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Redox Regulation of Cdc25B by Cell-Active Quinolinediones

Departments of Pharmacology (M.B., T.N., J.S.S., C.F., P.B., J.S.L.), Pharmaceutical Sciences (B.W.D, E.M.S.), Chemistry (P.W., B.J., B.W.D.), and the Center for Chemical Methodologies and Library Development (P.W., B.J.), University of Pittsburgh, Pittsburgh, Pennsylvania

Received for publication July 1, 2005.

Accepted for publication September 9, 2005.

	Abstract

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Intracellular reduction and oxidation pathways regulate protein functionality through both reversible and irreversible mechanisms. The Cdc25 phosphatases, which control cell cycle progression, are potential subjects of oxidative regulation. Many of the more potent Cdc25 phosphatase inhibitors reported to date are quinones, which are capable of redox cycling. Therefore, we used the previously characterized quinolinedione Cdc25 inhibitor DA3003-1 [NSC 663284 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] and a newly synthesized congener JUN1111 [7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] to test the hypothesis that quinone inhibitors of Cdc25 regulate phosphatase activity through redox mechanisms. Like DA3003-1, JUN1111 selectively inhibited Cdc25 phosphatases in vitro in an irreversible, time-dependent manner and arrested cells in the G₁ and G₂/M phases of the cell cycle. It is noteworthy that both DA3003-1 and JUN1111 directly inhibited Cdc25B activity in cells. Depletion of glutathione increased cellular sensitivity to DA3003-1 and JUN1111, and in vitro Cdc25B inhibition by these compounds was sensitive to pH, catalase, and reductants (dithiothreitol and glutathione), consistent with oxidative inactivation. In addition, both DA3003-1 and JUN1111 rapidly generated intracellular reactive oxygen species. Analysis of Cdc25B by mass spectrometry revealed sulfonic acid formation on the catalytic cysteine of Cdc25B after in vitro treatment with DA3003-1. These results indicate that irreversible oxidation of the catalytic cysteine of Cdc25B is indeed a mechanism by which these quinolinediones inactivate this protein phosphatase.

The Cdc25 phosphatases contain a structural feature that distinguishes them from other protein tyrosine phosphatases (Buhrman et al., 2005). The crystal structures of the catalytic domains of Cdc25A and Cdc25B reveal a shallow and open active site pocket, making structure-specific inhibitor design difficult (Fauman et al., 1998; Reynolds et al., 1999). However, like other tyrosine-specific protein phosphatases, the Cdc25 phosphatases have the signature catalytic domain comprising -H-C-X₅-R-, where X is any amino acid (Denu et al., 1996). The pK_a of the active site cysteine residue of Cdc25B is unusually low (5.6-6.3) as a result of the unique polar environment created by a proximal -helix, the amides of the five X residues, and the conserved arginine (Chen et al., 2000; Sohn and Rudolph, 2003). This ensures that the active site cysteine exists as a thiolate anion to form a phosphocysteine intermediate, allowing for removal of the phosphate from the substrate (Guan and Dixon, 1991; Cho et al., 1992; Rudolph, 2002). The low pK_a of the active site cysteine makes it a potential target for endogenous and exogenous oxidants (Salmeen et al., 2003; Wang et al., 2004). Reactive oxygen species (ROS), such as H₂O₂, have been shown to directly inhibit the activity of Cdc25B phosphatase in vitro with a second-order rate constant for oxidation that is 400 and 15 times faster than the oxidation of glutathione and tyrosine phosphatase 1B (PTP1B), respectively (Sohn and Rudolph, 2003).

Oxidative processes can profoundly influence both intracellular signaling and cell cycle progression (Tonks, 2005). For example, oxidative stress leads to mitotic cell arrest (Arrington et al., 2000; D'Agnillo and Alayash, 2001), which is reminiscent of the cell cycle arrest seen after addition of some Cdc25 phosphatase inhibitors (Lyon et al., 2002; Kristjansdottir and Rudolph, 2004). Addition of reducing agents (e.g., thioredoxin) or superoxide dismutase releases cells from mitotic arrest, coinciding with dephosphorylation of inhibitory tyrosine and threonine phosphates on Cdk1 (pCdk1), a known substrate for Cdc25 (Natsuyama et al., 1993). In addition, exposure of HeLa cells to H₂O₂ results in oxidation of the active site cysteine in Cdc25C and protein degradation, leading to cell cycle arrest (Savitsky and Finkel, 2002).

Several benzoquinones and fused quinones have previously been identified as potent in vitro Cdc25 inhibitors by high-throughput in vitro screening (Lyon et al., 2002; Kristjansdottir and Rudolph, 2004). One of these compounds, DA3003-1 [NSC 663284 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)quinoline-5,8-dione], is believed to inhibit the Cdc25A catalytic domain through covalent adduct formation between DA3003-1 and a serine residue adjacent to the catalytic cysteine in Cdc25A. This led to arylation of DA3003-1 and release of the halogen substituent (Kerns et al., 1995; Pu et al., 2002). However, based on our current studies, covalent adduct formation may not be the sole or even the major mechanism of Cdc25B inhibition by quinones.

