Requirement of ATM in UVA-induced signaling and apoptosis.

Solar UVA, but not UVC, reaches the earth's surface and therefore is an important etiological factor for the induction of human skin cancer. ATM kinase is an important regulator of cell survival and cell cycle checkpoints. Here, we observe that UVA, unlike UVC, triggers ATM kinase activity, and the activation may occur through reactive oxygen species produced after irradiation of cells with UVA. We also show that ATM activation is involved in the apoptotic response to UVA but not UVC. Furthermore, we provide evidence that ATM-dependent p53 and c-Jun N-terminal kinase (JNK) pathways are linked to UVA-induced apoptosis. On the other hand, UVC-induced apoptosis occurs through ATR-dependent p53 phosphorylation as well as the JNK pathway. Therefore, these results suggest that ATM, like p53, is involved in the UVA-induced apoptosis to suppress carcinogenesis.

The solar UV light that reaches the earth's surface is divided into ultraviolet A (UVA) (320 -400 nm) and ultraviolet B (UVB) (290 -320 nm) (1,2). UVB is absorbed mainly by the earth's ozone layer and can also be blocked efficiently by protective strategies such as sunscreens (2). Ultraviolet C (UVC) (200 -290 nm) is completely absorbed by the ozone layer and, therefore, is unlikely to have a major pathophysiological effect, although most of the reported UV-stimulated biological effects result from experiments with UVC (1-3). On the other hand, UVA constitutes over 90% of solar UV at ground level and can penetrate the skin. Both in vitro and in vivo experiments clearly confirmed that UVA, like UVB and UVC, is a mutagen and carcinogen (1,2). These observations, therefore, demonstrate that UVA is an important contributor to UV-induced carcinogenesis.
Carcinogenesis appears to arise as a consequence of a combination of disturbances in signal transduction pathways that control cell cycle checkpoints, cell survival, arrest, and apoptosis (2)(3)(4). Such disturbed signaling may result from mutation of key regulatory genes. For example, in many cancers, the tumor suppressor gene p53 has become dysfunctional or is completely lost and thus fails to repress carcinogenesis (5). Atm (ataxia telangiectasia mutated) is also shown to function as a tumor suppressor gene, and its mutation leads to ataxia telangiectasia (AT), 1 a multisystem genetic disorder with an exten-sive combination of somatic and physiological defects and high incidence of cancer (6 -8). Moreover, an accumulation of evidence, indicating that acquired resistance toward apoptosis is a hallmark of most and perhaps all types of cancer (4), suggests that apoptotic signaling pathways in cancer-prone AT cells may be disturbed by mutation of Atm. The gene product, ATM, has indeed been shown to be a serine/threonine protein kinase with a carboxyl-terminal domain significantly similar to the catalytic subunit of phosphatidylinositol 3-kinase and to play a vital role in multiple signal transduction pathways influencing cell cycle checkpoint controls (7, 9 -11). However, the role that ATM kinase may play in p53-dependent or -independent apoptosis pathways is less understood.
ATM was shown to be activated in the cellular response to ionizing radiation (IR), but not to UVC exposure (10 -13), and the activated ATM was recruited to double strand breaks (DSBs) for repair (14). These findings are frequently cited as evidence that UV-induced signal transduction does not require ATM activation. However, of the solar UV, UVA, and perhaps to a lesser extent UVB, but not UVC, are responsible for human skin diseases including cancer, and thus UVA-induced signal transduction should be most relevant to human skin carcinogenesis. Whether ATM kinase is activated and involved in the apoptotic response to UVA is as yet unknown. In this report, we provide evidence that ATM is activated by UVA, but not UVC, and is involved in the cellular decision to trigger p53-and c-Jun N-terminal kinase (JNK)-dependent apoptotic pathways in response to UVA.

