We devised two short peptides corresponding to amino acids 211–221 of human Cdc25C fused with a part of HIV1-TAT. These peptides inhibited hChk1 and Chk2/HuCds1 kinase activity in vitro and specifically abrogated the G2 checkpoint in vivo. These peptides sensitized p53-defective cancer cell lines to DNA-damaging agent to death without obvious cytotoxic effect on normal cells. Our results clearly indicate that the specific abrogation of the cell cycle G2 checkpoint is a feasible strategy for cancer therapy, and hChk1 and Chk2/HuCds1 are proper targets for that purpose.

Most of the cancer cells have mutations in genes involved in the G1 checkpoint such as impaired tumor suppressor genes including p53, Rb, p16INK4, and p19ARF or overexpression of relevant oncogenes including MDM-2 and cyclin D (1). In addition to these, excessive growth factor signaling caused by the overexpression of growth factors, together with the gain-of-function mutations of these genes, growth factor receptors, or the downstream signal-transducing molecules would give rise to cell transformation by overriding the G1 checkpoint. Only exceptionally, some cancer cells possess the disrupted G2 checkpoint instead of G1 checkpoint (2). This indicates the relative importance of G1 checkpoint compared with the G2 checkpoint for the normal human cell cycle. These checkpoint disruptions confer cells with the ability to accumulate mutations that eventually lead to carcinogenesis (3), although excessive accumulation of mutations would be lethal to the cells. Interestingly, the G2 checkpoint is usually retained in the cancer cells with impaired G1 checkpoint. If the G2 checkpoint was selectively disrupted by the treatment, the cancer cells with the impaired G1 checkpoint would become more sensitive to the DNA-damaging treatment compared with normal cells because normal cells still retain intact G1 checkpoint. For example, relatively nonspecific G2 checkpoint disruptions by caffeine or UCN-014 were reported to be effective in sensitizing the p53-defective cancer cells to DNA damage (4, 5). However, the effects of sensitizing cancer cells by these compounds were subtle, and in some reports, caffeine made cells even more resistant to genotoxic treatments (6), probably because of other irrelevant effects of these compounds to the cells (7).

The mechanism that promotes the cell cycle G2 arrest after DNA damage is conserved among species from yeast to human. In the presence of damaged DNA, Cdc2/Cyclin B is kept inactive because of inhibitory phosphorylation of threonine-14 and tyrosine-15 residues on Cdc2 (8). At the onset of mitosis, the dual phosphatase Cdc25 removes these inhibitory phosphates and thereby activates Cdc2/Cyclin B (9).

In fission yeast, the protein kinase Chk1 is required for the cell cycle arrest in response to damaged DNA (10). Chk1 acts downstream of several rad gene products and is modified by the phosphorylation upon DNA damage (11). The kinases Rad53 of budding yeast and Cds1 of fission yeast are known to conduct signals from unreplicated DNA (12). It appears that there is some redundancy between Chk1 and Cds1 because elimination of both Chk1 and Cds1 was culminated in disruption of the G2 arrest induced by unreplicated DNA (13). Interestingly, both Chk1 and Cds1 phosphorylate Cdc25 and promote Rad24 binding to Cdc25 (14, 15), which sequesters Cdc25 to cytosol and prevents Cdc2/Cyclin B activation (16). Therefore, Cdc25 appears to be a common target of theses kinases and presumably an indispensable factor in G2 checkpoint.

In human, both hChk1 (17, 18), a human homologue of fission yeast Chk1, and Chk2/HuCds1 (19), a human homologue of the budding yeast Rad53 and fission yeast Cds1, phosphorylate Cdc25C at serine-216, a critical regulatory site, in response to DNA damage (17, 18, 19, 20). This phosphorylation creates a binding site for small acidic proteins 14-3-3s, human homologues of Rad24 and Rad25 of fission yeast (20). Although serine-216 of Cdc25C can be constitutively phosphorylated by kinase(s) such as C-TAK1/Kp78/MARK3 (21, 22, 23), the regulatory role of this phosphorylation was clearly indicated by the fact that substitution of serine-216 on Cdc25C to alanine disrupted cell cycle G2 arrest in human cells (20).

