[CANCER RESEARCH 60, 6688–6695, December 1, 2000] Thioredoxin Nuclear Translocation and Interaction with Redox Factor-1 Activates the Activator -1 in Response to Ionizing Radiation1

S. Jack Wei,2 Ana Botero,2 Kiichi Hirota, C. Matthew Bradbury, Stephanie Markovina, Andrei Laszlo, Douglas R. Spitz,3 Prabhat C. Goswami,3 Junji Yodoi, and David Gius4 Section of Cancer Biology, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri [S. J. W., A. B., C. M. B., S. M., A. L., D. R. S., P. C. G., D. G.], and Departments of Anesthesia [K. H.] and Biological Responses, Institute for Virus Research [J. Y.], Kyoto University Hospital, Kyoto University, Kyoto 606- 8507, Japan

ABSTRACT naling pathways (2, 8, 9). Thus, these products may function in short-term changes in cellular phenotype by modulating the expres- Thioredoxin (TRX) is a cytoplasmic, redox-sensitive signaling factor sion of specific target involved in cellular defenses against believed to participate in the regulation of nuclear transcription factors exogenous cytotoxic agents, such as IR (6, 10). mediating cellular responses to environmental stress. Activation of the activator protein (AP)-1 transcription factor is thought to be mediated in A long-standing hypothesis in radiation biology states that the part by redox-sensitive interactions between the nuclear signaling protein IR-induced activation of cellular signaling pathways is initiated at the redox factor-1 (Ref-1) and TRX. In this study, the role of TRX and Ref-1 cytoplasmic membrane (4, 11, 12). In recent years, several studies in the activation of the AP-1 complex was examined in HeLa and Jurkat have demonstrated that genes encoding growth factors and cytokines cell lines exposed to ionizing radiation (IR). After exposure to IR, nuclear involved in cytoplasmic membrane signaling are targets of IR (4, 13, levels of immunoreactive TRX increased, accompanied by an increase in 14). Because c-Fos and c-Jun are nuclear that interact to form AP-1 DNA binding activity. It was shown that a physical interaction the activated AP-1 transcription factor, they are not likely to bridge between Ref-1 and TRX occurs within the nucleus and is enhanced after the physical barrier between the cytoplasm and the nucleus in the exposure to IR. Furthermore, TRX immunoprecipitated from irradiated cells was capable of activating AP-1 DNA binding activity in nonirradi- IR-induced signaling pathway (6, 15). Instead, upstream signaling ated nuclear extracts. In addition, immunodepletion of Ref-1 from nuclear factors probably exist to pass the cytoplasmic signal into the nucleus. extracts demonstrated that the increase in AP-1 DNA binding activity One potential candidate for such a role is TRX, a small, ubiquitous, after IR was also dependent upon the presence of Ref-1 from irradiated multifunctional protein containing a redox-active /dithiol cells. Finally, the ability of both TRX and Ref-1 from irradiated cells to within the conserved active site -Cys-Gly-Pro-Cys- (16, 17). TRX, stimulate AP-1 DNA binding in nonirradiated nuclear extracts was abol- also known as adult T cell leukemia-derived factor, has both intracel- ished by chemical oxidation and restored by chemical reduction. These lular and extracellular functions and is a key regulator of cellular results indicate that, in response to IR, TRX and Ref-1 undergo changes signaling in response to various cellular stresses (18–20). Similar to in redox state that contribute to the activation of AP-1 DNA binding activity. These experiments suggest that a redox-sensitive signaling path- AP-1, TRX is an inducible factor that shows cytoprotective activity way leading from TRX to Ref-1 to the AP-1 complex participates in the against -induced apoptosis (21, 22) and exhibits auto- up-regulation of DNA binding activity in response to ionizing radiation. crine growth-promoting effects (23–25). Agents such as phorbol 12- myristate 13-acetate and IR are known to generate intracellular free ⅐ ⅐Ϫ radicals such as OH and O2 (26–28) that are believed to mediate INTRODUCTION many of their biological effects. In tissue culture cells treated with phorbol 12-myristate 13-acetate, TRX is imported into the nucleus Eukaryotic cells have evolved adaptive responses to multiple forms and forms a physical interaction with the Ref-1 gene product, Ref-1 of environmental stress by initiating genetically preprogrammed sig- (29). Ref-1 (also designated APE, HAP-1, and APEX) functions as naling pathways (1, 2). These adaptive responses include the activa- both a nuclear DNA repair enzyme and as a reversible regulator of the tion of cellular machinery involved in DNA repair, cell cycle arrest, DNA binding activity of several nuclear transcription factors, includ- apoptosis, gene induction, and lethality (3, 4). In response to a wide ing AP-1, by altering the oxidation/reduction (redox) state of specific variety of environmental stresses including ionizing radiation, a class residues located in the basic DNA binding region of these of proto-oncogenes (including c-Fos and c-Jun) referred to as imme- diate-early response genes are activated (5–7). These genes encode transcription factors (15, 30–32). One possible role for the transient nuclear transcription factors (e.g., AP-15) involved in the transmission induction of AP-1 may be the initiation of protective or reparative of inter- and intracellular information through multiple cellular sig- cellular responses to the damaging effects of cellular stressing agents, including IR (6, 10). Therefore, the passage of redox signals through a series of sulfhydryl switches from TRX to Ref-1, finally resulting in Received 5/12/00; accepted 10/3/00. The costs of publication of this article were defrayed in part by the payment of page the activation of the AP-1 transcription factor, might represent an charges. This article must therefore be hereby marked advertisement in accordance with important pathway for the transmission of radiation-induced signaling 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grants 1 K08 CA72602-01 and PO1 CA75556 (to D. G.), Grant from the cytoplasm to the nucleus. R01HL51469 (to D. R. S.), Grants RO1 CA49018 and PO1 CA75556 (to A. L.), and In vitro Ref-1 activity is induced by chemical reducing agents such Grant CA69593 (to P. G.). Grants ACS-IRG-58-010-43 and ACS RPG-00-292-01-TBE as DTT; however, in the absence of such reducing agents, other (to D. G.) were from the American Cancer Society. 2 These two authors contributed equally to this work. cytoplasmic cellular signaling factors, such as TRX, TRX reductase, 3 Present address: B180 Medical Laboratories, Free Radical and Radiation Biology and NAD(P)H are hypothesized to activate the Ref-1 protein (15, 31, Program, University of Iowa, Iowa City, IA 52242. 4 To whom requests for reprints should be addressed, at Mallinckrodt Institute of 33). These findings implicate Ref-1 as a central target protein in Radiology, Washington University School of Medicine, 4511 Forest Park Boulevard, posttranslational, redox-sensitive signaling cascades that use thiol- Suite 411, St. Louis, MO 63108. Phone: (314) 362-9781; Fax: (314) 362-9790; E-mail: containing proteins to activate specific sets of redox-sensitive proteins [email protected]. 5 The abbreviations used are: AP-1, activator protein-1; IR, ionizing radiation; TRX, that could serve to induce alterations in in response to thioredoxin; Ref-1, redox factor-1; HI, heat inactivated; EMSA, electrophoretic mobility oxidative stress. Because IR generates intracellular free radicals and shift assay; IP, immunoprecipitate; NEM, N-ethylmaleimide; CMV, cytomegalovirus; ␤-gal, ␤-galactosidase; LUC, luciferase; tk, thymidine kinase; VP-16, etoposide; ROI, irradiated cells have been shown to respond to this insult by increasing reactive oxygen intermediate. the production of NADPH via the pentose cycle (34), it seems logical 6688