MATERIALS AND METHODS
Cell Lines, Cell Culture, and UV Sources-Mouse wild-type (p53 ϩ/ϩ or Jnk ϩ/ϩ ) and knockout (p53 Ϫ/Ϫ , Jnk1 Ϫ/Ϫ , or Jnk2 Ϫ/Ϫ ) embryonic fibroblasts (15,16) were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 10% fetal bovine serum (FBS; from Gemini Bio-Products, Inc., Calabasas, CA), 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin in a humidified atmosphere of 5% CO 2 at 37°C. Human cell lines stably expressing a wildtype or kinase-dead mutant of ATM or ATR (ATM)-and Rad3-related were used in this study because of the small amounts of ATM or ATR in normal mammalian cells (12,17). The AT cells transfected with an empty mammalian expression vector pEBS7 (Atm Ϫ ) or a construct, pEBS7-YZ5, containing recombinant full-length ATM (Atm ϩ ) (18) were maintained in the above described 10% FBS-DMEM supplemented with 100 g/ml of hygromycin B (Sigma) for selection. Human fibroblast GM847 lines stably expressing wild-type full-length (ATRwt) or a kinase-inactive allele (ATRkd) of ATR (17) were cultured in 10% FBS-DMEM containing 400 g/ml G418 (Gemini Bio-Products) for selection. The cell lines were treated with UVA or UVC irradiation, and nonirradiated cell samples were used as negative controls. For the detailed description of UVA or UVC sources, see our previous reports (15,16,19).
DNA Fragmentation Ladder Assay-The experimental cells (2.5 ϫ 10 6 to 3 ϫ 10 6 ) were seeded in 150-mm dishes and cultured for 12-24 h. The embryonic fibroblasts described above were not starved, but Atm ϩ , Atm Ϫ , ATRwt, and ATRkd cells were starved for 4 -8 h in 1% FBS-DMEM. The cells were harvested 14 -18 h after irradiation with UVA or UVC at the indicated doses. Subsequent DNA fragmentation laddering assays were performed according to previously described methods (19,20).
Immunoprecipitation Assay for p53-Following culture for 12-24 h, the experimental cells were starved for 24 h in 1% FBS-DMEM. The cells were harvested at the indicated times following irradiation and lysed in 250 l of IP buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerol phosphate, 1 mM Na 3 VO 4 , 10 g/ml leupeptin, 10 g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). The clarified supernatant fractions containing equal amounts of protein were subjected to immunoprecipitation followed by Western blot analysis according to the described methods (16,21). The immune complex beads were washed twice with IP buffer and twice with phosphate-buffered saline. A mouse monoclonal antibody against p53 (Ab-1) (Oncogene) was used for immunoprecipitation of p53 protein, and then rabbit polyclonal antibodies against phospho-p53 (Ser 15 or Ser 20 ) (Cell Signaling) were used for Western blotting.
Kinase Activity Assays for ATM or ATR-In vitro kinase activity assays for ATM or ATR were performed according to the reported methods (22) with some modification. Briefly, before irradiation as described above, the experimental cells were or were not preincubated for 60 min with ascorbic acid, catalase, N-acetyl cysteine, or L-ergothioneine (from Sigma). Then the cells were harvested at the indicated times after irradiation. The cells were disrupted in IP buffer (above) supplemented with 4 mM EDTA, 50 mM NaF, 0.2% (w/v) dodecyl ␤-Dmaltoside (Sigma), and 1 mM phenylmethylsulfonyl fluoride. The clarified supernatant fractions containing equal amounts of protein were subjected to preimmunoprecipitation for 2 h with normal IgG (Upstate Biotechnology, Lake Placid, NY) and protein A/G plus-agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and subsequent immunoprecipitation overnight with an antibody to ATM (Ab-3) or ATR (Ab-2) (Oncogene). The beads containing immune complexes of ATM or ATR were washed twice with the above-modified IP buffer and twice with kinase buffer A (50 mM Hepes (pH 7.5), 150 mM NaCl, 4 mM MnCl 2 , 6 mM MgCl 2 , 10% (v/v glycerol, 1 mM dithiothreitol, and 100 M Na 3 VO 4 (prepared fresh)). The immune complex of ATM was incubated for 15 min at 30°C with 1 g of PHAS-1 (phosphorylated heat-and acid-stable protein regulated by insulin; Stratagene, Los Angeles, CA) in 30 l of kinase buffer A containing 20 M unlabeled ATP and 10 Ci of [␥-32 P]ATP. The reaction was stopped by the addition of 3ϫ SDS sample buffer and then separated by 15% SDS-PAGE. After autoradiography, radioactive phosphate blots of PHAS-1 were quantified using the Im-ageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). After incubation of the ATR complexes with full-length p53 fusion protein (residues 1-393) conjugated with glutathione S-transferase (GST-p53) (Santa Cruz) in kinase buffer A containing 200 M ATP, the reaction was subjected to separation by 8% SDS-PAGE followed by Western blot analysis with phospho-p53 (Ser 15 ) antibody.
