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Mol. Cells, Vol. 12, No. 3, pp. 412-417 Communication Molecules and Cells KSMCB 2001 Creatine Kinase B Is a Target Molecule of Reactive Oxygen Species in Cervical Cancer Hyun Choi1, Chang Soo Park, Byoung Gie Kim, Jae Won Cho2, Jong-Bae Park3, Yun Soo Bae1, and Duk Soo Bae* Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul 135-710, Korea; 1 Division of Molecular Life Sciences, Center for Cell Signaling Research, Ewha Womans University, Seoul 120-750, Korea; 2 Department of General Surgery, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul 135-710, Korea; 3 Department of Molecular Life Sciences, Pohang University of Science and Technolgy, Pohang 790-784, Korea. (Received November 1, 2001; Accepted November 12, 2001) Recently, a procedure for detecting ROS-sensitive proteins that contain active cysteine residues was developed. The method is based on the fact that biotinconjugated iodoacetamide (BIAM) and ROS competitively and selectively react with the active cysteine residues in ROS-sensitive proteins. To investigate the role of ROS in cervical cancer, BIAM labeling on cytosolic proteins in normal and cancer tissues was performed, respectively. The BIAM labeling proteins are separated by 2-dimensional electrophoresis, and then identified by MALDI-TOF mass analysis. ROSsensitive protein is identified as creatine kinase B containing cysteine residue in active center. Activity of creatine kinase B in normal tissue is higher than that of oxidized form in cervical cancer tissues. The result suggests that ROS play an important role in metabolic regulation in cervical cancer cells. However, molecular mechanisms that ROS and creatine kinase B are integrated into a physiological signal leading to the cellular transformation remain to be elucidated. Keywords: 2-Dimensional Gel Electrophoresis (2DE); Biotinylation; Cervical Cancer; Creatine Kinase (CK); Hydrogen Peroxide; MALDI; Reactive Oxygen Species (ROS). Introduction Most reactive oxygen species (ROS) in cell are generated * To whom correspondence should be addressed. Tel: 82-2-3410-3511; Fax: 82-2-3410-0044 E-mail: [email protected] from results of aerobic respiration and various oxidase actions such as lipoxygenase and xanthin oxidase. Furthermore, many cell types transiently produce ROS in response to a variety of extracellular stimuli including growth factors and hormones (Finkel et al., 1998; Rhee et al., 2000). The term ROS encompasses many species including singlet oxygen, the superoxide anion radical (O2•), H2O2, lipid peroxides, nitric oxide (NO), peroxynitrite (ONOO-), the thiyl peroxyl radical (RSOO• ), the ferryl radical (FeO2+) and the hydroxyl radical (OH• ) (Yim et al., 1994). Although the chemical nature of ROS generated in response to the activation of various receptors has not been well characterized, H2O2 was shown to be a major component of ROS in cells (Bae et al., 1997; Sundaresan et al., 1995). Hydrogen peroxide is thought to contribute to various cellular functions, including the activation of transcription factors, phospholipases, and protein tyrosine kinases as well as the inactivation of protein tyrosine phosphatases and serine-threonine phosphatases (Bae et al., 1997; Kamata et al., 1999; Schreck et al., 1991; Sundaresan et al., 1995). A number of methods have been developed for the detection of such irreversibly oxidized products, including lipid-derived malondialdehyde (Luo et al., 1995), carbonyl group-containing proteins (Levine et al., 1995), 4hydroxynonenal protein (Yoritaka et al., 1996), and 8hydroxy-2′-deoxyguanosine derived from DNA (Teixeira et al., 1995). Recently, a procedure for identifying proteins Abbreviations: 2DE, 2-dimensional gel electrophoresis; BIAM, biotin-conjugated iodoacetamide; CK, creatine kinase; Cr, creatine; DTT, dithiothreitol; MALDI-TOF, Matrix-assisted laser desorption/ionization-time of flight; ROS, reactive oxygen species. Hyun Choi et al. teins that contain H2O2-sensitive Cys residues has been developed (Kim et al., 2000). This procedure is based on the facts that H2 O2 selectively oxidized Cys residues with a low acid constant (pKa), that these Cys residues are also selectively labeled at pH 6.5 with biotin-conjugated iodoacetamide (BIAM), that oxidized cysteines are not susceptible to labeling with BIAM, and then the decrease in BIAM labeling of proteins that results from oxidation can be monitored by streptavidin blot analysis. The method provides systematic