Previous studies with other protein tyrosine phosphatases and quinoid agents suggest that Cdc25B inhibition could be caused by redox cycling of the quinones in the presence of oxygen (Wardman, 1990; Koster, 1991; O'Brien, 1991). Bova et al. (2004) measured direct production of H₂O₂ by ortho-quinone inhibitors of protein tyrosine phosphatase . In addition, the active site cysteines of CD45 and protein tyrosine phosphatase 1B (PTP1B) can be irreversibly oxidized to sulfinic () and sulfonic acid () by quinones in the presence of oxygen, a course of action that is prevented by the addition of catalase or superoxide dismutase (Wang et al., 2004). Rudolph and colleagues (Sohn and Rudolph, 2003) have demonstrated the formation of a sulfenic acid (Cys-SO^-) intermediate after a brief H₂O₂ treatment of the catalytic domain of Cdc25B, and, more recently, the generation of sulfinic and sulfonic acid species after longer treatments with H₂O₂ (Buhrman et al., 2005).

In the current study, we have examined the essential nature of the halogen substituent in DA3003-1 in Cdc25 inactivation and the possible role of oxidation induced by the quinolinediones. We provide the first direct evidence that quinolinediones can directly inactive Cdc25B in cells by generating ROS, causing irreversible oxidization of the catalytic cysteine of Cdc25B.

	Materials and Methods

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Chemical Synthesis. The preparation of DA3003-1 has been reported previously (Lazo et al., 2001). To synthesize JUN1111 and JUN1120-2, we dissolved quinoline-5,8-dione (Barret and Daudon, 1990) (0.33 g; 2.1 mmol) in ethanol (20 ml) and added 4-(2-aminoethyl)-morpholine (0.27 ml; 2.1 mmol) at room temperature. The reaction mixture was stirred for 16 h and concentrated under reduced pressure. The crude residue was purified by chromatography on SiO₂ (methanol/CH₂Cl₂; 1:30) to give an 2:3 mixture of JUN1111 and JUN1120-2 (0.36 g; 60%). Pure JUN1111 and JUN1120-2 were obtained as red solids by further chromatographic separation on SiO₂ (MeOH/CH₂Cl₂; 1:100). JUN1111 had a melting point of 180°C (dec.); IR (neat) 3329, 2966, 2837, 1696, 1618, 1598 cm^-1; ¹H NMR 8.88 (dd, 1 H, J = 4.5, 1.6 Hz), 8.39 (dd, 1 H, J = 7.9, 1.6 Hz), 7.63 (dd, 1 H, J = 7.9, 4.5 Hz), 6.62 (bs, 1 H), 5.75 (s, 1 H), 3.80-3.60 (m, 4 H), 3.27-3.21 (m, 2 H), 2.69 (t, 2 H, J = 6.1 Hz), 2.55-2.35 (m, 4 H); ¹³C NMR 181.5, 180.1, 153.0, 148.3, 146.8, 134.3, 130.8, 128.3, 100.6, 66.9 (2C), 55.5, 53.3 (2C), 38.8; MS (EI) m/z (relative intensity) 289 ([M + 2H]^+·, 1), 189 (4), 160 (2), 100 (100); HRMS (EI) m/z calculated for C₁₅H₁₉N₃O₃ ([M + 2H]^+·) 289.1426, found 289.1427. JUN1120-2 had a melting point of 182°C (dec.); IR (neat) 3293, 2945, 2837, 1685, 1588, 1562, 1490 cm^-1; ¹H NMR 8.97 (dd, 1 H, J = 4.8, 1.6 Hz), 8.33 (dd, 1 H, J = 8.0, 1.6 Hz), 7.55 (dd, 1 H, J = 8.0, 4.8 H), 6.53 (bs, 1 H), 5.87 (s, 1 H), 3.80-3.60 (m, 4 H), 3.27-3.18 (m, 2 H), 2.69 (t, 2 H, J = 6.0 Hz), 2.55-2.35 (m, 4 H); ¹³C NMR 181.5, 181.2, 155.1, 149.3, 147.6, 134.2, 127.4, 126.3, 102.1, 66.9 (2C), 55.5, 53.2 (2C), 38.4; MS (EI) m/z (relative intensity) 289 ([M + 2H]^+·, 2), 261 (2), 100 (75), 91 (100); HRMS (EI) m/z calculated for C₁₅H₁₉N₃O₃ ([M + 2H]^+·) 289.1426, found 289.1429.

