Role of Human Cds1 (Chk2) Kinase in DNA Damage Checkpoint and Its Regulation by p53*

In response to DNA damage, mammalian cells adopt checkpoint regulation, by phosphorylation and stabilization of p53, to delay cell cycle progression. However, most cancer cells that lack functional p53 retain an unknown checkpoint mechanism(s) by which cells are arrested at the G2/M phase. Here we demonstrate that a human homolog of Cds1/Rad53 kinase (hCds1) is rapidly phosphorylated and activated in response to DNA damage not only in normal cells but in cancer cells lacking functional p53. A survey of various cancer cell lines revealed that the expression level of hCds1 mRNA is inversely related to the presence of functional p53. In addition, transfection of normal human fibroblasts with SV40 T antigen or human papilloma viruses E6 or E7 causes a marked induction of hCds1 mRNA, and the introduction of functional p53 into SV40 T antigen- and E6-, but not E7-, transfected cells decreases the hCds1 level, suggesting that p53 negatively regulates the expression of hCds1. In cells without functional ataxia telangiectasia mutated (ATM) protein, phosphorylation and activation of hCds1 were observed in response to DNA damage induced by UV but not by ionizing irradiation. These results suggest that hCds1 is activated through an ATM-dependent as well as -independent pathway and that it may complement the function of p53 in DNA damage checkpoints in mammalian cells.

The fidelity of chromosome segregation is achieved by checkpoint controls that prevent cell cycle progression when damage to DNA is encountered or key processes are not completed (1,2). In mammals, cell cycle arrest in response to DNA damage is mediated through the transcriptional activation of DNA damage-inducible genes by the p53 tumor suppressor gene. p53 has an essential role in the G 1 checkpoint in response to DNAdamaging agents such as ionizing radiation (3). p53, a tran-scription factor with sequence-specific DNA binding activity (4), activates the transcription of target genes, including p21 Cdk inhibitor (5), when DNA is damaged. Although cells lacking functional p53 are completely defective in the G 1 checkpoint in response to DNA damage, they retain an unknown checkpoint mechanism at G 2 /M, which may underlie the resistance of these cells to anti-cancer drugs (6). Interestingly, cells derived from patients with ataxia telangiectasia (AT) 1 are defective in both G 1 and G 2 checkpoint functions and display a high frequency of spontaneous as well as radiation-induced chromosomal aberrations (7,8). Although defects in the G 1 checkpoint function in AT cells appear to be the result of their inability to phosphorylate p53 in response to DNA damage (7,9), the molecular mechanism underlying the impaired G 2 checkpoint in AT cells is not known.
In yeast, several Rad-related proteins are thought to participate in the signaling as well as monitoring processes that detect DNA damage (10). Among these, Rad3, a yeast homolog of human ATM (AT mutated), appears to play a central role in the DNA damage checkpoint. Rad3 is a member of a subfamily of protein kinases that are large proteins with lipid kinaserelated domains at their carboxyl termini (11), and it is considered to be a transducer of a DNA damage signal in yeast. In fission yeast, the protein kinases Chk1 and Cds1 are required for cell cycle arrest in response to DNA damage (12)(13)(14)(15). These kinases act downstream of several Rad gene products and are phosphorylated upon DNA damage. Activated Chk1 and Cds1 kinases have been reported to phosphorylate Cdc25 (16,17), and phosphorylated Cdc25 is negatively regulated by binding to Rad24 and Rad25, which are homologs of human 14-3-3 protein (18). The inactive form of human and Xenopus Cdc25 before mitosis contains phosphorylated serine residues at 216 and 287, respectively, creating a consensus 14-3-3 binding site (19 -21). It has been shown that human homologs of Chk1 and Cds1 (hCds1) phosphorylate Cdc25C on Ser-216 (22)(23)(24) and act, at least in part, by regulating the binding of 14-3-3 to Cdc25C. A more recent study points to the involvement of hCds1 in the DNA damage checkpoint, by showing that hCds1 is activated through phosphorylation in an ATM-dependent manner in response to DNA damage induced by ionizing radiation (23). However, it remains unknown at which phase of the cell cycle hCds1 functions and how it cooperates with p53-dependent checkpoint pathways in response to DNA damage.