attempts to identify such target proteins of H2O2 in cells. Creatine kinase (CK) catalyzes the reversible transfer of γ-phosphate group of ATP to the guanidine group of creatine (Cr) to produce ADP and phosphorylcreatine (PCr). To date, four different isozymes of CK are identified in mammalian tissues (Wyss et al., 2000). Muscle CK (M-CK) and brain CK (B-CK) are located in cytosol and two mitochondrial CK isoforms (ubiquitous Mi-CK and sarcomeric Mi-CK) are located in mitochondrial intermembrane space. CK isozymes were known to contain a single active sulfhydryl group (Cys-278 in Mi-CK and Cys-283 in cytosolic CK) (Furter et al., 1993). Modification of sulfhydryl residue by ROS and NO resulted in a decrease of CK activity (Kaneko et al., 1993; Mekhfi et al., 1996; Suzuki et al., 1992). The result indicates that the active sulfhydryl group in CK plays an important role for catalytic activity. In this paper, it is demonstrated that the ROS target molecules in normal and cervical cancer tissue are identified using BIAM labeling method and MALDI-TOF mass analysis. Moreover, the modification of sulfhydryl group in CK by ROS resulted in a decrease of their activity. The result suggests that the inactivation of CK by ROS might be involved in energy metabolic regulation in cancer. Materials and Methods Materials and reagents Fresh cancer tissue and adjacent normal tissue from surgical specimens were divided and immediately frozen in liquid nitrogen and stored at −70°C until analyzed. N-(biotinoyl)-N′-(iodoacetyl)ethylenediamine (BIAM) was obtained from Molecular Probes. Horseradish peroxidase (HRP)conjugated antibodies to mouse and rabbit immunoglobulin G were from Upstate biotechnology. Enhanced chemiluminescence (ECL) reagents were from NEN. Horseradish peroxidase (HRP)-conjugated streptavidin was from Amersham. Rabbit anti creatine kinase-BB isoenzyme was from Fitzgerald. Aprotinin, leupeptin, 4-(2-aminoethyl)-benzene-sulfonyl fluoride hydrochloride were from ICN. Labeling with BIAM Human endometrial cervical cancer tissues were homogenated with lysis buffer [0.2 M Mes-NaOH (pH 6.5), 1 mM EDTA, aprotinin (1 µg/ml), leupeptin (1 µg/ml), 413 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (1 µg/ml) and 50 µM BIAM] (Kim et al., 2000). Bubbling with nitrogen gas at low flow rate for 15 min eliminated the oxygen from buffer. The cell lysates were incubated for 1 h at room temperature with nitrogen gas flush. The labeling reaction was terminated by the addition of β-mercaptoethanol to a final concentration of 50 mM. The reaction mixture was centrifuged 40,000 rpm for 1 h at 4°C. The supernatant was subjected to SDS-polyacrylamide gel electrophoresis (PAGE), and the separated proteins were transferred to a nitrocellulose membrane. Proteins labeled with BIAM were detected with HRPconjugated streptavidin and ECL. 2-Dimensional gel electrophoresis The supernatant was mixed for 2 h at room temperature with sample buffer (9.5 M urea, 2% Triton X-100, 5% β-mercaptoethanol) and electrofocused in 7 cm ImmobilineR DryStrips (pH 4−7) with the Amersham IPGphor. The following focusing protocol was used: 50 µA per strip at 20°C; (1) rehydration for 13 h; (2) 500 V for 1 h (step and hold); (3) 1000 V for 1 h (step and hold); (4) 8000 V for 3 h (step and hold). After electrofocusing, the strips were either stored at −70°C or shaken for 15 min with equilibration buffer (1.5 M Tris-Cl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 10 mg/ml DTT) and subjected to 10% SDS-PAGE. Protein identification by peptide mass fingerprinting analysis Protein peptide fingerprinting analysis was performed as described (Jensen et al., 1996). In brief, the candidate band was excised from the gel and digested with trypsin. One µl aliquot of the total digest (total volume 30 µl) was used for peptide mass fingerprinting. The masses of the tryptic peptides were measured with a Bruker Reflex III mass spectrometer. Matrixassisted laser desorption/ionization (MALDI) was performed with α-cyano-4-hydroxycinnamic acid as the matrix. Trypsin autolysis products were used for internal calibration. Accuracy in peptide masses was achieved with better than 50 ppm on average. Comparison of the mass values against the SWISS-PROT database was performed by Profound. Creatine kinase activity of tissues and cells Tissues were chopped and homogenized with PBS (pH 7.4) containing aprotinin (1 µg/ml), leupeptin (1 µg/ml), 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (1 µg/ml), and were