Involvement of the Interaction between p21 and Proliferating Cell Nuclear Antigen for the Maintenance of G2/M Arrest after DNA Damage*

Although a major effect of p21, a cyclin-dependent kinase inhibitor, is considered to be exerted during G1 phase of the cell cycle, p21 gene knock-out studies suggested its involvement in G2/M checkpoint as well. Here we demonstrate evidence that p21 is required for the cell cycle arrest at G2 upon DNA damage. We found that expression of wild-type p21 (p21WT), not mutant p21 (p21PCNA−) lacking the interaction with proliferating cell nuclear antigen (PCNA), caused G2 cell cycle arrest in p53-deficient DLD1 colon cancer cell line after the DNA damage by treatment with cis-diamminedichloroplatinum (II). We also found that p21WT was associated with Cdc2/cyclin B1 together with PCNA. Furthermore, coimmunoprecipitation experiments revealed that PCNA interacted with Cdc25C at the G2/M transition, and this interaction was abolished when p21WTwas expressed presumably due to the competition between p21WT and Cdc25C in the binding to PCNA. These findings suggest that p21 plays a regulatory role in the maintenance of cell cycle arrest at G2 by blocking the interaction of Cdc25C with PCNA.

The cell cycle is regulated by two major checkpoints at G 1 -S and G 2 /M transition. The fidelity of genomic replication during DNA synthesis and cell division is ensured by checkpoint controls that prevent cell cycle progression when the DNA damage or incomplete DNA replication is detected (1). Thus, checkpoint loss would result in genomic instability and has been implicated in carcinogenesis (2). In fact, the p53 tumor suppressor gene, a major gatekeeper of cell cycle checkpoints, is mutated in a large fraction of human cancers (3). Cell cycle arrest in G 1 caused by DNA damage or cellular senescence is mediated by p21, a cyclin-dependent kinase inhibitor, that is under the transcriptional control of p53 (4). Interestingly, although cells deficient in p21 proliferate normally, they are unable to maintain stable G 2 arrest and initiate cell death program when exposed to DNA-damaging agents such as irradiation and anti-cancer drugs (5,6). Circumstantial evidences indicate the involvement of p21 at the G 2 /M transition. For example, p21 mRNA in human fibroblasts show bimodal periodicity with peaks in G 1 and G 2 /M (7) and that p21 protein reaccumulates in the nucleus at the onset of mitosis (8). In addition, inducible p21 expression caused cell cycle arrest at G 1 and G 2 (9) and p21 induced G 2 arrest when it was induced at the beginning of S phase (10). These observations have suggested that p21 is also involved in G 2 /M checkpoint.
It is well established that p21 consists of at least two functional domains that bind to proliferating cell nuclear antigen (PCNA) 1 and Cdk/cyclins (11)(12)(13). PCNA was initially identified as an auxiliary protein for DNA polymerase ␦ that is essential for DNA replication and repair (14 -16). The role of PCNA in cell cycle regulation is suggested by the fact that polymerase ␦ is regulated by cell cycle proteins (17). In fact, PCNA was shown to interact with various Cdk-cyclin complexes (11,16,18). Thus, PCNA may act as a platform for multiple protein-protein interactions involved in replication, repair, recombination, and cell cycle regulation. Interestingly, the PCNA protein levels increased steadily through the entire cell cycle period and remained high at G 2 /M (19). Thus, PCNA may also be involved in G 2 cell cycle control.