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2000 American Association for Cancer Research. REDOX REGULATION OF THIOREDOXIN AND Ref-1 ACTIVATION OF AP-1 to investigate the relationship between IR and redox-sensitive signal- radiolabeled oligonucleotide (200,000 cpm of radiolabeled probe/reaction) and ing proteins in the cellular responses to IR. incubation at 25°C for 20 min. No reducing agents were added to the EMSAs The current study investigates the role of TRX and Ref-1 in the or the buffers used to make nuclear extracts. Samples were run on a 4.5% transduction of IR-induced signals from the cytoplasm to the nucleus, nondenaturing PAGE gel, dried, and exposed to a phosphorimager screen resulting in increased AP-1 DNA binding activity in HeLa and Jurkat using a STORM 840 Phosphorimager (Molecular Dynamics, Sunnyvale, CA). For immunodepletion experiments, cells were lysed by the addition of 1 ml of cells. IR-induced subcellular relocalization of TRX from the cyto- E1A lysis buffer [ELB: 50 mM HEPES (Sigma), pH 7.2, 250 mM NaCl (Fisher plasm to the nucleus in response to IR was demonstrated by Western Scientific), 2 mM EDTA (Sigma), and 0.1% NP40 (BDH Chemicals Ltd.)]. blot analysis of nuclear extracts and confirmed by indirect immuno- Cellular lysates were precleared with 25 ␮l of protein A (Santa Cruz Biotech- fluorescence cell staining. A physical interaction between TRX and nology, Inc). Immunodepletion of Ref-1 and TRX was performed immediately Ref-1 after exposure to IR was demonstrated using transient transfec- after the extracts were prepared by adding 25 ␮l of protein A (Santa Cruz tion two-hybrid assays. The addition of Ref-1 and TRX from irradi- Biotechnology, Inc.) and 5 ␮l of anti-Ref-1 antibody (polyclonal; Santa Cruz ated cells to nonirradiated nuclear extracts was shown to activate the Biotechnology, Inc.) or 5 ␮l of anti-TRX antibody (polyclonal; American DNA binding activity of AP-1. Furthermore, alterations in the redox Diagnostic, Inc.). These reactions were shaken slowly at 4°C for 2 h, and the state of TRX and Ref-1 immunoprecipitated from irradiated cells were IP was spun at 12,000 rpm for 1 min and washed in 750 ml of E1A buffer at shown to be involved in the ability of these proteins to activate AP-1 4°C three times. For the immunoprecipitation experiments where immunopre- cipitated TRX (Fig. 3B) or Ref-1 (Fig. 5B) was added to various cellular DNA binding activity. Immunodepletion of Ref-1 from nuclear ex- extracts, the immunoprecipitated complex was added to 20 ␮g of EMSA tracts showed inhibition of inducible AP-1 DNA binding activity in cellular extract for 45 min at 4°C with gentle shaking, and EMSA was response to IR. Overall, these results suggest that the molecular performed (27, 35). Chemical modification of immunoprecipitated Ref-1 and cascade leading to AP-1 activation in response to IR involves the TRX was performed by adding either diamide (80 ␮M; Sigma D-3648, 98% passage of redox signals through TRX from the cytoplasm to the pure), NEM (50 ␮M; Sigma E-3876, 98% pure), or DTT (8 mM; Sigma nucleus, followed by interaction with Ref-1, possibly through a phys- D-9163, 99% pure) to immunoprecipitation/protein A pellet for 30 min at 4°C. ical interaction between Ref-1 and TRX. All chemicals were filter sterilized and used without further purification. To remove the free residual chemicals from the immunoprecipitation reaction, the pellets were washed eight times in E1A buffer at 4°C. Briefly, for each wash MATERIALS AND METHODS 750 ml of E1A buffer were added to the immunoprecipitation/protein A pellet, followed by gentle shaking by tapping the Eppendorf tube against the coun- Cell Culture and IR Conditions. HeLa (human cervical carcinoma) cells tertop for 30 s, followed by spinning at 12,000 rpm for 1 min. The immuno- were grown in modified Eagle’s medium ␣ supplemented with 10% HI calf precipitation/protein A pellet was added to 20 ␮g of EMSA cellular extract for serum, Jurkat cells (human leukemia cell line) were grown in RPMI 1640 45 min at 4°C with gentle shaking, and EMSAs were performed (27, 35). supplemented with 5% HI fetal bovine serum, and Cos-1 (African green Indirect Immunofluorescence Cell Staining. Cells were grown on cov- monkey kidney) cells were grown in DMEM supplemented with 10% HI FCS erslips in 60-mm culture plates and fixed with 3.7% paraformaldehyde in PBS with penicillin and streptomycin (100 units/ml). Cells were grown in a humid- containing 10% FCS for 20 min at room temperature, followed by permeabi- lization for 10 min using 0.2% (wt/vol) Triton X-100 in PBS. After incubation ified 5% CO2 atmosphere at 37°C. Prior to irradiation, cells were serum starved for 48 h with 1% HI calf or fetal bovine serum and irradiated with 10 (1 h) with primary antibodies for TRX (American Diagnostica) slides were Gy using unfiltered X-rays from a GE Maxitron 250 kVp X-ray machine incubated (1 h) with either dye-labeled antimouse or antirabbit IgG. The slides containing an enclosed incubator. Control, nonirradiated cells were placed into with stained, and cells were mounted in 90% glycerol with 1 mg/ml p- a similar incubator. phenylenediamine. Cells were examined using a confocal microscope, MRC Nuclear and Cytoplasm Subcellular Fractionation. Cells at various time 600 (Bio-Rad Laboratories, Hercules, CA). points after exposure to IR were washed twice with PBS, scraped with 750 ␮l Transient Cotransfections and Mammalian Two-Hybrid Assay. A of PBS-2.5 mM EDTA, and centrifuged at 14,000 rpm for 2 min at 4°C. The cDNA of TRX fused in-frame with the transcription factor VP-16 downstream cell pellet was suspended in 400 ␮l of buffer A. After incubating for 15 min of the CMV promoter (pCMV-TRX-VP-16) and a cDNA of Ref-1 fused on ice, 25 ␮l of 10% NP40 were added. The cellular suspension was vortexed in-frame with the DNA binding domain of Gal-4 (pCMV-Ref-1-Gal-4) were 6 for 10 quick mixings and centrifuged at 14,000 rpm for 5 min at 4°C. The used (29). Cos-1 cells were plated at 2 ϫ 10 cells per 100-mm plate, serum supernatant (cytoplasmic fraction) was transferred to a new microcentrifuge starved (0.5% FCS) for 8 h, and transfected via calcium phosphate precipita- tube, and the supernatant and cell pellets were stored at Ϫ80°C overnight. The tion. In each transfection, 2 ␮g of pCMV-TRX-VP-16 and/or pCMV-Ref-1- nuclear pellet was thawed on ice for 15 min and suspended in 60 ␮lof Gal-4 were transfected with the reporter construct, ptk-3x-Gal-4-LUC contain- extraction buffer [10 mM HEPES (pH 7.2), 422 mM NaCl, 0.5 mM phenyl- ing three copies of the Gal-4 binding site upstream of the LUC gene in ␮ ␤ methylsulfonyl fluoride, 0.1 mM EGTA, and 5.3% glycerol], incubated at 4°C ptk-LUC (29). As an internal control, 1 gofthe -gal expression plasmid for 30 min, and spun for 10 min at 14,000 rpm. The supernatant (nuclear (pCMV-␤-gal) was used. Transfected cells were exposed to 10 Gy of IR 36 h fraction) was transferred to a new microcentrifuge tube and stored at Ϫ80°C. after transfection and harvested after 10 h. LUC activity was determined using Verification of the subcellular fractions was determined by immunoblotting a luminometer (Zylux Corp., Maryville, TN). ␤-gal activity was determined both the nuclear and cytoplasmic extracts with Ref-1, a nuclear protein, and (Promega Corp., Madison, WI), and the relative-fold induction of LUC activity I-␬B, a cytoplasmic protein (data not shown). Protein concentrations were was calculated by normalizing to the ␤-gal activity. determined using the Bradford method (per the manufacturer’s specification; Bio-Rad Laboratories, Hercules, CA). RESULTS SDS-PAGE and Western Blot Analysis. Equal amounts of protein (20 ␮g) from nuclear or cytoplasmic cellular extracts were mixed with 5ϫ Induction of AP-1 DNA Binding Activity after Irradiation. Laemmli lysis buffer (3) and boiled for 5 min. Samples were separated on c-Fos and c-Jun are members of a multigene family consisting of an denaturing SDS-polyacrylamide gels and transferred to nitrocellulose filter array of heterodimeric protein complexes that bind the specific AP-1 paper using a semidry apparatus (Owl Scientific Plastics, Inc., Portsmouth, cis-acting DNA regulatory elements. Given that AP-1 may be initiated NH). The nitrocellulose filter was prepared and developed as described pre- as part of the protective or reparative cellular responses to the dam- viously (27), and Western blot analysis was performed using an anti-TRX antibody (American Diagnostica, Inc., Greenwich, CT). aging effects of cellular stressing agents (6, 7, 9, 31), the effect of IR EMSA. EMSAs were performed as described previously using a 32P- on AP-1 DNA binding activity was examined. To investigate this, radiolabeled oligonucleotide corresponding to the consensus AP-1 DNA bind- AP-1 DNA binding activity was measured by performing EMSAs ing site (27). Nuclear extracts (10 ␮g) were incubated with poly(deoxyi- with extracts from HeLa and Jurkat cells. Serum-starved cells were nosinic-deoxycytidylic acid) for 10 min on ice, followed by the addition of irradiated with 10 Gy of IR and then allowed to recover for 1, 2, 3, 4, 6689