In this report, we demonstrate that hCds1 is specifically expressed at the S-to-M phase of the cell cycle and is rapidly activated through phosphorylation in response to DNA damage not only in normal cells but also in p53-deficient cancer cells. In addition, we present evidence that hCds1 expression is negatively regulated by functional p53, leading to a high level of expression in p53-deficient cancer cells. Thus, hCds1 may complement the function of p53 in the DNA damage checkpoint in p53-deficient cancer cells.

MATERIALS AND METHODS
Cell Lines-HeLa cells and human diploid fibroblasts (MJ90) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 100 g/ml penicillin and streptomycin. Human papilloma virus (HPV) type 16 E6 and E7 retrovirus vectors and LXSN-E6 and -E7 were kindly provided by Dr. D. Galloway (25). SV40 T antigen retrovirus vector pZipSV40 was kindly provided by Dr. P. Jat (26). Retroviruses encoding SV40 T antigen and HPV E6 and E7 were prepared using pZipSV40, LXSN-E6, LXSN-E7, respectively, as described previously (27). Normal human fibroblasts WI-38 (obtained from RIKEN Cell Bank) were used as the recipients for retroviral infection and were cultured under the same conditions as described above. Human p53 adenovirus vector was kindly donated by Drs. H. Hamada and J. Miyazaki. Fibroblasts derived from a patient with ataxia telangiectasia (AT2KY) were cultured in RPMI 1640 medium supplemented with 20% FBS and 100 g/ml penicillin and streptomycin. To induce DNA damage, cells were irradiated with UV at 322 nm using a UV cross-linker (Taitech, Tokyo).
Molecular Cloning of Human Cds1-Degenerate primers for the conserved motifs in the FHA and kinase domains of SpCds1 (28), T/AG/CNAAT/CGGNACCTTTTTNAAT and T/CTTA/GTTA/G/TATA/G/ TATT/C/TTA/G/TATGGC, were used to screen MDAH041 cDNAs by PCR. Sequence analysis of a PCR product revealed an incomplete open reading frame, which was highly homologous to SpCds1. Additional nucleotides of the novel 5Ј DNA sequence were obtained by 5Ј rapid amplification of the cDNA ends (RACE). The sequence of the 3Ј-end of hCds1 cDNA was identical with a partial sequence in the data base of an expressed sequence tag clone (AA773443).
Northern Blotting-mRNAs were isolated using a Quickprep mRNA Purification kit (Amersham Parmacia Biotech) according to the manufacture's instructions. RNA (2 g each) was denatured, separated by electrophoresis on 1.0% agarose-formaldehyde gels, and transferred onto nylon membranes. hCds1 cDNA was labeled with [␣-32 P]dCTP using a random primer labeling kit (Amersham Pharmacia Biotech). The hybridization signal was detected and quantitated using a Fuji imaging analyzer (BAS 1500).
Preparation of GST-fused hCds1 Protein in Escherichia coli-pGEX-hCds1 was generated by ligation of the EcoRI/XhoI fragment of hCds1 cDNA into pGEX 5X-1. Overnight cultures of E. coli transformed with plasmids encoding GST-fusion proteins were diluted 10-fold with fresh medium and cultured for 2 h at 37°C, followed by incubation with 0.1 mM isopropyl-␤-D-thiogalactopyranoside for an additional 2 h. Cells were harvested and lysed by sonication in NETN150 buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, and a mixture of protease inhibitors consisting of 20 g/ml soybean trypsin inhibitor, 2 g/ml aprotinin, and 100 g/ml phenylmethylsulfonyl fluoride (PMSF)). Recombinant proteins were purified on glutathione-Sepharose beads (Amersham Pharmacia Biotech).
Chromosomal Localization of hCds1 Gene-To determine the chromosomal localization of the hCds1 gene, fluorescence in situ hybridization was performed as described previously (30). A biotinylated cDNA probe for hCds1 was hybridized to R-banded chromosomes prepared from PHA-stimulated lymphocytes from normal donors. After overnight hybridization at 37°C, the slides were washed in 50% formamide/2ϫ SSC at 37°C for 10 min followed by a wash in 1ϫ SSC at room temperature for 15 min. Hybridization signals were amplified using rabbit antibiotin (Enzo) and fluorescein-labeled goat anti-rabbit IgG (Enzo). The chromosomes were counter-stained with propidium iodide.

