Role of ATM in Oxidative Stress-mediated c-Jun Phosphorylation in Response to Ionizing Radiation and CdCl2 *

Ionizing radiation-induced phosphorylation of the transcription factor c-Jun is impaired in cells derived from individuals with ataxia telangiectasia (AT), in which theATM gene is mutated. We demonstrate here that ATM modulates c-Jun phosphorylation following exposure to ionizing radiation as well as treatment with CdCl2, a potent pro-oxidant. Exposure of AT and control fibroblasts to CdCl2 induced a biphasic increase in c-Jun phosphorylation on serine residues 63 and 73, with the extent of the second phase being markedly greater in AT cells than in control cells. Heme oxygenase-1, a marker of oxidative stress, was also significantly induced in AT fibroblasts. Expression of recombinant ATM in AT fibroblasts, however, reduced the extent of the effects of CdCl2 on both c-Jun phosphorylation and heme oxygenase-1 induction. Our data suggest that ATM contributes to oxidative stress-mediated signaling that leads to c-Jun phosphorylation by acting as a sensor of ionizing radiation-induced oxidative stress and by modulating intracellular redox homeostasis.

Cells derived from individuals with AT show extreme sensitivity to ionizing radiation as well as defects in ionizing radiation-induced intracellular signaling (35)(36)(37). The gene (ATM) that is mutated in individuals with AT has been isolated, and the encoded protein (ATM) is thought to play a role in a variety of cellular processes including mitogenic signal transduction, cell cycle control, and repair of DNA damage (38 -40). Defects in ionizing radiation-induced signaling in AT cells include impaired activation of the transcription factors NF-B and c-Jun (37,(41)(42)(43).
The transcriptional activity of c-Jun is increased by phosphorylation of Ser 63 and Ser 73 residues in response to various stimuli, including ionizing radiation, ultraviolet light, cytokines, and oxidative stress (44 -48). We have previously shown that the ionizing radiation-induced phosphorylation of c-Jun on these two residues that is apparent in normal human fibroblasts does not occur in AT fibroblasts, whereas ultraviolet radiation-induced phosphorylation of these sites is intact in both types of cells (37). We have proposed that the c-Jun NH 2terminal kinase (JNK), which is responsible for the phosphorylation of c-Jun and is present in the nucleus of both normal and AT fibroblasts, is subjected to negative regulation that is relieved on exposure of normal cells to ionizing radiation (37). We now demonstrate a role for ATM in oxidative stress-induced JNK activation and c-Jun phosphorylation. Our data indicate that ATM functions as a sensor of ionizing radiation-generated oxidative stress and as a modulator of intracellular redox regulatory systems in cells exposed to ionizing radiation or the potent pro-oxidant CdCl 2 .

EXPERIMENTAL PROCEDURES
Materials-NAC, BSO, and CdCl 2 were obtained from Sigma. The GST-c-Jun-(1-89) fusion protein was obtained from Cell Signaling Technology. Antibodies to c-Jun and to HO-1 were from Transduction Laboratories; those specific for c-Jun phosphorylated on Ser 63 or on Ser 73 were from Cell Signaling Technology; those specific for JNK phosphorylated on Thr 183 and Tyr 185 were from Promega; and those to ATM were from Calbiochem.