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2000 American Association for Cancer Research. REDOX REGULATION OF THIOREDOXIN AND Ref-1 ACTIVATION OF AP-1 and6hat37°C before the cells were harvested using a subcellular fractionation technique. An increase in DNA binding activity in HeLa cells, as compared with nonirradiated cell extracts, was first noted at 1 h (Fig. 1A, Lane 1 versus Lane 2), reached a peak induction at 2 h (Lane 3), and returned to nearly baseline at6h(Lane 6). Supershift experiments with anti-Fos and anti-Jun antibodies and competition experiments with a cold AP-1 oligonucleotide confirmed the speci- ficity of the AP-1 complex in this system (27). These experiments were repeated in Jurkat cells with a similar induction of AP-1 binding at 2–4 h after IR (Fig. 1B, Lane 1 versus Lanes 2–6). Transient assays with 2x-AP-1-LUC were also done and confirmed a similar in induc- tion in AP-1-dependent gene expression (data not shown) as has been shown by others (10). Thus, an increase in AP-1 DNA binding activity is noted in both cell lines in response to IR. Radiation Induces Translocation of TRX from the Cytoplasm to the Nucleus. Nuclear localization of TRX has been shown previ- ously in cells treated with phorbol esters, which are known to induce oxidative stress (29). IR also produces free radical intermediates, such ⅐Ϫ ⅐ as superoxide (O2 ), hydroxyl radicals ( OH), and carbon centered radicals (⅐R) that are involved in the production of oxidative stress (26, 28, 36). In addition, TRX has been shown to increase AP-1 DNA binding activity of recombinant Fos and Jun proteins, as determined in a cell-free assay system (15, 31, 32,). These results suggest that translocation of TRX into the nucleus may be one mechanism by which IR results in the induction of AP-1 DNA binding activity. Fig. 2. The effect of IR on nuclear TRX protein levels. A, Western blot analysis of HeLa and Jurkat cells were irradiated, and the nuclear and cyto- nuclear TRX protein levels in response to IR using an anti-TRX antibody. HeLa (A, upper panel) and Jurkat (A, lower panel) cells were exposed to 10 Gy of IR, incubated at 37°C, plasmic cellular extracts were prepared by a subcellular fractionation. and harvested via a subcellular fractionation method at 1, 2, 3, 4, and 6 h. Equal protein Increases in nuclear TRX protein levels in response to IR were loading was determined using a Bradford protein assay. All results are presented as relative-fold increase above baseline control (C), nonirradiated cells. B, indirect immu- observed in both HeLa and Jurkat cells (Fig. 2A). These increases nofluorescence cell staining with anti-TRX antibody. Left image, nonirradiated HeLa cell were first seen at 1 h after radiation (Fig. 2A, Lane 2), reached a with TRX localized to the cytoplasm. Right images, HeLa cells 1 h after they were maximum at roughly3h(Lane 4), and returned to nearly baseline at exposed to 10 Gy of IR, where staining for TRX is localized to the nucleus. 6h(Lane 7). In addition, HeLa cells with or without exposure to IR were examined by an indirect immunofluorescence method using antibodies raised against TRX. TRX was located predominantly in the cytoplasm of nonirradiated cells and appeared to accumulate in the nucleus 1 h after exposure to IR (Fig. 2B). Nuclear TRX was no longer seen after 6 h (data not shown). There appears to be a close temporal correlation between the nuclear localization of TRX and the induction of AP-1 DNA binding, as shown in Fig. 2A. These results indicate that IR may activate a cellular signaling pathway(s) that results in the translocation of TRX into the nucleus. TRX Induces AP-1 DNA Binding in HeLa Cells. Although the results described above demonstrate TRX nuclear translocation in response to IR, they do not establish that TRX participates in the induction of AP-1 DNA binding activity after IR. The role of TRX in IR-induced activation of AP-1 DNA binding activity was initially examined by adding increasing amounts of cytoplasmic extract (con- taining TRX) from irradiated HeLa cells to a fixed amount of nuclear extract from nonirradiated HeLa cells. The presence of c-Fos, c-Jun, and Ref-1 protein in the nuclear fraction and the presence of TRX in the cytoplasmic fraction were confirmed using immunoblotting (data not shown). As shown in Fig. 3A, increasing the amount of the cytoplasmic fraction from irradiated cells appears to increase AP-1 DNA binding activity in a dose-dependent manner up to 4 ␮gof extract. The increase in DNA binding activity appears to be saturated above this amount. These results suggest that a cytoplasmic factor from irradiated cells increases AP-1 DNA binding activity in extracts from unirradiated cells. Because TRX is predominantly a cytoplasmic protein that is translocated to the nucleus upon exposure to IR (Fig. Fig. 1. Analysis of AP-1 DNA binding activity in response to IR by EMSA. HeLa (A) and Jurkat (B) cells exposed to 10 Gy of IR and incubated at 37°C until harvested are 2A), TRX may participate in the induction of AP-1 DNA binding shown. Ten ␮g of nuclear extracts were isolated and analyzed at 1, 2, 3, 4, and6hvia after IR. EMSA using an AP-1-specific, 32P-labeled oligonucleotide. Arrows, AP-1 complex, free AP-1 oligonucleotide, and nonspecific (NS) binding. All results are presented as relative- To further investigate the role of TRX in the activation of AP-1 fold increase above control, nonirradiated cell extracts (Lane 1). DNA binding activity, TRX was immunoprecipitated from the cyto- 6690