JNK Activity Assay-Activity assays for JNKs were carried out according to the protocol recommended by Cell Signaling. In brief, immunoprecipitates with the beads conjugated previously with c-Jun fusion protein (Cell Signaling) were incubated for 30 min at 30°C in kinase buffer B (50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM EGTA, 1 mM dithiothreitol, and 0.01% (v/v) Brij 35) containing 0.5 mM ATP. Then these reactive products were separated by 10% SDS-PAGE followed by Western blotting with a phospho-c-Jun (Ser 63 ) antibody.

ATM-dependent Apoptosis Induced by UVA but Not UVC-
DNA laddering is believed to be a significant marker of apoptosis, reflecting cleavage of internucleosomal DNA strands by the nuclease (known as caspase-activated DNase) to generate the multiple DNA fragments (23,24). The fragmentation laddering assays can thus be used to evaluate whether apoptosis is induced by UVA or UVC. Failure to induce apoptosis ( Fig. 1A) was observed in UVA-irradiated AT cells transfected with an empty vector pEBS7 (Atm Ϫ ). Conversely, UVA-induced apoptosis was restored (Fig. 1A) after ectopic expression of recombinant full-length ATM in AT cells stably transfected with a construct pEBS7-YZ5 (Atm ϩ ). On the other hand, no difference in UVC-induced apoptosis was observed in either Atm ϩ or Atm Ϫ cell lines (Fig. 1B). Caspase-3 has been shown to be one FIG. 1. Requirement of ATM for induction of apoptosis by UVA but not UVC. AT cells were transfected with an empty vector pEBS7 (Atm Ϫ ) or a construct pEBS7-YZ5 encoding wild-type ATM kinase (Atm ϩ ). The cells were grown in 10% FBS-DMEM and then starved for 8 h by replacing growth medium with 1% FBS-DMEM before irradiation. A, Atm ϩ and Atm Ϫ cells were harvested 18 h after irradiation with UVA at 20, 40, or 80 kJ/m 2 . B, Atm ϩ and Atm Ϫ cells were harvested 14 h following irradiation with UVC at 20, 40, 60, or 120 J/m 2 . DNA fragmentation laddering was visualized by 1.8% agarose gel electrophoresis. C, after UVA or UVC irradiation, total cell lysates containing equal amounts of protein were subjected to separation of activated caspase-3 on 10 -20% gradient PAGE gel followed by Western blotting with an antibody to cleaved caspase-3. These data are representative of at least three similar independent experiments. of the central executioners of apoptosis, being responsible for the proteolytic cleavage of many key proteins (24). Western blotting results using a cleaved caspase-3 antibody showed that caspase-3 was activated in Atm ϩ cells, but only very weakly in Atm Ϫ cells following UVA irradiation (Fig. 1C, upper panel). Similar activation of caspase-3 by UVC occurred in both Atm ϩ and Atm Ϫ cell lines (Fig. 1C, lower panel). Overall, these results suggest that ATM appears to be required for induction of apoptosis by UVA but not UVC.
UVA, Unlike UVC, Activates ATM Kinase-Previous reports (10 -13) showed that IR, but not UVC, activates ATM kinase, but whether UVA activates ATM kinase is as yet unknown. Here, Western blot analysis using an ATM antibody showed stable expression of ATM in Atm ϩ cells, but no significant change of ATM expression after irradiation with UVA or UVC ( Fig. 2A, upper panel). In contrast, weaker signals for ATM were observed in Atm Ϫ cells ( Fig. 2A), suggesting possible expression of an unstable protein product of mutated Atm (8,18). Additionally, no change in ␤-actin expression was found in the two cell lines ( Fig. 2A, lower panel). The identification of Atm ϩ and Atm Ϫ cell lines is consistent with previous reports (12,18). Interestingly, immune complex kinase activity assays showed that irradiation of Atm ϩ cells with UVA, but not UVC, stimulated a significant increase of ATM kinase activity toward PHAS-1 (Fig. 2, B and C), compared with the background level of kinase activity associated with protein A/G-agarose bead mock immunoprecipitates from nonirradiated Atm ϩ extracts. On the other hand, very low levels of ATM kinase activity in Atm Ϫ cells exposed to UVA or UVC were not significantly different from the background levels (Fig. 2, B and C). Moreover, ATM kinase activity stimulated by UVA increased to higher levels 5 min after irradiation and was maintained for 30 min and then decreased to a lower level until at least 120 min postirradiation (Fig. 2C). Conversely, within 5-120 min after UVC irradiation, ATM kinase activity was undetectable in either cell line (Fig. 2C). Taken together, our data suggest that activation of ATM kinase may be involved in apoptotic response pathways induced by UVA but not by UVC.