Cell Culture-Normal human fibroblasts (MRC5CV1) and AT fibroblasts (AT5BIVA, AT4BIVA, and AT3BIVA) were cultured under 5% CO 2 at 37°C in Eagle's minimum essential medium supplemented with 15% (normal cells) or 20% (AT cells) fetal bovine serum, 2 mM Lglutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, penicillin (100 units/ml), and streptomycin (100 g/ml). Hydrocortisone (5 g/ml) was also included in the culture medium for AT cells. MRC5CV1/pEAT22 (MRC5CV1 fibroblasts transfected with pEAT22, which encodes ATM antisense RNA) (49) and AT5BIVA/pMAT1 cells (AT5BIVA fibroblasts transfected with pMAT1, which contains the full-length ATM cDNA under the control of the inducible metallothionein II promoter) (50) were cultured in the presence of hygromycin B (200 g/ml). All fibroblast cell lines were immortalized with SV40 (51). Before exposure of cells to ionizing radiation or CdCl 2 , the culture medium was replaced with serum-free medium for 24 h. Irradiation was performed at room temperature with a JL Shepherd Mark I Radiator containing a 137 Cs source emitting at a fixed dose rate of 3.83 Gy/min. Subcellular Fractionation-Cells were washed three times with icecold phosphate-buffered saline, harvested by scraping from the culture dishes into ice-cold equilibration buffer (EB) (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl 2 , 2 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM EGTA, and protease inhibitors (52)), and collected by centrifugation at 12,000 rpm (18,000 ϫ g) for 1 min at 4°C. Cell pellets were resuspended in EB containing 0.1% Nonidet P-40 and incubated on ice for 15 min. The cytosolic fraction was then isolated by centrifugation at 14,000 rpm (25,000 ϫ g) for 1 min, and the resulting pellet was washed once with EB, resuspended in a nuclear extraction buffer (20 mM HEPES (pH 7.9), 0.42 M NaCl, 1 mM EDTA, 0.1 mM EGTA, 2 mM dithiothreitol, 20% glycerol, and protease inhibitors), and incubated on ice for 1 h with occasional vortexing. The extracted nuclear proteins were isolated by centrifugation at 50,000 ϫ g for 30 min. Protein concentration was determined with the Bradford assay (53), with bovine serum albumin as a standard.
Immunoblot Analysis-Subcellular fractions were denatured by boiling for 5 min in SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a polyvinylidene difluoride membrane, which was then incubated for 1 h at room temperature with 5% nonfat dry milk in phosphate-buffered saline before exposure overnight at 4°C to primary antibodies. Immune complexes were detected by enhanced chemiluminescence (Amersham Phamacia Biotech).
In Vitro Kinase Assay of JNK Activity-The kinase activity of JNK was assayed in vitro by incubation of cytosolic or nuclear proteins for 15 min at 30°C with 5 g of GST-c-Jun-(1-89) in 20 l of kinase buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM MnCl 2 , 0.1 mM Na 3 VO 4 , 2 mM dithiothreitol, 20 M ATP). Phosphorylated GST-c-Jun-(1-89) (as well as phosphorylated endogenous c-Jun) was detected by immunoblot analysis with antibody specific for c-Jun phosphorylated on Ser 73 .

Effect of CdCl 2 on c-Jun Phosphorylation-
The intracellular accumulation of Cd 2ϩ generates oxidative stress (4 -8, 10). To investigate whether c-Jun is activated in response to exposure of cells to Cd 2ϩ , we first examined c-Jun phosphorylation on Ser 63 and Ser 73 in normal human fibroblasts (MRC5CV1) and AT fibroblasts (AT5BIVA) after incubation with 10 M CdCl 2 for various times. Nuclear extracts were prepared and subjected to immunoblot analysis with antibodies specific for c-Jun phosphorylated on Ser 73 (Fig. 1A). A biphasic pattern of c-Jun phosphorylation was apparent in both MRC5CV1 and AT5BIVA cells. The first phase of c-Jun phosphorylation was characterized by a gradual increase that reached a maximum at ϳ4 h after exposure to CdCl 2 and had decreased to control values by 7 h in both normal and AT cells. Although the extent of the increase in c-Jun phosphorylation at 4 h was similar in both cell types, it was consistently slightly greater in AT5BIVA cells (6-fold) than in MRC5CV1 cells (5-fold). The onset of the second phase of c-Jun phosphorylation was apparent 12 h after exposure to CdCl 2 and reached a maximum at 16 -24 h. In contrast to the similar pattern of the first phase apparent in these two cell lines, the pattern of the second phase of c-Jun phosphorylation differed markedly between the normal and AT cells. Whereas MRC5CV1 cells exhibited only a 3-fold increase in c-Jun phosphorylation by 24 h, AT5BIVA cells showed an ϳ25-fold increase at both 16 and 24 h after exposure to CdCl 2 . A similar pattern of c-Jun phosphorylation on Ser 63 was also observed in both MRC5CV1 and AT5BIVA cells in response to CdCl 2 (Fig. 1B).
