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Department of Pathology and Laboratory Medicine, Center for Environmental Health and Susceptibility, and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599,1 Growth Control and Cancer Group, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 277092
Received 20 June 2002/ Returned for modification 12 July 2002/ Accepted 10 September 2002
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ABSTRACT |
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INTRODUCTION |
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DNA damage checkpoints operate in the G1, S, and G2 phases of the cell cycle, transducing signals from sensors of DNA damage to effector proteins that mediate the appropriate response (1, 21). Essential transducers of DNA damage checkpoint responses are the protein kinases ATM (ataxia telangiectasia [AT] mutated) and ATR (ATM and Rad3 related). Cells from AT patients are hypersensitive to the genotoxic effects of DNA damage induced by ionizing radiation (IR) and are defective in DNA damage checkpoint responses to IR (1, 21, 38). ATR, on the other hand, seems to be required for checkpoint responses to replication blocks caused by hydroxyurea (HU) and UV-induced DNA damage (13, 61, 64). Although the roles of ATM and ATR in DNA damage checkpoint responses in G1 and G2 are comparatively well characterized, the S checkpoint remains a topic of intense investigation (1).
The S checkpoint is activated in human cells in response to diverse forms of DNA damage (33-35, 53). The IR-induced S checkpoint response is inhibited or attenuated by mutations in ATM, Nbs1, and Mre11 (11, 53). Nbs1 and Mre11 are the gene products mutated in the familial genetic instability disorders Nijmegen breakage syndrome (NBS) and AT-like disorder (AT-LD), respectively (55). AT, NBS, and AT-LD are clinically distinct cancer-predisposing disorders, but they are phenotypically similar at the cellular level (55). Cells from these patients are hypersensitive to cell killing following exposure to IR, display increased risk of induced chromosomal aberrations, and fail to repress DNA synthesis in the presence of IR-induced DNA damage, a phenomenon termed radioresistant DNA synthesis (RDS) (11, 53). ATM has been shown to phosphorylate Nbs1 on several serine residues in response to IR. Changing any of these serines to alanine produces mutant Nbs1 proteins that fail to correct RDS in NBS cells (26, 42, 65, 67). The IR-induced S checkpoint has recently been reported to include two parallel ATM-dependent pathways (23). It appears that one pathway involves ATM signaling through Chk2, ultimately resulting in the ubiquitin-mediated proteolysis of Cdc25A (22). Loss of Cdc25A phosphatase activity results in a dramatic inhibition of cyclin E/Cdk2 activity and Cdc45 binding to origins of DNA replication (23). The second pathway is dependent on ATM signaling to the Nbs1/Mre11/Rad50 complex, which when activated localizes to sites of DNA double-strand breaks following exposure to IR (45, 49), and by unknown signaling pathways it also effects inhibition of replicon initiation (23). Although the role of ATM in the IR-induced S checkpoint is indisputable, the checkpoint pathway that mediates the inhibition of replicon initiation following UVC-induced DNA damage is less clear.
Normal S-phase cells respond to UVC-induced DNA damage by reducing the rate of DNA synthesis (34, 35). This inhibition is manifested at the level of chain elongation and replicon initiation (34, 35) and is the result of both passive and active cellular responses to DNA lesions. Passive inhibition of DNA replication is attributed to the physical obstruction of the DNA replication apparatus at sites of DNA damage. An example is the inability of the replicative DNA polymerases 
and 
to copy through UVC-induced template lesions such as cyclobutane pyrimidine dimers and 6-4 photoproducts (46, 47). Active inhibition is a trans effect mediated through checkpoint signals that emanate from sites of DNA damage and ultimately inhibit the initiation of distant replicons (56). This S checkpoint response imposes transient delays in S-phase progression and provides more time for DNA repair to remove lesions from unreplicated chromatin. A previous study suggested that AT cells display a reduced S checkpoint response to UVC (52). AT cells, while not hypersensitive to inactivation of colony formation by UVC, are nevertheless hypersensitive to S-dependent UV-induced chromosomal aberrations (20, 40). Less is known about the function of ATR, since it is an essential gene and its deletion results in embryonic lethality in mice (6, 16). However, overexpression of kinase-inactive ATR (ATRki) renders cells hypersensitive to cell killing by DNA damaging agents, including IR, HU, and UV (13, 64). Moreover, phosphorylation of checkpoint-associated proteins p53 and Brca1 has been shown to be ATM dependent following exposure to IR, but ATR dependent following exposure to HU and UV (3, 10, 15, 41, 60, 61).
