Defect in multiple cell cycle checkpoints in ataxia-telangiectasia postirradiation.

The recent description of a novel gene (ATM) mutated in ataxia-telangiectasia (A-T), with homologies to genes encoding proteins involved in both G1/S and G2/M checkpoint control, points to a common defect in cell cycle control in A-T operating through the cyclin-dependent kinases. In this report we demonstrate that cyclin-dependent kinases are resistant to inhibition by ionizing radiation exposure in A-T cells, and this appears to be due to insufficient induction of WAF1. Exposure of control lymphoblastoid cells to radiation during S phase and in G2 phase causes a rapid inhibition of cyclin A-Cdk2 and cyclin B-Cdc2 activities, respectively. Irradiation led to a 5-20-fold increase in Cdk-associated WAF1 in these cells, which accounts at least in part for the decrease in cyclin-dependent kinase activity. In contrast, radiation did not inhibit any of the cyclin-dependent kinase activities in S phase or G2 phase in A-T cells at short times after irradiation nor was there any significant change in the level of Cdk-associated WAF1 compared to unirradiated cells. These results are similar to those reported previously for the G1 checkpoint and provide additional evidence for the involvement of ATM at multiple points in cell cycle regulation.

The recent description of a novel gene (ATM) mutated in ataxia-telangiectasia (A-T), with homologies to genes encoding proteins involved in both G 1 /S and G 2 /M checkpoint control, points to a common defect in cell cycle control in A-T operating through the cyclin-dependent kinases. In this report we demonstrate that cyclin-dependent kinases are resistant to inhibition by ionizing radiation exposure in A-T cells, and this appears to be due to insufficient induction of WAF1. Exposure of control lymphoblastoid cells to radiation during S phase and in G 2 phase causes a rapid inhibition of cyclin A-Cdk2 and cyclin B-Cdc2 activities, respectively. Irradiation led to a 5-20-fold increase in Cdk-associated WAF1 in these cells, which accounts at least in part for the decrease in cyclin-dependent kinase activity. In contrast, radiation did not inhibit any of the cyclin-dependent kinase activities in S phase or G 2 phase in A-T cells at short times after irradiation nor was there any significant change in the level of Cdk-associated WAF1 compared to unirradiated cells. These results are similar to those reported previously for the G 1 checkpoint and provide additional evidence for the involvement of ATM at multiple points in cell cycle regulation.
Exposure of mammalian cells to ionizing radiation causes a delay at the G 1 /S phase transition, inhibition of DNA synthesis, and G 2 phase delay prior to mitosis (1)(2)(3)(4). Delay at the G 1 /S phase checkpoint appears to be mediated in part through the product of the tumor suppressor gene p53 (5). Induction of p53 in turn leads to the transcriptional activation of a number of genes including WAF1, which directly inhibits cyclin-dependent kinase activity and consequently produces a G 1 /S phase delay (5)(6)(7).
Accumulating evidence suggests that a major part of the phenotype in the human genetic disorder ataxia-telangiectasia (A-T) 1 is due to anomalies in cell cycle control (8 -10). This disorder is characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency, chromosomal instability, radiosensitivity, and increased propensity to develop cancer (11). Exposure of A-T cells to ionizing radiation fails to cause initial delays at the G 1 /S and G 2 /M phase transitions (12)(13)(14). These cells exhibit radioresistant DNA synthe-sis (15)(16), and at longer times postirradiation they are irreversibly delayed in G 2 phase (9,17,18). Kastan et al. (8) showed that A-T cells lacked the radiation induction of p53 observed in normal cells. Khanna and Lavin (19) subsequently revealed that the p53 response postirradiation is reduced and/or delayed in cells from the four major A-T complementation groups; a delay in p53 induction was also reported by Lu and Lane (20). More recently it has been demonstrated that a radiation signal transduction pathway operating through p53, its target gene WAF1, cyclin E-Cdk2 and cyclin A-Cdk2 kinases, and the retinoblastoma (Rb) protein is defective in A-T cells (21)(22)(23)(24). Together the above observations are consistent with defective checkpoint control at the G 1 /S phase transition after exposure of A-T cells to ionizing radiation (24).