Effects of CdCl 2 on JNK Phosphorylation and Activity-Activation of JNK is mediated by phosphorylation of the threonine and tyrosine residues within the TPY motif at positions 183-185 (54 -57). Substantial amounts of p46 and p55 isoforms of JNK are present in both nucleus and cytosol of MRC5CV1 and AT5BIVA cells (37). Furthermore, the dually phosphorylated form of p46 JNK, but not p55 isoform, is constitutively present in the nucleus of these cells (37). Treatment with 10 M CdCl 2 for 16 h had no effect on the abundance of JNK isoforms in either the nucleus or cytosol of these cells (data not shown). However, immunoblot analysis with antibody specific for the active, dually phosphorylated form of JNK revealed that phosphorylation of the p46 isoform present in the nucleus of AT5BIVA cells was increased within 10 h after exposure to CdCl 2 ( Fig. 2A). Furthermore, phosphorylation of the p55 isoform was induced at 16 h after exposure of these cells to CdCl 2 and sustained up to 24 h. These data suggest that both JNK isoforms are activated in response to CdCl 2 in AT5BIVA cells and that the marked, second phase phosphorylation of c-Jun in AT5BIVA cells (Fig. 1, A and B) is, at least in part, attributed to the phosphorylation of p55 isoform of JNK. The phosphorylation of an unidentified protein that reacted with the antibodies to dually phosphorylated JNK also appeared to be increased 10 and 16 h after exposure of AT5BIVA cells to CdCl 2 and sustained up to 24 h ( Fig. 2A).
We next examined the effect of CdCl 2 treatment of MRC5CV1 and AT5BIVA cells for various times to CdCl 2 on JNK activity measured in cytosolic and nuclear fractions by performing in vitro kinase assay with a glutathione S-transferase (GST) fusion protein containing residues 1-89 of c-Jun as substrate. Phosphorylated GST-c-Jun-(1-89) was detected by immunoblot analysis with antibodies specific for c-Jun phosphorylated on Ser 73 . Exposure of cells to CdCl 2 induced a biphasic increase in JNK activity in nuclear fraction of MRC5CV1 and AT5BIVA cells (Fig. 2B). In MRC5CV1 cells, the activity of JNK in the nucleus, as revealed by the phosphorylation of the GST-c-Jun-(1-89) fusion protein as well as by that of endogenous c-Jun, showed two peaks at 4 and 24 h. Cytosolic JNK activity showed a similar pattern of induction (data not shown). In AT5BIVA cells, the activity of JNK in the nucleus peaked at 4 and 16 -24 h. Overall, the pattern of JNK activity as revealed by phosphorylation of GST-c-Jun-(1-89) did not differ substantially between MRC5CV1 and AT5BIVA cells, suggesting that the greater extent of the second phase of CdCl 2 -induced phosphorylation of endogenous c-Jun apparent in AT5BIVA cells is not attributable to a greater increase in JNK activity in vitro. These data also implicate that different isoforms of JNK may participate in different phases for c-Jun phosphorylation in response to CdCl 2 . Alternatively, another mechanism, which involves residues other than amino acids 1-89, is associated with the second phase of c-Jun phosphorylation. Recently, JNK-independent phosphorylation of c-Jun on serine 63 and 73 residues has been demonstrated by a novel protein complex, which requires COOH-terminal amino acid residues including DNA binding and dimerization domains for its activity (58,59).
Combined Effects of Ionizing Radiation and CdCl 2 on c-Jun Expression and Phosphorylation-We have previously shown that ionizing radiation-induced phosphorylation of c-Jun is impaired in AT fibroblasts (37). We therefore investigated the effect of ionizing radiation on c-Jun phosphorylation as well as on c-Jun expression in AT cells that had been exposed to CdCl 2 , a treatment that increases c-Jun phosphorylation in these cells. We also examined further the role of ATM in Cd 2ϩinduced phosphorylation of c-Jun. For these experiments we used cell lines MRC5CV1, AT5BIVA, and AT5BIVA/pMAT1, the latter of which corresponds to AT5BIVA cells transfected with a plasmid (pMAT1) containing the full-length ATM cDNA under the control of a Cd 2ϩ -inducible promoter (50). Cells were incubated in the absence or presence of 10 M CdCl 2 for 16 h and then exposed to 20 Gy of ionizing radiation. Nuclear proteins were isolated from cells at various times after irradiation, and c-Jun expression (Fig. 3A) and phosphorylation (Fig. 3B) were examined by immunoblot analysis.