The G 2 checkpoint has been extensively studied in the fission yeast and is known to involve a number of proteins including Cdc2/cyclin B, Cdc25, 14-2-3, Wee1, Chk1, Cds1/Rad53, and DNA damage sensor proteins (20,21). Among these proteins, Cdc25, a dual phosphatase for Cdc2, plays a central role in the G 2 checkpoint by controlling the phosphorylation status, thus its kinase activity, of Cdc2 (22)(23)(24). In human cells, there are three Cdc25 homologues, Cdc25A, -B, and -C (25)(26)(27). Whereas Cdc25A is involved in the G 1 checkpoint, Cdc25B and Cdc25C are involved in the G 2 /M transition (28 -34). Because the activity of Cdc25C is induced at the onset of mitosis and regulates Cdc2/cyclin B1, it is regarded as a major regulator of the G 2 checkpoint, besides the fact that Cdc25C has the highest homology with the yeast Cdc25. Cdc2 is subject to multiple levels of regulation including periodic association with cyclin B, phosphorylation, dephosphorylation (35)(36)(37)(38), and intracellular compartmentation (39 -44). Cdc2 associates with cyclin B at the G 2 /M transition (38), and this complex is retained in an inactive state throughout S and G 2 phases by phosphorylation of Cdc2 at Thr-14 and Tyr-15 by another kinase Wee1 (22,36). Wee1 is activated by upstream kinases, Chk1 or Cds1 (45,46), that are activated by damaged DNA (47-49) and unreplicated DNA (45, 50, 51), respectively. Although Cdc25 activates Cdc2/ cyclin B by reversing the Wee1-mediated phosphorylation of Cdc2 (22,23), this activity is suppressed by Chk1 and Cds1 FIG. 1. Abolishment of cell cycle progression by p21 requires its interaction with PCNA. A, detection of p21 expression in DLD1 p21 WT and DLD1 p21 PCNAϪ cells. Cell lysates were prepared from cultured cells in the presence (ϩ Tet) of tetracycline or in the absence of tetracycline for 96 h (Ϫ Tet) and probed with anti-HA antibody (detecting both wild type and PCNA (Ϫ) mutant form p21 proteins) by Western blotting. B, quantitation of cells expressing p21 proteins by flow cytometry. C, cell cycle analysis. Cells were stained with propidium iodide (PI) and subjected to the DNA content analysis using fluorescence-activated cell sorter. The percentage of cells accumulated at each cell cycle stage is indicated. Induction of p21 protein was done by culturing cells in the medium without tetracycline for 96 h. Note that expression of wild-type p21 (DLD1 p21 WT , Ϫ Tet) arrested the cell cycle progression at both G 1 and G 2 /M with a significant decrease in cells at S phase, whereas expression of mutant p21 PCNAϪ (DLD1 p21 PCNAϪ , Ϫ Tet) failed to do so. through phosphorylation of Cdc25 at Ser-216 in the yeast (50,52,53). The phosphorylated Cdc25 is sequestered in the cytoplasm by the interaction with 14-2-3 (54 -59). However, a small amount of Cdc25 was shown to reside in the nucleus at interphase, and moreover, most of Cdc25 stays in the nucleus throughout cell cycle in some cells (46,47). Therefore, an additional mechanism is required to ensure the inactivation of Cdc2-cyclin B complex by preventing coincidental contacts with Cdc25.
In this study, we attempted to examine the role of p21 in G 2 /M transition particularly in G 2 DNA damage checkpoint by utilizing the p53-deficient human colon cancer cell lines inducibly expressing the wild-type or the mutant p21 PCNAϪ lacking the interaction with PCNA. We demonstrate evidence that p21 PCNAϪ cannot confer cell cycle arrest at G 2 upon DNA damage, and we suggest that the p21 may interfere with the interaction between PCNA and Cdc25 thus preventing Cdc25 from the activation of Cdc2. The possible role of PCNA as a platform for regulatory protein-protein interactions for the cell cycle regulation is discussed.
