Redox regulation of transcriptional activators

Free Radical Biology & Medicine,Vol. 21, No. 3, pp. 335-348, 1996 Copyright © 1996 ElsevierScienceInc. Printed in the USA. All rights reserved 0891-5849/96 $15.00 + .00 ELSEVIER

PII S0891-5849(96)00109-8

-J~ Review Article REDOX REGULATION OF TRANSCRIPTIONAL ACTIVATORS YI SUN * and LARRY W. OBERLEY * • Department of Cancer Research, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105, USA; and *Radiation Research Laboratory, University of Iowa, Iowa City, IA 52242, USA

(Received 7 September 1995; Revised 7 February 1996; Accepted 7 February 1996) Abstract--Transcription factors/activators are a group of proteins that bind to specific consensus sequences (cis elements) in the promoter regions of downstream target/effector genes and transactivate or repress effector gene expression. The up- or downregulation of effector genes will ultimately lead to many biological changes such as proliferation, growth suppression, differentiation, or senescence. Transcription factors are subject to transcriptional and posttranslational regulation. This review will focus on the redox (reduction/oxidation) regulation of transcription factors/activators with emphasis on p53, AP-1, and NF-KB. The redox regulation of transcriptional activators occurs through highly conserved cysteine residues in the D N A binding domains of these proteins. In vitro studies have shown that reducing environments increase, while oxidizing conditions inhibit sequence-specific D N A binding of these transcriptional activators. When intact cells have been used for study, a more complex regulation has been observed. Reduction/oxidation can either up- or downregulate D N A binding and/or transactivation activities in transcriptional activator-dependent as well as cell type-dependent manners. In general, reductants decrease p53 and NF-KB activities but dramatically activate AP-1 activity. Oxidants, on the other hand, greatly activate NF-KB activity. Furthermore, redox-induced biochemical alterations sometimes lead to change in the biological functions of these proteins. Therefore, differential regulation of these transcriptional activators, which in turn, regulate many target/ effector genes, may provide an additional mechanism by which small antioxidant molecules play protective roles in anticancer and antiaging processes. Better understanding of the mechanism of redox regulation, particularly in vivo, will have an important impact on drug discovery for chemoprevention and therapy of human diseases such as cancer. K e y w o r d s - - F r e e radicals, Redox regulation, p53, AP-1, NF-KB, Transcription factors/activators

INTRODUCTION

III. G e n e r a l l y , R N A p o l y m e r a s e I c o n t r o l s the trans c r i p t i o n o f r i b o s o m a l p r e R N A (45S R N A ) , R N A p o l y m e r a s e III t r a n s c r i b e s the g e n e s e n c o d i n g a series o f s m a l l R N A species s u c h as t R N A and r i b o s o m a l 5S R N A , w h i l e R N A p o l y m e r a s e II c o n t r o l s the transcrip-

E u k a r y o t i c g e n e e x p r e s s i o n is g o v e r n e d b y a g r o u p o f R N A p o l y m e r a s e s , n a m e l y R N A p o l y m e r a s e I, II, and Address correspondence to: Yi Sun, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Co., 2800 Plymouth Road, Ann Arbor, MI 48105. Dr. Yi Sun is a native of China. He completed his medical training in People's Republic of China in 1983. In 1989, Dr. Sun received a Ph.D. from the University of Iowa where he developed his interests in free radical biology and roles of antioxidant enzymes in multistage carcinogenesis. He then spent 5 years, first as a postdoctor, later as a senior staff fellow, at the National Cancer Institute. During that time, he has studied potential involvement of the tumor suppressor genes, including p53, Rb, p21, p16, and VHL in nasopharyngeal carcinogenesis and has cloned the mouse tissue inhibitor of metalloproteinases-3 gene, a gene specifically not expressed in neoplastic JB6 cells. Dr. Sun has been a senior scientist at the Parke-Davis Pharmaceutical Research and an associate member in the Comprehensive Cancer Center, University of Michigan since early 1995. He is the sole author or first/last author of 43 with a total of 53 scientific