Pretreatment of cells with CdCl 2 for 16 h markedly increased the abundance of c-Jun in all three cell lines, and subsequent exposure to ionizing radiation did not further increase c-Jun expression (Fig. 3A). Although c-Jun expression was maximally increased after treatment with CdCl 2 , such treatment induced only a small increase in c-Jun phosphorylation in MRC5CV1 cells (Fig. 3B). Subsequent exposure of these cells to ionizing radiation further increased c-Jun phosphorylation, although not to the extent greater than that apparent in MRC5CV1 cells not pretreated with CdCl 2 . In AT5BIVA cells, the marked increase in c-Jun phosphorylation induced by CdCl 2 treatment was not further increased by subsequent exposure of the cells to ionizing radiation (Fig. 3B). Other AT fibroblast lines (AT4BIVA and AT3BIVA) exhibited a similar pattern of c-Jun phosphorylation in response to CdCl 2 and ionizing radiation (Fig. 3C). In the absence of CdCl 2 pretreatment, ionizing radiation increased both expression (Fig. 3A) and phosphorylation (Fig. 3B) of c-Jun in MRC5CV1 cells but not in AT5BIVA cells. In AT5BIVA/pMAT1 cells not treated with CdCl 2 , ionizing radiation induced a slight increase in c-Jun expression ( Fig. 3A) but had no effect on c-Jun phosphorylation (Fig. 3B). However, pretreatment of AT5BIVA/pMAT1 cells with CdCl 2 , which induces ATM expression in these cells, resulted in a substantial induction of c-Jun phosphorylation that was further increased by exposure to ionizing radiation; subsequent irradiation did not further increase c-Jun expression in Cd 2ϩ -pretreated AT5BIVA/pMAT1 cells. The increases in c-Jun expression and phosphorylation induced by CdCl 2 pretreatment in AT5BIVA/ pMAT1 cells were smaller than those apparent in AT5BIVA cells.
These data suggest that CdCl 2 and ionizing radiation activate two distinct signaling pathways that converge upstream of activation of c-Jun and that ATM contributes to the regulation of c-Jun phosphorylation by both CdCl 2 and ionizing radiation. The combined effects of these two pathways are most apparent in the pattern of c-Jun phosphorylation observed in AT5BIVA/ pMAT1 cells exposed to both CdCl 2 and ionizing radiation.
Role of ATM in c-Jun Phosphorylation Induced by CdCl 2 -To investigate further the role of ATM in CdCl 2 -induced c-Jun phosphorylation, MRC5CV1 cells were transfected with plasmid containing antisense ATM cDNA under the control of the Rous sarcoma virus promoter (49). Immunoblot analysis revealed that the amount of ATM in MRC5CV1/pEAT22 cells was reduced to ϳ40% of that in the parental MRC5CV1 cells (data not shown). The extent of c-Jun phosphorylation in MRC5CV1/ pEAT22 cells at 4 h after exposure to CdCl 2 was similar to that in MRC5CV1 and AT5BIVA cells (Fig. 4). However, at 16 h after exposure to CdCl 2 , c-Jun phosphorylation in MRC5CV1/ pEAT22 cells was markedly greater than that apparent in MRC5CV1 cells but less than that observed in AT5BIVA cells.
Role of Oxidative Stress in c-Jun Phosphorylation Induced by CdCl 2 or Ionizing Radiation-Induction of c-jun gene expression by Cd 2ϩ is thought to result from the oxidative stress induced by these metal ions (29). Cadmium ions react readily with thiol groups of proteins and thereby disrupt intracellular sulfhydryl homeostasis (9 -11). Ionizing radiation also induces oxidative stress by generating reactive oxygen species that result from the ionization of intracellular water (30). Therefore, we examined the role of oxidative stress in c-Jun phosphorylation induced by ionizing radiation or CdCl 2 in normal and AT cells. Pretreatment of cells with 30 mM N-acetylcysteine (NAC), an intracellular precursor of glutathione, markedly reduced the extent of CdCl 2 -induced phosphorylation of c-Jun in both MRC5CV1 and AT5BIVA cells (Fig. 5A). Whereas NAC alone had no effect on c-Jun phosphorylation in MRC5CV1 cells, it induced c-Jun phosphorylation in AT5BIVA cells, possibly reflecting a difference in intracellular sulfhydryl homeostasis and basal oxidative stress between these two cell types (31). Pretreatment of MRC5CV1 cells with NAC also inhibited the increase in c-Jun phosphorylation induced by ionizing radiation (Fig. 5B). These data suggest that oxidative stress contributes to the increase in c-Jun phosphorylation in response to CdCl 2 treatment or ionizing radiation exposure.