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2000 American Association for Cancer Research. REDOX REGULATION OF THIOREDOXIN AND Ref-1 ACTIVATION OF AP-1

treated with diamide (a sulfhydryl oxidizing agent), NEM (a sulfhy- dryl alkylating agent), or DTT (a sulfhydryl reducing agent) for 30 min on ice and washed eight times to remove any residual chemical. The effects of adding TRX IP from HeLa cells to the control nuclear extract from nonirradiated cells that were not treated with chemicals are shown as a control (Fig. 4A, Lane 1). The addition of diamide or NEM to IP TRX decreased AP-1 DNA binding activity (Fig. 4A, Lanes 2 and 3). In contrast to treatment with diamide and NEM, treatment of the IP TRX with DTT increased AP-1 DNA binding compared with the control sample (Fig. 4A, Lane 1 versus Lane 4). Finally, a fraction of IP TRX was treated with diamide, washed eight times to remove any free chemical, and subsequently treated with DTT. This treatment appeared to restore the ability of the IP TRX to induce AP-1 DNA binding (Fig. 4A, Lane 1 versus Lane 5). These results suggest that alterations in the redox status of TRX play a central role in the regulation AP-1 DNA binding after IR. TRX Physically Interacts with Ref-1 and This Interaction Is Enhanced by IR. To determine whether TRX interacts with Ref-1 after IR, a transient transfection two-hybrid system using Cos-1 cells

Fig. 3. A, a cytoplasmic factor induces AP-1 DNA binding activity. Increasing amounts of cytoplasmic extract from irradiated HeLa cells were added to the nuclear extract from nonirradiated cells. Nuclear extract alone is shown as a control (Lane 1). Increasing amounts of cytoplasmic (1–8 ␮g) were added to 10 ␮g of nuclear extract (Lanes 2–6), and EMSA was performed as above. EMSA with cytoplasmic extract alone are shown as controls (Lanes 7 and 8). EMSA for AP-1 was performed as described. Arrows in B, AP-1 complex and free unbound AP-1 oligonucleotide. B, AP-1 DNA binding activity after addition of TRX to nuclear extracts from HeLa cells. IP TRX/protein A pellets from the cytoplasmic fraction of irradiated HeLa cells were added to nuclear extracts from control, nonirradiated cells (Lane 4). Nuclear extract alone (Lane 2) and IP TRX/protein A alone (Lane 3) are shown as controls. plasmic extracts obtained from irradiated HeLa cells. The IP TRX/ protein A pellet was combined with 10 ␮g of nuclear extract from nonirradiated HeLa cells, and EMSA was performed 40 min later. Control, IP protein A (Fig. 3B, Lane 1) and IP TRX (Lane 3), both without any nuclear extract, are shown as controls. Nonirradiated nuclear extract was also run as a control to establish the baseline AP-1 DNA binding activity (Lane 2) and is similar to the result shown in Fig. 3A, Lane 1. When cytoplasmic TRX IP from irradiated cells was added to nonirradiated nuclear cell extract, a marked increase in AP-1 DNA binding activity was seen (Fig. 3B, Lane 4). Western blot analysis of the IP TRX using anti-Ref-1 antibody demonstrated an absence of Ref-1 protein in the IP TRX (data not shown). This result combined with the results showing translocation of TRX to the Fig. 4. A, the ability of IP TRX to regulate AP-1 DNA binding activity is altered by chemical agents that alter the oxidation or reduction status of TRX. Either diamide (Lane nucleus (Fig. 2) is consistent with the concept that cytoplasmic TRX 2), NEM (Lane 3), or DTT (Lane 4) were added to the TRX/protein A complex IP from activates AP-1 DNA binding by passing an IR-induced signal from the cytoplasm of irradiated HeLa cells (2 h after IR) after pelleting. A fraction of IP TRX the cytoplasm to the nucleus. was treated with diamide, washed eight times, and then treated with DTT (Lane 5). IP TRX that did not receive any treatment with oxidizing or reducing agents is shown as Redox Alterations in the TRX Protein That Regulates AP-1 control (Lane 1). EMSA for AP-1 was performed as described. Arrows, AP-1 complex and DNA Binding Activity. To determine whether the oxidation/reduc- free unbound AP-1 oligonucleotide. B, TRX forms a physical association with Ref-1 that tion status of TRX protein regulates AP-1 DNA binding activity, TRX is enhanced by IR. Cells were transfected with pCMV-TRX-VP-16, pCMV-Gal-4-Ref-1, or both with ptk-3x-Gal-4-LUC as a reporter plasmid. pUC was used to standardize each was immunoprecipitated from irradiated cells and split into five equal transfection to a total of 30 ␮g of plasmid DNA. The relative-fold induction of LUC fractions that were subsequently treated with various chemicals that activity was normalized by ␤-gal activity via cotransfection of pSV-40-␤-gal. The results are presented as relative-fold induction in LUC activity over the baseline (Lane 1). All alter protein thiol oxidation/reduction (redox) status. After three wash- results are the means of three separate experiments, all done in duplicate. The results of ing steps with 750 ml of E1A buffer and repelleting, the IP TRX was individual transfections varied by Ͻ25%. 6691