RESULTS AND DISCUSSION
Molecular Cloning of hCds1 cDNA-A human homolog of Schizosaccharomyces pombe Cds1 (spCds1), which functions in both DNA replication and damage checkpoints, was cloned by degenerate PCR using primers designed on the basis of spCds1 sequences. The human cDNA predicts a translation product of 543 amino acids with a calculated molecular mass of 60 kDa, and the nucleotide sequence of hCds1 gene is identical with that of human Chk2, which has recently been reported by Matsuoka et al. (23).
hCds1 Is Expressed at Late G 1 -to-M Phase of the Cell Cycle-To examine at which stage(s) of the cell cycle hCds1 functions in mammalian cells, we first determined the expression of hCds1 mRNA during cell cycle progression by Northern blotting. Normal human fibroblasts (MJ90) were synchronized at the G 0 phase of the cell cycle by serum starvation for 72 h and then stimulated with fresh medium containing 15% FBS. Flow cytometric analysis showed that the S phase started approximately 18 h after serum stimulation and that maximal cell division occurred at 24 -36 h (data not shown). As shown in Fig.  1A, Northern blot analysis with an hCds1 cDNA probe revealed that an hCds1 transcript of approximately 2.2 kilobases was weakly detected in serum-starved, quiescent cells (time 0). The steady-state level of hCds1 mRNA increased as cells approached the G 1 /S transition (18 h), remained elevated until 36 h, and declined during the next G 1 phase (48 h in Fig. 1A).
To examine whether the level of hChk1 protein also changes in a cell cycle-dependent fashion, we prepared antiserum that specifically interacts with full-length hCds1 protein expressed in insect cells, and extracts from synchronized MJ90 cells were analyzed by Western blotting using affinity-purified anti-hCds1 antibody. As shown in Fig. 1A (lower panel), using the antibody, a predominant band was detected at 60 kDa in the lysates of MJ90 cells, and this completely disappeared after preabsorption of the antibody with GST-fused hCds1 protein expressed in E. coli (data not shown). In agreement with the results of the Northern analysis, Western analysis revealed that hCds1 protein was expressed from late G 1 through M phases of the cell cycle. Although cell cycle-dependent expression of Cds1 and Rad53 has not been described in yeast, the expression pattern of hCds1 is very similar to that of hChk1, another checkpoint kinase in mammalian cells (29).
Nuclear Localization of hCds1-We next attempted to determine the subcellular localization of hCds1 protein. As shown in Fig. 1B, whereas almost no signal was detected using normal rabbit serum (Control Ab), indirect immunofluorescence revealed clearly detected signals with our anti-hCds1 antibody in the nucleoplasm of HeLa cells, indicating that hCds1 is present in the nucleoplasm. The nuclear localization of hCds1 was not affected by DNA damage (data not shown).
hCds1 Gene Exists on Chromosome 22q11.2-Very recently, Cahill et al. (31) reported that mutations of a mitotic checkpoint gene, hBUB1, can cause chromosomal instability in human cancers. To see whether this is also the case with hCds1, we determined the chromosomal localization of hCds1 and analyzed the chromosomal aberrations in the region. The results of fluorescence in situ hybridization showed that the hCds1 gene is localized to human chromosome 22q11.2 (Fig. 2). It is also noteworthy that the hCds1 locus is adjacent to the gene encoding hSNF5/INI1, which has been implicated in the etiology of malignant rhabdoid tumors (32), raising the possibility that a mutation or deletion in the hCds1 gene may be involved in the oncogenesis of rhabdoid tumors.
Activation of hCds1 through Phosphorylation in Response to DNA Damage-In yeast, Rad53 and spCds1 have been reported to be phosphorylated in response to DNA damage, in a Mec1/ Rad3-dependent manner (33,34). To see whether hCds1 functions in DNA damage response in mammalian cells under p53-deficient conditions, modification of hCds1 protein following UV-induced DNA damage was determined in HeLa cells that lack functional p53. As shown in Fig. 3A, the band corresponding to hCds1 protein was shifted upward on SDS-PAGE in extracts of HeLa cells exposed to UV compared with those from untreated cells. This mobility shift was completely reversed by phosphatase treatment, indicating that the modification following UV irradiation was because of phosphorylation of hCds1 protein. Although hCds1 protein was mostly phosphorylated at 2 h after UV irradiation, a time course experiment revealed that phosphorylation of hCds1 protein occurred as early as 10 min, when an intermediate pattern was observed (data not shown).
