose-dependent manner, involving oxidative DNA damage (formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG)). Singlet oxygen quenchers significantly inhibited the formation of 8-oxo-dG, whereas hydroxyl radical and superoixide anion radical scavengers showed no effect. N-Acetyl-l-cysteine prevented the formation of 8-oxo-dG. These findings suggested the possibility of increased carcinogenesis in the skin via singlet oxygen produced by sunlight plus environmental BaP contamination and the efficiency of anti-oxidant agents for its prevention.

Change of estrogenic activity and release of chloride ion in chlorinated bisphenol a after exposure to ultraviolet B.

Bisphenol A (BPA) and chlorinated bisphenol A (ClBPAs) were detected in wastewater from waste paper recycling plants. In previous study, we showed the acute cytotoxicity of oxidized products of BPA and ClBPAs generated by ultraviolet (UV) irradiation. However, estrogenic activities of these photoproducts have not been studied. Therefore, we investigated change of estrogenic activities of BPA and ClBPAs [3-chlorobisphenol A (3-ClBPA), 3,3'-dichlorobisphenol A (3,3'-diClBPA) and 3,3',5-trichlorobisphenol A (3,3',5-triClBPA)] after UVB irradiation using yeast two-hybrid assay. The agonist activities of ClBPAs were higher than that of BPA in the absence of S9. ClBPAs irradiated with UVB lost agonist activities. The addition of S9 also completely erased the activity. The antagonist activities of BPA and ClBPAs with or without UVB irradiation were not detected both in the absence or presence of S9. UVB irradiation (0-100 J/cm2) decreased the agonist activity of 3,3'-diClBPA in proportion to increase of released chloride ion. The agonist activity was completely lost at 50 J/cm2 of UVB, of which dose could dissociated almost all chlorine. These findings suggested that UVB irradiation could decrease the estrogenic activity of chlorinated compounds, which was due to the selective release of chloride ion.

Hydrogen peroxide is critical for UV-induced apoptosis inhibition.

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O2*-) and hydrogen peroxide (H2O2), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H2O2-eliminating enzyme, catalase, but not in the presence of an O2*--eliminating enzyme, superoxide dismutase. Treatment with H2O2 itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H2O2 treatment decreased in H2O2-resistant cells. Although both UVB and H2O2 are known to induce DNA damage, other DNA damaging agents, like gamma-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H2O2 was essential to the inhibition of apoptosis, in which DNA damage had no role.

Coexposure to benzo[a]pyrene plus UVA induced DNA double strand breaks: visualization of Ku assembly in the nucleus having DNA lesions.

Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant with potential carcinogenicity. It has been shown that BaP, upon UVA irradiation, synergistically induced oxidative DNA damage, but other DNA damage was not confirmed. In this study, we examined whether coexposure to BaP plus UVA induces double strand breaks (DSBs) using xrs-5 cells, deficient in the repair of DSBs (Ku80 mutant), and whether Ku translocates involving the formation of DSBs. BaP plus UVA had a significant cytotoxic effect on CHO-K1 cells and an even more drastic effect on Ku80-deficient, xrs-5 cells, suggesting that the DSBs were generated by coexposure to BaP plus UVA. The DSBs were repaired in CHO-K1 cells within 30 min, but not in xrs-5 cells, indicating the involvement of a non-homologous end joining, which needs Ku proteins. Furthermore, we succeeded in visualizing that Ku80 rapidly assembled to the exposed region, in which DSBs might be generated, and clarified that the presence of both Ku70 and Ku80 was important for their accumulation.