Amplified genes in cancer cells reside on extrachromosomal double minutes (DMs) or chromosomal homogeneously staining regions (HSRs). We used a plasmid bearing a mammalian replication initiation region to model gene amplification. Recombination junctions in the amplified region were comprehensively identified and sequenced. The junctions consisted of truncated direct repeats (type 1) or inverted repeats (type 2) with or without spacing. All of these junctions were frequently detected in HSRs, whereas there were few type 1 or a unique type 2 flanked by a short inverted repeat in DMs. The junction sequences suggested a model in which the inverted repeats were generated by sister chromatid fusion. We were consistently able to detect anaphase chromatin bridges connected by the plasmid repeat, which were severed in the middle during mitosis. De novo HSR generation was observed in live cells, and each HSR was lengthened more rapidly than expected from the classical breakage/fusion/bridge model. Importantly, we found massive DNA synthesis at the broken anaphase bridge during the G1 to S phase, which could explain the rapid lengthening of the HSR. This mechanism may not operate in acentric DMs, where most of the junctions are eliminated and only those junctions produced through stable intermediates remain.
A Frequent association of side effects has been a long-standing dilemma in clinical glucocorticoid therapy. Recent progress in molecular biology of glucocorticoid hormone action, however, has prompted researchers to tackle the dissociation of side effects and therapeutic effects based on the assumption that selective modulation of its receptor function could be achieved by as yet unknown compounds. Already a number of selective modulators of the glucocorticoid receptor (SGRMs) have been reported, and certain compounds have dissociating characteristics in vivo. We have addressed ligand-dependent modular recruitment of AF-1 function using a phenylpyrazologlucocorticoid cortivazol, suggesting the possibility of developing tissue-specific SGRMs. It should also be emphasized that SGRMs do not always have a steroid structure.
Reduction-oxidation (redox) regulation has been implicated in the activation of the transcription factor NF-κB. However, the significance and mechanism of the redox regulation remain elusive, mainly due to the technical limitations caused by rapid proton transfer in redox reactions and by the presence of many redox molecules within cells. Here we establish versatile methods for measuring redox states of proteins and their individual cysteine residues in vitro and in vivo, involving thiol-modifying reagents and LC-MS analysis. Using these methods, we demonstrate that the redox state of NF-κB is spatially regulated by its subcellular localization. While the p65 subunit and most cysteine residues of the p50 subunit are reduced similarly in the cytoplasm and in the nucleus, Cys-62 of p50 is highly oxidized in the cytoplasm and strongly reduced in the nucleus. The reduced form of Cys-62 is essential for the DNA binding activity of NF-κB. Several lines of evidence suggest that the redox factor Ref-1 is involved in Cys-62 reduction in the nucleus. We propose that the Ref-1-dependent reduction of p50 in the nucleus is a necessary step for NF-κB activation. This study also provides the first example of a drug that inhibits the redox reaction between two specific proteins. Reduction-oxidation (redox) regulation has been implicated in the activation of the transcription factor NF-κB. However, the significance and mechanism of the redox regulation remain elusive, mainly due to the technical limitations caused by rapid proton transfer in redox reactions and by the presence of many redox molecules within cells. Here we establish versatile methods for measuring redox states of proteins and their individual cysteine residues in vitro and in vivo, involving thiol-modifying reagents and LC-MS analysis. Using these methods, we demonstrate that the redox state of NF-κB is spatially regulated by its subcellular localization. While the p65 subunit and