Introduction

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Glutathione (GSH) is the most prevalent nonprotein thiol in eukaryotic cells, with intracellular levels frequently in the range of 1 to 10 mM (Tew, 1994). Although involved in numerous endogenous processes, GSH also plays a critical role in protecting important cellular nucleophiles against exogenous free radical and electrophilic chemical damage. Either by spontaneous or enzyme-mediated catalysis, a thioether product is formed. In most cases, this metabolite has a neutralized electrophilic center, is more polar, and is less toxic. Such GSH conjugates have affinity for the multidrug resistance-associated protein (MRP), a member of the ATP-binding cassette transporter family (Jedlitschky et al., 1994; Müller et al., 1994; Shen et al., 1996), which actively transports the conjugate across the membrane, ultimately completing the efflux process.

The significance of GSH-based detoxification mechanisms is supported by numerous examples of drug-resistant phenotypes. For example, an ethacrynic acid (EA)-resistant human colon cancer cell line exhibits an increased expression of -glutamylcysteine synthetase (-GCS) (rate-limiting enzyme in de novo GSH biosynthesis), glutathione S-transferase (GST) P1-1 (catalyzes the conjugation of a wide variety of endogenous and exogenous compounds to GSH) and MRP (Shen et al., 1996). The coordinated increase in expression of these proteins permits the cell to accelerate the metabolism and efflux of this drug (Shen et al., 1997). The EA-SG conjugate has GST-inhibitory properties per se (Ploemen et al., 1994), emphasizing the importance of cellular efflux in controlling toxicity. In addition, the GSH conjugates of alkylhalides (e.g., ethylene dibromide or methylene chloride) have greater toxicity than the unconjugated chemicals (Dekant et al., 1989), suggesting that GSH conjugates can have important consequences to a number of cellular processes.

A number of endogenous eicosanoids are also GSH conjugates. Previous studies have made use of photoaffinity ligands of leukotrienes (LTs) to identify cellular binding proteins. In particular, the GSH conjugate LTC4 has been labeled and shown to bind to LTC4 synthetase (Nicholson et al., 1992), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Puder and Soberman, 1997), and MRP (Jedlitschky et al., 1994). These proteins have respective roles in the synthesis, transport, and secretion of this eicosanoid.

Recently, novel anticancer drugs have been designed premised upon the basic structure of GSH conjugates. The GSH peptidomimetic -glutamyl-S-(benzyl)cysteinyl-R()-phenylglycine diethyl ester (TER199) permeates membranes and delivers the active drug moiety into cells (Lyttle et al., 1994). After cellular uptake, deesterification of TER199 releases the active form, -glutamyl-S-(benzyl)cysteinyl-R()-phenyl glycine (TER117). This drug is a site-directed inhibitor of GSTP1-1 (Flatgaard et al., 1993; Morgan et al., 1996a) and, at the same time, interferes with MRP-mediated efflux of other drugs (M. L. O'Brien, B. Vulevic, S. Freer, J. Boyd, H. S., and K. D. T. submitted). Although designed to be a modulator of anticancer drug cytotoxicity, TER199 has also been shown to stimulate myeloproliferation (Broxmeyer et al., 1996; Morgan et al., 1996b).

Because cells are continuously exposed to GSH conjugates (both endogenous and exogenous), it may be important to identify GSH conjugate-binding protein(s) that may affect response to drug treatment. To this end, we synthesized an isotope-labeled drug-SG conjugate, ³⁵S-labeled azidophenacyl-glutathione (APA-SG) (Fig. 1A) and used it as a high, specific activity probe. The catalytic subunit of DNA-dependent protein kinase (DNA-PK) was identified as a novel binding protein. DNA-PK is a serine/threonine kinase, composed of a 460-kDa polypeptide catalytic subunit (DNA-PKcs) and a Ku autoimmune antigen that is a heterodimer of an 86-kDa (Ku80) and a 70-kDa (Ku70) subunit (Hartley et al., 1995). DNA-PK is involved in DNA double-strand break repair and phosphorylates transcription factors in vitro (Finnie et al., 1995; Lees-Miller, 1996; Chu, 1996; Weaver, 1996). In this study, we demonstrate that various GSH conjugates bind to DNA-PKcs and inhibit its kinase activity. Therefore, we suggest that the inhibitory effects of GSH conjugates on DNA-PK-mediated phosphorylation may be a useful lead in the design of agents that target this enzyme.