Cellular responses to UVC-induced DNA damage are dose dependent. The inhibition of replicon initiation in response to UVC is best observed after exposure to 1 J/m2, a dose which produces little reduction in cell colony-forming efficiency and very little inhibition of DNA chain elongation or mitotic entry. Cytotoxic doses between 5 and 10 J/m2 UVC, which have been shown to saturate nucleotide excision repair (NER) (39), significantly inhibit DNA chain elongation and mitotic entry (7, 36) and typically reduce colony formation by 50 to 70% (5). UVC doses of >40 J/m2, which have been used to study cellular stress responses to UVC, inhibit DNA chain elongation severely and inactivate colony formation by >95% of normal human fibroblasts (5). This study examined the role of checkpoint proteins in the response of cells to 1 J/m2 UVC, thereby negating potential contributions associated with saturation of NER, induction of stress responses, and cytotoxicity. The results strongly implicate ATR and Chk1 as important signaling molecules for the UVC-induced inhibition of replicon initiation in human cells.
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MATERIALS AND METHODS |
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Immortalization of the NHF1, GM02052A, W1799, and AT-LD2 cell strains with telomerase was accomplished by infection with a replication-defective retrovirus containing hTERT cDNA in a Moloney murine leukemia virus-based retroviral backbone containing the puromycin resistance gene (hTERT cDNA was provided by Robert Weinberg, MIT) (17). Puromycin-resistant cells expressed telomerase and displayed an indefinite in vitro proliferative life span. NHF1, GM02052A, W1799, and AT-LD2 fibroblasts were transduced with the hTERT retrovirus at population doubling levels (PDL) of 23.5, 31.6, 23.6, and 30, respectively. NHF1-hTERT, GM02052A-hTERT, W1799-hTERT, and AT-LD2-hTERT fibroblasts were maintained in continuous culture up to PDL of 186, 80, 75, and 64, respectively, without reduction in their proliferation rate. Throughout this report, hTERT-expressing normal and mutant fibroblasts are designated NHF1 (the NHF1-hTERT fibroblasts), ATM-/- (GM02052A-hTERT fibroblasts), Nbs1-/- (W1799-hTERT fibroblasts), and Mre11-/- (AT-LD2-hTERT fibroblasts).
Cytogenetics. Metaphase spreads were prepared from hTERT-immortalized human fibroblasts as previously described (25). Twenty-five metaphases were G-banded according to standard protocols (62) and scored for chromosome number and structure.
Cell irradiation. Prior to treatment with UVC, culture medium was removed and reserved. Cultures were washed once with warm Hank's balanced salt solution (Gibco BRL) and then placed uncovered under a UV lamp emitting primarily 254 nm radiation at a fluency rate of 0.5 J/m2/s. Following irradiation, reserved medium was replaced and the cultures were incubated for the indicated periods of time. Sham-treated cultures were handled exactly the same way, except that they were not exposed to UVC. Cells treated with gamma rays were maintained in their culture medium and exposed to a 137Cs source at a dose rate of 0.86 Gy/min.
Western analyses. Logarithmically growing cells were seeded at 106 per 100-mm dish and incubated for 40 h. Cultures were irradiated as described above and incubated in reserved medium for 30 min at 37°C. Cells were harvested by trypsinization, washed once in phosphate-buffered saline, and resuspended in lysis buffer (10 mM sodium phosphate buffer [pH 7.2], 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, and 1% NP-40, supplemented with 10 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 10 mM ß-glycerophosphate, 10 mM sodium orthovanadate, and 10-µg/ml concentrations of leupeptin and aprotinin). Protein concentrations were determined using the Bio-Rad DC protein assay (Bio-Rad Laboratories). Samples containing equal amounts of protein were mixed with an equal volume of 2x Laemmli sample buffer (125 mM Tris-HCl [pH 6.8], 4% sodium dodecyl sulfate [SDS], 20% glycerol) containing 5% ß-mercaptoethanol, boiled, and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose and probed with antibodies against ATR (PA1-450 [ABR] or Ab-2 [Oncogene]), Mre11 (NB 100-142 [Novus]), Nbs1 (NB 100-143 [Novus]), Chk1 (G4 [Santa Cruz]), and phospho-serine 317 and phospho-serine 345 Chk1 (Cell Signaling). Production of affinity-purified anti-ATM antibody was previously described (58).