In Vitro Enzyme Assays. Epitope-tagged His₆Cdc25A₁, His₆Cdc25B₂, and GST-Cdc25C₁ were expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid (His₆) or glutathione-Sepharose resin (glutathione S-transferase) as described previously (Lazo et al., 2001). Human recombinant VHR and PTP1B were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). A serine-to-alanine point mutation in the Cdc25B₂ catalytic domain cDNA at Ser⁴⁵⁰ (GenBank accession no. NM_021872 [GenBank] ) was accomplished using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and complementary custom oligonucleotide primers from Invitrogen (Carlsbad, CA). The resulting DNA was sequenced by the University of Pittsburgh Core DNA Sequencing Facility to ensure proper integration of primers. Enzyme activities in the absence and presence of inhibitors were measured using the artificial substrate 3-O-methylfluorescein phosphate (Sigma-Aldrich, St. Louis, MO) at concentrations equal to the K_m of each enzyme and at the optimal pH for individual enzyme activity in a 96-well microtiter plate assay based on methods described previously (Lazo et al., 2001). Fluorescence emission from the product was measured after a 20-min (VHR and PTP1B) or 60 min (Cdc25) incubation period at ambient temperature with a multiwell plate reader (Cytofluor II; Applied Biosystems, Foster City, CA; excitation filter, 485 nm/20-nm bandwidth; emission filter, 530 nm/30-nm bandwidth). IC₅₀ concentrations were determined using Prism 3.0 (GraphPad Software Inc., San Diego, CA). For dithiothreitol (DTT) studies, enzyme activities for full-length Cdc25B₂ in the absence or presence of quinones was assessed in the presence of 2 to 100 mM DTT in the assay buffer. The pH of the assay buffer was adjusted from pH 7.0 to 8.3 to determine the effect of pH on compound inhibition. The effect of glutathione concentration on in vitro inhibition was measured by the addition of 0, 5, or 10 mM glutathione to the assay buffer. For reversibility studies with inhibitors, we used a protocol similar to a dilution method described previously (Sohn et al., 2003). Cdc25B₂ full-length enzyme (60 mM Tris, 2 mM EDTA, and 150 mM NaCl, pH 8.0) was preincubated with 3 times the IC₅₀ concentration for each inhibitor: DA3003-1 (3 µM) or 6 µM JUN1111 for 0, 5, or 20 min at room temperature. The enzyme was also incubated separately with the DMSO vehicle. After preincubation, the enzyme from each treatment was diluted >10-fold, and remaining enzyme activity was determined by the above-mentioned phosphatase assay using 3-O-methyl fluorescein phosphate (OMFP).

Flow Cytometric Analysis. tsFT210 cell synchronization and flow cytometry assays were performed as described previously (Osada et al., 1997) on a FACSCalibur flow cytometer (BD Biosciences PharMingen, San Diego, CA), and data were analyzed using ModFit LT cell-cycle analysis software (Verity Software House, Topsham, ME).

Western Blotting and Immunoprecipitation of pCdk1. tsFT210 cells were arrested at G₂/M by incubation at 39.4°C for 17 h. DA3003-1 (1 or 10 µM) or JUN1111 (10 or 30 µM) or DMSO vehicle was added to cells for 1 h at 32°C. Cells were vortexed every 10 min in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, containing 250 mM NaCl, 5 mM EDTA, and 0.1% Triton X-100) supplemented with various protease and phosphatase inhibitors. Total protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA), and lysates were incubated with 50 µl of an anti-Cdc2 p34 IgG_2A mouse monoclonal antibody-agarose conjugate (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C on an orbital rocker. Immunocomplexes were washed three times in ice-cold PBS supplemented with protease and phosphatase inhibitors. Immunocomplexes were boiled in SDS electrophoresis loading buffer and supernatants were resolved on a 12% Tris-glycine gel. Proteins were transferred to a nitrocellulose membrane and blotted with anti-phospho-Cdc2 (Tyr15) rabbit polyclonal antibody (Cell Signaling Technology, Beverly, MA) for detection of hyperphosphorylated Cdk1. Membranes were stripped and reprobed with an anti-Cdk1 mouse monoclonal antibody (Santa Cruz Biotechnology) for detection of total levels of Cdk1 (loading control).

Direct Inhibition of Cdc25B in HeLa Cells. The pCMV-myc-Cdc25B₂ vector was constructed by subcloning Cdc25B₂ cDNA from the pQE30-H25B plasmid and subsequent ligation into the BamHI/HindIII sites of the pCMV-Tag 2-5 vector from Stratagene (La Jolla, CA). The pCMV-myc-Cdc25B₂ plasmid was expressed in HeLa cells for 24 h followed by the addition of the vehicle control (DMSO), 10 µM DA3003-1 or JUN1111 for 2 h, or 1 mM H₂O₂ for 15 min. A pCMV-myc-vector was expressed in HeLa cells and treated with DMSO as a negative control. Cells were lysed as stated above and ectopically expressed myc-Cdc25B₂ was immunoprecipitated with an agarose-conjugated mouse monoclonal anti-myc (9E10) antibody (Santa Cruz Biotechnology). The beads were washed three times in ice-cold phosphate-buffered saline (PBS) supplemented with protease inhibitors. Remaining enzyme activity was determined with an in vitro OMFP assay similar to methods described previously (Lazo et al., 2001). Percentage of inhibition was calculated using the following formula for enzyme activity in the immunoprecipitates: [100 - (Cdc25B-transfected cells after treatment with DA3003-1 or JUN1111 - vector-transfected cells)/(Cdc25B-transfected cells after treatment with DMSO - vector-transfected cells)] x 100%.