	Materials and Methods

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Preparation of Cell Lysate. HL-60 and HL-60/ADR cells (Krishnamachary and Center, 1993) were cultured in RPMI 1640 with 10% fetal bovine serum, harvested by centrifugation at 1200g for 10 min, and washed twice in ice-cold PBS. The cell pellet was diluted 1:10 with lysis buffer (10 mM Tris-HCl, pH 7.4, 250 mM sucrose), supplemented with protease inhibitors [0.1 mM phenylmethyl sulfonyl fluoride (PMSF), 1 mM leupeptin, and 0.3 mM aprotinin]. After sonication, samples were centrifuged at 4000g at 4°C for 10 min, and the supernatants are hereafter referred to as whole-cell lysate (WCL).

Synthesis of ³⁵S-Labeled APA-SG. ³⁵S-labeled APA-SG was prepared as described elsewhere (Shen et al., 1996). Briefly, direct exposure to light was avoided during operations, and steps before TLC were conducted under nitrogen or with nitrogen-saturated solutions. ³⁵S-labeled GSH (250 µCi, 517.3 Ci/mmol; Dupont NEN, Boston, MA) was freed of dithiothreitol by ethyl acetate extraction and added to a reaction mixture containing potassium phosphate buffer (50 mM, pH 7.4), 4-azidophenacylbromide (10 mM), GSH reductase (120 mU), and NADPH (1 mM). The reaction was allowed to proceed at room temperature for 1 h, and the products were separated by Silica G TLC using 1-propanol/water (7:3, v/v) as developer. Radiolabeled APA-SG was located by autoradiography, scraped off the plate, and extracted with water. After filtration through a 0.2-µm Gelman filter (Gelman Science, Ann Arbor, MI), the extract was concentrated under nitrogen.

Labeling of Proteins with ³⁵S-Labeled APA-SG. WCL (150 µg) was incubated with 3 µCi of ³⁵S-labeled APA-SG in 10 mM Tris-HCl (pH 7.4)/250 mM sucrose containing 100 µM AT-125 (a specific -glutamyl transpeptidase inhibitor) for 30 min at 4°C. For competition studies, 2 µg of purified DNA-PK (purchased from Promega, Madison, WI) was preincubated for 30 min at 4°C in the presence of various concentrations of unlabeled competitors. After 30 min of incubation at 4°C in the dark, the samples were photolyzed in a Stratagene (La Jolla, CA) UV Stratalinker using standard settings to achieve 200,000 µJ. The labeled proteins were then analyzed by SDS-polyacrylamide gel electrophoresis (PAGE).

Western Blot Analysis. After separation by SDS-PAGE, proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Immunoblotting was accomplished using an enhanced chemiluminescence kit (Amersham, Buckinghamshire, England). DNA-PKcs monoclonal antibody mAb42-27 was a generous gift from Dr. Thomas Shenk (Department of Molecular Biology, Princeton University, Princeton, NJ).

DNA-PK Enzyme Activity Assay. Standard assay mixture contained 25 mM HEPES (pH 7.5), 0.25 mM peptide substrate (EPPLSQEAFADLWKK) (purchased from Promega), 75 mM KCl, 10 mM MgCl₂, 1 mM dithiothreitol, 0.05% Nonidet NonidetP-40, 0.1 mM EDTA, 10 µg/ml sonicated salmon sperm DNA, and 0.25 mM ATP containing 5 µCi of [-³²P]ATP (3000 Ci/mmol; Dupont NEN), and reactions were started by the addition of enzyme. Reactions were continued at 30°C for 10 min and stopped by adding 30% acetic acid. An aliquot was spotted on Whatman P-81 phosphocellulose paper. After washing, incorporated radioactivity was counted. To test the effects of drugs on DNA-PK activity, the above assay was performed using purified DNA-PK (Promega). Drugs were preincubated with DNA-PK in reaction buffer without DNA, ATP, [-³²P]ATP, and substrate for 5 min at 4°C. The reaction was started by the addition of DNA, ATP, [-³²P]ATP, and substrate and was allowed to react for 10 min at 30°C.