Here we demonstrate a novel finding that inactivation of hChk1 and Chk2/HuCds1 kinase activities can be achieved by small peptides corresponding to amino acids 211–221 of human Cdc25C. We also show that these peptides can efficiently disrupt the cell cycle G2 checkpoint that is activated by DNA damage and thus sensitize cancer cells but not normal cells to anticancer reagents.

Chemicals and Reagents.

Bleomycin and colchicine were purchased from Wako Pure Chemical Co. (Osaka, Japan). Hydroxyurea was purchased from Sigma Chemical Co. (St. Louis, MO). These chemicals were dissolved in distilled H2O to 10, 5, and 50 mg/ml, respectively, and stored at 4°C. Antibodies against 14-3-3β were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and antirabbit IgG horseradish peroxidase-conjugated secondary antibodies were purchased from Amersham Life Sciences (Arlington Heights, IL). Antibodies against HA and c-myc and protein G-Sepharose were purchased from Santa Cruz Biotechnology and Amersham Pharmacia Biotech (Uppsala, Sweden), respectively.

Cell Culture and Plasmid.

A human T-cell leukemia-derived cell line, Jurkat, was cultured in RPMI 1640 (Sigma) supplemented with 10% FCS (IBL, Gunma, Japan) at 37°C/5% CO2. Human pancreatic epithelioid carcinoma-derived cell lines, MIAPaCa2 and PANC1, were cultured in Eagle’s MEM (Iwaki, Chiba, Japan) and DMEM with 4 mm l-glucose (Sigma) and 1.0 mm sodium pyruvate (Life Technologies, Inc., Grand Island, NY), respectively, and supplemented with 10% FCS at 37°C/5% CO2. Normal human peripheral blood lymphocytes were collected by Ficoll-Paque (Amersham Pharmacia Biotech) density gradient. Two million cells/ml were cultured in RPMI 1640 supplemented with 10% FCS at 37°C/5% CO2 in the presence of 5 μg/ml PHA (Life Technologies, Inc.) for 1 week. Baculovirus lysates that include HA-tagged hChk1 or c-myc-tagged Chk2/HuCds1 and plasmid for GST-Cdc25C (amino acids 200–256) were kindly provided by Dr. Makoto Nakanishi (Department of Biochemistry, Nagoya City University; Ref. 19).

Peptides.

TAT-S216 peptide was synthesized so that it contained an NH2-terminal 11 amino acid TAT protein transduction domain (YGRKKRRQRRR; Ref. 24), followed by corresponding amino acids 211–221 derived from the Cdc25C amino acid sequence (S216; LYRSPASMPENL). Serine-216 residue was changed to alanine in TAT-S216A (S216A; LYRSPSMPENL). The Cdc25C portion was partially deleted and substituted with glycine in TAT-Control (GGRSPAMPE). All peptides were synthesized by Sawady Technology Co. (Tokyo, Japan).

Purification of Recombinant GST-Cdc25C Proteins.

Escherichia coli DH5α cells were transformed by GST-Cdc25C (200–256) plasmid. The cells were incubated with 0.1 mm isopropyl β-d-thiogalactoside for 2 h, harvested, and lysed with a buffer containing 50 mm Tris-HCl (pH 8.0), 100 mm NaCl, 0.5% NP40, 5 μg/ml aprotinin, 5 μg/ml pepstatin A, and 5 μg/ml leupeptin. The lysate was sonicated, centrifuged for clarification, and incubated with glutathione-Sepharose 4B beads for 1 h at 4°C and washed five times.

Kinase Assay.

HA-tagged hChk1 and c-myc-tagged Chk2/HuCds1 expressed in insect cells using recombinant baculovirus (Ref. 18) were purified by immunoprecipitation using anti-HA or anti-c-myc antibodies and protein G-Sepharose. Immune complex kinase reaction was done in PBS with 1 mm DTT, 1 mm MgCl2, and 100 μCi of [γ-32P]ATP (Amersham; 6000 Ci/mmol) plus purified 1 μm GST-Cdc25C or 10 μm Cdc25C peptide (amino acids 211–221 of Cdc25C; LYRSPSMPENL; Sawady Technology Co.) substrates at 30°C for 15 min in the presence of 10 μm TAT-S216, TAT-S216A; or TAT-Control. After the reaction, samples were separated in 12 or 15% SDS-PAGE and autoradiographed to detect GST-Cdc25C or peptide phosphorylation.