Furthermore, UVA-stimulated ATM kinase activity was markedly inhibited (Fig. 3A) by preincubation of Atm ϩ cells with ascorbic acid (AA) or catalase (CAT), two effective scavengers of reactive oxygen species (ROS) including singlet oxygen or hydrogen peroxide (H 2 O 2 ), respectively. N-Acetylcysteine (NAC), an intracellular precursor of glutathione, or L-ergothioneine (LET), a novel radioprotective and antioxidant agent, also inhibited UVA-stimulated ATM kinase activity ( Fig. 3A). Additionally, exposure of cells to H 2 O 2 also activated ATM kinase, and the activation was blocked by preincubation with catalase or L-ergothioneine (Fig. 3B) and therefore was used as an internal positive control (25). On the other hand, treatment with ascorbic acid, catalase, N-acetylcysteine, or L-ergothioneine alone had no effect on ATM kinase activity (Fig. 3A). These data suggest that UVA activation of ATM kinase is through reactive oxygen species generated after exposure of cells to UVA irradiation.
Involvement of ATM-dependent p53 in Apoptosis Induced by UVA-Phosphorylation of p53 at Ser 15 and Ser 20 in an aminoterminal nuclear export signal was confirmed to be crucial for activation of p53 signaling pathways (26,27). Following UVA irradiation, phosphorylation of Ser 15 and Ser 20 in p53 was significantly induced in Atm ϩ cells, but a poorer induction occurred in Atm Ϫ cells (Fig. 4A, upper two panels), indicating a requirement of ATM kinase activation for UVA-induced p53 phosphorylation at Ser 15 and Ser 20 . In contrast, a weaker or almost undetectable phosphorylation of p53 at Ser 15 and Ser 20 was induced by UVC and was not different in either Atm ϩ or Atm Ϫ cell lines (Fig. 4A, lower two panels), suggesting that UVC-stimulated p53 phosphorylation may be ATM-independ-ent. Furthermore, our data showed that UVA-or UVC-induced apoptosis was significantly blocked in p53 Ϫ/Ϫ cells compared with corresponding induction in p53 ϩ/ϩ cells (Fig. 4B). Taken together with data in Fig. 1, these results indicate that UVAinduced apoptosis may be mediated by activation of an ATMdependent p53 pathway, but UVC-induced apoptosis appears to occur through an ATM-independent p53 pathway.
JNKs Link ATM to the Apoptotic Response Induced by UVA-Our previous report suggested that a sphingomyelinasedependent and -independent signaling pathway might be involved in JNKs-mediated apoptosis in the UVA response (19). Here, phosphorylation (Fig. 5A) and activation (Fig. 5C) of JNKs was stimulated strongly in Atm ϩ cells exposed to UVA irradiation, but the stimulation of JNKs by UVA was defective in Atm Ϫ cells (Fig. 5, A and C). On the other hand, a strong phosphorylation of JNKs induced by UVC was not different in either Atm ϩ or Atm Ϫ cell lines (Fig. 5B). Additionally, no change of the basal JNK levels was observed in the experimented cells (Fig. 5, A and B), suggesting that expression of JNKs was unaffected by deficiency of ATM. Therefore, the results indicate that ATM kinase activation is required for stimulation of JNKs by UVA, but not UVC. Interestingly, UVAor UVC-induced apoptosis was inhibited partially in Jnk 1 Ϫ/Ϫ

cells, but was attenuated completely in Jnk 2
Ϫ/Ϫ cells, compared with corresponding controls in Jnk ϩ/ϩ cells (Fig. 5D). Together with the results in Fig. 1, these data suggest that an ATMmediated JNKs pathway may be required for apoptosis induced by UVA, but UVC-induced apoptosis appears to occur through an ATM-independent JNK pathway.