Glutathione Reduction in tsFT210 Cells. Cellular glutathione was reduced 50% in 1 x 10⁶ tsFT210 cells after a 24-h incubation at 32°C with 50 µM L-buthionine sulfoximine. Glutathione levels were tested using the ApoAlert glutathione detection kit from BD Biosciences Clontech (Palo Alto, CA). Cells were then treated with DMSO, DA3003-1 (0.1-10 µM), or JUN1111 (1-30 µM) for 48 h followed by counting of live and dead cells by trypan blue exclusion. Percentage of inhibition of cell growth at each inhibitor concentration was calculated by 100 - [number of live cells treated with inhibitor/number of live cells treated with DMSO] x 100%. IC₅₀ concentrations were determined using Prism 3.0 (GraphPad Software Inc.).

Measurement of Cellular ROS Generation and Calculation of Reduction Potential. tsFT210 cells (1 x 10⁶) were suspended in PBS and preloaded with chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester dye (Invitrogen). Cells were washed in PBS and resuspended in PBS buffer containing 3 µM PI. Cells were then treated for 10 min with DMSO, 1 mM H₂O₂,or10 µM DA3003-1 or JUN1111. DCF and PI fluorescence was measured by flow cytometry using the FACSCalibur flow cytometer (BD Biosciences PharMingen). The one-electron reduction potential was calculated using the quantum chemical modeling program Spartan from Wavefunction, Inc. (Irvine, CA), based on Austin-model 1/lowest unoccupied molecular orbital energies.

Mass Spectrometry. Recombinant Cdc25B₂ catalytic domain was purified as described previously in Guan and Dixon (1991), with one exception: the size exclusion buffer included 50 mM Tris-HCl, pH 7.5, containing 300 mM NaCl, 1 mM EDTA, and 1 mM DTT. Recombinant Cdc25B₂ catalytic domain protein in size exclusion buffer was treated with the DMSO vehicle or a 30-fold molar excess of DA3003-1 for 1 h at 25°C. An aliquot from each reaction was removed, diluted 3-fold in 1:1 H₂O-CH₃CN containing 0.1% CF₃CO₂H, mixed with an equal volume of a sinapinic acid-saturated solution of the same solvents, spotted on a stainless steel target, and analyzed by MALDI-TOF-MS in the linear mode on a Voyager DE Pro spectrometer (Applied Biosystems). Another aliquot of each solution was treated with iodoacetamide, precipitated in cold (-20°C) acidic acetone, resuspended in loading buffer and segregated by SDS-polyacrylamide gel electrophoresis on a 12% Tris-glycine gel with Coomassie Blue staining. Bands containing the protein as well as a band from a lane in which no protein was loaded, were excised, destained with dilute methanolic aqueous acetic acid, dried, and treated with sequencing grade trypsin (Promega, Madison, WI) in aqueous NH₄HCO₃ for 12 h at 37°C. The gel pieces were subjected to centrifugation, and the supernatant was removed and lyophilized. The digest was resuspended in 1% aqueous HCO₂H. One portion was diluted 3-fold in 1:1 H₂O-CH₃CN containing 0.1% CF₃CO₂H, mixed with an equal volume of a -cyano-4-hydroxycinnamic acid-saturated solution of the same solvents, spotted on a stainless steel target, and analyzed by MALDI-TOF-MS and MALDI-TOF/TOF-MS/MS in reflector mode on a spectrometer (model 4700; Applied Biosystems). The remainder of each sample was analyzed by nanoflow LC-electrospray ionizationion trap MSⁿ (where n = 1 or 2, for single or tandem MS) on either an LCQ DECA XP Plus or an LTQ system with a Surveyor LC (all from Thermo Electron Corporation, Waltham, MA).