publications. He has a beloved wife, Hua and two children, Steven and Grace. Larry W. Oberley received his B.A. degree in physics from Northwestern University in 1968. In his 4 years at Northwestern, Dr. Oberley did not see as many victories in football as this year's Rose Bowl bound team accomplished in one season! Go, Northwestern! Dr. Oberley received his M.S. and Ph.D. degrees in physics from the University of Iowa in 1970 and 1974, respectively. After obtaining the M.S. Degree in 1970, Dr. Oberley joined the Radiology Department as a Research Assistant. He has stayed in this department since that time, progressing to the rank of Professor. Dr. Oberley's research interests have also progressed, from physics to biology to biochemistry to molecular biology. Dr. Oberley's longtime research interests have been on the role of free radicals and antioxidant enzymes in cancer. He is particularly interested in the effect of modulation of superoxide dismutase on cancer therapy. 335

336

Y. SUN and L. W. OBERLEY

tion of all protein-encoding genes.~ In a particular time point of development or differentiation in a given cell type, expression of some nonrequired genes is repressed; others, such as housekeeping genes are constitutively expressed; and still others, which respond to environmental stimuli are induced. The on-and-off switch of gene expression is precisely controlled by transcription factors. Transcription factors (TFs) are regulatory proteins that recognize specific DNA sequences, bind them, and recruit the correct RNA polymerase to carry out RNA synthesis. The transcription factors can be generally divided into two categories: general transcription factors and sequence-specific transcription factors or transcriptional activators. The general transcription factors for RNA polymerase II include TFIIA, TFIIB, TFIIC, TFIID, TFIIE, TFIIF, TFIIG, TFIIH, TFIIS. They are part of RNA polymerase II transcription machinery and direct the basal transcription through the minimal promoter elements consisting of a TATA box and the initiation region. ~-3 The transcription activators, on the other hand, contain two main domains, a sequence-specific DNA-binding domain and a transactivation domain. The DNA binding domain of many transcriptional activators contains one of the four motifs: helix-turn-helix, helix-loop-helix, zinc finger, or leucine zipper. It binds specifically to consensus cis elements in the promoter region of the effector genes and the transactivation domain facilitates the stimulation of transcription in cooperation with the basic transcription machinery. 3 Transcriptional control of gene expression is, therefore, the result of a complex cross-talk between transcriptional activator and the basic transcription machinery. The transcription factors/activators are subject to regulation at both transcriptional and posttranslational levels. 4'5 At the transcriptional level, many transcriptional activators are inducible by various factors. Examples include the induction of jurdfos and NF-I
regulation of several important transactivators. Small antioxidant molecules, such as glutathione, and certain vitamins have been long believed to have anticancer and antiaging activities by scavenging reactive oxygen radicals produced in normal respiratory processes or after oxidative insults] 9 The data shown in this review suggest another mechanism of action by which small antioxidants/reductants play protective roles through differential regulation of transcriptional activator activities. REDOX REGULATION OF THE P53 TUMOR SUPPRESSOR GENE

p53 is a 393-amino acid nuclear phosphoprotein. It consists of a transactivation domain in the amino terminus, an oligomerization domain in the carboxyl terminus and a specific DNA binding domain in the center portion of the molecule. 2° By binding to its consensus binding site 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3', 21 p53 functions as a transcriptional activator to transactivate the target genes. As a tumor suppressor gene, p53 inhibits tumor cell growth and suppresses transformation. 22 As a "genome guard," p53 induces blocks in the cell cycle and prevents gene amplification (refs. 20, 23, 24, and references therein). Mutational inactivation of p53 has been found to be the most frequent molecular alteration in human cancers, indicating the importance of p53 in human carcinogenesis. 25

Structural basis for redox regulation of p53 Redox regulation at a posttranslational level often occurs through the cysteine residues of a protein by disruption (reduction) or formation (oxidation) of a disulphide bond. There are 10 cysteine residues in p53 protein. 26 They all are localized in the specific DNA binding region at the central part of the molecule where most human mutations are clustered. Eight of the 10 cysteines are evolutionarily conserved (see Table 1), implying the importance of cysteine residues in p53. 27 There are two types of p53 mutations found in human cancers involving the substitution of cysteine residues. As summarized (based upon refs. 27, 28) in Tables 1 and 2, mutations could either replace cysteine residues (9 out of 10 cysteine residues were found mutated in human cancers, see Table 1) or cause substitution of cysteine residues from other amino acids (there are 20 cases in human cancers, see Table 2). Either type of mutation would lead to the disruption of p53 conformation and possibly abolish wildtype p53 activity. The structure of the central portion of human p53 in complex with DNA has been recently crystallized. 29 The core domain structure consists of a sandwich that serves