Cell Cycle Analysis.

The cell cycle status of the cells treated with peptides and/or bleomycin or colchicine was analyzed by FACS (2). Two million Jurkat cells were resuspended and incubated in 300 μl of Krishan’s solution (0.1% sodium citrate, 50 μg/ml propidium iodide, 20 μg/ml RNase A, and 0.5% NP40; Ref. 2) for 1 h at 4°C and analyzed by FACScan (Becton Dickinson, Mountain View, CA) with the program CELLQuest (Becton Dickinson).

Histone H1 Kinase Assay.

Ten million Jurkat cells were treated with hydroxyurea (100 μg/ml), bleomycin (10 μg/ml), or colchicine (5 μg/ml) with or without addition of TAT-S216A, TAT-S216, or TAT-Control (10 μm) for 6 h. The cells were washed in cold PBS and lysed at 4°C in 1 ml of buffer A (50 mM Tris pH8. 2 mM DTT, 5 mM EDTA, 100 mM NaCl, 0.5% NP40, 20 mM Na3VO4, 50 mM NaF, 4μm Okadaic Acid, 5 μg/ml aprotinin, 5 μg/ml pepstatin, and 5μg/ml leupeptin). Twenty μl of p13suc1 agarose beads (Upstate Biotechnology, Saranac, NY) were added to the cleared lysates, incubated for 4 h at 4°C, and washed five times with buffer A without 5 mM EDTA, 20 mM NO3VO4, 50 mM NaF and 4μm Okadaic Acid. Histone H1 kinase activity on the beads were analyzed by using Cdc2 kinase assay kit (Upstate Biotechnology) with [γ-32P]ATP, followed by 12% SDS-PAGE electrophoresis and autoradiographed to detect the phosphorylated Histone H1.

Cell Cytotoxicity Assay.

MIAPaCa2 and PANC1 cells (3 × 103/well) were plated in 96-well microtiter plates. After an overnight adherence, cells were treated with bleomycin (10 μg/ml) with or without the indicated TAT-peptides at various time points up to 96 h. Cytotoxicity and cell survival were determined by the 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate assay (Cell Proliferation Kit II; Boehringer Mannheim, Mannheim, Germany), which was done according to the company’s protocol (25).

TAT-S216 and TAT-S216A Peptides Inhibit hChk1 and Chk2/HuCds1 Kinase Activities.

In our initial attempts to inhibit hChk1 and Chk2/HuCds1 kinase activities and to abrogate DNA damage-induced G2 arrest, we generated synthetic peptide consisted with amino acids 211–221 of Cdc25C (TAT-S216). TAT-S216A peptide in which serine-216 was substituted by alanine to stabilize the transient status of its interaction with hChk1 and Chk2/HuCds1 (Fig. 1,A). To efficiently transduce these peptides into cells, a part of the HIV-1 TAT peptide sequence was included (Ref. 24; Fig. 1 A). This sequence is known to facilitate the uptake of heterologous proteins across the cell membrane. As a control peptide, part of the Cdc25C portion of this peptide was deleted (TAT-Control).

As shown in Fig. 1,B, hChk1 was capable of phosphorylating a Cdc25C protein (residues 200–256) fused to GST. Serine-216 on Cdc25C is the major phosphorylation site of this fusion protein in vivo(14, 17, 20). In Fig. 1,B, both TAT-S216 and TAT-S216A inhibited the phosphorylation of Cdc25C by baculovirus produced hChk1. TAT-S216 but not TAT-S216A was efficiently phosphorylated by hChk1, suggesting that serine-216 on TAT-S216 was phosphorylated by hChk1, and TAT-S216 would competitively inhibit substrate phosphorylation at excess molar ratio if present in great enough quantity. TAT-Control peptide did not inhibit hChk1 kinase activity (data not shown). As shown in Fig. 1 C, TAT-S216A significantly inhibited phosphorylation of Cdc25C peptide mediated by hChk1 and Chk2/HuCds1, even at a low stoichiometry (at four times more molar excess of TAT-S216A peptide against substrate Cdc25C peptide).