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2000 American Association for Cancer Research. REDOX REGULATION OF THIOREDOXIN AND Ref-1 ACTIVATION OF AP-1 was performed. These transfections were performed using Ref-1/ Gal-4 and TRX/VP-16 fusion proteins expressed by a CMV expres- sion vector and a LUC reporter cassette containing three Gal-4 DNA binding sites (p3x-Gal-4/tk-LUC; Ref. 29). Cotransfection of p3x- Gal-4/tk-LUC with either CMV-Ref-1/Gal-4 or CMV-TRX/VP-16 showed no increase in LUC activity when compared with transfection with p3x-Gal-4/tk-LUC alone (Fig. 4B, Lanes 3–4 and 5–6 versus Lanes 1–2). Cotransfection of p3x-Gal-4/tk-LUC with both CMV- Ref-1/Gal-4 and CMV-TRX/VP-16 in nonirradiated cells resulted in a 6-fold increase in LUC activity (Fig. 4B, Lane 7). Ten h after exposure to 10 Gy of IR, an additional increase in LUC activity to 11-fold above baseline was observed (Fig. 4B, Lane 8). These results suggest that a physical interaction between TRX and Ref-1 occurs and that this interaction is enhanced in response to IR. Immunodepletion of Ref-1 Impairs Inducible, but not Basal, AP-1 DNA Binding Activity. Ref-1 has been shown to regulate AP-1 activity through a redox-sensitive mechanism (27, 31, 32, 35); there- fore, the role of Ref-1 in the regulation of IR-induced increases in AP-1 DNA binding activity was examined. Irradiated cellular extracts were depleted of Ref-1 by immunodepletion with anti-Ref-1 antibody to determine the role of Ref-1 in the induction of AP-1 DNA binding activity after IR. HeLa cell extracts from the experiments shown above (Fig. 1) were treated with the addition of protein A and anti-Ref-1 antibody, followed by gentle shaking at 4°C for2hand spinning at 12,000 rpm for 1 min to pellet out the Ref-1 antibody complex. Western blot analysis confirmed the removal of Ref-1 protein from the extracts as well as the presence of Ref-1 in the IP complex (data not shown). The continued presence of c-Fos/c-Jun proteins in the Ref-1-depleted extracts as well as the lack of c-Fos/c- Jun protein in the IP complex was established by Western analysis (data not shown). Immunodepletion of Ref-1 from nonirradiated cells did not alter the levels of AP-1 DNA binding activity detected by EMSA (Fig. 5A, Lane 1 versus Lane 3). These results suggest that the presence of Ref-1 in the extracts was not required for AP-1 DNA binding activity in nonirradiated cells. Nuclear extracts from cells harvested 1 h after exposure to IR and immunoprecipitated with protein A only (Fig. 5A, Lane 2) were used as a positive control, and similar to Fig. 1 (Lanes Fig. 5. Impact of immunodepletion of Ref-1 and re-addition of Ref-1 to immunode- 2–4), a significant increase in AP-1 DNA binding activity was ob- pleted extracts upon AP-1 DNA binding activity in response to IR. A, Ref-1 without extracts were either harvested from cells 2 h after exposure to IR or harvested from served. In contrast, no increase in AP-1 DNA binding activity was untreated cells. ϩ, addition of IP Ref-1 from irradiated or control nuclear extracts added observed in nuclear extracts from irradiated cells immunodepleted of back to immunodepleted nuclear extracts. Ϫ, no Ref-1 immunodepleted extracts from the Ref-1 and isolated at 1 and 2 h after irradiation (Fig. 5A, Lanes 4 and respective nuclear extract was added. Arrows in A and B, AP-1 complex, free unbound AP-1 oligonucleotide, and nonspecific binding. B, restoration of AP-1 DNA binding by 5). These results indicate that the presence of Ref-1 is required for the the addition of IP Ref-1. Lanes 1 and 2 are controls and are identical to Lanes 3 and 5 from IR-induced increase in AP-1 DNA binding activity. A. Immunoprecipitated Ref-1/protein A from irradiated cells was added back to immu- Restoration of IR-induced AP-1 DNA Binding Activity after nodepleted (Ref-1-Ϫ) cell extracts from irradiated samples (Lane 3), IP Ref-1/protein A pellets from irradiated cells were added back to immunodepleted (Ref-1-Ϫ) cell extracts Addition of Ref-1 IP to Immunodepleted Extracts. To confirm that from nonirradiated samples (Lane 4), and IP Ref-1/protein A pellets from control (non- Ref-1 from irradiated cells must be present in the HeLa cell extracts irradiated) cells were added back to immunodepleted (Ref-1-Ϫ) cell extracts from irra- diated samples (Lane 5). The IP Ref-1 and immunodepleted extracts were analyzed via for IR-induced increases in AP-1 DNA binding activity to occur, IP EMSA as described above. C, the ability of IP Ref-1 to regulate AP-1 DNA binding containing Ref-1 from irradiated or nonirradiated cells was reintro- activity is altered by chemical agents that alter the oxidation or reduction status of Ref-1. duced to the Ref-1-immunodepleted extracts from irradiated cells Either diamide (Lane 2), NEM (Lane 3), or DTT (Lane 4) were added to the IP Ref-1/protein A complex from irradiated cells after pelleting (using HeLa cell extracts). harvested 2 h after IR (Fig. 5B). When Ref-1 IP from irradiated cells Control, nontreated IP Ref-1 is shown as control (Lane 1). EMSA for AP-1 was performed was added back to Ref-1 without extracts from irradiated cells (Fig. as described. Arrows in A and B, AP-1 complex and free unbound AP-1 oligonucleotide. 5B, Lane 3), the IR-induced increase in AP-1 DNA binding was restored (Fig. 5B, compare Lane 3 with Lane 2). When Ref-1 IP from nonirradiated cells was added to Ref-1 without irradiated cell extracts binding was attributable to increased levels of IP Ref-1 protein. In (Fig. 5B, Lane 5), a small, but reproducible increase in AP-1 DNA addition, Western blot analysis confirmed the continued presence of binding was observed (Fig. 5B, compare Lane 5 with Lane 2). Inter- c-Fos and c-Jun proteins in the Ref-1-depleted extracts as well as the estingly, when Ref-1 IP from irradiated cells was added to Ref-1 absence of c-Fos and c-Jun protein in the IP complex (data not without extracts from nonirradiated cells (Fig. 5B, Lane 4), AP-1 shown). Finally, we confirmed that recombinant bacterially expressed DNA binding increased to levels seen in cells exposed to IR (Fig. 5B, His-tagged Ref-1 protein also activates AP-1 DNA binding activity compare Lane 4 with Lane 1). Equal amounts of IP Ref-1 from control (data not shown), as shown by others (31, 32). These results indicate and irradiated cells were confirmed by Western analysis (data not that the presence of Ref-1 from IR-treated cells is required to activate shown). This ruled out the possibility that the induction of AP-1 DNA inducible, but not basal, AP-1 DNA binding. These experiments are 6692