centrifuged at 40,000 rpm for 1 h at 4°C. The creatine kinase activities of supernatants were measured using CK kit according to the manufacturer’s instructions (Diagnostica Merck) (Alterio et al., 1990). Results and Discussion Identification of ROS-sensitive molecules in cervical cancer tissues A few proteins are identified as a ROS target protein. Those proteins contain cystein thiolate anion (Cys-S-) form in active center. The cystein thiolate 414 ROS-Target Molecules A A B B Fig. 1. Detection of ROS target molecules. A. Human cervical cancer and normal tissues were homogenated with lysis buffer containing BIAM. The labeling reaction was terminated by the addition of β-mercaptoethanol to a final concentration of 50 mM. The reaction mixture was centrifuged 40,000 rpm for 1 h at 4°C. The supernatant was subjected to 2-dimensional gel electrophoresis, and the separated proteins were transferred to a nitrocellulose membrane. Proteins labeled with BIAM were detected with HRP-conjugated streptavidin and ECL. Detection of ROS target molecules in normal tissue (left, inset) and cervical cancer tissue (right, inset). B. Coomasie staining of normal (left) and cervical cancer tissue (right) extracts of 2-D electrophoresis. anion is more readily oxidized by ROS than is the cysteine sulfhydryl group (Cys-SH). Moreover, sulfhydryl modifying agent, biotin-conjugated iodoacetamide (BIAM) is also more reactive with cystein thiolate ion than sulfhydryl cystein residues (Kim et al., 2000). However, the oxidized cystein residues (sulfenic or sulfinic form) by ROS do not react with BIAM. Therefore, BIAM labeling amount of cysteine residues might be decreased in cells after exposure to ROS. The extent of BIAM labeling on proteins can be readily measured by SDS-PAGE and blot analysis of the biotinylated proteins with HRPconjugated streptavidin and ECL. We investigated whether differential BIAM labeling of protein in between normal and cervical cancer tissues. Figure 1A showed that BIAM labeling of one spot (approximately 42 kDa, and PI point is 5.3) is markedly decreased. The protein spot from 2-D gel electrophoresis are subjected into trypsin digestion and tryptic peptides are analyzed by MALDI-TOF mass spectroscopy (Jensen et al., 1996). Mass spectrum is analyzed by using of database of peptide map from University of Rockefeller. Nine peptides are matched with creatine kinase B (Fig. 2 and Fig. 2. MALDI-TOF mass analysis of ROS target molecule. A. The mass spectrum was produced using the peptide supernatant obtained after ‘in -gel’ digestion of the excised band with trypsin as described under “Experimental Procedure”. Database search for the measured tryptic peptide masses uniquely identified as creatine kinase. The peaks labelled with an arrow matched the calculated tryptic peptide masses from creatine kinase within 50 ppm. B. The sequences of peptides derived from MALDI-TOF mass analysis are contained within a creatine kinase B sequence. Table 1). Result from 2-D electrophoresis is also similar to physicochemical parameter of creatine kinase B. Recently, Kim et al. (2000) demonstrated that creatine kinase B identified as a ROS-sensitive molecule and Cys283 of the enzyme is the major site of modification by BIAM at pH 6.5 as well as the site of oxidation by H2O2 in PC12 cells (Kim et al., 2000). Effect of oxidation on creatine kinase B activity Several lines of evidence indicate that ROS can oxidize active cysteine residue in proteins to regulate their activity (Kim et al., 2000; Rhee et al., 2000). To investigate the effect of oxidation on creatine kinase B activity, the activity assay of total cytosolic fraction from cancer and normal tissues was performed, respectively. Although normal and cancer tissues contain similar amount of creatine kinase B, the enzyme activity from cytosolic fraction of normal tissue is higher than that of cancer tissues (Fig. 3). HeLa cells contain high creatine kinase B activity and expression level. Creatine kinase B isozymes were known to contain a single active sulfhydryl group (Cys-283 in cytosolic CK) (Furter et al., 1993). The cysteine residue is essential for catalytic activity. The result suggests that the regulation of creatine kinase B by ROS might be involved in energy metabolism in cancer cells. Next we explored whether the oxidatively inactivated creatine kinase B by ROS is restored by the addition of reducing agent, DTT. Reduction of crude extracts from Hyun Choi et al. 415 Table 1. MALDI-TOF mass spectrometry