	Results

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Previous investigations with DA3003-1 proposed the potential importance of Ser⁴³⁵ (accession no. NM_001789 [GenBank] ) in adduct formation and halogen release from DA3003-1, resulting in Cdc25A inhibition (Pu et al., 2002). To investigate this mechanism further, we generated an S450A mutant Cdc25B construct (equivalent to Ser⁴³⁵ in Cdc25A), expressed the recombinant protein, and evaluated inhibition with DA3003-1. The Ser⁴⁵⁰ mutation produced no significant difference in the Cdc25B catalytic activity and, remarkably, did not alter the ability of DA3003-1 to inhibit the enzyme activity in vitro (data not shown). In addition, we synthesized the dehalogenated congener of DA3003-1, namely, JUN1111, and its regioisomer JUN1120-2 as small molecule tools (Table 1). A comparison of the IC₅₀ values for all three Cdc25 isoforms for DA3003-1 and JUN1111 revealed that the chloride substituent was not essential for inhibition (Table 1). JUN1111 was slightly more selective than DA3003-1, with higher in vitro IC₅₀ values against VHR and PTP1B. We have observed a substantial reduction in inhibition with the regioisomer of DA3003-1 and proposed that this reflected different steric or electrostatic effects (Lazo et al., 2001). This effect also extended to the regioisomer of JUN1111, namely, JUN1120-2, which was markedly less active against the Cdc25, VHR, and PTP1B phosphatases (Table 1). Although it has been suggested by others (Ham et al., 2004) that reduction potentials can be a factor in quinone-based inhibition of Cdc25 phosphatases, the potency for in vitro inhibition of Cdc25 did not correlate with the calculated one electron reduction potentials (E 1/7) for these compounds (Table 1). Thus, the difference in E 1/7 values between JUN1111 and JUN1120-2 (-193 mV - (-146 mV) = -47 mV) was much smaller than that between JUN1111 and DA3003-1 (-193 mV - (-78 mV) = -115 mV), although the difference in IC₅₀ values for Cdc25 phosphatases was much greater between the two regioisomers.

The halogen was also dispensable for cell cycle arrest. We demonstrated previously that DA3003-1 caused both a G₁ and G₂M arrest in tsFT210 cells, consistent with its ability to inhibit the Cdc25 family of phosphatases (Pu et al., 2002). JUN1111 also caused both G₁ (Fig. 1) and G₂/M (Fig. 2) phase arrest. The G₂/M arrest induced by DA3003-1 and JUN1111 coincided with Cdk1 hyperphosphorylation, further supporting cellular inhibition of Cdc25 phosphatase activity (Fig. 3). Overall, JUN1111 was slightly less potent in the tsFT210 mouse cell line compared with DA3003-1. Nonetheless, JUN1111 was more potent than DA3003-1 in inhibiting human MDA-MB-435 breast (JUN1111, IC₅₀ = 0.1 µM; DA3003-1, IC₅₀ = 0.5 µM) and PC-3 prostate (JUN1111, IC₅₀ = 0.8 µM; DA3003-1, IC₅₀ = 1 µM) cell growth.

View larger version (20K): [in this window] [in a new window]   Fig. 1. G1 phase arrest with DA3003-1 and JUN1111. tsFT210 cells were cultured at the permissive temperature of 32°C and then incubated at the nonpermissive temperature (39.4°C) for 17 h to synchronize cells at G2/M. Cells were then released for 4 to 6 h at 32°C to arrest cells at the G1 checkpoint. DMSO or inhibitor was added for an additional 6 h at 32°C. A, asynchronous control. B, G2/M-arrested cells after temperature shift for 17 h at 39.4°C. C, G1-arrested cells after temperature shift back to 32°C. D, DMSO-treated cells. E, cells treated with 50 µM roscovitine (positive control). F, cells treated with 10 µM DA3003-1. G and H, cells treated with 10 or 30 µM JUN1111. Data are representative of three independent experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 2. G2/M arrest with DA3003-1 and JUN1111. tsFT210 cells were cultured at the permissive temperature of 32°C and then incubated for 17 h at the nonpermissive temperature (39.4°C). DMSO or drug was added to cells for an additional 6 h at 32°C. A, asynchronous control. B, G2/M-arrested cells after temperature shift for 17 h at 39.4°C. C, DMSO-treated cells. D, cells treated with 1 µM nocodazole (positive control). E, cells treated with 10 µM DA3003-1. F and G, cells treated with 10 or 30 µM JUN1111. Data are representative of three independent experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 3. Cdk1 is hyperphosphorylated in the presence of DA3003-1 and JUN1111. G2/M-arrested tsFT210 cells were released for 1 h in the presence of 10 and 30 µM JUN1111 or 1 and 10 µM DA3003-1 or DMSO vehicle followed by immunoprecipitation with anti-Cdk1 mouse monoclonal antibody. Western blots were probed with phospho-Cdk1 (Tyr15) antibody. Blots were stripped and reprobed with total Cdk1 antibody as a loading control.

To confirm direct cellular inhibition of Cdc25B by DA3001-1 and JUN1111, we transfected HeLa cells with a vector containing or lacking myc-Cdc25B, before treatment of cells for 2 h with DMSO vehicle, DA3003-1, or JUN1111 or a 15-min treatment with H₂O₂. Ectopically expressed Cdc25B activity was measured by immunoprecipitation of myc-Cdc25 followed by in vitro phosphatase assays using OMFP as a substrate. DA3003-1 and JUN1111 inhibited the etopically expressed Cdc25B in HeLa cells by 77 and 56%, respectively, which was comparable with the 73% inhibition seen with 1 mM H₂O₂ (Fig. 4). The loss of cellular enzyme activity in the immunoprecipitates of Cdc25B from cells treated with DA3003-1 and JUN1111 is consistent with either prolonged or irreversible enzyme inhibition.