Immunoprecipitation Assay. Adriamycin-resistant HL-60 (HL-60/ADR) cells, grown to 50% confluency, were harvested by centrifugation at 1200g for 10 min and washed twice in ice-cold PBS. The cell pellet was resuspended in 100 µl of extraction buffer (50 mM NaF, 20 mM HEPES, pH 7.8, 450 mM NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol) supplemented with protease inhibitors (2 mM PMSF, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 0.7 µg/ml pepstatin), and then frozen on dry ice and thawed at 37°C three times. After microcentrifugation at full speed for 5 min at 4°C, the supernatant was used for subsequent assays. Protein concentration was determined using the Bio-Rad assay. Immunoprecipitation experiments were performed with 2 mg of protein incubated with different concentrations of APA-SG (Sigma, St. Louis, MO) for a total volume of 25 µl at 4°C for 30 min. Fifty microliters of protein G-agarose (Sigma) was incubated with 2 µg of DNA-PKcs antibody targeted against the C terminus (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or 2.12 µg of Ku70 antibody (Ab) (QED Bioscience Inc., San Diego, CA) at 4°C for 3 hr. After incubation, drug-treated cell extracts were combined with antibody-linked protein G-agarose; the volume was increased to 1 ml with immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1% Nonidet P-40, 0.5% sodium deoxycholate, 5 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM PMSF 0.7 g/ml pepstatin, 1 mM EDTA), and immunoprecipitation was performed at 4°C overnight. After washing, beads were resuspended in SDS-PAGE sample buffer, incubated at 100°C for 5 min, and spun at full speed in a microcentrifuge. The supernatant was resolved on SDS-7.5% PAGE gel and transferred to a nitrocellulose membrane. Immunoblotting was performed using the various DNA-PK antibodies (Kamiya Biomedical Co., Seattle, WA), and bands were visualized using enhanced chemiluminescence (Amersham).

	Results

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To identify GSH conjugate-binding protein(s), WCL from HL-60 wild-type, HL-60/ADR cell lines were labeled with the ³⁵S-labeled APA-SG photoaffinity analog (Fig. 1) by photolyzing with UV light, separated by SDS-PAGE, and subjected to autoradiography. Fig. 2A shows that a high-molecular-weight band was apparent. In the absence of photoactivation, no protein binding was detected. The kinetically favorable activation of the phenyl azide by broad-wavelength UV produces a single nitrene that is unstable, reacting with a rapid half-life with acceptor nucleophiles in target amino acids. The arrows indicate those proteins which were reproducibly labeled. Following semipurification through Q-Sepharose, the ~460-kDa protein was excised from the gel, and partial amino acid sequences were analyzed. The sequenced fragments were identical to DNA-PKcs (Table 1). Confirmation of the identity of the protein was achieved by Western blot analysis using anti-DNA-PKcs MAb 42-27 (Fig. 2B). Both photoaffinity labeling and Western blot analysis showed that DNA-PKcs is overexpressed in drug-resistant HL-60 cell line, consistent with our previous report (Shen et al., 1998). In addition to DNA-PKcs and MRP, several other lower molecular weight bands are also labeled by ³⁵S-labeled APA-SG. We have identified some of these bands as proteolytic products of DNA-PKcs (Shen et al., 1998), whereas others were identified by partial amino acid sequences as pyruvate kinase, actin, and GAPDH (Fig. 2A, a-c, respectively). These identities were confirmed by Western blot using specific antibodies (data not shown). The identity of DNA-PKcs as an APA-SG-binding protein was further confirmed by immunoprecipitation analysis (Fig. 2C). The ³⁵S-labeled APA-SG-labeled WCL from the HL-60/ADR cell line was incubated with DNA-PKcs antibody-linked protein G-agarose. After extensive washing, the samples were subjected to SDS-PAGE analysis and autoradiography after drying. The ³⁵S-labeled APA-SG-labeled protein was detected in the precipitates (lane 1) but not in the supernatants (lane 2).