The recent description of a novel gene (ATM) mutated in A-T provides further support for a defect in signal transduction in this syndrome (25). The product of this gene shows homology to TOR proteins of yeast and their mammalian homologs RAFT and FRAP, which are involved in G 1 phase control (26 -27). This homology is in a lipid kinase domain, which is also present in the catalytic subunit of phosphoinositide 3-kinase, a mediator of signal transduction (28 -29). The ATM protein also shows significant homology to the yeast RAD3 and MEC1 proteins required for coupling G 2 /M checkpoint control to completion of S phase (30). ATM is also homologous to ME1-41 (Drosophila), TEL1 (yeast), and DNA-dependent protein kinase (DNA-PK), all of which play a role in cell cycle control and/or DNA damage recognition (31)(32)(33). These observations, together with cell cycle kinetic data demonstrating anomalies at several checkpoints in A-T cells, suggest that the defective signaling in this syndrome is responsible for the cell cycle abnormalities. We have tested this by investigating the effects of ionizing radiation on members of the cyclin-dependent kinase (Cdk) family that control distinct checkpoints in the cell cycle. Our results clearly demonstrate that in A-T, radiation fails to inhibit the members of Cdk examined, which control progression through distinct phases of the cell cycle. Furthermore, WAF1 appears to play a role not only in the inhibition of Cdk activity observed at the G 1 /S phase transition but also in S phase and at the G 2 /M transition in control cells. Failure of WAF1 induction and Cdk association in A-T cells can explain both the lack of Cdk inhibition and concomitant cell cycle delay and the characteristic radioresistant DNA synthesis observed in A-T cells. In addition, the accumulation of A-T cells in G 2 /M at 24 h postirradiation correlated with increased association of WAF1 with Cdc2.

MATERIALS AND METHODS
Cell Culture-All cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics at 37°C in a humidified atmosphere of 5% CO 2 . Nomenclature for control and A-T lymphoblastoid cell lines is the same as that described previously (15). The control cells used were C3ABR and C30ABR and the A-T cell lines AT1ABR and AT3ABR, which have been assigned to complementation groups E and D, respectively (34), and AT3LA, complementation group C (received from Richard Gatti, UCLA, Los Angeles, CA). Doubling times were calculated by plating cells at a density of 2 ϫ 10 5 /ml in 2-ml dishes and counting at regular intervals. All lymphoblastoid cell lines used had a doubling time of 24 Ϯ 4 h.
To study events in S phase, cells were synchronized in late G 1 with mimosine (300 M for 18 h) and then returned to media containing 2.5 g/ml aphidicolin for an additional 4 h to enhance the degree of synchronization. The cells were washed twice with PBS and returned to media and irradiated with 3 Gy when the peak of cells was in mid-S phase (4 h after release from aphidicolin). Movement of cells through S phase was monitored by fluorescence-activated cell sorter analysis in order to establish the optimum time for assays. For protein assays used in this study, time points were taken at 0.25 and 2 h post-treatment. A similar approach was used to study G 2 phase cells. After release from aphidicolin (5 g/ml), cells were monitored in their passage through S phase and into G 2 phase. Cells were exposed to 3 Gy of ionizing radiation in G 2 phase as determined by flow cytometry and sampling carried out at 0.25 and 2 h postirradiation. For asynchronous studies, the cells were plated at a density of 2 ϫ 10 5 /ml, irradiated with 3 Gy of ionizing radiation after 24 h of growth, and subsequently harvested 24 h postirradiation. In order to determine the mitotic indices, treated and untreated cells were washed twice with PBS before fixing in methanolglacial acetic acid (3:1). The fixed cells were spread on glass slides, air-dried, and stained with Giemsa. Mitotic figures were counted (ϫ400) by analyzing at least 1000 cells for each sample.
Immunoblotting-Levels of cyclin, Cdk, and additional proteins were measured at times indicated after cells were exposed to 3 Gy of ␥-radiation in a 137 Cs irradiator at 3 Gy/min as described (19). Cells were washed twice in cold PBS, transferred to Eppendorf tubes and lysed in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 25 mM ␤-glycerol phosphate, 0.1 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, 1 g/ml aprotinin, 0.2% Triton X-100, 0.3% Nonidet P-40). Lysis was carried out on ice for 30 min with inversion every 5 min. Cell debris was removed by centrifugation at 10,000 ϫ g at 4°C. The supernatant was placed in an Eppendorf microcentrifuge and the protein concentration determined according to the manufacturer's instructions (Bio-Rad). Proteins were separated on 12% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (DuPont), blocked with 5% skim milk powder in PBS-Tween 20 (0.5%), and incubated with primary antibodies as indicated by the supplier, followed by secondary antibody (horseradish peroxidase/alkaline phosphatase-labeled anti-rabbit/mouse Ig). Protein bands were visualized using the ECL system (Amersham Corp.) as per manufacturer's instructions for the horseradish peroxidase-labeled secondary antibodies or as described previously (19) for the alkaline phosphatase-labeled secondary antibodies.