UVC Induces ATR Signaling to p53/JNKs-mediated Apoptosis-The above-mentioned data (Figs. 1B, 4B, and 5D) appear to suggest that UVC-induced apoptosis may require ATMindependent signaling through p53 or JNKs. ATR, being functionally and structurally similar to ATM (11), was shown to be involved in the regulation of cell survival and cell cycle checkpoints (17,28), but a role of ATR in activation of the apoptotic response pathways is as yet unknown. To clarify the role, therefore, we used human fibroblast lines stably expressing wild-type full-length (ATRwt) or a kinase-inactive allele (ATRkd) of ATR. Both ATRwt and ATRkd cell lines were previously established and shown to have wild-type active or dominant negative effects on ATR kinase activity, respectively (17,28). The full-length p53 protein conjugated with glutathione S-transferase (GST-p53) was used as a substrate in kinase activity assays for studying ATR activity. Results showed a rapidly stimulated increase of ATR kinase activity toward GST-p53 in UVC-irradiated ATRwt cells, but the ATR activity was significantly inhibited in ATRkd cells (Fig. 6A, lower panel). In addition, a strong phosphorylation of Ser 15 in endogenous p53 was detected in ATR immune complexes isolated from UVC-irradiated ATRwt cells, but a poorer phosphorylation was observed in the complex from ATRkd cells exposed to UVC (Fig.  6A, lower panel). Conversely, neither the ATR kinase activity nor the endogenous p53 phosphorylation was detected in the complexes immunoprecipitated from UVA-stimulated ATRwt or ATRkd cells (Fig. 6A, upper panel). These data suggest that UVC, but not UVA, activates ATR kinase and that the activated ATR may co-precipitate with the p53 protein. Furthermore, UVC-induced apoptosis was significantly inhibited in ATRkd cells compared with ATRwt cells (Fig. 6B). Additionally, no typical apoptosis was observed in either ATRwt or ATRkd cell lines exposed to UVA (data not shown). Together with Fig. 4B and previous reports (17,28), these data indicate a requirement of ATR-dependent p53 signaling activation for induction of apoptosis by UVC, but not UVA.
However, UVC-induced apoptosis was not completely atten- FIG. 4. A link between ATM-dependent p53 phosphorylation and UVA-induced apoptosis. A, inhibition of UVA-stimulated phosphorylation of p53 at Ser 15 and Ser 20 by deficiency of ATM. Atm ϩ and Atm Ϫ cells were starved for 24 h in 1% FBS-DMEM and then harvested at the indicated times following irradiation with UVA (80 kJ/m 2 ; upper two panels) or UVC (60 J/m 2 ; lower two panels). The cell lysates containing equal amounts of protein were subjected to immunoprecipitation with a mouse p53 monoclonal antibody followed by Western blotting with a rabbit phospho-p53 (Ser 15 or Ser 20 ) polyclonal antibody. p, phosphorylated. B, inhibition of UV-induced apoptosis by deficiency of p53. Mouse wild-type (p53 ϩ/ϩ ) and knockout (p53 Ϫ/Ϫ ) embryonic fibroblasts were or were not irradiated with UVA (80 kJ/m 2 ) or UVC (60 J/m 2 ). DNA fragmentation laddering was assayed by 1.8% agarose gel electrophoresis. These results are representative of at least three similar independent experiments. uated in p53 Ϫ/Ϫ or ATRkd cells (Figs. 4B and 6B), indicating that besides ATR-mediated p53 signaling, other pathways may also be involved in this process. For example, protein kinase C was previously reported to activate a JNK-mediated apoptotic pathway in the UVC response (20). Interestingly, phosphorylation of JNKs corresponding with activation of JNKs was observed to be significantly inhibited in UVC-irradiated ATRkd cells, in comparison with correspondingly treated ATRwt cells (Fig. 6C, lower panel). On the other hand, UVA-induced phosphorylation of JNKs was not affected in ATRkd cells compared with that in ATRwt cells (Fig. 6C, upper panel). Additionally, no change of basal levels of JNKs was observed in these experimental cells (Fig. 6C). Together with data in Figs. 5D and 6A, these results suggest that activation of ATR kinase may be involved in JNK mediation of apoptotic responses induced by UVC but not by UVA.