RDS assay. Logarithmically growing cells were plated at a density of 2.5 x 105 cells per 60-mm dish and grown at 37°C for 30 to 40 h in medium containing 10 nCi of [14C]thymidine (ICN Radiochemicals) per ml to uniformly label DNA. Radioactive medium was replaced with fresh medium to chase [14C]-labeled precursors into DNA for at least 3 h. To determine the ability of cells to repress DNA synthesis in the presence of DNA damage, cells were either sham treated or exposed to gamma rays (2.5, 5.0, and 7.5 Gy). Cells were incubated at 37°C for 1 h and then for 30 min with 20 µCi of [3H]thymidine/ml. Radioactive medium was removed and cells were washed twice in cold phosphate-buffered saline and harvested by scraping with a rubber policeman into 0.5 ml of 0.1 M NaCl containing 0.01 M EDTA (pH 8) per plate. An aliquot (200 µl) was added to a separate tube containing 200 µl of lysis buffer (1 M NaOH, 0.02 M EDTA), and acid-insoluble DNA was collected on GF/C microfiber glass filters (34). Net 3H radioactivity corrected for 14C spillover was normalized for cell number (total 14C radioactivity).
Alkaline sucrose gradient centrifugation. The methodology used to determine the steady-state distribution of sizes of nascent DNA 30 to 45 min after irradiation of log-phase cultures with either 1 J/m2 UVC or 1.5 Gy IR has been described previously (12, 14). When indicated, caffeine (Sigma) and UCN-01 (provided by the Drug Synthesis and Chemistry Branch, National Cancer Institute [NCI]) were added to the culture medium 30 min prior to irradiation at concentrations of 2 to 3 mM and 100 nM, respectively.
Adenovirus infection. Recombinant adenoviruses encoding wild-type murine Chk1 or kinase-inactive Chk1 were generously provided by Cyrus Vaziri (28). Adenovirus encoding green fluorescent protein (GFP) was obtained from the UNC Adenovirus Vector Core Facility. Cells were infected with a multiplicity of infection (MOI) of 50 for 15 h, and adenovirus-encoded protein expression was measured by Western immunoblot analysis (Chk1) or fluorescence microscopy (GFP).
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RESULTS |
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The phenotypes of normal and mutant diploid fibroblasts used in this study were verified by Western immunoblot analysis and DNA synthesis assays following treatment with IR (Fig. 1). Cells from AT, NBS, and AT-LD patients lacked expression of ATM, Nbs1, and Mre11 proteins, respectively. However, all of these cell lines expressed ATR (Fig. 1A). Diploid fibroblasts containing mutations in ATM, Nbs1, or Mre11 are defective in the IR-induced S checkpoint (11, 23, 53). We confirmed the RDS phenotype of these mutant cell lines by measuring the degree of inhibition of [3H]thymidine incorporation into DNA after exposing the cultures to gamma rays (2.5, 5, and 7.5 Gy). Whereas the NHFs inhibited DNA synthesis in a dose-dependent manner, this response was severely compromised and not dose dependent in fibroblasts lacking ATM, Nbs1, or Mre11 (Fig. 1B).
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UVC-induced DNA damage inhibits replicon initiation. A low dose (1 J/m2) of UVC (254 nm), causing very little cytotoxicity in NHFs, also inhibits DNA synthesis by reducing the rate of initiation of new replicons (34, 35). The experiment illustrated in Fig. 2 shows how the inhibition of synthesis of low-molecular-weight intermediates of DNA replication, observed 30 to 45 min after irradiation, spread progressively to higher-molecular-weight intermediates, as the incubation time prior to the [3H]thymidine pulse was extended from 30 to 90 min. Note that the point of divergence between irradiated and sham profiles moved progressively to higher molecular weights as the length of incubation postirradiation was increased. These results illustrate both the transient nature of the inhibition of DNA synthesis by low-dose UVC and the precursor-product relationship of newly synthesized DNA from single replicons to multireplicon size. This phenomenon of inhibition of low-molecular-weight DNA (Fig. 2A) that sweeps out towards higher-molecular-weight intermediates (Fig. 2B and C) lends support to the interpretation that low doses of IR (Fig. 1C) and UVC (Fig. 2) induced an inhibition of initiation of those replicons that were programmed to be activated shortly after irradiation. By 90 min after irradiation, some recovery of replicon initiation was apparent as increased synthesis of low-molecular-weight DNA (Fig. 2C). As previously published (34), recovery of replicon initiation rate was nearly complete 3 h after exposing NHFs to low-dose UVC.