Exposure of cells to L-buthionine sulfoximine (BSO), a potent inhibitor of ␥-glutamyl-cysteine synthetase, reduces the intracellular level of glutathione (29). We therefore examined the effect of BSO on c-Jun phosphorylation in response to CdCl 2 and ionizing radiation in MRC5CV1, AT5BIVA, and AT5BIVA/ pMAT1 cells (Fig. 5C). Incubation with 5 M BSO alone for 24 h had little or no effect on c-Jun phosphorylation in these cells. In AT5BIVA cells, however, BSO pretreatment potentiated the increase in c-Jun phosphorylation induced either by CdCl 2 alone or by CdCl 2 and ionizing radiation. In contrast, pretreatment of MRC5CV1 or AT5BIVA/pMAT1 cells with BSO had no effect on c-Jun phosphorylation induced by CdCl 2 either alone or combined with ionizing radiation. These data are consistent with the notion that AT cells are more susceptible to disruption of intracellular redox homeostasis and more sensitive to oxidative stress than are cells in which ATM function is intact.

Possible Role of ATM as a Sensor of Ionizing Radiationinduced Oxidative Stress and as a Modulator of Intracellular
Redox Homeostasis-Heme oxygenase (HO) oxidatively cleaves heme, a pro-oxidant, into carbon monoxide and biliverdin, which is subsequently converted to bilirubin, an antioxidant. Three HO isozymes encoded by three distinct genes have been identified (60,61). The expression of HO-1 is induced in a wide variety of mammalian tissues by agents that elicit cellular injury, and induction of HO-1 has been implicated as a generalized cellular response to oxidative stress in cultured mammalian cells (62). We therefore examined the effect of CdCl 2 on HO-1 expression in MRC5CV1, AT5BIVA, and MRC5CV1/ pEAT22 cells (Fig. 6). None of these cells expressed HO-1 at a detectable level under normal physiological conditions. However, exposure of these cells to CdCl 2 resulted in an induction of HO-1 expression in both nucleus and cytosol (Fig. 6, A and  B). Whereas MRC5CV1 cells showed a low level of HO-1 expression that was first apparent at 7 h after exposure to CdCl 2 and was sustained for up to 24 h (Fig. 6A), AT5BIVA cells exhibited a marked induction of HO-1 that was first apparent at 4 h and continued to increase for up to 24 h. The extent of  HO-1 induction in MRC5CV1/pEAT22 cells, which express ATM antisense RNA, was intermediate between those apparent in MRC5CV1 and AT5BIVA cells (Fig. 6B), suggesting an inverse relation between the level of CdCl 2 -induced oxidative stress and the abundance of ATM in these cells. These data thus indicate that ATM plays an important role in modulating intracellular oxidative stress generated by the potent pro-oxidant, CdCl 2 .