Redox regulation of transcriptional activators

337

Table 1. Human Cancer Mutations That Replace Cysteine Residues in the p53 Gene Cysteine Residues Codons

Mutation Hot Spot

Evolution Conservation a

135

II II

All All

141

II

All

1 2 4

Mutation Substitution

Cancers

Cys ~ Tyr Trp Ser Cys -- Tyr Phe Gly Cys -- Phe Ser Tyr -- Trp Cys -- Ser -- Stop Cys -- Stop ~ Ser

Leukemia, breast, colon, lung, ovary

Cys -- Phe -- Tyr lie Ser -- Arg Gly -- Stop Cys -- Ser -- Phe -- Trp Arg Tyr Cys ~ Gly Tyr -- Trp Arg Cys ~ Phe Gly

Ovary, pancreas, leukemias, melanoma, head/neck, skin, breast, colon, lung

--

Ovary, colon, bladder, Breast, leukemia, lung, kidney

--

176

III

All

IV

Human, monkey, mouse, chicken Human, monkey, chicken, xenopus, trout All

182 229

238

Breast, colon, leukemia, esophagus, lung, liver, pancreas, head/neck, ovary

Stomach, Brain Lung

--

--

--

242

IV

All

275

V

All

277

V

All

--

a Compared among species of human, monkey, rat, mouse, chicken, xenopus gene A,

as a scaffold for two large loops and a loop-sheet-helix motif. Together with the loop-sheet-helix motif, these loops form the DNA-binding surface of p53. 29 Although no disulphide bonds are found in the structure, the loops are found to be connected by a tetrahedrally coordinated zinc atom bound on residues Cys 176,His 179, Cys 238, and Cys 242. The mutation rate of these cysteine residues in human cancer is 1.5% for Cys 176, 1.8% for Cys 2~8, and 1.4% for Cys 242, respectively. 28'29The crystal structure along with the high mutation rate found in human cancer of these cysteine residues indicate their importance in coordination with zinc in stablizing the DNA-binding domain of p53.

In vitro cell-free study Milner and co-workers have systematically studied posttranslational regulation of p53 conformation by redox, metals/metal chelators, salt, and temperature using in vitro-translated p53 protein. 3°-34 The concept was that if cysteines and zinc are important in stablizing the DNA-binding of p53, disruption of them by reductants/

Ovary, lung, breast, brain, colon, head/neck, leukemia, liver, uterus

Brain, kidney, lung, stomach, breast, colon

Lung, ovary, skin

Xenopus gene B, R. trout.

oxidants or metal chelators should abolish DNA binding activity. Indeed, they found that copper ion, the metal chelator 1,10-phenanthroline (OP) and oxidation (by diamide) all disrupt wildtype p53 conformation as detected by conformation-specific p53 antibodies, and inhibit sequence specific DNA binding demonstrated by a mobility-shift assay. The reductant 1,4-dithio-lthreitol (DTT) favors the folding of p53 into wild-type conformation and increases DNA binding) °'31 DTT also restores p53 binding activity disrupted by diamide. 35 Similar observations have been reported by the others using metal chelators (OP and EDTA); 36-38 the alkylating reagent N-ethyl-maleimide (NEM) and oxidant diamide; 35'3s and the reducing reagent DTT. 38 Removal of reducing agents by dialysis completely inactivated p53 DNA binding activity, suggesting the importance of a reducing environment in ceils for active p53 protein. 35Many tumor cells are in a prooxidant status resulting from increased production of oxygen radicals or decreased expression of antioxidant enzymes (ref. 39 and references therein). The oxidizing enviroments in tumor cells may, therefore, render struc-