Histone H1 Kinase-Cells were synchronized as described above prior to exposure to 3 Gy of ␥-rays or mock-irradiated, incubated for various times as indicated, and lysed as for immunoblotting. Each kinase reaction was carried out on 400 g of lysate, which was precleared prior to immunoprecipitation by the addition of 40 l of Protein A-agarose beads (Pharmacia Biotech Inc.), followed by rocking at 4°C for 30 min. The protein A-agarose was removed by centrifugation at 15,000 ϫ g for 1 min. To the precleared lysate, 1 g (or as indicated by the manufacturer) of the appropriate antibody was added and then rocked at 4°C for 60 min; in addition, during each histone H1 kinase assay at least one precleared lysate was incubated without the immunoprecipitating antibody, which acted as a negative control. For cyclin A histone H1 kinase assays, a specific immunoprecipitating antibody was used (AM5.39; Ref. 35). The antibody complex was captured by the addition of 50 l of protein G or A-agarose for 30 min at 4°C and washed twice with 1 ml of lysis buffer and once with 1 ml of kinase buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol). Kinase reactions were carried out on the antibody-protein G/A complex at 30°C for 30 min in the kinase buffer in the presence of 5 g of histone H1, 40 mM MgCl 2 , 100 M ATP, and 0.2 Ci of [␥-32 P]ATP. Reactions were terminated by the addition of SDS-polyacrylamide gel electrophoresis sample buffer (60 mM Tris-HCl pH 7.5, 40 mM dithiothreitol, 1.6% SDS, 10% glycerol). The extent of phosphorylation of the histone H1 protein was assessed following SDS-polyacrylamide gel electrophoresis and autoradiography. In addition, gels were quantitated using Image Quant software on a PhosphorImager (Molecular Dynamics).
Coupled Immunoblotting and Immunoprecipitation-Cells synchronized in S and G 2 phases were irradiated and cell lysates were prepared 2 h post-treatment. One milligram of precleared protein extracts was incubated with 2 g of Cdk2 antibody in the case of S phase and anti-Cdc2 for G 2 phase overnight at 4°C. The antibody complex was captured by the addition of 50 l of protein A-agarose for 30 min at 4°C, pelleted by centrifugation at 400 ϫ g for 1 min, and washed three times with 1 ml each of lysis buffer. Samples were boiled in sample loading buffer for 5 min and then run on 12% SDS-polyacrylamide gel electrophoresis. The proteins were immunoblotted as described previously and screened with Cdc2/Cdk2 specific antibodies (1:1000 dilution, a gift from F. Hall, Children's Hospital, Los Angeles, CA). The same Western blots were stripped by heating to 50°C in buffer (62.5 mM Tris, pH 6.8, 1 mM ␤-mercaptoethanol, 2% SDS) for 30 min. The filters were then washed with copious amounts for PBS-Tween and rescreened with phosphotyrosine-specific antibodies (1:1000 dilution, UBI). This process was essentially the same for WAFI (1:1000 dilution, Oncogene Science Inc.), cyclin A (CHLA.1; Ref. 35), and cyclin B (1:1000 dilution, UBI). The resulting ECL blots were overlaid to ensure the correct positioning of the bands.