most cysteine residues of the p50 subunit are reduced similarly in the cytoplasm and in the nucleus, Cys-62 of p50 is highly oxidized in the cytoplasm and strongly reduced in the nucleus. The reduced form of Cys-62 is essential for the DNA binding activity of NF-κB. Several lines of evidence suggest that the redox factor Ref-1 is involved in Cys-62 reduction in the nucleus. We propose that the Ref-1-dependent reduction of p50 in the nucleus is a necessary step for NF-κB activation. This study also provides the first example of a drug that inhibits the redox reaction between two specific proteins. The redox states of cysteine residues, which can change reversibly within cells, often greatly influence the various properties of proteins, such as protein stability, chaperone activity, enzymatic activity, and protein structure (1Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C. Cell. 1999; 98: 217-227Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 2Jakob U. Muse W. Eser M. Bardwell J.C. Cell. 1999; 96: 341-352Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 3Mannick J.B. Hausladen A. Liu L. Hess D.T. Zeng M. Miao Q.X. Kane L.S. Gow A.J. Stamler J.S. Science. 1999; 284: 651-654Crossref PubMed Scopus (706) Google Scholar, 4Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 5Eu J.P. Sun J., Xu, L. Stamler J.S. Meissner G. Cell. 2000; 102: 499-509Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). It has also been suggested that several transcription factors bind to their cognate sites in a redox-regulated manner. Well characterized cases include the prokaryotic transcription factors SoxR and OxyR, which function as oxidative stress sensors, their DNA binding activated through oxidation of critical cysteine residues (6Hidalgo E. Ding H. Demple B. Cell. 1997; 88: 121-129Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 7Zheng M. Aslund F. Storz G. Science. 1998; 279: 1718-1721Crossref PubMed Scopus (979) Google Scholar). In most cases, however, the roles and mechanisms of redox regulation are not fully defined because it is difficult to monitor the alteration of redox states of proteins mainly due to the rapid proton transfer in redox reactions. A few have directly quantified the redox state of cysteine clustered with iron or amounts of oxidized cysteines using physicochemical or biochemical techniques (3Mannick J.B. Hausladen A. Liu L. Hess D.T. Zeng M. Miao Q.X. Kane L.S. Gow A.J. Stamler J.S. Science. 1999; 284: 651-654Crossref PubMed Scopus (706) Google Scholar, 8Demple B. Methods. 1997; 11: 267-278Crossref PubMed Scopus (21) Google Scholar, 9Ding H. Demple B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8445-8449Crossref PubMed Scopus (115) Google Scholar), but these methods cannot describe the whole picture of redox states of a protein and are not widely applicable to other proteins. Therefore, most researchers have chosen an indirect way of using cysteine-substitution mutant proteins (3Mannick J.B. Hausladen A. Liu L. Hess D.T. Zeng M. Miao Q.X. Kane L.S. Gow A.J. Stamler J.S. Science. 1999; 284: 651-654Crossref PubMed Scopus (706) Google Scholar, 4Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 5Eu J.P. Sun J., Xu, L. Stamler J.S. Meissner G. Cell. 2000; 102: 499-509Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 7Zheng M. Aslund F. Storz G. Science. 1998; 279: 1718-1721Crossref PubMed Scopus (979) Google Scholar). NF-κB 1The abbreviations used are: NF-κB, nuclear factor κB; IκB, inhibitor of NF-κB; Ref-1, redox factor-1; Trx, thioredoxin; TrxR, thioredoxin-reductase; AP-1, activator protein-1; E3330, (2E)-3-[5-(2,3-dimethoxy-6-methyl-1, 4-benzoquinoyl)]-2-nonyl-2-propenoic acid; PMA, phorbol 12-myristate 13-acetate; F5M, fluorescein-5-maleimide; NEM, N-ethyl-maleimide; DTT, dithiothreitol; TCEP, tris-(2-carboxyethyl)phosphine-hydrochloride; EMSA, electrophoretic mobility shift assay; LC-MS, liquid chromatography mass spectrometry; MI, mass intensity; RNP, ratio of NEM-labeled peptide is a eukaryotic transcription factor that regulates a wide variety of genes involved in immune function and development (10Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2935) Google Scholar). NF-κB is composed of two subunits, p50 and p65, both of which are members of the Rel family of transcription factors. NF-κB normally exists in the cytoplasm, forming an inactive ternary complex with the inhibitor protein IκBα. Following the application of appropriate stimuli, NF-κB is released from IκBα and translocates into the nucleus, where it binds DNA and activates transcription of target genes. Mechanisms of NF-κB activation have been extensively studied; however, it is largely unknown if, and how, the DNA binding step is activated in cells. Some reports have described that the DNA binding activity of NF-κB is regulated by redox potential in vitro(11Toledano M.B. Leonard W.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4328-4332Crossref PubMed Scopus (579) Google Scholar, 12Matthews J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar, 13Hayashi T. Ueno Y. Okamoto T. J. Biol. Chem. 1993; 268: 11380-11388Abstract Full Text PDF PubMed Google Scholar). Recently, we reported an antiinflammatory drug that could allow us to solve this issue. The synthetic quinone derivative, (2E)-3-[5-(2,3-dimethoxy-6-methyl-1, 4-benzoquinoyl)]-2-nonyl-2-propenoic acid (E3330), is a novel anti-NF-κB drug that specifically suppresses DNA binding activity of NF-κB but not those of other inflammatory transcription factors, such as activator protein-1 (AP-1) and nuclear factor of activated T cell (NF-AT), in phorbol 12-myristate 13-acetate (PMA)- stimulated Jurkat cells (14Hiramoto M. Shimizu N. Sugimoto K. Tang J. Kawakami Y. Ito M. Aizawa S. Tanaka H. Makino I. Handa H. J. Immunol. 1998; 160: 810-819PubMed Google Scholar). Interestingly, E3330 did not affect the DNA binding activity of purified NF-κB or several steps of NF-κB activation, including IκB degradation, p65 phosphorylation, and nuclear translocation of NF-κB (14Hiramoto M. Shimizu N. Sugimoto K. Tang J. Kawakami Y. Ito M. Aizawa S. Tanaka H. Makino I. Handa H. J. Immunol. 1998; 160: 810-819PubMed Google Scholar). This led us to hypothesize that E3330 may target an unknown nuclear factor that stimulates the DNA binding activity of NF-κB. Consistent with this assumption, we purified and identified redox factor-1 (Ref-1) from Jurkat nuclear extracts as an E3330-binding protein that enhances DNA binding activity of NF-κB in an E3330-sensitive manner (15Shimizu N. Sugimoto K. Tang J. Nishi T. Sato I. Hiramoto M. Aizawa S. Hatakeyama M. Ohba R. Hatori H. Yoshikawa T. Suzuki F. Oomori A. Tanaka H. Kawaguchi H. Watanabe H. Handa H. Nat. Biotechnol. 2000; 18: 877-881Crossref PubMed Scopus (231) Google Scholar). Considering the specificity and affinity of the E3330-Ref-1 interaction (15Shimizu N. Sugimoto K. Tang J. Nishi T. Sato I. Hiramoto M. Aizawa S. Hatakeyama M. Ohba R. Hatori H. Yoshikawa T. Suzuki F. Oomori A. Tanaka H. Kawaguchi H. Watanabe H. Handa H. Nat. Biotechnol. 2000; 18: 877-881Crossref PubMed Scopus (231) Google Scholar), E3330 is likely a selective inhibitor of Ref-1 activity. Ref-1 is a nuclear protein that was originally identified as a DNA repair enzyme with an apurinic (AP)-endonuclease activity (16Demple B. Herman T. Chen D.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11450-11454Crossref PubMed Scopus (478) Google Scholar, 17Robson C.N. Hickson I.D. Nucleic Acids Res. 1991; 19: 5519-5523Crossref PubMed Scopus (293) Google Scholar, 18Xanthoudakis S. Miao G. Wang F. Pan Y.C. Curran T. EMBO J. 1992; 11: 3323-3335Crossref PubMed Scopus (826) Google Scholar). Ref-1 has been reported to stimulate activities of AP-1 as well as other transcription factors including p53 and hypoxia inducible factor-1α (18−24). In case of AP-1, it was well investigated by using in vitro cysteine point mutagenesis analysis that DNA binding activity of AP-1 is stimulated by the reducing activity of Ref-1. We have recently shown that Ref-1 enhances the DNA binding activity of NF-κB in vitro as well as NF-κB-dependent transcriptional activation in vivo in an E3330-sensitive manner (15Shimizu N. Sugimoto K. Tang J. Nishi T. Sato I. Hiramoto M. Aizawa S. Hatakeyama M. Ohba R. Hatori H. Yoshikawa T. Suzuki F. Oomori A. Tanaka H. Kawaguchi H. Watanabe H. Handa H. Nat. Biotechnol. 