Abrogation of DNA Damage-induced G2 Checkpoint by TAT-S216 and TAT-S216A Peptides.

We then analyzed the cell cycle status of the cells treated with TAT-S216A or TAT-S216 upon the DNA damage-induced G2 arrest by FACS analysis. Histone H1 kinase activities of theses cells were simultaneously monitored. Jurkat cells arrested exclusively at G2 by bleomycin (10 μg/ml) treatment, because it does not have functional p53. As shown in Fig. 2,A, G2 arrest was completely abrogated by the addition of TAT-S216A or TAT-S216 in response to bleomycin. G2 arrest was abrogated at any time point between 12 and 48 h by the treatment with TAT-S216A or TAT-S216 (data not shown). Jurkat cells treated with bleomycin, together with TAT-Control, arrested at G2 similarly to the cells treated with bleomycin alone. We also observed that either TAT-S216A or TAT-S216 also abrogated G2 arrest induced by γ-irradiation and cisplatin (γ-irradiation, 5 Gy; cisplatin, 1 μg/ml; for 1 h treatment; data not shown). To further analyze the effect of these peptides on G2-M transition, histone H1 kinase activity was monitored. Consistently with the above findings, although histone H1 kinase activity was decreased by the treatment with bleomycin or hydroxyurea, it was unchanged or rather increased by the treatment with bleomycin in the presence of TAT-S216A or TAT-S216 (Fig. 2,B). In the presence of TAT-Control peptide, the bleomycin treatment did not affect with H1 kinase activity (data not shown). As shown in Fig. 2 C, the M-phase arrest of Jurkat cells induced by colchicine was not affected by the addition of TAT-S216 or TAT-S216A. These results strongly suggested that TAT-S216A and TAT-S216 specifically abrogated the DNA damage-activated cell cycle G2 checkpoint by inhibiting hChk1 and/or Chk2/HuCds1 kinase activities.

Sensitization of Jurkat Cells to Bleomycin-induced Cell Death by TAT-S216A and TAT-S216 Peptides.

We then examined the effect of TAT-S216A and TAT-S216 on the cell death induced by bleomycin. As shown in Fig. 3,A, the addition of TAT-S216A and TAT-S216 efficiently sensitized Jurkat cells to the bleomycin-induced cell death. Whereas bleomycin treatment at 5 or 10 μg/ml killed Jurkat cells by only 27–30%, the addition of 10 μm TAT-216A or TAT-S216 killed Jurkat cells by nearly 80%. In contrast, these peptides by themselves did not show any significant cytotoxicity. In addition, a control peptide TAT-Control did not affect the viability of bleomycin-treated Jurkat cells (data not shown). Moreover, as expected from the result in Fig. 2,C, either TAT-S216A or TAT-S216 did not affect the cytotoxicity by colchicine (Fig. 3 B). This observation indicates that the cell death induced by these peptides in the presence of bleomycin was not attributable to the nonspecific cytotoxic effect.

TAT-S216 and TAT-S216A Peptides Did Not Affect the Viability of Normal Cells.

To confirm the specificity of the effect of these peptides on cancer cells in which the G1 checkpoint is abrogated, we investigated the effect of these peptides on normal human cells. We prepared mitogen-activated normal human T lymphocytes (PHA blasts) by stimulating peripheral blood mononuclear cells obtained from a healthy donor with PHA for 1 week. These cells were treated with bleomycin (5 and 10 μg/ml) in the presence or absence of either TAT-S216A or TAT-S216. As shown in Fig. 3,C, these peptides did not augment the cytotoxic effect of bleomycin, although these cells replicated as fast as Jurkat cells. As shown in Fig. 3 D, PHA blasts treated with bleomycin (5 μg/ml) arrested at G1 and S phase but not G2, presumably because of the activity of wild-type p53. When these cells were treated with TAT-S216 or TAT-S216A in addition to bleomycin, no further alteration of cell cycle pattern was observed.