Effect of Radiation on Cyclin-dependent Kinase Activity in S
Phase Cells-Radioresistant DNA synthesis is a universal characteristic of A-T cells (15,16,36). A-T cells exposed to ionizing radiation in S phase do not experience a delay in that phase of the cycle but proceed to the subsequent G 2 /M phase, where they undergo a prolonged delay and presumably die at that transition (9). While the defective p53 response in A-T cells would be expected to result in a failure to inhibit Cdk activity at the G 1 /S phase transition, it would not be expected to influence progress through S phase (5). Nevertheless, since A-T cells fail to exhibit inhibition of DNA synthesis, it was of interest to determine the effects of radiation on Cdk activity in S phase cells. Enrichment for S phase cells was obtained by synchronization in late G 1 with mimosine, release into an early S phase block with aphidicolin, followed by release and incubation for 4 h, at which time 60% of cells were in mid-S phase prior to exposure to 3 Gy of ionizing radiation (Fig. 1a, C and F). At later times it was evident that control cells are delayed in their passage through S phase (Fig. 1a, E), but A-T cells were not delayed (Fig. 1a, J). Immunoprecipitates of Cdk2 and cyclin A from irradiated control cells (C3ABR and C30ABR) exhibited reduced Cdk activity in the first 2 h postirradiation (Fig. 1b). In contrast, cyclin A-Cdk 2 activity was not diminished by radiation exposure in S phase A-T cells over that period (Fig. 1b). Changes in the cyclin and Cdk proteins did not account for the differences between A-T and control (Fig. 1c). In order to investigate the possible role of WAF1 in Cdk inhibition in S phase, these complexes were immunoprecipitated with anti-Cdk2 antibody. This represents an appropriate approach to measuring S phase Cdk activity, since a considerable enrichment (60%) of S phase cells was achieved in the synchronization and release protocol, and the contribution from cyclin B-Cdc2 would be negligible due to the specificity of the Cdk2 antibodies. Radiation exposure led to a marked increase in Cdk2-associated WAF1 in control cells by 2 h but failed to increase the levels above the basal level in irradiated A-T cells (Fig. 2). The increase is somewhat exaggerated due to the low basal level in the unirradiated control in this experiment. These results are in keeping with failure to observe inhibition of cyclin A-Cdk2 kinase activity in A-T cells at this time (Fig.  1b) and would support a role for WAF1 in radiation-induced inhibition of S phase cyclin A kinase activity. Induction of WAF1 is delayed but is not completely deficient in AT1ABR (peaks by 4 -6 h postirradiation), and this will become evident when 24 h postirradiation data are compared. Tyrosine phosphorylation has also been shown to contribute to Cdk2 inhibition postirradiation (22), but since the extent of phosphorylation was similar in A-T and control, it would not appear to account for appreciable inhibition of the cyclin A-Cdk2 kinase in control cells (Fig. 1b).
Radiation and the G 2 /M Transition-Exposure of normal cells to radiation in the G 2 phase of the cell cycle leads to a dose-dependent transient delay in progression to mitosis (2,37). This suppression of mitotic index by radiation is not observed in A-T cells (38), and these cells proceed into the following G 1 phase unimpeded (9). The failure of A-T cells to observe this checkpoint might also be explained by a defect in the functioning/inhibition of cyclin B-Cdc2 kinase postirradiation. In order to obtain an enriched G 2 population, cells were incubated in aphidicolin for 18 h, released from that block, and monitored for movement into G 2 phase prior to irradiation (Fig.  3a, 0 h point). In order to verify that these were G 2 phase cells we measured mitotic indices and showed that only 2.7% of C3ABR and 2.6% of AT1ABR were in mitosis at this time point. The corresponding values for irradiated cells were 1.9% (C3ABR) and 0.6% (AT1ABR). Irradiation of control cells (Fig.  3a, B) caused a delay in progression from G 2 /M into G 1 phase (G 2 /G 1 ϭ 1.7) compared to a G 2 /G 1 ratio of 1.0 for unirradiated cells (Fig. 3a, A). No such delay was observed in A-T (Fig. 3a, C  and D). Cyclin B-Cdc2 kinase activity was inhibited by radiation 2.6 -2.9-fold in control cells (2 h postirradiation), but as observed in S phase Cdk activity was not