To examine whether the phosphorylation of hCds1 protein affects its kinase activity, we measured the kinase activity of hCds1 from HeLa cell extracts, with or without DNA damage, using the GST-Cdc25C fragment as a substrate. As shown in Fig. 3B, the kinase activity in the anti-hCds1 immunoprecipitates increased approximately 5-fold following UV irradiation. The kinase activity was not detected when a Cdc25C mutant (S216A) with a substitution of alanine at serine 216 was used as a substrate (data not shown), which is consistent with a previous report that hChk2 phosphorylates serine 216 of Cdc25C (23). Interestingly, autophosphorylation of hCds1 was detected in the anti-hCds1 immunoprecipitates following UV treatment (Fig. 3B). These results suggest that hCds1 is activated by phosphorylation in response to DNA damage induced by UV irradiation and that activated hCds1 phosphorylates Cdc25C on serine 216. Taken together with the recent report that phosphorylation of Cdc25C on serine 216 is critical for the DNA damage checkpoint in mammals (19), this finding suggests that hCds1 plays a pivotal role in this checkpoint pathway.
We next examined whether hCds1 is phosphorylated by other DNA-damaging agents. As shown in Fig. 3C, hCds1 was phosphorylated following x-ray irradiation and MMS treat-

FIG. 1. Cell cycle-dependent expression (A) and nuclear localization (B) of hCds1.
A, MJ90 cells were cultured in serum-deprived medium for 3 days and then restimulated with fresh medium containing 15% FBS. mRNA and protein extracts were prepared at the indicated times after serum stimulation, and hCds1 mRNA and protein were detected by Northern and Western blot analysis, respectively. AS, asynchronous culture. B, HeLa cells cultured on glass coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. The permeabilized cells were incubated with anti-hCds1 antibody (1:200, right) or normal rabbit IgG (left) and then with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Immunotech, 1:100).
FIG . 2. Chromosomal localization of the hCds1 gene. A biotinylated hCds1 cDNA probe was hybridized to R-banded chromosomes from activated lymphocytes. The arrow indicates the locus of hCds1 at 22q11.2 ment, as well as UV irradiation, not only in HeLa cells but in normal fibroblasts, suggesting that hCds1 is involved in DNA damage checkpoint pathways induced by a wide variety of DNA damage. Interestingly, in AT2KY cells, which lack functional ATM, the band shift of hCds1 was observed in response to UV irradiation or MMS treatment but not following x-ray irradiation (Fig. 3C). Taken together with the previous observations that AT cells show hypersensitivity to ionizing radiation but not to UV or alkylating agents (35,36), it is likely that hCds1 is regulated through an ATM-dependent as well as -independent mechanism, depending on the type of DNA damage.
Negative Regulation of hCds1 by p53-Although the data so far suggest the role of hCds1 in the DNA damage response, the functional relationship between the hCds1-mediated G 2 checkpoint and the p53-mediated G 1 checkpoint is largely unknown. To address this question, we first surveyed the expression of hCds1 in various cell lines with and without functional p53.
Interestingly, the level of hCds1 mRNA was very low (Cds1 mRNA/GAPDH mRNA ratio, 0.12-0.23) in cells with wild-type p53, such as MJ90 (37), AT2KY (38), and A172 (39) cells, whereas it was relatively higher (hCds1 mRNA/GAPDH mRNA ratio, 0.7-3.7) in cells without functional p53, including Raji (40), MDAH041 (41), HeLa (42), U937 (43), and T98G (44) cells (Fig. 4A), raising the possibility that wild-type p53 negatively regulates the expression of the hCds1 gene. The status of p53 and its content in these cell lines were confirmed by Northern blot analysis using p21 cDNA as a probe, which showed a higher expression of p21 in cells containing functional p53 and a lower expression in cells lacking functional p53 (data not shown). These results, that the levels of hCds1 mRNA varied among cancer cells lacking functional 53, raise the possibility that additional genetic alterations may be involved in the regulation of hCds1 expression.