Cell Lines and Culture Conditions-Parental DLD1 human colon carcinoma cell line constitutively expresses very low levels of endogenous p21 because of mutations of both alleles of p53 (p53Ϫ/Ϫ) (60). DLD1 cell lines containing exogenous genes for wild-type (WT) or mutant (PCNAϪ) p21 proteins tagged with HA epitope were as described (9). The p21 PCNAϪ mutant contains amino acid substitutions (M147A, D149A, and F150A) to abolish the interaction with PCNA specifically. Expression of p21 in these cells is under the tight control of a tetracycline-regulated promoter and can be induced by eliminating tetracycline (Tet) from the culture medium ("Tet-OFF" system). These cell lines were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum (IBL, Maebashi, Japan), 4 mM L-glutamine (Life Technologies, Inc.), 100 units/ml penicillin, 100 g/ml streptomycin (Life Technologies, Inc.), and 2 g/ml tetracycline in a 5% CO 2 incubator. Subcloning of these cell lines was repeatedly done in selective medium containing 50 g/ml hygromycin. Western blotting and flow cytometry was performed to ensure that ϳ90% of the cells expressed p21 when tetracycline was eliminated. There was no significant difference in the sensitivity to CDDP among parental DLD1, DLD1 p21 WT , and DLD1 PCNAϪ cells. for 60 min at room temperature in the presence of 0.1% bovine serum albumin (Sigma) and 0.5 g/ml ribonuclease A (Roche Molecular Biochemicals). The cells were then washed three times with TPBS, stained with propidium iodide (Sigma), and incubated overnight at 4°C in TPBS containing 200 g/ml propidium iodide (PI). The cell cycle status of the cells was determined as described previously (24). Briefly, cells (5 ϫ 10 6 ) were collected, washed twice with cold PBS, and resuspended in Krisham's solution (0.1% sodium citrate, 50 g/ml PI, 20 g/ml ribonuclease A, and 0.3% Nonidet P-40) prior to flow cytometric analysis.
Co-immunoprecipitation and Immunoblotting-DLD1 cells (2 ϫ 10 6 ) were collected, washed twice with PBS, and suspended in 200 l of lysis buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM EDTA, 10 M NaF, 1 mM Na 3 VO 4 , 1 M okadaic acid, 2 mM dithiothreitol, 0.25% Nonidet P-40, and 0.5 mM phenylmethylsulfonyl fluoride) according to the method described previously (62). The lysate was clarified by centrifugation at 15,000 ϫ g for 20 min. The supernatant was collected and incubated with either biotin-conjugated anti-PCNA antibody or anti-HA antibody (for detecting p21) for immunoprecipitation at 4°C for 1.5 h with gentle rotation. Twenty l of streptavidin-Sepharose beads (Amersham Pharmacia Biotech) or protein G-Sepharose beads (Amersham Pharmacia Biotech) were added and further incubated for 1 h.
The beads were washed 5 times with 1 ml of lysis buffer. Antibodybound complexes were eluted by boiling in 2ϫ Laemmli sample buffer and resolved by 12% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Hybond-C, Amersham Pharmacia Biotech). Membranes were blocked in TPBS including 2% non-fat milk 4°C overnight, probed with various primary antibodies for 1 h at room temperature, washed three times with TPBS, and probed with the secondary antibody for 40 min at room temperature. The immunoreactive proteins were visualized by enhanced chemiluminescence (Super-Signal; Pierce).

Conditional Expression of Wild-type and Mutant p21 Proteins in p53-deficient Human Colon Cancer Cell-Repeated
subcloning of cells, DLD1 p21 WT and DLD1 p21 PCNAϪ , was carried out to enrich cell lines conditionally expressing wildtype or mutant (PCNAϪ; in which the PCNA-interacting amino acid residues were mutated) p21, respectively. As shown in Fig.  1, A and B, levels of p21 protein expression in these cells were virtually null in the presence of tetracycline but became readily detectable when tetracycline was eliminated from the culture medium. In Fig. 1B, cells were fixed and p21 expression was examined by flow cytometry. Upon induction, p21 protein expression was detected in 94% of DLD1p21 WT and in 87% of DLD1p21 PCNAϪ cells. To induce p21 proteins, DLD1 p21 WT and DLD1 p21 PCNAϪ cells were treated with CDDP (12 g/ml) for 