Finally, we investigated the role of ATM in modulation of oxidative stress generated by ionizing radiation in MRC5CV1, AT5BIVA, and AT5BIVA/pMAT1 cells (Fig. 6C). Ionizing radiation induced a low level of HO-1 expression in MRC5CV1 cells but had no effect on this parameter in AT5BIVA or AT5BIVA/ pMAT1 cells. Exposure of AT5BIVA/pMAT cells to 10 M CdCl 2 for 16 h resulted in an induction of HO-1 expression that was greater than that apparent in MRC5CV1 cells but smaller than that apparent in AT5BIVA cells. Furthermore, whereas the induction of HO-1 in AT5BIVA or MRC5CV1 cells in response to CdCl 2 treatment was not further increased by exposure to ionizing radiation, that apparent in AT5BIVA/pMAT1 cells in which ATM expression was induced by pretreatment with CdCl 2 was potentiated by irradiation. These data suggest that MRC5CV1 cells, but not AT5BIVA cells, were able to sense and to respond to ionizing radiation-induced oxidative stress and that expression of recombinant ATM in AT5BIVA cells restored the cellular response to ionizing radiation-induced oxidative stress. Taken together, our results suggest a role for ATM as a sensor of ionizing radiation-induced oxidative stress and as a modulator of intracellular redox regulatory pathways in cells exposed to ionizing radiation or CdCl 2 . DISCUSSION Intracellular accumulation of Cd 2ϩ results in a variety of cellular responses including transcriptional activation of the proto-oncogene c-jun (29,33) as well as oxidative damage to cellular macromolecules (4 -11). Disruption of intracellular sulfhydryl homeostasis and reaction with protein thiol groups are thought to contribute to the Cd 2ϩ -induced oxidative stress (10). Ionizing radiation also damages cellular macromolecules as a result of the generation of reactive oxygen species (30). We Nuclear proteins were subsequently subjected to immunoblot analysis with antibodies specific for c-Jun phosphorylated on Ser 73 . B, MRC5CV1 cells were incubated for 1 h in the absence or presence of 30 mM NAC and subsequently exposed to 20 Gy of ionizing radiation. Cells were harvested at 2 h thereafter, and nuclear proteins were analyzed by immunoblot analysis as in A. C, MRC5CV1, AT5BIVA, and AT5BIVA/ pMAT1 cells were incubated for 24 h in the absence or presence of 5 M BSO and then for 16 h with or without 10 M CdCl 2 (in the continuous absence or presence of BSO); cells were exposed to 20 Gy of ionizing radiation 1 h before the end of the 16-h incubation period. Nuclear proteins were analyzed for c-Jun phosphorylation as in A.

FIG. 6. Induction of HO-1 expression by CdCl 2 and ionizing radiation in normal human fibroblasts and AT cells.
A, MRC5CV1 and AT5BIVA cells were incubated with 10 M CdCl 2 for the indicated times, after which nuclear and cytosolic proteins were subjected to immunoblot analysis with antibodies to HO-1. B, MRC5CV1, AT5BIVA, and MRC5CV1/pEAT22 cells were incubated with 10 M CdCl 2 for the indicated times, after which the abundance of HO-1 in the nucleus was evaluated as in A. C, MRC5CV1, AT5BIVA, and AT5BIVA/ pMAT1 cells were incubated in the absence or presence of 10 M CdCl 2 for 16 h and exposed (or sham) to 20 Gy of ionizing radiation 1 h before the end of the incubation. The amount of HO-1 in the nucleus was then determined by immunoblot analysis.
have now shown that CdCl 2 induces a biphasic phosphorylation of c-Jun on Ser 63 and Ser 73 residues in both normal and AT fibroblasts. Furthermore, this Cd 2ϩ -induced c-Jun phosphorylation was mediated, at least in part, through a signaling pathway activated by oxidative stress. Thus, preincubation of cells with the antioxidant NAC markedly reduced the extent of CdCl 2 -induced phosphorylation of c-Jun. Our data also revealed that the extent of the second phase c-Jun phosphorylation induced by CdCl 2 is much greater in AT cells than in normal cells, whereas the extent of the first phase is similar in the two types of cells. Cadmium chloride has previously been shown to induce a biphasic increase in the abundance of c-jun mRNA in mouse liver cells (63); whereas the first phase was suggested to result from an early cellular response to Cd 2ϩ itself, the second phase was attributed to Cd 2ϩ -induced cytotoxicity. Similarly, the exaggerated second phase of c-Jun phosphorylation in AT cells exposed to CdCl 2 may represent a cellular response to unchecked Cd 2ϩ -induced cytotoxicity, presumably attributable to the excessive generation of oxidative stress.
We have previously shown that the p46 isoform of JNK contributes to the ionizing radiation-induced phosphorylation of c-Jun in normal human fibroblasts, and that p55 isoform of JNK, which is constitutively expressed in both normal and AT human fibroblasts, is not phosphorylated in response to ionizing radiation (37). We have now shown that CdCl 2 induces a delayed phosphorylation of p55 as well as phosphorylation of p46 in AT cells. Phosphorylation of p55 was not induced by CdCl 2 in MRC5CV1 cells (data not shown). However, CdCl 2 treatment induced a biphasic increase in JNK activity in both MRC5CV1 and AT5BIVA cells even though the greater extent of the second phase of CdCl 2 -induced phosphorylation of endogenous c-Jun apparent in AT5BIVA cells is not attributable to a greater increase in JNK activity in vitro. We interpret these data to show that different isoforms of JNK, p55 and p46, may participate in different phases of c-Jun phosphorylation in response to CdCl 2 .