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2000 American Association for Cancer Research. REDOX REGULATION OF THIOREDOXIN AND Ref-1 ACTIVATION OF AP-1 consistent with previous studies investigating Ref-1 activation of AP-1 in response to oxidative stress, including 12-O-tetradecanoyl- phorbol-13-acetate and H2O2 (4, 10, 14). Redox Alterations in the Ref-1 Protein from Irradiated Cells Regulates AP-1 DNA Binding Activity. In Fig. 4A, it was demon- strated that TRX regulation of AP-1 DNA binding is altered by chemical oxidation and reduction. It was also shown that TRX phys- ically interacts with Ref-1. Thus, the role of redox status in Ref-1 regulation of AP-1 DNA binding activity was examined. Ref-1 from irradiated cells was immunoprecipitated and treated in the presence of either diamide (a sulfhydryl oxidizing agent), NEM (a sulfhydryl specific alkylating agent), or DTT (a sulfhydryl reducing agent) and washed eight times to remove any free chemical. The IP Ref-1 was then added to nonirradiated Ref-1-immunodepleted cell extracts, and AP-1 DNA binding activity was examined. Ref-1 IP from irradiated cells without chemicals is shown as a control (Fig. 5C, Lane 1). The addition of diamide or NEM to IP Ref-1 from irradiated cells (Fig. 5C, Lanes 2 and 3) decreased AP-1 DNA binding activity. Ref-1 IP from irradiated cells and treated with DTT significantly increased AP-1 DNA binding compared with Ref-1 IP from irradiated cells that were not treated with chemicals (Fig. 5C, Lane 1 versus Lane 4). These results suggest that alterations in redox status of the Ref-1 protein from irradiated cells play a central role in the enhancement of AP-1 DNA binding activity after exposure to IR. Fig. 6. Schematic model of a signaling pathway involved in IR induction of AP-1 DNA binding activity. IR forms ROIs, which act in the cytoplasm to induce a signaling cascade that results in the reduction of TRX. IR increases steady-state levels of NADPH by stimulating pentose cycle activity. Reduction of TRX by TR and NADPH allows trans- DISCUSSION location of reduced TRX from the cytoplasm to the nucleus. There, TRX physically interacts with Ref-1 that activates the AP-1 complex in a redox-dependent manner. AP-1 Stress-activated signaling cascades are tightly regulated cellular DNA binding occurs at critical cysteine residues, which are flanked by lysine and arginine processes that allow eukaryotic cells to respond to environmental amino acids. Binding of this transcription factor leads to transcription in a number downstream target genes involved in cellular response to environmental stress. TR, TRX stress, including therapeutic modalities such as chemotherapy and reductase. radiotherapy (37–39). These signaling pathways and factors appear to sense stress-induced changes in the intracellular oxidation/reduction (redox) status, providing the cell the ability to mount a response to stressing agents through a series of thiol-containing molecules, such TRX becomes reduced and is transported from the cytoplasm to the as TRX and Ref-1 (21, 29). These signaling pathways and transcrip- nucleus in response to IR, resulting in the induction of AP-1 DNA tion factors appear to play a major role in maintaining the steady-state binding activity. For many years, it has been known that exposure to intracellular balance between prooxidant production, ca- IR and other oxidants results in an immediate increase in NADPH pacity, and the repair of oxidative damage. pools caused by stimulation of glucose metabolism through the pen- Key contributors in altering the intracellular redox potential are tose cycle (34). Because NADPH pools coupled with TRX reductase ROIs. ROIs can be formed by a variety of extracellular stressing provide the reducing equivalents for TRX, these results are consistent agents including multiple exogenous genotoxic agents, such as in- with the hypothesis that IR-induced stimulation of NADPH produc- flammatory cytokines, chemical carcinogens, chemotherapeutic tion could be linked to radiation-induced through agents, and irradiation (9, 26, 28). Alterations in steady-state levels of redox changes in TRX (Fig. 6). ROIs and the subsequent alteration of the intracellular redox potential EMSA extracts immunodepleted of Ref-1 protein demonstrated that are considered to be a primary mechanism regulating cellular signal- the increase in AP-1 DNA binding activity after IR is dependent upon ing factors that link external stimuli with signal transduction in the presence of Ref-1 and that Ref-1 regulates inducible, but not basal, cellular response to stress (40). It has been shown that IR results in the AP-1 DNA binding activity. This result was confirmed by the resto- ⅐Ϫ ration of IR-inducible AP-1 DNA binding upon addition of Ref-1 IP generation of multiple ROIs including superoxide (O2 ), hydrogen ⅐ from irradiated cells to immunodepleted extracts. Similar to TRX, the peroxide (H2O2), hydroxyl radical ( OH), and organic hydroperoxides (ROOH; Refs. 26 and 28). Because IR generates ROIs and activates ability of Ref-1 to stimulate AP-1 DNA binding was abolished by redox-sensitive signaling factors, such as AP-1, the role of TRX chemical oxidation and increased by chemical reduction. These results and/or Ref-1 in bridging the physical barrier between the cytoplasm suggest that changes in the oxidation/reduction status of both TRX and the nucleus and regulating the induction of AP-1 in response to IR and Ref-1 play a role in the induction of AP-1 DNA binding activity was examined. in response to IR but does not show a linear pathway from TRX to The current work confirms that IR induces AP-1 DNA binding Ref-1 regulating the DNA binding of AP-1. To support this, transient activity. In addition, subcellular fractionation demonstrated that in transfection two-hybrid assays were performed that demonstrated response to IR, TRX is transported into the nucleus, and this result physical interaction between TRX and Ref-1 in the nucleus and that was confirmed via indirect immunofluorescence cell staining. Fur- this interaction is increased after IR. The increase in LUC activity thermore, TRX IP from irradiated cells added to nonirradiated nuclear observed in the cotransfection with p3x-Gal-4/tk-LUC, CMV-Ref-1/ extracts resulted in activation of AP-1 DNA binding activity. It was Gal-4, and CMV-TRX/VP-16 in nonirradiated cells may result from also shown that the ability of TRX to stimulate AP-1 DNA binding leakage of the TRX/VP-16 fusion protein from the cytoplasm into the activity was abolished by chemical oxidation and increased by chem- nucleus attributable to the VP-16 nuclear localization sequence. Over- ical reduction. The results of these experiments strongly suggest that all, these results suggest that in response to IR a change in the redox 6693