1 h, and tetracycline was eliminated (Ϫ Tet) from the culture at 48 h after CDDP treatment (Tx). Cells were washed and further cultured for additional 24 (72 h) or 72 h (120 h). At 72 h, both Ϫ Tet and ϩ Tet cells entered G 2 . At 120 h, whereas Ϫ Tet DLD1 p21 WT cells remained at G 2 , ϩ Tet cells and Ϫ Tet DLD1 p21 PCNAϪ cells entered G 1 because of the lack of functional p21 (for the details of cell cycle status, see Fig. 2). B, detection of proteins co-immunoprecipitated with p21 WT (left panel) or p21 PCNAϪ (right panel) by Western blot (WB). Cells were harvested, and the protein complexes containing p21 were immunoprecipitated (IP) from the total cell lysate by anti-HA (epitope for the exogenous p21 WT and p21 PCNAϪ ) antibody followed by Western blot analyses as described under "Experimental Procedures" using the indicated antibodies including anti-PCNA, anti-Cdc2, anti-Cdk7, anti-cyclin B1, and anti-cyclin H antibodies. Equal amounts of cell lysates were used in each immunoprecipitation. The similar experiments were carried out for three times with essentially the same results. The representative results are demonstrated. Note that cyclin B1 was degraded in DLD1p21 PCNAϪ cells (Ϫ Tet) at 120 h after the DNA damage. p21 Binding to PCNA at G 2 /M Checkpoint proliferation and cell cycle progression by flow cytometry. In the absence of p21 expression, the cell cycle distribution was similar for both cell lines. For example, in DLD1p21 WT ϳ49.3, 15.0, and 35.7% of cells were in G 1 , S, and G 2 phases, respectively (Fig. 1C). When p21 WT expression was induced in DLD1 cells, a striking decrease in the number of cells in S phase (1.8%) was observed, whereas the majority of cells accumulated at G 1 (39.6%) or G 2 /M (58.6%). On the other hand, p21 PCNAϪ expression did not significantly change the cell distribution (Fig. 1C, lower panel). These findings confirmed those by Cayrol et al. (9) that p21 was involved in the cell cycle arrest at G 1 and G 2 /M through binding to PCNA.
Requirement of Wild-type p21 in the G 2 DNA Damage Checkpoint-To examine the effects of p21 expression on the DNA damage-induced G 2 cell cycle arrest, DLD1 p21 WT and DLD1 p21 PCNAϪ cells were treated with CDDP (12 g/ml) for 1 h, and p21 proteins were induced by removing Tet, and the cell cycle analysis was carried out by flow cytometry. As shown in Fig.  2A, because most of the cells were accumulated at G 2 /M 48 -72 h after the DNA damage induced by CDDP, we induced p21 expression by Tet withdrawal ("Ϫ Tet") at 48 h after the CDDP treatment. Cells not expressing p21 or expressing p21 mutant progressed into G 1 , and significant numbers of cells underwent cell death (detected as cells in sub-G 1 ). Fig. 2B indicates that 48 h after the DNA damage ϳ74% of cells expressing p21 WT entered G 2 /M, and these cells remained in G 2 /M at 120 h. On the other hand, cells not expressing p21 or expressing p21 mutant immediately progressed into G 1 . For example, although ϳ87% of DLD1 cells expressing p21 PCNAϪ entered G 2 /M at 48 h after the DNA damage, only 50% of the cells were detected at G 2 /M at 120 h. Similar observations were obtained with cells not expressing p21. These observations indicate that p21 is involved in the DNA damage-induced G 2 cell cycle arrest and that the ability of p21 to bind PCNA is crucial.
Binding of p21 WT to the PCNA-Cdc2-Cyclin B1 Complex in the DNA Damage G 2 Checkpoint-It is known that p21 not only inhibits Cdk4/cyclin D1 and Cdk6/cyclin D2 in G 1 checkpoint but also inhibits Cdc2/cyclin B1 during the G 2 /M transition at least in vitro (63). We then examined if p21 associates with PCNA at the G 2 /M transition together with Cdc2/cyclin B1. In Fig. 3, DLD1 cells were treated with CDDP for 1 h, and expression of either wild type (p21 WT ) or mutant (p21 PCNAϪ ) was induced at 48 h after the DNA damage. The interaction of p21 with the Cdc2-cyclin B1 complex and PCNA at the DNA damage G 2 checkpoint was examined by immunoprecipitation (with anti-HA antibody detecting p21 WT and p21 PCNAϪ ) followed by Western blotting (with antibodies to PCNA, cyclin B1, and Cdc2).