2000; 18: 877-881Crossref PubMed Scopus (231) Google Scholar). However, few analyses have been performed to determine if these processes involve redox reaction, and how these processes occur in cells. The existence of many redox regulatory molecules, such as thioredoxin (Trx), thioredoxin-reductase (TrxR), glutaredoxin, nucleoredoxin, and glutathione, makes it difficult to explore the specific redox regulation between proteins in eukaryotic cells. In order to overcome the difficulties associated with redox studiesin vivo, we utilized E3330 as a useful tool because E3330 is a Ref-1-specific inhibitor that specifically inhibits NF-κB activity but not the other transcription factors potentially regulated by Ref-1 as mentioned above. Using irreversible thiol-modifying reagents and LC-MS analysis, we evaluated here the redox states of NF-κB during its activation step in vivo. We provide evidence that Cys-62 of NF-κB p50 is selectively reduced by Ref-1 in the nucleus and that this reduction is a prerequisite for NF-κB activation in vivo. Thiol-modifying reagents, fluorescein-5-maleimide (F5M) and N-ethylmaleimide (NEM), were obtained from Molecular Probes (Eugene, OR) and Nacalai Tesque (Kyoto, Japan), respectively, dissolved in dimethylformamide to a final concentration of 200 mm and stored at −20 °C until use. LysC was obtained from Wako (Osaka, Japan). pET14b (Novagen)-based expression plasmids of full-length human p50 and Ref-1 have previously been described (15Shimizu N. Sugimoto K. Tang J. Nishi T. Sato I. Hiramoto M. Aizawa S. Hatakeyama M. Ohba R. Hatori H. Yoshikawa T. Suzuki F. Oomori A. Tanaka H. Kawaguchi H. Watanabe H. Handa H. Nat. Biotechnol. 2000; 18: 877-881Crossref PubMed Scopus (231) Google Scholar). An expression vector of full-length human Trx was prepared by inserting the entire open reading frame amplified by polymerase chain reaction into the NdeI-BamHI sites of pET14b (Novagen). Each His-tagged protein was expressed inEscherichia coli strain BL21(DE3) and purified under native conditions using Ni-NTA-agarose beads (Qiagen) as instructed by the manufacturer. Purified proteins were dialyzed against dialysis buffer (HEPES pH 7.9, 100 mm KCl, 10% glycerol, 0.2 mm EDTA). For oxidation, p50 was further treated with 10 mm diamide for 30 min at 16 °C. For reduction, Ref-1 and Trx were treated with 0.3 mmTris-(2-carboxyethyl)phosphine-hydrochloride (TCEP) for 10 min at 37 °C. Diamide and TCEP were removed by an additional Ni-NTA agarose chromatography, and purified proteins were dialyzed against dialysis buffer. Native TrxR purified from bovine (4 unit/mg) was purchased from American Diagnostica Inc. (Greenwich, Connecticut). Recombinant proteins were mixed in 100 μl of reaction buffer (20 mm HEPES pH 7.9, 100 mm KCl, 10% glycerol, 0.2 mm EDTA). As a carrier protein, 100 μg of carbonic anhydrase was included in the reactions to help stabilize the recombinant proteins and to normalize the difference in protein concentrations. After incubation for various times at 37 °C, the reactions were used in electrophoretic mobility shift assay (EMSA) or fluorescence assays. The DNA probe containing an NF-κB binding sequence was prepared as described (14Hiramoto M. Shimizu N. Sugimoto K. Tang J. Kawakami Y. Ito M. Aizawa S. Tanaka H. Makino I. Handa H. J. Immunol. 1998; 160: 810-819PubMed Google Scholar). Reactions (8 μl) containing 2–4 μl of recombinant proteins, 10 μg of bovine serum albumin, 1 ng of32P-labeled DNA probe, and 300 ng of poly (dI-dC) in a buffer containing 20 mm HEPES pH 7.9, 0.1 mKCl, 0.2 mm EDTA, 15% glycerol, and 0.1% Nonidet P-40 were incubated for 15 min at 4 °C. Reactions were then subjected to native 4% polyacrylamide gel electrophoresis (PAGE) as described (14Hiramoto M. Shimizu N. Sugimoto K. Tang J. Kawakami Y. Ito M. Aizawa S. Tanaka H. Makino I. Handa H. J. Immunol. 