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We overexpressed either ATRwt or ATRki in U2OS cells to test the role of ATR in the UVC-induced S checkpoint directly. As seen in Fig. 5A, addition of doxycycline for 48 h resulted in a 
5-fold increase in ATRwt or ATRki expression over endogenous levels. Additionally, velocity sedimentation analyses revealed that U2OS cells responded to low-dose UVC by inhibiting replicon initiation (Fig. 5B). However, overexpression of ATRki or pretreatment with 2 mM caffeine abolished this response, suggesting that ATR signaling is required to inhibit replicon initiation following UVC-induced DNA damage (Fig. 5B). U2OS cells induced to overexpress ATRwt inhibited replicon initiation to the same extent as uninduced controls following exposure to 1 J/m2 UVC. Figure 5C illustrates the average degree of inhibition of replicon initiation in U2OS cells, as determined from several velocity sedimentation profiles (Fig. 5B, fractions 14 to 23). Whereas uninduced U2OS cells inhibited replicon initiation by 36% 30 min after irradiation with 1 J/m2, overexpression of ATRki resulted in a significant reversal of this response (9% inhibition). Further, overexpression of ATRwt did not affect the response to UVC, and [3H]thymidine incorporation was inhibited to the same extent as in uninduced cells (Fig. 5C).
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70%). These findings suggest that not only high doses of UVC but also doses as low as 1 J/m2 result in ATR-dependent Chk1 phosphorylation. Evidence for activation of Chk1 after 1 and 8 J/m2 was also obtained in NHF1 and HeLa cells (results not shown).
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10-fold increase in Chk1 expression over endogenous levels (Fig. 7E). Analysis of adenovirus-GFP-infected cells with fluorescence microscopy revealed that 90% were GFP positive. Whereas uninfected and adenovirus-GFP-infected cells responded to UVC by inhibiting DNA synthesis, overexpression of Chk1ki abolished this response (Fig. 7F), suggesting that Chk1 is the effector kinase in the UVC-induced S checkpoint. Interestingly, overexpression of Chk1wt inhibited global DNA synthesis by nearly 95%, while overexpression of Chk1ki stimulated DNA synthesis above levels detected in the uninfected controls. These observations suggest that Chk1 may function at some level in the regulation of DNA synthesis, even in the absence of induced DNA damage.
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DISCUSSION |
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Since ATR is an essential gene and embryos lacking expression of this protein are not viable (6, 16), we examined the contribution of ATR to the UVC-induced inhibition of replicon initiation by pretreating cells with caffeine, an inhibitor of ATM and ATR kinases (4, 57). Treatment of normal and ATM-/- fibroblasts with caffeine fully reversed the UVC-induced inhibition of replicon initiation. Moreover, overexpression of ATRki in U2OS cells reversed the S checkpoint response to low-dose UVC, as revealed by velocity sedimentation. Taken together, these data indicate that the UVC-induced S checkpoint response of inhibition of replicon initiation is mediated by ATR.
Like ATR, Chk1 is an essential gene, and null mutations result in embryonic lethality (43, 59). Nonetheless, blastocytes or embryonic stem cells from Chk1-/- mice are deficient in the IR-induced G2 DNA damage checkpoint, as well as the UV- and aphidicolin-induced replication checkpoint (43, 59). Because ATR-/- and Chk1-/- mice have the same phenotype and are equally deficient in checkpoint responses to DNA damage, an association between the two has been hypothesized. Mammalian Chk1 has recently been shown to be phosphorylated on serines 317 and 345 following exposure to UV and HU (43, 66). Additionally, overexpression of ATRki completely abolishes Chk1 phosphorylation in 293T cells (43). Nghiem et al. (50) recently demonstrated the requirement of ATR and Chk1 in the replication checkpoint. Overexpression of ATRki, or Chk1ki, reversed the inhibition of mitosis and produced premature chromatin condensation in U2OS cells treated with HU or UVB (50). Immunodepletion of ATR from Xenopus egg extracts also abolished XChk1 phosphorylation induced by UV-damaged DNA (29, 31). Further, XChk1 containing nonphosphorylatable residues at conserved ATR phosphorylation sites was deficient in the DNA replication checkpoint (29). As a group, these studies suggest that ATR and Chk1 are required for checkpoint responses to UV-induced DNA damage and incomplete DNA synthesis.