DISCUSSION
Mutation of the Atm gene is known to cause AT disorder with ploiotropic biological markers including high cancer risk, hypersensitivity to DNA-damaging agents, cell cycle checkpoint alterations, increased chromosomal breakage, and telomere end fusion (7, 9 -11), indicating that a role of ATM in multiple response pathways is probably altered or even lost in AT cells. ATM kinase was shown to be activated in the cellular responses to DSBs (14,29) and such breaks induced by IR (12,13,30), radiomimetic drugs (12), or arsenite (31) but not to UVB/ UVC or base-damaging agents (11,13,17,28,30), suggesting that ATM may act specifically in response to DSBs. However, broken DNA is not required for in vitro activation of ATM kinase (30), suggesting that besides DSBs, other mechanisms may be involved in ATM activation. In addition, various abnormalities other than hypersensitivity to IR in AT have raised the possibility of a role for ATM in other cellular responses. Indeed, recent reports showed activation of ATM kinase by oxidative stresses including H 2 O 2 (25) and CdCl 2 (32,33) and even by insulin (34). Here, evidence is provided that UVA particularly induces rapid activation of ATM kinase, but ATM activation was not observed within at least 2 h following UVC. This observation supports the idea that the photobiochemical effects of UVA (e.g. free radical or reactive oxygen species production and other damage by UVA-absorbing proteins and lipids) are different from those induced by UVC (e.g. base damage by UVC-absorbing DNA) (1)(2)(3)35). Indeed, the damage induced by UVA has been shown to occur largely via reactive oxygen intermediates or radicals generated on endogenous UVA-absorbing non-DNA chromophores (called photosensitizers), because of the low absorption of DNA (1-3). Here, our data showed that ATM kinase was activated by oxidative stresses including UVA and H 2 O 2 , suggesting that ATM kinase is an oxidative stress sensor.
The high predisposition of AT cells to progress toward malignant transformation suggests possible resistance of AT cells to apoptosis. The resistance appears to result from defective apoptotic responses in ATM-deficient cells (5, 36 -38). However, whether ATM mediates apoptosis is not fully understood, because contradictory observations have been reported. Some reports showed either no effect or an inhibitory effect of ATM kinase on the apoptotic responses to IR (39) or oxidative stress (40); conversely, other reports indicated a requirement of ATM kinase for induction of apoptosis (5, 36 -38, 41). The latter assumption is supported by our demonstration that ATM kinase activation may be involved in UVA-induced apoptosis. The discrepancies among these reported apoptotic responses, although not fully understood, may be associated with cell types, even cellular states (e.g. proliferative or quiescent) or extracellular microenvironments of the same target, as well as with the kind and intensity of the apoptotic stimuli.
A role of ATM in mediating the p53-and JNK-activated apoptotic pathways is not well clarified, although p53 (36 -39) and JNKs (19,(41)(42)(43) have been shown to be involved in activation of the apoptotic response pathways. Previous experiments with IR showed p53 phosphorylation of Ser 15 by ATM directly (10 -14, 29) and of Ser 20 by ATM-dependent Chk2 indirectly (11,44,45). However, a link of the ATM-mediated apoptotic response to UVA is as yet unknown. Here, we present evidence that ATM-dependent p53 activation is required for FIG. 6. Involvement of ATR kinase activation in UVC-induced apoptosis. A, activation of ATR kinase by UVC but not UVA. GM847 cell lines expressing wild-type ATR kinase (ATRwt) or the kinase-dead mutant (ATRkd) were starved for 24 h in 1% FBS-DMEM and then were or were not irradiated with UVA (80 kJ/ m 2 ) or UVC (60 J/m 2 ). The cell lysates were subjected to immunoprecipitation with an ATR antibody followed by assays for ATR activity using a full-length p53 fusion protein (GST-p53) as the substrate. Phosphorylation of Ser 15 in GST-p53 (p-GST-p53 Ser 15 ) (upper band in lower panel), reflecting ATR activity, or in endogenous p53 (p-p53 Ser 15 ) (lower band in lower panel) forming a possible complex with ATR, was detected with Western blotting. B, inhibition of UVC-induced apoptosis by a dominant negative ATR kinase. ATRwt and ATRkd cells were starved for 4 h in 1% FBS-DMEM and then were or were not irradiated with UVC (60 J/m 2 ). Apoptosis was assayed by DNA fragmentation laddering. C, inhibition of phosphorylation of JNKs by the dominant negative ATR kinase in the cellular responses induced by UVC, but not by UVA. ATRwt and ATRkd cells were treated as described for Fig. 5, A and B, and then harvested at the indicated times following irradiation with UVA (80 kJ/m 2 , upper two panels) or UVC (60 J/m 2 , lower two panels). Total cell lysates were subjected to Western blotting with antibodies to phosphorylated (p) or nonphosphorylated (np) JNKs. The above data are representative of at least three similar independent experiments.