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Structure of 35S-labeled APA-SG (A) and TER117 (B).

View larger version (47K): [in this window] [in a new window]   Fig. 2.   Identification of APA-SG-binding proteins. A, APA-SG labeling of cellular proteins. 35S-labeled APA-SG (3 µCi) was incubated with 150 µg of WCL at 4°C in the dark for 30 min and then photolyzed in Stratagene UV Stratalinker using standard settings to achieve 2,000,000 µJ. The labeled proteins were then fractionated on SDS-PAGE. Lane 1, HL-60 cell line; lane 2, HL-60/ADR cell line. Top arrow band was purified and sequenced to identify this protein. Result is shown in Table 1. Bands labeled a, b, and c were also identified as pyruvate kinase, actin, and GAPDH, respectively, using the same methods (sequence data not shown). B, Western blot analysis. WCL (100 µg) from HL-60 wild-type (lane 1) and HL-60/ADR (lane 2) cells with anti-DNA-PKcs MAb 42-47. C, immunoprecipitation analysis. The 35S-labeled APA-SG labeled WCL from HL-60/ADR cells was incubated with DNA-PKcs Ab-linked protein G-agarose. After extensive washing, the proteins were removed from the beads by adding 1× SDS sample buffer and boiling for 3 min. The proteins were separated on a SDS-7.5% PAGE gel followed by autoradiography. Lane 1, precipitates; lane 2, supernatant.

To confirm specificity of binding, isotope dilution analyses were performed using purified DNA-PK and ³⁵S-labeled APA-SG. Figure 3 shows that excess unlabeled APA-SG or oxidized glutathione (GSSG) could compete with the labeling for the protein and that the competition was concentration-dependent. The ability of the DNA-PK holoenzyme complex to phosphorylate an in vitro peptide substrate based on p53 primary sequence is a measure of the kinase activity of the enzyme (Chan and Lees-Miller, 1996). This function is generally associated with the transcriptional activation and/or signal-transduction role ascribed to DNA-PK. As shown in Fig. 4A, APA-SG caused a concentration-dependent inhibition of this phosphorylation. The azide group of APA-SG is "activatable" and forms a covalent bond with target amino acids only when exposed to high-intensity UV light. The use of S-(p-chlorophenacyl)-glutathione, in which the azide group was replaced with chlorine, excluded the possibility that this contingency would have resulted from the present experimental conditions. S-(p-chlorophenacyl)-glutathione inhibited DNA-PK-mediated phosphorylation in the same fashion as APA-SG (Fig. 4A). The overall involvement of GSH in the inhibition was also demonstrated by the experiments carried out with GSSG (Fig. 4A). These data are consistent with the fact that GSSG dose-dependently competed the labeling of ³⁵S-labeled APA-SG (Fig. 3). In addition, a GSH peptidomimetic agent, TER117 (Fig. 1B) also inhibited DNA-PK activity in a concentration-dependent fashion (Fig. 4B) and at much lower concentrations.

View larger version (44K): [in this window] [in a new window]   Fig. 3.   Isotope dilution assays with purified DNA-PK. Purified DNA-PK (1 µg) was incubated with various concentrations of unlabeled APA-SG or GSSG before incubation with 35S-labeled APA-SG followed by separation on SDS-PAGE and autoradiography (top panel). Lanes 1 and 5, without APA-SG or GSSG; lanes 2 to 4 and 6 to 8, 0.01, 0.1, and 1.0 mM APA-SG and GSSG, respectively. Bottom panel, protein staining of the same gels for equal gel loading.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Inhibition of in vitro kinase activity (for experimental details, see Materials and Methods) of DNA-PK by GSH conjugates (A) and TER117 (B). All data points are calculated by subtracting background values (assays without DNA). The data are mean ± S.D. of three experiments except GSSG which is of two experiments.