View larger version (12K): [in this window] [in a new window]   Fig. 4. DA3003-1 and JUN1111 directly inhibit transfected Cdc25B in HeLa cells. Transfected cells were treated with DA3003-1 (10 µM), JUN1111 (10 µM), or H2O2 (1 mM) followed by immunoprecipitation of myc-Cdc25B. The remaining enzyme activity was tested in vitro using OMFP as a substrate. Percentage of inhibition was calculated by comparing to DMSO-treated myc-Cdc25B transfected cells (positive control) and DMSO-treated myc-vector transfected cells (negative control). Results are mean ± S.E.M. (n = 4).

View larger version (10K): [in this window] [in a new window]   Fig. 5. DA3003-1 and JUN1111 are irreversible inhibitors of Cdc25B. Recombinant Cdc25B was preincubated with 3 times the IC50 of DA3003-1 (2.5 µM) or JUN1111 (6 µM) for 0, 5, or 20 min in the absence of substrate and then diluted 10-fold and assayed for enzyme activity. Results are mean ± S.E.M. (n = 3). Statistical significance was assessed by one-way analysis of variance. ***, p < 0.001 compared with DMSO control.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Effect of DTT concentration and pH on phosphatase inhibition. A, Cdc25B phosphatase inhibition by DA3003-1 and JUN1111 was tested using our in vitro enzyme assay as described under Materials and Methods at DTT concentrations of 2, 10, 25, and 100 mM. Results are mean ± S.E.M. (n 3). B, Cdc25B phosphatase inhibition by DA3003-1 and JUN1111 was tested using our in vitro enzyme assay as described under Materials and Methods under various pH conditions (pH 7-8.3). Results are mean ± S.E.M. (n = 3). IC50 values were calculated using GraphPad Prism software. A positive correlation of +1 (effect of DTT on IC50) and a negative correlation of -1 (effect of pH on IC50) were determined by Spearman's rank correlation.

View larger version (13K): [in this window] [in a new window]   Fig. 7. Glutathione decreases the potency of DA3003-1 and JUN1111. In vitro inhibition of Cdc25B phosphatase by DA3003-1 (1.56 µM) and JUN1111 (3.13 µM) was tested in the presence of increasing concentrations of glutathione (0, 5, and 10 mM). Results are mean ± S.E.M. (n = 6). A negative correlation of -1 (effect of glutathione on percentage of inhibition of enzyme) was determined by Spearman's rank correlation.

The above-mentioned data are consistent with the hypothesis that the Cdc25 inhibition by DA3003-1 and JUN1111 could be due to oxidation via production of ROS. To determine whether H₂O₂ was involved in quinone inhibition, we added catalase (80 U/ml) to the in vitro phosphatase assay. Catalase eliminated the inhibitory activity of DA3003-1 and JUN1111 against Cdc25B, indicating that H₂O₂ was at least one potential ROS involved in Cdc25B inhibition. To assess direct ROS production by the quinone inhibitors, we preloaded tsFT210 cells with dichlorodihydrofluorescein diacetate (chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester) dye, a cell-permeant indicator of ROS. A 10-min treatment with 10 µM DA3003-1 or JUN1111 produced a 3-fold increase in DCF fluorescence, reflecting a rapid and marked oxidative burst (Fig. 8). Simultaneous staining with PI confirmed that no significant cell death was caused by compound exposure (data not shown). Therefore, both DA3003-1 and JUN1111 are capable of rapidly generating ROS in cells.

View larger version (11K): [in this window] [in a new window]   Fig. 8. DA3003-1 and JUN1111 produce ROS in cells. tsFT210 cells were preloaded with DCF dye (to measure ROS) and propidium iodide (to measure cell death) before treatment with DMSO, DA3003-1 (10 µM), or JUN1111 (10 µM) for 10 min. Resulting DCF and PI fluorescence was measured by flow cytometry. Results are mean ± S.E.M. (n = 3). Statistical significance was assessed by one-way analysis of variance. ***, p < 0.001 compared with DMSO control.

	Discussion

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The physiological role of redox regulation of protein phosphatases including PTP1B, low molecular weight phosphatases PTEN and SHP-2 has gained considerable support (Lee et al., 1998; Mahadev et al., 2001; Meng et al., 2002; Cho et al., 2004). Mild oxidation of protein tyrosine phosphatases at low concentrations of ROS leads to reversible oxidation of the catalytic cysteine residue to Cys-SO^-, which is readily reversible by cellular reductants. Further oxidation of the catalytic cysteine to or leads to irreversible inactivation of the enzyme. This mechanism of oxidation has been demonstrated with Cdc25B upon exposure to H₂O₂ (Sohn and Rudolph, 2003; Buhrman et al., 2005). Cdc25B can be protected from irreversible oxidation through formation of the reversible sulfenic acid species followed by rapid formation of a disulfide bridge between the active-site cysteine and a neighboring, backdoor cysteine residue. The oxidized sulfenic acid and the disulfide bridge forms of the enzyme can be reduced by DTT and cellular reductants, such as thioredoxin/thioredoxin reductase, with concomitant enzyme reactivation. With increased exposure or higher concentrations of ROS (H₂O₂), further oxidation of Cdc25B occurs, resulting in irreversible deactivation.