Consistent with the findings mentioned above, Chk1 was phosphorylated on serine 317 and serine 345 in response to a low fluency of UVC in U2OS cells. At a dose that inhibited replicon initiation but not chain elongation (1 J/m2), modest Chk1 phosphorylation was observed. A toxic dose of UVC (8 J/m2) that caused a more severe inhibition of DNA chain elongation induced a significant increase in Chk1 phosphorylation. These findings suggest that the phosphorylation status of Chk-1 and/or the effective concentration of activated Chk1 might determine the checkpoint-signaling pathway that is triggered. The UVC-induced S checkpoint response of inhibition of replicon initiation may only require a low degree of Chk1 phosphorylation to inhibit origin firing. Lethal doses of UVC that severely block DNA chain elongation and activate the replication checkpoint may need a higher concentration of active Chk1 to signal to a more diverse group of downstream effectors, such as those that block premature chromosome condensation. Additionally, pretreatment of cells with UCN-01 or overexpression of Chk1ki abolished the inhibition of DNA synthesis by a low dose of UVC. Since the UVC-induced S checkpoint response is caffeine and UCN-01 sensitive, independent of ATM, and can be reversed by overexpressing ATRki or Chk1ki, our data strongly suggest that the upstream transducers that respond to low levels of UVC-induced DNA damage are ATR and Chk1. The chemical carcinogen BPDE produces the same stereotypic inhibition of replicon initiation as UVC and IR (33). Consistent with the data reported here, Guo et al. (28) recently demonstrated that a caffeine-sensitive, Chk1-dependent pathway mediates the BPDE-induced inhibition of replicon initiation.
Cellular responses to UVC are significantly affected by dose. The low dose of 1 J/m2 used in these studies, which does not saturate NER and inhibits DNA chain elongation minimally, produces about 1 pyrimidine dimer per 4 x 107-Da replicon (5). Nucleotide excision and postreplication repair activities, in concert with inhibition of replicon initiation, can largely ameliorate the genotoxic effects of such a low dose. The protective influence of NER is seen in the greater inhibition of DNA chain elongation 30 min after irradiation of repair-defective xeroderma pigmentosum (XP) cells (34). Pol 
-dependent replicative bypass is also evident as XP variant cells display significant inhibition of DNA chain elongation after 1 J/m2 (14, 35). Chk1 is activated modestly by low-dose UVC to induce the transient inhibition of replicon initiation. Higher doses of UVC in the range of 5 to 10 J/m2 saturate NER (39), severely inhibit DNA chain elongation due to the large numbers of photoproducts blocking DNA polymerases (5), and activate Chk1 more strongly. This greater level of activation might reflect the larger number of blocked growing points and may be required to sustain the inhibition of replicon initiation for a longer period and to block premature chromosome condensation as progression through S phase is significantly impeded. Survival is less certain after higher damage levels, and mutations and chromosomal aberrations are measurably increased in the survivors (40). Still-higher doses, in the range of 25 to 50 J/m2, can cross-link receptor tyrosine kinases and activate cytoplasmic kinase cascades (18, 19), in addition to the above-mentioned effects deriving from nuclear DNA damage. Activation of JNK appears to be a high-dose phenomenon, with a threshold of no response evident at UVC doses lower than 20 J/m2 (2). The severe inhibition of DNA chain elongation after such high fluencies of UVC produces a state of replicative arrest similar to that induced by depletion of DNA precursors using HU or aphidicolin. Under these conditions, ATR and Chk1 appear to be required to prevent activation of late origins (24, 56) and premature chromosome condensation (50) as elements of replication checkpoint function. It is evident from our studies that inhibition of replicon initiation is phenomenologically equivalent to inhibition of late origins. Because the inhibition of replicon initiation is triggered rapidly after UVC, the effect may not be limited to those replicons that initiate late in S phase but could include any replicon or replicon cluster that is scheduled to initiate replication after the DNA damage is incurred.
In summary, the S checkpoint that inhibits replicon initiation following exposure to low-dose UVC is distinct from the IR-induced S checkpoint (Fig. 8). ATM, Nbs1, and Mre11 are required for the S checkpoint response to IR. ATM signals to activate Chk2 and thereby to inhibit Cdc25A and cyclin E/Cdk2 kinase activity. The contribution of Nbs1 and Mre11 to inhibition of replicon initiation by IR is independent of Chk2 and Cdc25A signaling, through a still-undetermined pathway (23). ATR and Chk1 are required for the S checkpoint response to UVC, and these transducers may also inactivate Cdc25A (44). Thus, human cells express at least two independent signaling mechanisms that regulate the replicon initiation rate in response to environmental stress.
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ADDENDUM IN PROOF |
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ACKNOWLEDGMENTS |
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This work was supported primarily by PHS grant CA55065 (M.C.S.). This study was also supported by PHS grant CA81343 (W.K.K.) and Center grants P30-CA16086 and P30-ES10126. T.P.H. is supported by Environmental Pathology Training Grant ES07017.
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FOOTNOTES |
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