As demonstrated in Fig. 3B (left panel), p21 WT coimmunoprecipitated PCNA, Cdc2, and cyclin B1 when cells were arrested at G 2 /M transition due to CDDP-induced DNA damage, indicating the formation of p21 WT -PCNA-Cdc2-cyclin B1 complex in cells arrested at G 2 /M. Interestingly, although Cdc2cyclin B1 complex interacts with p21 PCNAϪ at the G 2 /M transition (72 h after DNA damage) (Fig. 3, right panel), cells could not maintain the G 2 /M arrest (Fig. 2B). In these cells, cyclin B1 was proteolytically degraded and cells entered M and then progressed to G 1 (120 h after DNA damage) (Fig. 3B, right  panel). These findings suggest that in order for p21 to induce the cell cycle arrest at G 2 in response to DNA damage, the interaction of p21 with PCNA is crucial. Because p21 was previously shown to inhibit CAK-mediated Cdc2 phosphorylation and promote cell cycle arrest at G 2 /M (10), we also examined the interaction of p21 with cyclin H and Cdk7 (CAK). However, neither cyclin H nor Cdk7 was coimmunoprecipitated with p21 (Fig. 3B).
Involvement of p21 WT in G 2 /M Checkpoint, Alternative Binding of PCNA to p21 WT and Cdc25C-During the cell cycle interphase, Wee1HU inactivates Cdc2-cyclin B1 complex by phosphorylation of Cdc2 at Tyr-15 (64), which is subsequently activated by Cdc25 through dephosphorylation at Tyr-15 when cells enter mitosis (65). We thus hypothesized that, in human cells, the Cdc25C-mediated activation of Cdc2/cyclin B1 would occur at the nascent DNA following the completion of DNA synthesis or DNA repair, and actions of p21 and Cdc25C would be exclusive. We then examined whether Cdc25C associates with PCNA in the absence of p21, and the PCNA binding of p21 and that of Cdc25C are mutually exclusive (Fig. 4). This pos- FIG. 4. Alternative binding of PCNA to p21 and Cdc25C during G 2 /M progression. Similar co-immunoprecipitation and Western blot (WB) determination was performed with DLD1 p21 WT (left panel) and DLD1 p21 PCNAϪ (right panel) after the treatment with CDDP as in Fig. 3. The cell lysate was immunoprecipitated with anti-PCNA antibody, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to a membrane. Western blot determination of the PCNA precipitates was performed with antibodies to p21 (HA), Cdc25C, cyclin H, Cdk7, and PCNA. Equal amounts of cell lysates were used in each immunoprecipitation. The similar experiments were carried out for three times with essentially the same results. The representative data are demonstrated here.
p21 Binding to PCNA at G 2 /M Checkpoint sibility was also prompted by a previous report by Saha et al. (66) that p21 and Cdc25A competitively bind to Cdk2 presumably at the G 1 -S transition.