1998; 160: 810-819PubMed Google Scholar). To label reduced cysteines in vitro, protein samples were incubated with 0.3 mm F5M for 5 min on ice. F5M labeling was stopped by the addition of 100 mm dithiothreitol (DTT). Proteins were acetone-precipitated, suspended in SDS sample buffer, and separated by SDS-PAGE. Fluorescence intensities of the proteins were quantified by scanning gels at an excitation wavelength of 490 nm using a fluorescence-image analyzer FLA2000 (Fuji Film). Data were analyzed with the Image Gauge software (Fuji Film). For fluorescence labeling in vivo, J-50 cells cultured under different conditions were incubated in medium containing 10 mm F5M for 5 min at 37 °C. All the subsequent steps were performed at 4 °C. After washing twice with phosphate-buffered saline containing 10 mm F5M, cells were harvested by centrifugation, and pellets were resuspended in 8 packed cell volumes of hypotonic buffer containing 10 mm HEPES (pH 7.9), 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 0.5 mm phenylmethylsulfonyl fluoride, and 1 mmF5M. After a 10-min incubation period, Nonidet P-40 was added to a final concentration of 0.6%, and tubes were mixed vigorously for 10 s. Tubes were centrifuged for 5 s at 10,000 rpm, and supernatants (cytoplasmic fractions) were removed to separate vials. Nuclei were resuspended in 4 packed cell volumes of high salt buffer containing 20 mm HEPES (pH 7.9), 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, 0.5 mmphenylmethylsulfonyl fluoride, and 1 mm F5M. After a 20-min incubation period, tubes were centrifuged for 2 min at 15,000 rpm, and supernatants (nuclear fractions) were saved. F5M labeling reactions were stopped by adding 100 mm DTT to the cytoplasmic and nuclear fractions. Following immunoprecipitation using an anti-FLAG M2 affinity gel, eluates were subjected to SDS-PAGE, and fluorescence intensities of proteins were analyzed as described above. NEM labeling was performed similarly to F5M labeling, except that the experiments were performed with 1 mm (in vitro) and 20 mm(in vivo) NEM. Proteins were acetone-precipitated, resuspended in a small volume of 8 m urea, and digested overnight at 37 °C with LysC in digestion buffer containing 25 mm Tris-HCl (pH 9.0), 2 m urea, and 1 mm DTT. The high-performance liquid chromatography (HPLC)-coupled electron spray ionization time-of-flight MS system Mariner TK5000 (PerkinElmer) was used to analyze protein digests. Protein digests were initially separated on a protein/peptide ODS column with an inner diameter of 0.5 mm (PerkinElmer), using a linear gradient of 5–55% acetonitrile plus 1% formic acid at a flow rate of 5 μm/min. Peptide masses were analyzed online, using instrument settings for positive ion polarity at 3800 V spray tip potential, 130 V nozzle potential, 11.5 V skimmer 1 potential, 7.6 V quadrupole DC potential, 0.6 V deflection voltage, −37.5 V einzel lens potential, 700 V quadrupole RF voltage, 140 °C quadrupole temperature, 140 °C nozzle temperature, 700 V push pulse potential, 278 V pull pulse potential, 5 V pull bias potential, 4000 V acceleration potential, 1400 V reflector potential, and 2150 V detector voltage. RNP is defined as (MIred)/[(MIred) + (MIox)] × 100 (%), where MIred and MIox represent the mass intensities of NEM-labeled (MIred) and -unlabeled (MIox) peptides. MIred partially comprises the mass intensity of hydrolytic-NEM-labeled peptide (∼10% total NEM). For a single peptide, a series of RNP values were determined with respect to the number of protons within the peptide (1-5H+). The RNP values obtained from peptides protonated differently were usually fairly similar. Figs. 2 B and 4 B show the averages of three or more RNP values.Figure 4The reduced form of Cys-62 is critical for DNA binding by p50. Oxidized forms of p50 wild type (WT) and its mutants were incubated with increasing concentrations of TCEP for 1 h at 37 °C. Their DNA binding activities and redox states were analyzed by EMSA and fluorescence assays. C62S, C119S, C262S, and C273S represent p50 mutants carrying cysteine to serine substitutions at positions 62, 119, 262, and 273.View Large Image Figure ViewerDownload (PPT) pCAGGS/FLAG-His-p50 (5 μg) and