To further substantiate negative regulation of hCds1 expression by p53, normal human fibroblasts (WI-38) were transfected with retroviruses encoding the viral oncoproteins, SV40 large T antigen, and HPV E6 or E7 protein to abrogate the FIG. 3. Phosphorylation and activation of hCds1 in response to DNA damage. A, HeLa cells were irradiated with UV light at 0.1 J/cm 2 . After 2 h, cell lysates were prepared, and hCds1 protein was immunoprecipitated with a specific antibody. After washing, the immunoprecipitates were treated with 2000 units of phosphatase (PPase) at 30°C for 1 h in the presence or absence of phosphatase inhibitor and were subjected to SDS-PAGE and Western blot analysis to detect hCds1. B, HeLa cells were irradiated with UV light at 0.07 J/cm2. After 8 h, cell lysates were prepared, immunoprecipitation was performed as in A, and the kinase activity in the anti-hCds1 immunoprecipitates was determined using a GST-hCdc25C fragment as a substrate. In this experiment, a lower dose of UV light (0.07 J/cm 2 ) was used because a higher dose (0.1 J/cm 2 ) appeared to be slightly toxic for a longer culture. C, HeLa cells, normal human fibroblasts (MJ90), and fibroblasts derived from a patient with ataxia telangiectasia (AT2KY) were subjected to ionizing radiation (X-ray; 10 Gy), UV irradiation (0.07 J/cm 2 ), and MMS (0.03%) treatment for 2, 8, and 16 h, respectively. Cell lysates were prepared, and hCds1 protein was detected by Western blotting (HeLa cells and MJ90 cells) or by immunoprecipitation followed by Western blotting, in the case of AT2KY cells, because of the presence of a nonspecific band recognized by the secondary antibody.  38) and derivatives that express SV40 large T antigen or HPV E6 or E7 were cultured overnight with (ϩ) or without (Ϫ) infection with p53expressing adenovirus (Ad-p53, multiplicity of infection ϭ 10). Total cellular RNA was extracted, and Northern blot analysis was performed with a hCds1 cDNA probe. The levels of hCds1 mRNA with standard deviations determined by three independent experiments, corrected for those of GAPDH mRNA, are shown below. kb, kilobase pair. function of both p53 and pRb (45,46), p53 only (47), or pRb only (48), respectively, and the expression of hCds1 mRNA was determined. As shown in Fig. 4B, parent WI-38 cells expressed a low level of endogenous hCds1 mRNA. Expression of SV40 large T antigen, E6 or E7 protein in WI-38 cells caused a marked induction of hCds1 mRNA, with T antigen being more potent than E6 or E7 protein in inducing hCds1 expression (4.5-fold by T antigen, 2.0-fold by E6, and 2.1-fold by E7). Interestingly, as shown in Fig. 4B, infection of an adenovirus vector encoding wild-type p53 significantly decreased the level of hCds1 mRNA in SV 40 large T antigen (both p53 and pRb disrupted)-and E6-transformed cells (only p53 function disrupted) but not in E7-transformed cells (only pRb function disrupted). These results indicate that the induction of hCds1 expression in oncoprotein-transduced cells (Fig. 4B) is, at least in part, because of the inhibition of p53 function. In addition, on the basis of our finding that the expression of hCds1 was also increased in E7-transformed (pRb function disrupted) cells, compared with parent WI-38 cells, and that the level was higher in cells transformed by SV 40 large T-than in E6transduced cells, it is tempting to speculate that the pRb pathway may also negatively regulate hCds1 expression. Taken together with the recent report that pRb plays an essential role in the DNA damage checkpoint during the G 1 phase (49), our results are consistent with the hypothesis that the expression of hCds1 is enhanced when the G 1 checkpoint function is compromised by either p53 or pRb dysfunction.
In conclusion, we propose the existence of a counter-regulatory mechanism; when cells lose functional p53 and thus G 1 checkpoint control, hCds1 plays a pivotal role in the DNA damage checkpoint during the G 2 phase of the cell cycle. In normal cells, p53 and/or pRb, rather than hCds1, are thought to play the main role in DNA damage checkpoint. Once mutations disrupt the p53-and/or pRb-dependent pathways, cells respond with increased expression of hCds1, which in turn plays a compensatory role in the G 2 checkpoint in the case of DNA damage. It is conceivable that activation of the hCds1-dependent checkpoint pathway may underlie the resistance of p53-deficient cancer cells to ionizing irradiation and anti-cancer drugs. Thus, the development of specific inhibitors of hCds1 may provide a novel strategy for cancer therapy by enhancing the sensitivity of cancer cells to DNA damage induced by anticancer drugs.