In Fig. 4, DNA damage was induced in DLD1 cells, and expression of either wild type (p21 WT ) or mutant (p21 PCNAϪ ) was induced as in Fig. 3. The cell lysate was analyzed for the protein-protein interaction by immunoprecipitation (with anti-PCNA antibody) followed by Western blotting to detect the PCNA-associated p21 and Cdc25C. As demonstrated in Fig. 4 (left panel), Cdc25C was detected in the PCNA complex when p21 was not induced (ϩ Tet). Cdc25C was coimmunoprecipitated with PCNA in the absence of DNA damage, i.e. in the absence of p21 (data not shown). In the presence of p21 WT , PCNA coimmunoprecipitated p21 WT but not Cdc25C. However, when p21 PCNAϪ was expressed, PCNA coimmunoprecipitated Cdc25C irrespective of the presence of p21 PCNAϪ , indicating that p21 and Cdc25C interact with the same region of PCNA. DISCUSSION We have explored the role of p21 in the G 2 DNA damage checkpoint using p53-deficient cells in which p21 WT or p21 PCNAϪ was complemented. Our findings clearly indicate the crucial role of p21 in the G 2 checkpoint upon DNA damage and that the interaction of p21 with Cdc2-cyclin B1 complex is mediated by PCNA. We assume that the p21-PCNA interaction is probably required for recognition of the repaired DNA. Because the p21-PCNA interaction appeared to be mutually exclusive with the Cdc25C-PCNA interaction at G 2 /M transition, p21 may prevent the incorporation of Cdc25C into the Cdc2cyclin B1 complex and thus induce cell cycle arrest at G 2 . In fact, the formation of ternary complex involving p21, PCNA, and Cdc2/cyclin B1 at G 2 was previously demonstrated (7,19,63). In addition, Dulic et al. (8) and Medema et al. (67) independently showed the formation of p21-Cdc2-cyclin B1 complex and inhibition of Cdc2 kinase activity at G 2 . Moreover, we observed that cells failed to arrest at G 2 /M because of the absence of functional p21 and underwent cell death (Fig. 2). In agreement with this finding, Bunz et al. (5) demonstrated with cells defective for p53 or p21 genes that ␥-irradiation induced cell death associated with the lack of cytokinesis after entering into M phase, again indicating the crucial importance of p21 for the maintenance of the G 2 /M arrest.
Expression of p21 is transcriptionally regulated by p53 upon DNA damage or cellular senescence and is known to induce cell cycle arrest at G 2 as well as at G 1 by inhibiting Cdc2 (4, 68, 69).  (74). AA denotes the position of the starting amino acid residues. Dark boxes represent identical amino acid residues of previously described PCNA-binding proteins to p21. Shaded boxes represent homologous amino acid residues to those of p21. B, involvement of PCNA and its binding proteins in cell cycle and DNA damage checkpoints. From our data presented in this study, p21 appears to participate in G 2 checkpoint upon DNA damage by the interaction with PCNA. During normal S-G 2 /M transition, PCNA interacts with Fen1 (during S phase) and subsequently with Cdc25C. However, upon the DNA damage, PCNA interacts with p21 instead of Cdc25C and induced cell cycle arrest at G 2 /M by recruiting Cdc2/cyclin B1. PCNA may act as a platform for protein-protein interactions for the cell cycle regulation and checkpoints.
In addition to the binding with Cdc2-cyclin B1 complex, p21 is also shown to bind PCNA (70). PCNA acts as an auxiliary factor for DNA polymerase ␦ and stimulates DNA replication (71). It was shown that p21 inhibited PCNA-dependent DNA replication in vitro by binding to PCNA through its PCNAinteracting region (11,12,72,73). We found that DLD1 p21 PCNAϪ , expressing a mutant p21 defective for the interaction with PCNA, could not arrest at G 2 /M checkpoint even after DNA damage (Fig. 2). Thus, it is possible that, through the interacting with PCNA, p21 inhibits DNA synthesis and maintains G 2 /M arrest.
These findings also suggest an important role for PCNA as a platform for the interaction of various cell cycle regulator proteins that occur adjacently to the nascent DNA or the repaired DNA. Among these proteins, p21, Fen1 (flap endonuclease 1), and xeroderma pigmentosum G are known to contain similar sequences (called PIP box) that interact with PCNA (74). It was shown that Fen1 and p21 compete for binding to the same site of PCNA (75). Similarly, xeroderma pigmentosum G and p21 were shown to compete for the PCNA binding at least in vitro (76). We also found a similar PCNA-binding motif in Cdc25C (Fig. 5A). It is thus conceivable that competitive binding among PCNA-interacting proteins plays an important role in the coordinated DNA replication and repair. Although further studies are needed, it is likely that p21 inhibits cell cycle progression to mitosis by regulating the Cdc25C interaction with the Cdc2/cyclin B1 (Fig. 5B). Considering that fact that PCNA forms a stable homotrimer when it binds to DNA upon DNA synthesis and possibly DNA repair, it remains to be clarified whether these protein-protein interactions with PCNA can occur concomitantly to some extent or are mutually exclusive.