pCV/neo (0.5 μg) were co-transfected into Jurkat cells (5 × 105cells) by electroporation. Cells were plated onto dishes containing RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum, 0.8% methylcellulose, and 300 μg/ml G418. After 3–4 weeks, G418-resistant clones were isolated, and expression of FLAG-His/p50 was examined by Western blotting using antibodies against p50 and FLAG. A FLAG-His/p50-expressing cell line of J-50 was maintained in RPMI 1640 containing 10% fetal calf serum and 200 μg/ml G418. To measure the redox state of proteins, F5M, a maleimide derivative with a fluorescein group was employed as a thiol-modifying reagent. Maleimides irreversibly modify reduced, but not oxidized, cysteines rapidly at 4 °C at the physiological pH range (pH 6.5–8.0) where normal redox reactions can barely proceed. The thiol selectivity of maleimides is higher than those of other thiol-modifying reagents, such as iodoacetamides (25Richard P.H. Handbook of fluorescent probes and research chemicals. 6th Ed. Molecular probes, Inc., Eugene, OR1998: 47-62Google Scholar). Using F5M, the amount of proteins in the reduced state can be quantified as fluorescence intensity on an SDS-polyacrylamide gel (Fig.1 A). To assess this F5M-mediated thiol-trapping system, we initially examined a well characterized redox exchange reaction between Trx and TrxR in vitro (26Holmgren A. Bjornstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (820) Google Scholar). In the presence of NADPH, TrxR specifically reduces Trx at the two conserved cysteine residues, Cys-32 and Cys-35. A constant amount of oxidized Trx was incubated with increasing concentrations of TrxR for various times at 37 °C, and reactions were processed for F5M labeling at 4 °C. As shown in Fig. 1,B and C, the fluorescence intensity of Trx increased with time, depending on the concentration of TrxR. When both Cys-32 and Cys-35 of Trx were mutated to serine residues, fluorescence intensity did not increase to a detectable level over time. These results strongly suggest that the F5M labeling reaction is specific and permits the quantitative measurement of the protein redox states. We have previously shown using EMSA that Ref-1 stimulates DNA binding activity of p50/p50 homodimers and p50/p65 heterodimers, but not p65/p65 homodimers (15Shimizu N. Sugimoto K. Tang J. Nishi T. Sato I. Hiramoto M. Aizawa S. Hatakeyama M. Ohba R. Hatori H. Yoshikawa T. Suzuki F. Oomori A. Tanaka H. Kawaguchi H. Watanabe H. Handa H. Nat. Biotechnol. 2000; 18: 877-881Crossref PubMed Scopus (231) Google Scholar). These findings suggest that Ref-1 activates DNA binding of NF-κB by affecting the p50 subunit. To directly determine whether Ref-1 reduces p50, we used the above F5M labeling method. Oxidized and reduced forms of p50 were prepared by incubating recombinant p50 with the oxidizing reagent diamide and the reducing reagent TCEP, as described under “Experimental Procedures.” As shown in Fig.2 A, the reduced form of p50 (lane 1) but not the oxidized form (lane 2) strongly bound to the DNA probe containing an NF-κB binding site. When oxidized p50 was incubated with increasing amounts of recombinant Ref-1 (lanes 3–5), Trx (lanes 6–7), or TrxR (lanes 9–11) for 1 min prior to EMSA or F5M labeling, only Ref-1 reduced p50 and reactivated its DNA binding activity. Even in the presence of NADPH, TrxR was unable to reduce p50 under these conditions (lane 12). The level of reduction correlated well with the level of DNA binding. To substantiate that redox exchange reactions occur between Ref-1 and p50, the redox states of both Ref-1 and p50 were monitored at different time points after mixing Ref-1 and oxidized p50. As shown in Fig.1 B, after 1 min, p50 was reduced to ∼50% of the level achieved after a 60-min incubation. Conversely, fluorescence intensity of Ref-1 was decreased to ∼60% in 1 min. We thus concluded that Ref-1 directly and rapidly reduces p50 by a redox exchange reaction. It has been suggested, but not proven, that inhibition of Ref-1 by E3330 occurs through the block of its redox activity (15Shimizu N. Sugimoto K. Tang J. Nishi T. Sato I. Hiramoto M. Aizawa S. Hatakeyama M. Ohba R. Hatori H. Yoshikawa T. Suzuki F. Oomori A. Tanaka H. Kawaguchi H. Watanabe H. Handa H. Nat. Biotechnol. 