inhibited in A-T cells (Fig. 3b). No significant change in cyclin or Cdk protein level was observed (Fig. 3c). We also determined the levels and phosphorylation states of wee1 kinase and Cdc25C phosphatase, which are implicated in controlling the transition from G 2 to mitosis (39). There was no alteration in the level or phos- FIG. 3. a, flow cytometric analysis of enriched G 2 phase cell populations subsequently exposed to ionizing radiation. Cells were synchronized in early S phase with aphidicolin (5 g/ml) for 18 h and released into fresh medium. When the cells were found to be in the G 2 phase as determined by flow cytometric analysis they were irradiated with 3 Gy of ␥-rays (0 h) and subsequently harvested at the indicated times. A, C3ABR, unirradiated; B, C3ABR, irradiated; C, AT1ABR, unirradiated; D, AT1ABR, irradiated. b, effect of irradiation on cyclin B-Cdc2 kinase activity in G 2 phase enriched cells. The control cell line, C3ABR, and the A-T cell line, AT1ABR, were synchronized in G 2 phase as described under "Materials and Methods." Cells were lysed at 0.25 and 2 h postirradiation, and the Cdc2 and cyclin B histone H1 kinase activity was assayed. c, Western blot analysis was carried out on C3ABR and AT1ABR at 0.25 and 2 h post-treatment. The levels of Cdc2, cyclin B, wee1, and Cdc25 C proteins were examined. phorylation state of these proteins as indicated by band intensity and a lack of shift in migration (40) in response to radiation in A-T and control cells (Fig. 3c). Immunoprecipitation of the Cdc2 complex and subsequent immunoblotting with anti-WAF1 antibody revealed a 10-fold increase in WAF1 associated with Cdc2 in irradiated control cells (Fig. 4). In agreement with the lack of radiation-induced inhibition of cyclin B-Cdc2 kinase in A-T cells, there was no increase in Cdc2-associated WAF1 above that of the basal level in unirradiated cells (Fig. 4). Again the increase in Cdc2 tyrosine phosphorylation, which was evident in both cell types, does not appear to contribute significantly to inhibition of activity.
Cyclin Kinase Activity in Cells Accumulating in G 2 /M Postirradiation-When A-T cells are irradiated while in G 1 or S phases and allowed to proceed through the cycle, they accumulate in G 2 /M where the majority die (9,14,41). G 2 /M accumulation is also observed in control cells postirradiation but to a lesser extent than in A-T. This prolonged delay contrasts with the failure to observe checkpoints at short times after irradiation in A-T cells. It might be predicted that such a G 2 /M accumulation would be caused at least in part by inhibition of cyclin B-Cdc2 kinase, contrary to what is observed at short times postirradiation. In order to provide a meaningful comparison between control and A-T, control cells were exposed to an equitoxic dose (10 Gy compared to 3 Gy for A-T cells), which resulted in approximately 60% of cells accumulating in G 2 /M at 24 h postirradiation for both cell types (Fig. 5a, C and E). Again in this case the bulk of cells were in G 2 phase, and mitotic indices ranged from 0.5 to 1.85% in A-T and control cells. Cyclin B-Cdc2 kinase activity was inhibited in C3ABR (10-fold), AT1ABR (4-fold), and AT3ABR (5-fold) cells exposed to equitoxic doses of radiation (Fig. 5b). Inhibition of kinase activity was also evident in control cells exposed to the lower dose of 3 Gy (data not shown). The extent of inhibition may be underestimated in these experiments, since only 16% of unirradiated cells are in G 2 phase at the time of assay compared to 60 -67% of irradiated cells (Fig. 5a). The extent of induction of Cdc2associated WAF1 in control cells was 11-and 9-fold for the 3and 10-Gy doses, respectively (Fig. 5c), and WAF1 bound to Cdc2 was also shown to increase 8-fold in irradiated AT1ABR and 5-fold in AT3ABR at 3 Gy. Immunoblotting for Cdc2 in the Cdc2 immunoprecipitates revealed the presence of a second slower migrating isoform in all irradiated cells, which suggests an inactivating phosphorylation. Blotting with an antiphosphotyrosine antibody confirmed an increase in tyrosine phosphorylation in a band that comigrated exactly with this slower migrating isoform of Cdc2 (Fig. 5c). Comparable levels of cyclin B in the unirradiated (16% of cells in G 2 ) and irradiated cells (60% in G 2 ) are most likely due to the inhibition of cyclin B synthesis by ionizing radiation as observed previously (42).