Quinoid arenes are highly electrophilic compounds that participate in redox cycling, producing ROS that can inactivate phosphatases at their active site cysteine. The most potent and selective Cdc25 inhibitors reported to date are quinones. Most seem to be irreversible inhibitors of Cdc25 phosphatases (Lyon et al., 2002), with the possible exception of the naphthofurandione 5169131 (Brisson et al., 2004) and some indolylhydroxyquinones (Sohn et al., 2003). It is noteworthy that not all quinones are potent in vitro Cdc25 inhibitors, indicating that there is some specificity in the chemical structure of the quinones found to inhibit this enzyme. Previous studies with DA3003-1 (Pu et al., 2002) indicated that dehalogenation of the inhibitor occurred, with formation of a covalent ether adduct with a serine residue corresponding to Ser⁴³⁵ of Cdc25A (equivalent to Ser⁴⁵⁰ in Cdc25B). Therefore, it is interesting that in the current study, the S450A mutation in Cdc25B did not affect DA3003-1 inhibition (data not shown) and that the chlorine moiety was not obligatory for inhibition of Cdc25A, Cdc25B, or Cdc25C (JUN1111; Table 1). We also saw no evidence of a covalent adduct after incubation of Cdc25B with DA3003-1 (Table 2). This difference may be caused by possible alternate confirmations of the active sites between the two Cdc25 isoforms as revealed in the X-ray crystal structures (Reynolds et al., 1999). It is also possible that redox regulation and adduct formation occur under different reaction conditions. A low concentration of reducing agent is required to reduce the quinone inhibitor and initiate redox cycling and ROS production (Bova et al., 2004). In the previous investigations of adduct formation, a reductant was not included in the buffer conditions when incubating DA3003-1 with the enzyme; therefore, in the absence of redox cycling, it is possible that arylation of DA3003-1 and adduct formation was favored. Because DTT was added in the current experiment, oxidation probably became the dominant mechanism of inhibition.

We have provided considerable experimental evidence suggesting that quinolinediones participate in redox cycling, leading to Cdc25B inactivation. First, increasing concentrations of DTT (Fig. 6A) and glutathione (Fig. 7) caused a subsequent decrease in Cdc25B inhibition by DA3003-1 and JUN1111. Second, when DA3003-1 and JUN1111 were incubated with Cdc25B phosphatase in the absence of reducing agents, the quinones were not capable of inhibiting Cdc25B, presumably because redox cycling was not initiated. Therefore, the presence of high concentrations of reducing agents interfered with Cdc25B oxidation and inactivation by quinolinediones, whereas low reductant levels were required to induce inhibition by initiating quinone redox cycling and subsequent production of ROS. Third, the presence of catalase abolished inhibition by DA3003-1 and JUN1111, indicating that H₂O₂ production was important in inhibiting Cdc25B phosphatase activity. Fourth, the pH of the reaction also directly affected enzyme oxidation. Raising the reaction pH from pH 6.0 up to pH 8.0 enhances oxygen consumption by quinones, thereby increasing the rate of oxidation of enzymes (Molina Portela and Stoppani, 1996; Wang et al., 2004). Furthermore, increasing the pH above 7.0 increases hydroquinone oxidation by superoxide, resulting in increased production of H₂O₂ (Ordonez and Cadenas, 1992; Bova et al., 2004). Thus, the IC₅₀ concentrations of DA3003-1 and JUN1111 decreased with increasing pH from pH 7.0 to 8.3 (Fig. 6B). Finally, we observed rapid ROS formation in cells treated with both DA3003-1 and JUN1111 (Fig. 8). Although DA3003-1 seemed to participate in redox cycling in cells, cell lines differing in levels of NAD(P)H:quinone oxidoreductase-1 are equally sensitive to DA3003-1 cytotoxic effects (Han et al., 2004). Therefore, these compounds may be substrates for other quinone reductases or dehydrogenases, such as carbonyl reductase and xanthine dehydrogenase.