Sensitization of Pancreatic Cancer Cells to the Bleomycin-induced Cell Death by TAT-S216A and TAT-S216 Peptides.

We then examined the effect of these peptides on two other p53-defective cancer cell lines, MIAPaCa2 and PANC1 cells. Although these pancreatic cancer cells are known to be resistant to various anticancer reagents, these cells could also be sensitized to the bleomycin-induced cell death by TAT-S216A and TAT-S216 (Fig. 4). Similarly, these peptides could sensitize these cells to the cell death induced by other DNA-damaging agents including cisplatin and γ-irradiation (data not shown).

Discussion

The effects of abrogating cell cycle G2 checkpoint on the sensitization of the G1-abrogated cancer cells to cell death induced by DNA-damaging agents have long been investigated (4, 5, 6, 7). For example, effects of caffeine have been studied extensively (4, 6). However, these results were considered to be controversial because of its various pharmacological actions other than its potential effects on cell cycle checkpoint disruption (7). Similarly, the use of UCN-01, initially developed as a protein kinase C inhibitor, has been claimed to work by disrupting the G2 checkpoint (5). However, UCN-01 also shows a variety of activity other than the G2 checkpoint abrogation. Thus, it is still controversial whether the abrogation of the G2 checkpoint is effective to sensitize cancer cells to the DNA damage-induced cell death.

In this report, we have demonstrated for the first time that short peptides that inhibit both hChk1 and Chk2/HuCds1 kinase activities specifically abrogated DNA damage-induced G2 checkpoint. We also demonstrated that the specific abrogation of the G2 checkpoint sensitized cancer cells to bleomycin, a DNA-damaging agent, without obvious effect on normal cell cycle and its viability. These observations indicate that these kinases involved in G2 cell cycle checkpoint are ideal targets for the specific abrogation of G2 checkpoint and that these peptides or their derivatives could be practical feasible candidates for novel cancer therapy.

In addition to the applicability to cancer treatment, our findings have provided novel insights into the DNA damage-induced G2 checkpoint mechanism. Whereas yeast Chk1 is required for the cell cycle arrest caused by damaged DNA (10), yeast Rad53/Cds1 is shown to be involved in the cell cycle arrest induced by unreplicated DNA (12). It was required to disrupt both Chk1 and Cds1 to abrogate DNA replication checkpoint in fission yeast (13). In humans, it has not yet been fully clarified whether hChk1 and/or Chk2/HuCds1 are involved in the cell cycle DNA damage-induced G2 checkpoint of human cells. Our results shown in Figs. 1 and 2 show clearly that at least in human cells, the kinases that phosphorylate serine-216 of Cdc25C, such as hChk1 and Chk2/HuCds1, are involved in the cell cycle G2 checkpoint. In addition, it is suggested that the protein moiety around serine-216 of Cdc25C could be a common target to block the G2 checkpoint and thus sensitize cancer cells to the DNA damage-induced cell death. Because the cell cycle pattern and the viability of normal cells (PHA blasts) were not affected by the peptides containing this structural moiety, application of these inhibitory peptide would not have any effect in killing normal cells, unlike other anticancer drugs. Whereas these peptides by themselves are candidates for efficient anticancer agents, when used with known DNA-damaging agents, smaller molecular compounds that can inhibit both hChk1 and Chk2/HuCds1 kinase activities are needed for more cost-effective and practical candidates for anticancer medicine. We believe that these findings should facilitate the development of a novel therapy against intractable cancers that are resistant to conventional anticancer therapies.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

      
1

This work was supported by a part of grants from the Ministry of Health and Welfare, the Ministry of Education, Science and Culture of Japan and from the Human Science Foundation.

                  
4

The abbreviations used are: UCN-01, 7-hydroxystaurosporine; PHA, phytohemagglutinin; HA, hemagglutinin; GST, glutathione S-transferase; FACS, fluorescence-activated cell sorter.

We thank Dr. M. Nakanishi for the kind gift of HA-hChk1, c-myc-Chk2/HuCds1, and GST-Cdc25C plasmid. We also thank K. Aoyama, H. Sato, and M. Kimura for assistance.

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