To gain further insight into the APA-SG-induced inhibition mechanism, a series of immunoprecipitation experiments was undertaken. Figure 5 shows the results of two such approaches. Using a polyclonal antibody to the C terminus of DNA-PKcs (Fig. 5A), the complex of the catalytic subunit, Ku70, and Ku80 were coimmunoprecipitated with the catalytic subunit in approximately equivalent amounts (Fig. 5A, lane 1). When increasing concentrations of APA-SG were added to the complex before the immunoprecipitation, Ku70 and Ku80 did not coprecipitate with DNA-PKcs (lanes 2-4; see also Table 2). Similarly, when precipitation was achieved with an antibody to Ku70, in the absence of APA-SG, the other components of the holoenzyme complex were coimmunoprecipitated (Fig. 5B, lane 1). In the presence of increasing concentrations of APA-SG (Fig. 5B, lanes 2-4), although a proportion of the total Ku80 was coprecipitated, there was a significant reduction in equivalence of DNA-PKcs coprecipitation (Table 2). These data suggest that APA-SG interferes with protein-protein interactions between DNA-PKcs and the Ku heterodimer complex. This is consistent with the fact that APA-SG binds to DNA-PKcs but not to either Ku70 or Ku80.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Immunoprecipitation of DNA-PK with anti-DNA-PKcs Ab (A) and anti-Ku70 Ab (B). Two milligrams of protein was preincubated with increasing concentrations of APA-SG followed by incubation with antibody-linked protein G-agarose. After washing, beads were resuspended in SDS sample buffer and fractionated on a SDS-7.5% PAGE gel (for experimental details, see Materials and Methods). Lane 1, control (no APA-SG); lanes 2 to 4, 1, 2, and 5 mM APA-SG, respectively. For quantitation of absorbance, see Table 2.

	Discussion

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Cellular redox balance is heavily biased toward a reducing environment. Under normal physiological conditions, GSH:GSSG ratios are usually >20:1 (Meister and Anderson, 1983). When levels of GSSG or GSH conjugates are elevated, cells actively efflux them to maintain a physiological environment. Because there is significant biological advantage in maintaining thiol homeostasis and removing GS-thioether conjugates from the cellular environment, it follows that selective pressures must have resulted in the evolution of an efficient machinery for dealing with such products. As a first step in understanding this process, we have used photoaffinity analog techniques to identify GSH conjugate-binding proteins.

We have shown previously that a HL-60 Adriamycin-resistant cell line overexpresses DNA-PKcs and that the increase is functionally linked to drug resistance (Shen et al., 1998). Our present results showed that DNA-PKcs is also a binding protein for APA-SG. The absence of apparent ³⁵S-labeled APA-SG labeling of DNA-PKcs in HL-60 wild-type (Fig. 2) reflects low levels of expression in this cell line. The critical role of the DNA-PK holoenzyme complex in DNA repair and transcriptional regulation (Jin et al., 1997a) suggests that APA-SG binding may impair a critical enzyme activity. Indeed, our data showed that the binding of GSH conjugates, including APA-SG, inhibited the capacity of the enzyme to phosphorylate a p53-based peptide substrate.