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2000 American Association for Cancer Research. REDOX REGULATION OF THIOREDOXIN AND Ref-1 ACTIVATION OF AP-1 state of TRX may result in the regulation of the DNA binding activity suggest that an upstream factor such as TRX reductase reduces TRX of the AP-1 transcription factor via the interaction of Ref-1 and TRX. prior to nuclear translocation. Reduced TRX interacts with Ref-1 (Fig. Although the activation of transcription factors, such as AP-1, 4B) and passes a redox signal to Ref-1, which must also be in a ultimately occurs within the nucleus, a long-standing hypothesis in reduced state to activate AP-1 DNA binding (Fig. 5C). Finally, Ref-1 radiation biology states that the initiation of cellular signaling path- appears to pass a redox signal to the AP-1 complex to induce DNA ways resulting from irradiation exposure occurs at the cytoplasmic binding activity (Fig. 6). The results of these experiments suggest a membrane (4, 9, 14). This raises several interesting questions regard- stress-activated, redox-sensitive signaling pathway involving proteins ing the cytoplasmic factors and pathways activated by IR as well as containing critical cysteine residues that are required for functional the specific factors that pass the signal from the cytoplasm to the activity (Fig. 6). The potential linkage between stress-activated met- nucleus. One mechanism of transcriptional regulation involves the abolic pathways for increasing NADPH synthesis and TRX/Ref-1 also movement of specific signaling factors from one subcellular location provides a plausible explanation for why IR and other oxidants can to another. Two examples of this are the extracellular signal-regulated activate transcription factors (i.e., AP-1) that require reduction to kinases, also known as mitogen-activated protein kinases, and nuclear become activated in vitro. factor-␬B, both of which are rapidly transported to the nucleus in The discovery that TRX is a redox-sensitive cytoplasmic signaling response to exposure to IR (3, 41). The results presented in this report factor activating AP-1 in response to IR identifies TRX as a potential identify TRX as another signaling factor that is transported to the target for radio-modifying agents. Furthermore, the subsequent deter- nucleus in response to IR, resulting in the activation of nuclear mination of signaling factors upstream of TRX may also identify transcription factors. cellular targets for altering the inherent radiosensitivity of tumor cells. One of the intracellular functions of TRX is to initiate protein- Finally, TRX nuclear translocation and interaction with Ref-1 firmly nucleic acid interactions of nuclear transcription factors (29). TRX has establishes a link between cytoplasmic events after radiation, resulting two redox-active cysteine residues, -Cys-Gly-Pro-Cys-, in an active in the activation of nuclear transcription factors that potentially gov- center and participates in redox reactions through the reversible oxi- ern cellular responses to IR. Taken together with previous investiga- dation/reduction of these thiol residues (16, 42, 43). In this regard, tions (21), these results support the concept that a common central TRX serves as a source of reducing equivalents to alter protein pathway(s) mediating cellular responses to IR or other types of function and subsequently regulate nuclear transcription factor activ- environmental or metabolic oxidative stress may involve redox- ity. This suggests that alterations in intracellular oxidation/reduction sensitive signaling pathways leading to AP-1 activation via Ref-1 and reactions may induce alterations in the redox state of TRX initiating redox-sensitive cytoplasmic signaling factors such as TRX and TRX a signaling cascade (19, 44) in response to a variety of environmental reductase. stresses. Once TRX has reached the nucleus, at least one mechanism regu- REFERENCES lating AP-1 DNA binding activity involves the nuclear redox-sensi- tive signaling protein Ref-1 (3, 35). This signal transduction appears 1. Halliwell, B., and Gutteridge, J. M. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol., 186: 1–85, 1990. to occur through a critical cysteine residue (motif), located in the basic 2. Hill, C. S., and Treisman, R. Transcriptional regulation by extracellular agents: DNA binding domain of c-Fos/c-Jun, that is flanked by the basic mechanisms and specificity. Cell, 80: 199–211, 1995. amino acids lysine and arginine (KCR). The redox status of this 3. Curry, H. A., Clemens, R. A., Shah, S., Bradbury, C. M., Botero, A., Goswami, P., and Gius, D. Heat shock inhibits radiation-induced activation of NF-␬B via inhibition critical cysteine plays a central role in the regulation of c-Fos/c-Jun of I-␬B kinase. J. Biol. Chem., 274: 23061–23067, 1999. (AP-1) DNA binding activity (3, 15, 31, 32, 45). Interestingly, genetic 4. Maity, A., McKenna, W. G., and Muschel, R. J. The molecular basis for cell cycle delays following ionizing radiation: a review. Radiother. Oncol., 31: 1–13, 1994. analysis of Ref-1 using site-directed mutagenesis has identified a 5. Abate, C., Marshak, D. R., and Curran, T. Fos is phosphorylated by p34cdc2, cysteine located at position 65 in the redox domain that is critical for cAMP-dependent protein kinase and protein kinase C at multiple sites clustered the redox-sensitive activation of c-Fos/c-Jun DNA binding (46). within regulatory regions. Oncogene, 6: 2179–2185, 1991. 6. Kerppola, T. K., and Curran, T. Zen and the art of Fos and Jun. Nature (Lond.), 373: Therefore, these critical , located in the functional domains 199–200, 1995. of TRX and Ref-1, appear to be targets for the passage of redox- 7. Gius, D., Cao, X. M., Rauscher, F. J., III, Cohen, D. R., Curran, T., and Sukhatme, sensitive cellular signals to transcription factors in response to stress. V. P. Transcriptional activation and repression by Fos are independent functions: the C terminus represses immediate-early gene expression via CarG elements. Mol. Cell. In this model, the redox status of these critical cysteine residue(s) Biol., 10: 4243–4255, 1990. is strongly influenced by its local protein microenvironment (47). 8. Kerr, L. D., Inoue, J., and Verma, I. M. Signal transduction: a nuclear target. Curr. Subtle changes in cellular redox potentially alter the ionization state of Opin. Cell Biol., 4: 496–501, 1992. Ϫ 9. Holbrook, N. J., and Fornace, A. J., Jr. Response to adversity: molecular control of the cysteine molecule (-CH2-SH to -CH2-S ), resulting in gene activation following genotoxic stress. New Biol., 3: 825–833, 1991. profound changes in protein activity (23). The critical cysteine(s) 10. Hallahan, D. E., Dunphy, E., Virudachalam, S., Sukhatme, V. P., Kufe, D. W., and Weichselbaum, R. R. c-jun and Egr-1 participate in the DNA synthesis and cell would act as a redox-sensitive “sulfhydryl switch” that reversibly survival in response to ionizing radiation exposure. J. Biol. Chem., 270: 30303– modulates protein activity (31, 32). The redox potential then could be 30309, 1995. passed along specific signal transduction cascades using proteins with 11. Weichselbaum, R. R., Hallahan, D., Fuks, Z., and Kufe, D. Radiation induction of immediate early genes: effectors of the radiation-stress response. Int. J. Radiat. Oncol. redox-sensitive sites (1, 15). Biol. Phys., 30: 229–234, 1994. The results presented in this report are consistent with the model 12. Karin, M., and Smeal, T. Control of transcription factors by signal transduction pathways: the beginning of the end. Trends Biochem. Sci., 17: 418–422, 1992. that IR results in environmental stress and the formation of ROIs that 13. Datta, R., Hallahan, D. E., Kharbanda, S. M., Rubin, E., Sherman, M. L., Huberman, initiates a cellular response pathway at the level of the cytoplasmic E., Weichselbaum, R. R., and Kufe, D. W. Involvement of reactive oxygen interme- membrane. IR has also been shown to stimulate NADPH production diates in the induction of c-jun gene transcription by ionizing radiation. Biochemistry, 31: 8300–8306, 1992. through the pentose cycle and as such represents a possible mecha- 14. Hallahan, D. E., Gius, D., Kuchibhotla, J., Sukhatme, V., Kufe, D. W., and nism linking IR-induced changes in metabolism to activation of Weichselbaum, R. R. Radiation signaling mediated by Jun activation following transcription factors via TRX and Ref-1 (Fig. 6; Ref. 28). In this dissociation from a cell type-specific repressor. J. Biol. Chem., 268: 4903–4907, 1993. model, IR induction of NADPH levels may activate a pathway in- 15. Abate, C., Patel, L., Rauscher, F. J., and Curran, T. Redox regulation of fos and jun volving TRX reductase that is upstream of TRX (Fig. 6). Because DNA-binding activity in vitro. Science (Washington DC), 249: 1157–1161, 1990. 16. Buchanan, B. B., Schurmann, P., Decottignies, P., and Lozano, R. M. Thioredoxin: a reduced, but not oxidized, TRX activates AP-1 DNA binding activity multifunctional regulatory protein with a bright future in technology and medicine. (Fig. 4A, Lanes 2 and 3 versus Lane 4), it would seem logical to Arch. Biochem. Biophys., 314: 257–260, 1994. 6694

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2000 American Association for Cancer Research. REDOX REGULATION OF THIOREDOXIN AND Ref-1 ACTIVATION OF AP-1