These results are compatible with the inhibition of cyclin B-Cdc2 activity in both control and A-T cells mediated by tyrosine phosphorylation of the Cdc2 subunit and/or WAF1 association. DISCUSSION We have demonstrated that the movement of cells through S and G 2 phases of the cell cycle, each controlled by specific cyclin-dependent kinase complexes, is resistant to delay by ionizing radiation in A-T cells. Under conditions in which cell cycle progression is delayed in irradiated control cells, there is a close correlation with a reduction in activity of these cyclindependent kinases. While it is well established that A-T cells fail to undergo initial delays at the G 1 /S and G 2 /M phases postirradiation (12, 14) and exhibit radioresistant DNA synthesis in S phase (15,16), the nature of the defect is best understood at the G 1 /S phase transition. Kastan et al. (5) provided evidence that wild type p53 delayed the passage of cells from G 1 to S phase and inhibited DNA synthesis after exposure of cells to radiation. The same group subsequently showed that this cell cycle checkpoint, which utilized both p53 and gadd45, was defective in A-T cells (8). Khanna and Lavin (19) confirmed that the postirradiation p53 response was reduced and/or delayed in cells from the four major complementation groups of A-T. Further investigation has revealed that a radiation signal transduction pathway operating through p53 is defective in A-T (21)(22)(23). This pathway involves the induction of several genes by p53, including the Cdk inhibitor WAF1, which in turn binds in excess with Cdk complexes, thereby preventing the phosphorylation of critical substrates (such as the retinoblastoma protein) and ultimately leading to a delay in the passage of cells from G 1 to S phase (22). A-T cells have been shown to be defective at all levels in this pathway (24). Dulic et al. (22) found that ␥-radiation blocked the activation of cyclin D-and cyclin E-associated kinases (G 1 phase) without altering the accumulation of either the cyclins or their associated Cdk subunits. Activation was blocked by p53-dependent accumulation of WAF1.
While it appears likely that WAF1 plays an important role at the G 1 /S transition, evidence in favor of a role at other cell cycle checkpoints is mixed. Dulic et al. (22) reported that failure to detect significant levels of cyclin A-associated kinase activity was due to inhibition of accumulation of cyclin A rather than interaction with WAF1. Furthermore, Kastan et al. (5) showed that cells with altered p53 failed to arrest in G 1 postirradiation but exhibited an increase in the percentage of cells in G 2 phase as observed in cells with wild type p53. This was interpreted to mean that p53 participated in the inhibition of DNA synthesis via G 1 arrest but probably did not contribute significantly to G 2 arrest. However, different cell lines were used in that study and a more meaningful comparison might be made between cell lines varying only in their p53 content. Where a single fibroblast cell line was used with and without transfection with the adenovirus E6 gene, radiation (in G 2 phase) inhibited Cdc2cyclin B kinase activity in the transfected cells by a mechanism independent of WAF1 association (43). However, it is possible that the E6 protein may induce another delay pathway independent of p53. More recently Aloni-Grinstein et al. (44) have demonstrated that p53 plays a role in ␥-radiation-induced G 2 arrest in pre-B cells. In addition, isolated G 2 fractions from cells expressing mutant p53 protein as well as the endogenous wild type p53 were not blocked and were capable of undergoing mitosis. Control of both the G 2 /M and G 1 cell cycle checkpoints by p53 has also been shown by Agarwal et al. (45). When p53 was induced in S phase, cells were blocked predominantly in G 2 /M and an appreciable induction of WAF1 was observed in these cells. Li et al. (46) have reported an increase in WAF1 mRNA as cells enter G 2 and M phase, which leads to the speculation of a possible function for WAF1 at the G 2 /M checkpoint as well as the G 1 /S checkpoint. It has been shown that while WAF1 expression can be regulated independently of p53 during tissue development and differentiation, most tissues require p53 for WAF1 induction following exposure of whole animals to ␥-radiation (47). On the other hand, WAF1 directly inhibits proliferating cell nuclear antigen-dependent DNA rep-lication independent of cyclin-dependent kinases (48,49), and p53 interacts with the single-stranded DNA binding protein complex RPA to inhibit DNA replication (50). Xiong et al. (51) have revealed that WAF1 and proliferating cell nuclear antigen serve as universal components of cyclin-Cdk complexes and this association occurs