UVA-induced apoptosis, inasmuch as a defective UVA-stimulated phosphorylation of p53 at Ser 15 and Ser 20 in ATM-deficient cells was restored in ATM-expressing cells, and the apoptotic response to UVA was significantly inhibited in ATM-or p53-deficient cells. Interestingly, another link between ATM and UVA-induced apoptosis was here found to be through an ATM-dependent JNK response pathway. This indication is supported by our experiments showing inhibition of UVA-induced apoptosis and JNK activation by deficiency of ATM or JNKs. In fact, phosphorylation of c-Jun was shown to activate the apoptotic response pathway (42,43), and the phosphorylation induced by IR or other stimuli was reported to occur via ATM-dependent signaling (25,32,46). However, a possible link of ATM signaling to activation of the c-Jun-mediated apoptotic pathway is as yet unclear. This connection is postulated to be the tumor suppressor protein Brca1 (breast cancer gene 1) or c-Abl tyrosine kinase, based on the findings that Brca1 (20,47) and c-Abl (48,49) were confirmed to be direct substrates of ATM kinase and upstream signal molecules of JNKs in the cellular apoptotic response to IR (41,50). Altogether, our results indicate that UVA-induced apoptosis is mediated by ATMdependent JNKs and p53 signaling pathways. However, the lack of a total abrogation of UVA-induced apoptosis by deficiency of ATM suggests possible participation of ATM-independent signaling in this process, for example, the sphingomyelinase pathway as reported previously (19).
Moreover, previous reports (17,28) showed that ATR, a kinase homologous to ATM, was activated by IR or UV and was essential for cell survival, but whether ATR is involved in activation of the apoptotic pathways is as yet unclear. Our studies demonstrate neither activation nor involvement of ATR kinase in the apoptotic response to UVA. Contrary to UVA, UVC can stimulate a particularly rapid activation of ATR kinase, and the ATR activation is involved in UVC-induced apoptosis based on significant inhibition of the apoptotic response by the dominant negative ATR. Furthermore, activated ATR has been shown to directly phosphorylate Ser 15 (11,17,28) and Ser 37 (28) in p53 and to mediate Ser 20 phosphorylation of p53 indirectly via Chk1 (11,26). Additionally, a possible complex of ATR with p53 protein was observed in our experiments. Therefore, ATR-dependent p53 may be involved in the cellular apoptotic pathway induced by UVC. Interestingly, evidence that ATR kinase is required for JNK mediation of the apoptotic response to UVC is also provided in this study, but the mechanism by which ATR regulates this process remains to be determined.
In summary (Fig. 7), ATM and ATR, two related protein kinases sitting at the top of the signaling networks, are activated rapidly in the cellular apoptotic responses to UVA or UVC, respectively. UVA activation of ATM kinase occurs through the generation of ROS. Activation of JNKs and p53 links ATM signaling to apoptosis induced by UVA. On the other hand, UVC-induced apoptosis occurs through ATR-dependent p53 and JNK signaling pathways. Additionally, sphingomyelinase or protein kinase C signaling to JNKs was suggested to be involved in induction of apoptosis by UVA (19) or UVC (20), respectively. FIG. 7. A proposed model for involvement of ATM or ATR in activation of the apoptotic pathway. UVA or UVC activates ATM or ATR, respectively. In the UVA response, p53 and JNKs link ATM to activation of apoptotic pathways. ATR is linked by p53 and JNKs to the cellular apoptotic response induced by UVC. Sphingomyelinase or protein kinase C signaling to JNKs is also suggested to mediate activation of apoptosis by UVA or UVC, respectively. The arrows indicate direct (thick arrow) or indirect activation (thin arrow).