Alternatively, the apparent mechanistic dissociation of JNK activity and c-Jun phosphorylation may be due to another mechanism, which involves residues other than amino acid 1-89. This observation is consistent with recent reports demonstrating JNK-independent phosphorylation of c-Jun on serine 63 and 73 residues by a novel protein complex, which requires COOH-terminal amino acid residues including DNA binding and dimerization domains for its activity (58,59).
Evidence suggests that AT cells exhibit an increased basal state of oxidative stress. ATM has been suggested to function as a sensor of reactive oxygen species and oxidative damage to cellular macromolecules (31,64,65). Thus, ATM-deficient mice show evidence of oxidative damage to proteins and lipids in organs such as the brain and testes, and they exhibit an increased abundance and activity of HO-1 in various cell types including Purkinje cells of the cerebellum (65). Induction of HO-1 expression has been associated with a generalized cellular response to oxidative stress induced by a variety of stimuli in mammalian cells (62). We have now shown that CdCl 2 generates a greater extent of oxidative stress in AT cells than in normal cells, and our data suggest that ATM functions as a sensor of ionizing radiation-induced oxidative stress and as a modulator of intracellular redox regulatory systems. We examined induction of HO-1 expression as a marker for the generation of and cellular response to oxidative stress. The extent of HO-1 induction in response to CdCl 2 was markedly greater in AT cells than in normal cells. The pattern and kinetics of CdCl 2 -induced expression of HO-1 in AT cells and in normal cells resembled those of the second phase of c-Jun phosphoryl-ation in the respective cells, supporting the notion that the second phase of c-Jun phosphorylation is indeed mediated by CdCl 2 -induced oxidative stress. In contrast to the marked induction of HO-1 by CdCl 2 in AT cells, HO-1 expression was not induced by ionizing radiation in these cells. Irradiation of normal cells induced expression of HO-1 at a very low level. These observations indicate that AT cells are unable to detect ionizing radiation-induced oxidative stress; however, these cells are able to detect oxidative stress generated by CdCl 2 and exhibit a hypersensitive response, presumably because of impaired intracellular antioxidant function.
Expression of full-length wild-type ATM in AT cells corrects various aspects of the AT phenotype, including extreme radiosensitivity and impaired activation of JNK in response to ionizing radiation (50). Conversely, expression of full-length ATM antisense RNA in normal lymphoblastoid cells was shown to convert the normal phenotype of these cells to the one resembling that of AT cells (49). Our results with cells transfected with ATM cDNA in the normal or antisense orientation further support a role for ATM as a sensor and modulator of oxidative stress. Thus, AT5BIVA/pMAT1 cells, which express ATM in a CdCl 2 -inducible manner, exhibit induction of HO-1 expression in response to ionizing radiation after pretreatment with Cd 2ϩ . Expression of HO-1 was not increased further by ionizing radiation in CdCl 2 -pretreated AT5BIVA cells, indicating that Cd 2ϩ -induced expression of ATM was responsible for the induction of HO-1 in response to ionizing radiation in AT5BIVA/ pMAT1 cells and that ATM functions as a sensor of ionizing radiation-induced oxidative stress. MRC5CV1/pEAT22 cells, in which the abundance of ATM is reduced as a result of expression of ATM antisense RNA, the extent of HO-1 induction in response to CdCl 2 was greater than that apparent in MRC5CV1 cells but smaller than that observed in AT5BIVA cells. These observations indicate that suppression of ATM function in normal human fibroblasts results in an increased sensitivity to CdCl 2 -induced oxidative stress. Taken together, our results indicate ATM as a sensor of ionizing radiationinduced oxidative stress and as a modulator of intracellular sulfhydryl homeostasis.