2000; 18: 877-881Crossref PubMed Scopus (231) Google Scholar). Consistent with the previous results, E3330 suppressed Ref-1-dependent activation of DNA binding by oxidized p50 in a dose-dependent manner (Fig. 2 C, upper panel, lanes 1–6). Similarly, E3330 also inhibited the Ref-1-dependent reduction of oxidized p50 (lower panel). In contrast, E3330 did not affect redox states or DNA binding activity of the reduced form of p50. From these results, we conclude that E3330 inhibition is mediated by the block of Ref-1 redox activity. The above method is a convenient way to measure changes in the redox status of proteins. However, it may not be suitable in cases where proteins contain multiple reduced cysteines of which only a subset is subject to redox regulation because F5M labeling of non-target cysteines may cause a high basal level of fluorescence. Human p50 has seven cysteine residues at positions 62, 88, 119, 124, 162, 262, and 273. To identify cysteine residues of p50 that are reduced by Ref-1, we improved the above thiol-trapping method. The modified method comprises three steps: 1) irreversible labeling of reduced cysteines with another maleimide derivative, NEM, 2) digestion of NEM-labeled proteins by Lys-C endopeptidase, and 3) LC-MS analysis for the identification of NEM-labeled (reduced) and -unlabeled (oxidized) peptides. NEM was employed because F5M-labeled peptides could not be fully recovered from liquid chromatography, probably due to the hydrophobicity as a result of F5M labeling (data not shown). From the mass intensities of NEM-labeled and -unlabeled peptides (MIred and MIox), the fraction of reduced cysteine residues was estimated using the equation: MIred/(MIred + MIox) × 100 (%) or the ratio of NEM-labeled peptide (RNP). From the results shown in Fig. 2 A, we presumed that residues reduced by TCEP and Ref-1 should be functionally important. Recombinant p50 previously oxidized with diamide was incubated with TCEP or Ref-1 and processed as described above. Lys-C endopeptidase digests of p50 include three peptides containing a single cysteine residue and two with two cysteine residues (TableI). Fig.3 A shows representative mass spectra of the Cys-62-containing peptide. When oxidized p50 was left untreated, a large fraction of Cys-62 appeared to be fully oxidized because only the unlabeled peptide (53-77) was detected. Prior incubation with TCEP or Ref-1 resulted in the NEM-labeled peptide (53-77-NEM), indicating that Cys-62 was reduced by these reagents. The RNPs, obtained from their mass intensities, suggest that treatment of p50 with TCEP or Ref-1 resulted in ∼60 or ∼50% reduction of Cys-62 (Fig. 3 B).Table ICysteine-containing peptides of p50 resulting from Lys-C digestionPositionsSequenceNumber of NEM labeling (NEM Mw: 125.12)Theoretical mass (Z1)6253–77QRGFRFRYVCEGPSHGGLPGAS02722.33SEK12847.45880964.4987–95ICNYVGPAK11089.62119 12401430.64118–131HCEDGICTVTAGPK11555.7721680.90162150–194VFETLEARMTEACIRGYNPGLLV04988.48HPDLAYLQAEGGGDRQLGDREK15113.60262 27202542.25253–275IVRMDRTAGCVTGGEEIYLLCDK12667.3722792.50 Open table in a new tab Similarly, all cysteine-containing peptides were identified by LC-MS analysis. RNP values for these peptides are presented in Fig.3 B. For peptides containing two cysteines, RNPs were calculated for singly and doubly labeled peptides, respectively. All the cysteine residues were reduced by Ref-1 by varying degrees; however, the reduction of Cys-62 by Ref-1 was significant and highly sensitive to E3330 inhibition. For comparison, EMSAs for an identical set of reactions were performed (Fig. 3 C). The DNA binding activity of p50 correlated well with the redox states of Cys-62, indicating that Cys-62 plays a determinant role in DNA binding. Unexpectedly, TCEP did not reduce all the cysteine residues of p50. TCEP may be capable of reducing only the exposed cysteine residues under the non-denaturing condition. Consistent with this hypothesis, structural data show that thiol groups of Cys-88 and Cys-162, which were not reduced by TCEP in our experiments, are buried in the p50 protein (27Chen F.E. Huang D.B. Chen Y.Q. Ghosh G. Nature. 