in a cell cycle-dependent manner (46). Enforced expression has demonstrated that WAF1 can act as a Asynchronous control (C3ABR) and A-T (AT1ABR and AT3ABR) cells were exposed to 10 and 3 Gy of ionizing radiation, respectively. At 24 h postirradiation, cells were lysed and cyclin B immunoprecipitates were assayed for histone H1 kinase activity. c, association of WAF1 with the cyclin B-Cdc2 complex after exposure of control and A-T cells to equitoxic doses of radiation. Control (C3ABR) and A-T (AT1ABR, AT3ABR) cells were treated as described previously. Coupled immunoprecipitation (anti-Cdc2) and immunoblotting with antibodies to Cdc2, phosphotyrosine, cyclin B, and WAF1 were carried out. Numbers indicate protein standards in kilodaltons. potent and universal inhibitor of Cdk activity and simultaneously induce cell cycle arrest (7,45,52,53). It is evident from our data that WAF1 induction by radiation occurs at all stages of the cell cycle, providing the opportunity to associate with the complexes of Cdk that are activated at that transition. We have shown that the amount of WAF1 associating with cyclin A-Cdk2 and cyclin B-Cdc2 increases from 5-to 20-fold in irradiated control cells in both the short (2 h) and long term (24 h) and correlates with radiation-induced inhibition of these kinases and the concomitant delay of cells at distinct cell cycle checkpoints. In contrast we observed neither an inhibition of any of these Cdks in A-T cells in the short term postirradiation nor any significant increase in the amount of Cdk-associated WAF1, and delay was not seen at any of the cell cycle checkpoints as determined by flow cytometry. We interpreted these data to mean that WAF1 has some inhibitory effect at all stages of the cell cycle in normal cells, but in A-T cells the defective p53/WAF1 response accounts at least in part for the failure to detect cyclin-dependent kinase inhibition at multiple cell cycle checkpoints. Furthermore, overexpression of WAF1 in A-T cells using an Epstein-Barr virus-based vector not only causes inhibition of cyclin E-Cdk2 activity and restoration of the G 1 /S checkpoint but also leads to inhibition of cyclin B-Cdc2 activity postirradiation, providing further supportive evidence for a role for WAF1 at multiple checkpoints. 2 Further evidence for a role for WAF1 in G 2 /M delay is presented here by the large accumulation of A-T cells at this checkpoint 18 -24 h postirradiation. A-T cells irradiated in G 1 and S phases traverse these stages of the cycle with no significant delay and are subsequently irreversibly blocked at G 2 /M (9). This is reflected in an inhibition of cyclin B-Cdc2 kinase activity to approximately the same extent as in control cells exposed to an equitoxic radiation dose, and in both cases there is a parallel increase of Cdk-associated WAF1. The inhibition of kinase activity, Cdc2 tyrosine phosphorylation, and association of WAF1 with Cdc2 in A-T cells are consistent with the delayed but detectable p53/WAF1 response at longer times postirradiation.
The failure of A-T cells to respond to the various cell cycle checkpoints together with the resistance of the different cyclin-Cdk combinations to radiation suggests that the defective A-T protein either controls more than one signal transduction pathway or that the commonality of the cyclin-dependent kinase response is derived from a defect in a single pathway. The recent description of a novel gene mutated in ataxia-telangiectasia (ATM) and the homologies of the ATM gene product to proteins with phosphoinositide 3-kinase domains (25,33) provides further insight into the nature of the cell cycle defect in A-T. Part of this gene product bears considerable homology to the mammalian FKBP-rapamycin-associated protein (FRAP) (26) and the Saccharomyces cerevisiae proteins TOR1 and TOR2, which function as targets for G 1 arrest mediated by the rapamycin-FKBP12 complex (54). Rapamycin interferes with mitogenic signaling by binding with the intracellular receptor FKBP12 (55) and prevents progression from G 1 to S phase by interfering with the activation of p70 S6k (56) and cyclindependent kinases (57,58). The phosphoinositide 3-kinase domain and an adjacent region of the ATM gene product also show similarity to rad3, mec1, and Mei-41 gene products, all of which are required for the G 2 /M cell cycle checkpoint (25). Both rad3 and mec1 are essential for correct coupling of DNA synthesis to mitosis, and mutants in these genes are characterized by sensitivity to both ␥ and ultraviolet irradiation (30,59).
Clearly, a defect in a protein controlling both G 1 /S and G 2 /M transitions and the coupling of mitosis to successful completion of DNA synthesis could explain the variety of cell cycle anomalies observed in A-T.