identification of the peptide. Mass (m/z) (theoretical) Mass (m/z) (observed) Difference (theoretical-observed) Amino acid residues 1030.547 1231.608 1302.718 1585.830 1656.825 1863.964 1963.923 2120.024 1030.547 1231.604 1302.722 1585.873 1656.904 1864.024 1963.949 2120.050 0.000 0.004 -0.005 -0.043 -0.079 -0.059 -0.027 -0.026 LLIEMEQR366 DLFDPIIEDR96 33 VLTPELYAELR43 157 LAVEALSSLDGDLAGR172 224 TFLVWVNEEDHLR236 342 LGFSEVELVQMVVDGVK358 321 GTGGVDTAAVGGVFDVSNADR341 320 RGTGGVDTAAVGGVGGVFDVSNADR341 359 87 A B Fig. 4. Effect of DTT on creatine kinase B activity in cervical tissues. Tissue extracts were preincubated with 500 µM DTT for 10 min on ice, and measured kinase activity according to the manufacturer’s instructions (Diagnostica Merck). Fig. 3. Creatine kinase activity in extract of normal and tumor tissues, respectively. A. Tissues were homogenized with phosphate-buffered saline (pH 7.4) containing aprotinin (1 µg/ml), leupeptin (1 µg/ml), 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (1 µg/ml) and then were centrifuged at 40,000 rpm for 1 h at 4°C. The creatine kinase activity of supernatants was measured using CK kit according to the manufacturer’s instructions (Diagnostica Merck). B. Immunoblot analysis of cell extracts with antibody against creatine kinase B or actin. Tissue extracts were subjected to SDS-PAGE on 10% gel, transferred to a nitrocellulose membrane, and incubated with antibody to creatine kinase B or actin. cervical cancer tissues with DTT did not affect the creatine kinase B activity (Fig. 4). It is likely that the failure of activation by DTT suggests the irreversible oxidation of creatine kinase B by ROS in tumor cells. Cys sulfhydryl groups (-SH) oxidized to sulfenic (-SOH) or disulfide (-S-S-) can be reduced back to sulfhydryl groups by the reduction of DTT. Further oxidation of sulfenic acid to sulfinic (-SOOH) and sulfonic acid (-SOOOH) by ROS such as hydrogen peroxide has been well characterized (Davis et al., 1981). Due to the irreversible nature of such oxidation, proteins underwent that kind of severe oxidation become inactivated and their activity cannot be recovered even by the treatment of strong reductant such as DTT. Creatine kinase B is expressed in a wide range of tissues, such as brain, cervix, kidney, and prostate. Several lines of evidence indicate that the enzyme might be involved in the metabolic events that take place in oncogenic activation (Lillie et al., 1993). The highest CK levels were found in mesotheliomas, brain tumors, and small cell lung cancer cells, whereas the lowest levels are observed in kidney cancer, lymphomas, and non-small cell lung cancer cells. The tumors that show low CK activity were not able to form colonies in soft agar. This result suggested that the enzyme might be required for tumor establishment. It is known that the infection of papilloma virus to normal cervical cells is a major risk factor for cellular trans- 416 ROS-Target Molecules formation. It has been reported that E6 protein from papilloma virus promotes the degradation of p53 tumor suppressor protein resulting in increased creatine kinase B expression (Tamir et al, 1998). The pathway suggested that the enzyme is an important factor for the metabolic process after oncogenic process. In our results, creatine kinase B expression in tumor tissues comparing to normal tissues is very similar or lower rather than overexpression. It is well known that growth of cancer cells is very rapid compared to that of normal cells, because cancerous cells produce various growth factors and angiogenesis-related factors. These factors stimulated the generation of ROS in cells, resulting in the production of large amounts of ROS in several human cancer cell lines (Shackelford et al., 2000). Furthermore, activity of antioxidants such as catalase and superoxide dismutase in cancer cells is very low compared to normal cells (Mates et al., 1999). Although many efforts for the understanding of ROS function have been done, precise role of ROS in cancer cells is still unclear. Our results suggest that intracellular generated ROS in tumor cells inhibit creatine kinase activity resulting in the regulation of energy metabolism. However, molecular mechanisms that ROS and creatine kinase B are integrated into a physiological signal leading to the cellular transformation remain to be elucidated. Acknowledgments We thank Drs. Chae HZ and Ryu SH for valuable discussions. 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