1998; 391: 410-413Crossref PubMed Scopus (339) Google Scholar). The above results do not exclude the possibility that, in addition to Cys-62, other cysteines may play some role in redox regulation of p50. To address this issue, we carried out EMSA and fluorescence assays for point mutants of p50, whose cysteine residues at positions 62, 119, 262, and 273 were individually changed to serines (C62S, C119S, C262S, and C273S). Serine was chosen because it may mimic a reduced form of cysteine. Similar sets of the point mutants were previously described and analyzed by EMSA (12Matthews J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar, 28Toledano M.B. Ghosh D. Trinh F. Leonard W.J. Mol. Cell. Biol. 1993; 13: 852-860Crossref PubMed Google Scholar). As observed for wild type p50, DNA binding activities of C119S, C262S, and C273S were not detectable under oxidized conditions and were strongly stimulated by TCEP in a concentration-dependent manner (Fig.4). In sharp contrast, C62S showed strong DNA binding even in its oxidized form and was not affected by TCEP,i.e. by the redox states of the other cysteine residues. The results of EMSA are consistent with those of earlier reports (12Matthews J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar, 28Toledano M.B. Ghosh D. Trinh F. Leonard W.J. Mol. Cell. Biol. 1993; 13: 852-860Crossref PubMed Google Scholar,29Matthews J.R. Kaszubska W. Turcatti G. Wells T.N. Hay R.T. Nucleic Acids Res. 1993; 21: 1727-1734Crossref PubMed Scopus (125) Google Scholar). Taken together, it is very likely that Cys-62 alone is responsible for Ref-1-mediated reduction and activation of p50. Using these redox-monitoring methods, we investigated the possibility that the redox state of NF-κB is regulated during the activation processin vivo. We established a Jurkat cell line expressing p50 fused to FLAG and 6× His tags at the N terminus (J-50 cells) to obtain a sufficient quantity of homogenous NF-κB complex from cells. FLAG-His-p50 (FLAG-p50) behaved similarly to endogenous p50,i.e. it formed a ternary complex with p65 and IκBα in the cytoplasm, was translocated into the nucleus on PMA treatment concomitant with IκB degradation, and bound DNA as a heterodimer with p65 in a sequence-specific manner (data not shown). To visualize the overall redox state of NF-κB in cells, we modified the F5M labeling protocol as follows: J-50 cells were maintained with or without PMA for 30 min, labeled in situwith membrane-permeable F5M, lysed, an
Abstract Gene amplification plays a pivotal role in malignant transformation. Amplified genes often reside on extrachromosomal double minutes (DMs). Low‐dose hydroxyurea induces DM aggregation in the nucleus which, in turn, generates micronuclei composed of DMs. Low‐dose hydroxyurea also induces random double‐strand breakage throughout the nucleus. In the present study, we found that double‐strand breakage in DMs is sufficient for induction of DM aggregation. Here, we used CRISPR/Cas9 to introduce specific breakages in both natural and artificially tagged DMs of human colorectal carcinoma COLO 320DM cells. Aggregation occurred in the S phase but not in the G1 phase within 4 hours after breakage, which suggested the possible involvement of homologous recombination in the aggregation of numerous DMs. Simultaneous detection of DMs and the phosphorylated histone H2AX revealed that the aggregation persisted after breakage repair. Thus, the aggregate generated cytoplasmic micronuclei at the next interphase. Our data also suggested that micronuclear entrapment eliminated the DMs or morphologically transformed them into giant DMs or homogeneously staining regions (HSRs). In this study, we obtained a model explaining the consequences of DMs after double‐strand breakage in cancer cells. Because double‐strand breakage is frequently involved in cancer therapy, the model suggests how it affects gene amplification.