17. Holmgren, A. Thioredoxin. Annu. Rev. Biochem., 54: 237–271, 1985. 32. Xanthoudakis, S., Miao, G. G., Wang, F., Pan, Y-C. E., and Curran, T. Redox 18. Biguet, C., Wakasugi, N., Mishal, Z., Holmgren, A., Couaib, S., Tursz, T., and activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. Wakasugi, H. Thioredoxin increases the proliferation of human B-cell lines through EMBO J., 11: 3323–3335, 1992. a protein kinase C-dependent mechanism. J. Biol. Chem., 269: 28865–28870, 1994. 33. Powis, G., Briehl, M., and Oblong, J. Redox signaling and the control of cell growth 19. Nakamura, H., Nakamura, K., and Yodoi, J. Redox regulation of cellular activation. and death. Pharmacol. Ther., 68: 149–173, 1995. Annu. Rev. Immunol., 15: 351–369, 1997. 34. Tuttle, S. W., Varnes, M. E., Mitchell, J. B., and Biaglow, J. E. Sensitivity to chemical 20. Okamoto, T., Ogiwara, H., Haysashi, T., Mitsui, A., Kawabe, T., and Yodoi, J. oxidants and radiation in CHO cell lines deficient in oxidative pentose cycle activity. Human thioredoxin/adult T cell leukemia-derived factor activates the enhancer bind- Int. J. Radiat. Oncol. Biol. Phys., 22: 671–675, 1992. ing protein of human immunodeficiency virus type 1 by thiol redox control mecha- 35. Yao, K. S., Xanthoudakis, S., Curran, T., and O’Dwyer, P. J. Activation of AP-1 and nism. Int. Immunol., 4: 811–819, 1992. of a nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to 21. Nonogaki, T., Noda, Y., Narimoto, K., Umaoka, Y., and Mori, T. Protection from hypoxia. Mol. Cell. Biol., 14: 5997–6003, 1994. oxidative stress by thioredoxin and superoxide dismutase of mouse embryos fertilized 36. Freeman, B. A., and Crapo, J. D. Biology of disease: free radicals and tissue injury. in vitro. Hum. Reprod., 6: 1305–1310, 1991. 22. Tanaka, T., Nishiyama, Y., Okada, K., Hirota, K., Matsui, M., Yodoi, J., Hiai, H., and Lab. Investig., 47: 412–426, 1982. Toyokuni, S. Induction and nuclear translocation of thioredoxin by oxidative damage 37. Mathias, S., Pena, L. A., and Kolesnick, R. N. Signal transduction of stress via in the mouse kidney: independence of tubular necrosis and sulfhydryl depletion. Lab. ceramide. Biochem. J., 335: 465–480, 1998. Investig., 77: 145–155, 1997. 38. Yokomizo, A., Ono, M., Nanri, H., Makino, Y., Ohga, T., Wada, M., Okamoto, T., 23. Gallegos, A., Gasdaska, J. R., Taylor, C. W., Paine-Murrieta, G. D., Goodman, D., Yodoi, J., Kuwano, M., and Kohno, K. Cellular levels of thioredoxin associated with Gasdaska, P. Y., Berggren, M., Briehl, M. M., and Powis, G. Transfection with drug sensitivity to cisplatin, mitomycin C, doxorubicin, and etoposide. Cancer Res., human thioredoxin increases cell proliferation and a dominant-negative mutant thi- 55: 4293–4296, 1995. oredoxin reverses the transformed phenotype of human breast cancer cells. Cancer 39. Yarnold, J. Molecular aspects of cellular responses to radiotherapy. Radiother. On- Res., 56: 5765–5770, 1996. col., 44: 1–7, 1997. 24. Powis, G., Oblong, J. E., Gasdaska, P. Y., Berggren, M., Hill, S. R., and Kirkpatrick, 40. Bergendi, L., Benes, L., Durackova, Z., and Ferencik, M. Chemistry, physiology and D. L. The thioredoxin/ redox system and control of cell growth. pathology of free radicals. Life Sci., 65: 1865–1874, 1999. Oncol. Res., 6: 539–544, 1994. 41. Chen, Z. J., Parent, L., and Maniatis, T. Site-specific phosphorylation of I␬-Ba by a 25. Powis, G., Kirkpatrick, D. L., Angulo, M., and Baker, A. Thioredoxin redox control novel ubiquitination-dependent protein kinase activity. Cell, 84: 853–862, 1996. of cell growth and death and the effects of inhibitors. Chem.-Biol. Interact., 111–112: 42. Holmgren, A. Thioredoxin and systems. J. Biol. Chem., 264: 13963– 23–34, 1998. 13966, 1989. 26. Del Maestro, R. F. An approach to free radicals in medicine and biology. Acta 43. Ren, X., Bjornstedt, M., Shen, B., Ericson, M. L., and Holmgren, A. Mutagenesis of Physiol. Scand., 492 (Suppl.): 153–168, 1980. structural half-cysteine residues in human thioredoxin and effects on the regulation of 27. Diamond, D. A., Parsian, A., Hunt, C. R., Lofgren, S., Spitz, D. R., Goswami, P. C., activity by selenodiglutathione. Biochemistry, 32: 9701–9708, 1993. and Gius, D. Redox factor-1 (Ref-1) mediates the activation of AP-1 in HeLa and NIH 44. Sasada, T., Sono, H., and Yodoi, J. Thioredoxin/adult T-cell leukemia-derived factor 3T3 cells in response to heat shock. J. Biol. Chem., 274: 16959–16964, 1999. (ADF), and redox regulation. J. Toxicol. Sci., 21: 285–287, 1996. 28. Mitchell, J. B., and Russo, A. The role of in IR and drug induced cytotoxicity. Br. J. Cancer, 55: 5096–5104, 1987. 45. Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J., and Hay, R. T. Thioredoxin ␬ 29. Hirota, K., Matsui, M., Iwata, S., Nishiyama, A., Mori, K., and Yodoi, J. AP-1 regulates the DNA binding activity of NF- B by reduction of a disulphide bond transcriptional activity is regulated by a direct association between thioredoxin and involving cysteine. Nucleic Acids Res., 20: 3821–3830, 1992. Ref-1. Proc. Natl. Acad. Sci. USA, 94: 3633–3638, 1997. 46. Walker, L. J., Robson, C. N., Black, E., Gillespie, D., and Hickson, I. D. Identification 30. Holmgren, A. Thioredoxin and glutaredoxin: small multi-functional redox proteins of residues in the human DNA repair enzyme HAP-2 (Ref-1) that are essential for with active-site disulphide bonds. Biochem. Soc. Trans., 16: 95–96, 1988. redox regulation of jun DNA-binding. Mol. Cell. Biol., 13: 5370–5376, 1993. 31. Xanthoudakis, S., and Curran, T. Identification and characterization of Ref-1, a 47. Clancey, C. J., and Gilbert, H. F. Thiol/disulfide exchange in the thioredoxin- nuclear protein that facilitates AP-1 DNA-binding activity. EMBO J., 11: 653–664, catalyzed reductive activation of spinach chloroplast fructose-1,6-bisphosphatase. 1992. J. Biol. Chem., 262: 13545–13549, 1987.

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S. Jack Wei, Ana Botero, Kiichi Hirota, et al.

Cancer Res 2000;60:6688-6695.

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