Other inhibitors of DNA-PK have been identified. For example, Henkels and Turchi (1997) described a cisplatinum-mediated alteration in the DNA template that acts as a cofactor in the kinase reaction. This results in a decrease in Ku binding to DNA, with concomitant reduction in kinase activity when compared to undamaged DNA controls. The platinum adduct-binding to the DNA template produced a large decrease in the V_max for Ku binding. Our data suggest a different inhibition mechanism for APA-SG. In the presence of APA-SG, the capacity of the C-terminal antibody to DNA-PKcs to immunoprecipitate the three-component holoenzyme complex was impaired. When antibodies to either Ku70 (Fig. 5B) or Ku80 (data not shown) were used, only the catalytic subunit of DNA-PK showed impaired coprecipitation. These results, along with the fact that no APA-SG binding to Ku70 and Ku80 was found, suggest an inhibition mechanism involving conjugate binding to DNA-PKcs and subsequent prevention of the formation of a complex between the catalytic subunit and Ku heterodimer. The inactivation mechanism of DNA-PK caused by dissociation of DNA-PKcs from the Ku heterodimer has been attributed to autophosphorylation of DNA-PKcs but not the Ku components (Chan and Lees-Miller, 1996). Phosphorylation of DNA-PKcs by c-Abl protein tyrosine kinase also inhibits the ability of DNA-PKcs to form a complex with Ku and DNA (Kharbanda et al., 1997; Jin et al., 1997b). Using immunoprecipitation experiments with anti-Ku antibody following coincubation of in vitro translation products of various fragments of DNA-PKcs with Ku-containing cell lysates, Jin et al. (1997b) reported that amino acids 3002-3850 of DNA-PKcs associate with Ku. This region is the N-terminal to conserved residues potentially involved in phosphotransfer catalysis for DNA-PKcs. Ku binding to this region can contribute to structural changes in DNA-PKcs, stimulating its kinase activity (Jin et al., 1997b). Interestingly, the same region also binds to c-Abl protein kinase and is phosphorylated by c-Abl. Therefore, c-Abl activity dissociates DNA-PKcs from the Ku components, inactivating DNA-PK activity (Jin et al., 1997b). Recently, a similar down-regulation of DNA-PK activity by another protein, Lyn tyrosine kinase, has been reported (Kumar et al., 1998). The SH3 domain of Lyn interacts directly with a leucine zipper region in DNA-PKcs (amino acids 1520-1975), and induces the dissociation of DNA-PKcs from the Ku-DNA complex, resulting in inactivation of DNA-PK (Kumar et al., 1998). The different binding regions to DNA-PKcs of c-Abl and Lyn suggest that multiple regulatory domains in DNA-PKcs may exist. Recent atomic-force microscopy studies using highly purified DNA-PKcs also show that DNA-PKcs binds directly to DNA and displays kinase activity in the absence of Ku components (Yaneva et al., 1997; Hammarsten and Chu, 1998). Our present data suggest another level of DNA-PK activity regulation by GSH conjugate binding. Therefore, it will be interesting to define the specific GSH conjugate binding region in DNA-PKcs.

DNA-PK is a member of the phosphatidylinositol-3-kinase (PI3-kinase) family. Chemical inhibitors of the PI3-kinase enzyme family have been described, although generally they lack specificity for DNA-PK. Wortmannin has inhibitory activity for a range of PI3-kinases, including DNA-PK, with K_i values at nanomolar concentrations (Cao et al., 1998). The compound reacts covalently with the ATP-binding site, at the Lys802 residue of PI3-kinase. The equivalent lysine residue in DNA-PKcs is at position 3771. The covalent bond with the -amino group occurs through nucleophilic attack of the C-20 of wortmannin, leading to furan ring opening and the formation of an enamine (Wymann et al., 1996). A similar reaction mechanism is predicted for demethoxyviridin, another nanomolar PI3-kinase inhibitor (Bonser et al., 1991). Other PI3-kinase inhibitors include quercetin and derivatives such as LY294002 (Davidson, 1995). These compounds lack electrophilic sites, do not bind covalently to PI3-kinases, and inhibit at micromolar concentrations. These inhibition concentrations are equivalent to wortmannin analogs with hydrolyzed furan rings (Baggioline et al., 1987; Yano et al., 1993), again, lacking the electrophilicity of the parent drug. Our data with APA-SG show DNA-PK inhibition within the millimolar range (Fig. 4A) consistent with a noncovalent binding mechanism. However, the GSH peptidomimetic TER117 has inhibitory activity in the micromolar range (Fig. 4B) and competed ³⁵S-labeled APA-SG labeling (data not shown). TER117 is released from the prodrug TER199 by the action of esterase within cells. This drug is also a low micromolar inhibitor of GSTP1-1 and binds to the MRP-mediated drug transporter (M. L. O'Brien, B. Vulevic, S. Freer, J. Boyd, H. S. and K. D. T., submitted). These observations suggest that using the general GSH conjugate backbone, the design of more effective inhibitors of DNA-PK may be plausible.