The oxidation caused by quinolinediones may exploit the extreme reactivity of the Cdc25 phosphatases to oxidants and their susceptibility to form higher, irreversibly oxidized sulfinic and sulfonic acid states, compared with other protein tyrosine phosphatases (PTP1B and VHR). This may be due, in part, to the open, exposed active site pocket of Cdc25 (Denu and Tanner, 1998; Reynolds et al., 1999; Rudolph, 2004). For example, the deeper active site pocket in PTP1B causes the rate of inactivation of the PTP1B to be 15-fold less than that of Cdc25B (Sohn and Rudolph, 2003). This differential reactivity provides a possible explanation for the selectivity of our quinolinediones for inhibition of Cdc25 phosphatases over VHR and PTP1B (Table 1). ROS have also been shown to oxidize and inactivate mitogen-activated protein kinase phosphatases or MKPs resulting in c-Jun NH₂-terminal kinase (JNK) activation (Kamata et al., 2005). Therefore, we examined the effects of DA3003-1 and JUN1111 on MKP-1 both in vitro and in cells. Both compounds had no effect on in vitro MKP-1 activity up to 100 µM. Furthermore, addition of DA3003-1 (10 µM) and JUN1111 (30 µM) to HeLa cells for 1 h did not induce activation of JNK when lysates were probed with an anti-phospho-stress-activated protein kinase/JNK (Thr183/Tyr185) antibody (data not shown). Although these studies indicate some selectivity of the compounds for Cdc25 phosphatases, we recognize that more of the almost 100 human protein tyrosine phosphatases would need to be evaluated to securely state a high degree of selectivity both in vitro and in vivo.

View larger version (22K): [in this window] [in a new window]   Fig. 9. Fragmentation patterns for peptides generated from tryptic digests of Cdc25B2 incubated with DMSO vehicle or with a 30-fold molar excess of DA3003-1 for MALDI-TOF-MS spectra analysis. RVILIFHC446EFSSER ( is Cys-sulfonic acid) treated with DA3003-1. VILIFHC446*EFSSER (* is carbamidomethylated) containing the iodoacetamide-blocked active site cysteine.

We have provided direct evidence of irreversible inactivation and oxidation of Cdc25B after exposure to DA3003-1. DA3003-1 and JUN1111 irreversibly inactivated Cdc25B enzyme activity in a time-dependent manner, similar to inactivation seen with H₂O₂ (Figs. 4 and 5). Mass spectrometry analysis of the Cdc25B catalytic domain after a 1-h in vitro incubation with DA3003-1 revealed that the catalytic cysteine was irreversibly oxidized to sulfonic acid (Fig. 9; Table 2). Figure 9 shows the fragmentation patterns of the tryptic peptide containing the iodoacetamide-blocked active site cysteine (^* is carbamidomethylated) as well as the DA3003-1-treated peptide that revealed the oxidized catalytic cysteine ( is Cys-sulfonic acid). A total of seven fragments as well as an internal ion fragment—H₂O of mass 584 (Table 2)— confirmed an increase in mass coinciding with conversion of the catalytic cysteine to its sulfonic acid form. These data are in agreement with irreversible oxidation of the catalytic cysteines of other phosphatases by ortho- and para-quinone inhibitors producing ROS (Bova et al., 2004; Wang et al., 2004). We recognize that additional post-translational modifications, such as S-glutathionylation, may regulate Cdc25 phosphatase activity as seen with regulation of PTP1B (Barrett et al., 1999). This possibility should be addressed in future studies.

In conclusion, in vitro inhibition of Cdc25B by para-quinolinediones DA3003-1 and JUN1111 seems to be due, at least in part, to ROS production and irreversible oxidation of the catalytic cysteine thiol to sulfonic acid. Differences in Cdc25 phosphatase inhibition are not directly correlated to the redox potentials of quinones, allowing for the possibility that interactions controlled by steric and electronic factors may confer some specificity for inhibition. Future synthesis of analogs of the quinolinediones to promote selective inhibition of Cdc25 could entail the addition of moieties that would better direct the generated ROS to the active site of Cdc25.

	Acknowledgements

 
We thank Johannes Rudolph (Duke University Medical Center, Durham, NC) for generously providing the Cdc25B₂ catalytic domain construct that we used for our spectrometry analysis and for helpful advice.

	Footnotes

 
This work was supported in part by U.S. Public Health Service National Institutes of Health grants CA78039 and GM06585 and the Fiske Drug Discovery Fund.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.016360.

ABBREVIATIONS: Cdk, cyclin-dependent kinase; ROS, reactive oxygen species; PTP1B, tyrosine phosphatase 1B; VHR, vaccinia H1-related phosphatase; DA3003-1, 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione; JUN1111, 7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione; JUN1120-2, 6-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione; DTT, dithiothreitol; DMSO, dimethyl sulfoxide; OMFP, O-methyl fluorescein phosphate; PBS, phosphate-buffered saline; PI, propidium iodide; DCF, 2',7'-dichlorodihydrofluorescein; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; MS, mass spectrometry; MS/MS, tandem mass spectrometry; LC, liquid chromatography; MKP, mitogen-activated protein kinase phosphatase; JNK, c-Jun NH₂-terminal kinase; VHR, vaccinia virus VH1-related human phosphatase.

Address correspondence to: Dr. John S. Lazo; Department of Pharmacology, University of Pittsburgh, Biomedical Science Tower 3-Suite 1032, 3501 Fifth Ave, Pittsburgh, PA 15260. E-mail: lazo{at}pitt.edu

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