We have shown that ionizing radiation-induced c-Jun phosphorylation in normal fibroblasts is also mediated, at least in part, by oxidative stress. Preincubation of cells with NAC markedly reduced the extent of ionizing radiation-induced c-Jun phosphorylation. AT cells are not able to sense ionizing radiation-induced oxidative stress and do not exhibit c-Jun phosphorylation in response to irradiation. Our data indicate that intact ATM function is required for oxidative stress-mediated c-Jun phosphorylation in response to ionizing radiation. AT5BIVA/pMAT1 cells in which full-length wild-type ATM is expressed are able to sense ionizing radiation-generated oxidative stress and exhibit c-Jun phosphorylation in response to irradiation. Conversely, in MRC5CV1/pEAT22 cells, which express ATM antisense RNA, the extent of c-Jun phosphorylation in response to ionizing radiation was reduced compared with that apparent in MRC5CV1 cells. We also showed that ATM modulates the oxidative stress and the resulting c-Jun phosphorylation induced in cells by CdCl 2 . The excess oxidative stress generated in AT cells as well as the resulting c-Jun phosphorylation in response to CdCl 2 were markedly reduced in the ATM-expressing AT cell line AT5BIVA/pMAT1 relative to those apparent in the parental AT5BIVA cells. Furthermore, the synergistic effect of BSO on CdCl 2 -induced c-Jun phosphorylation apparent in AT5BIVA cells was not observed in AT5BIVA/pMAT1 cells. Conversely, MRC5CV1/pEAT22 cells showed an increased sensitivity to CdCl 2 -induced oxidative stress and an increased extent of c-Jun phosphorylation in response to CdCl 2 compared with those apparent in the parental MRC5CV1 cells.
ATM has been shown to associate with DNA in normal human cells, and ionizing radiation-induced double-strand DNA breaks potentiates this association, suggesting a role for ATM as a sensor of DNA damage (66). Cadmium ion has been shown to induce single-strand DNA breaks (12,13). Therefore, it would be noteworthy to clarify whether a role for ATM in CdCl 2 -induced c-Jun phosphorylation was to sense DNA lesion or to modulate intracellular sulfhydryl homeostasis. Cadmium ion-induced single-strand DNA breakage has been shown to occur in a variety of cell systems at relatively high concentrations of CdCl 2 (at 500 M to 5 mM). A significant line of evidence suggests that this DNA damage is the result of generation of reactive oxygen species in cells exposed to CdCl 2 (67)(68)(69). The inability of CdCl 2 to induce single-strand DNA breakage under anaerobic conditions, as measured by alkaline elution technique, and also inhibition of this DNA damage by antioxidants have been demonstrated (68). However, low concentrations of CdCl 2 (less than 10 M) can effectively induce oxidative stress in cultured cells by disrupting intracellular sulfhydryl homeostasis (10, 11). Recently, inhibition of proteins involved in the intracellular reduction of protein glutathionyl-mixed disulfides, such as thiotransferase, GSSG reductase, thioredoxin, and thioredoxin reductase, has been demonstrated in the presence of very low concentrations of CdCl 2 (70). The concentration of CdCl 2 used in our study was 10 M. At this concentration, the effect of CdCl 2 on single-strand DNA breakage would be considered as minimal, if at all (68 -70).
Unlike the abnormal c-Jun phosphorylation apparent in AT cells in response to ionizing radiation or CdCl 2 , AT cells have been shown to exhibit normal c-Jun phosphorylation in response to UV-C light (37) and also normal JNK activation in response to anisomycin or UV-C light (39). There is evidence that ribostressor anisomycin does not induce oxidative stress in cells (71) and that oxidative damage generated by UV-C light is shown to be minimal in cells (72). Furthermore, pretreatment with NAC has been shown to inhibit the activation of JNK1 by various oxidative stressors but not by UV light or anisomycin (71). In our study, we have shown that c-Jun phosphorylation induced by ionizing radiation or CdCl 2 , which generates oxidative stress in cells, was significantly reduced by NAC pretreatment. Therefore, our data support that ATM plays a role in c-Jun phosphorylation in response to ionizing radiation and CdCl 2 through sensing and/or modulating oxidative stress generated in cells by these stimulants.
In conclusion, our data indicate that ATM is required for the oxidative stress-mediated signaling that leads to c-Jun phosphorylation in response to ionizing radiation and that ATM plays an important role in modulating the oxidative stress and the resulting c-Jun phosphorylation induced by CdCl 2 . The biochemical mechanisms by which ATM senses ionizing radiation-induced oxidative stress and modulates intracellular sulfhydryl homeostasis remain to be elucidated.