338

Y. SUN and L. W. OBERLEY Table 2. Human Cancer Mutations That Form Cysteine Residues in the p53 Gene

Codons

MutationHot Spot

CysteineSubstitution

III

Trp ~ Arg ~ Tyr ~ Tyr -Arg ~ Arg -Gly -Tyr ~ Tyr ~

Cys Cys Cys Cys Cys Cys Cys Cys Cys

234 236 241 244 245 270 273

IV IV IV IV IV V V

Tyr ~ Tyr -Ser -Gly -Gly -Phe ~ Arg --

Cys Cys Cys Cys Cys Cys Cys

283 285 286 337

V V V

Arg -Glu ~ Glu ~ Arg ~

Cys Cys Cys Cys

53 110 126 163 175 181 187 205 220

Cancers Leukemia Liver, uterus Head/neck, lung Stomach, breast, lung, ovary, sarcoma, leukemia, esophagus, brain Colon, uterus Breast, leukemia, uterus Breast Head/neck, lung, ovary, uterus, leukemia, brain, breast Esophagus, colon, pancreas, ovary, liver, stomach, lung, head/neck, Li-Fraumeni, breast Ovary, leukemia, head/neck, lung, uterus, breast Leukemia, ovary, stomach, colon, liver Lung, bladder, colon, kidney Liver, brain, leukemia, esophagus, breast, lung Sarcoma, bladder, esophagus, head/neck, Li-Fraumeni, liver, lung, skin, breast Brain, esophagus, leukemia Esophagus, leukemia, lung, breast, colon, brain, kidney, prostate, uterus, stomach, liver, skin, ovary Lung, colon, Breast Colon Liver

turally wild-type p53 conformationally mutant and give rise to the same biological outcome as p53 mutation.

Cell culture studies To extend the observation regarding redox regulation of p53 from the test tube to intact cells, we have used transient transfection/luciferase reporter transactivation assay to assess regulation of p53 by redox in tissue culture cells. Several luciferase reporter plasmids driven by a p53-specific binding site derived from the mouse MDM-2 gene, Waf-1 gene, or synthetic p53 binding sites (PG13, 13 repeats of p53 binding site) 4w42 were used for transfection. The recipient cells include L-RT101, a mouse JB6 tumor line, and Sp-Tx, a spontaneous transformed mouse liver line, both harboring endogenous wild-type p53, 43'44and human Saos2 cells lacking endogenous p53 (cotransfected with wildtype or mutant p53 in this case). 45"46 Transient transfectants were then exposed to reducing (DTT, GSH, NAC) or oxidizing agents (H202, paraquat, oxidized DTT, GSSG, diamide, NEM), to metals (zinc sulfate, cuprous chloride, p-chloromercurphenylsulfonic acid, mercury chloride), or metal chelators (OP, dipicolinic acid, diethylenetriamine-acetic acid), to compounds that inhibit endogenous GSH synthesis [L-buthionine sulfoximine (BSO)], or simply to antioxidants [butylated hydrozy-anisole (BHA), pyrrolidine dithiocarbamate (PDTC)]. Attempts were made to identify compounds that regulate wildtype p53 activity (either up or down) or that restore mutant p53 to a wild-type p53 conformation. These active compounds, if identi-

fled, may have potential use in cancer therapy. To our astonishment, most reducing reagents, but not their oxidant forms (which showed no significant effect), decreased p53-induced transactivational activity by twoto threefold. The metal chelator, OP, however, increased p53 transactivation activity by 2-10-fold in a cell type specific and dose-dependent manners. These effects were more obviously seen in cells harboring wild-type p53 (Bian, Wang, Jacobs, Sun, manuscript submitted). The results obtained in cultured cells are inconsistent with the observations made in the test tube. Disagreement in redox regulation studies between cellfree and intact cells has also been observed with other transcriptional activators such as AP-1 and NF-~cB (ref. 47, also see below). Our current efforts are directed to identifying whether these regulations occur at transcriptional or posttranslational level and whether this induced activation/inactivation of p53 transactivation activity has biological significance. Overall, our results show, on one hand, a complex picture of p53 regulation by redox in intact cells and offer, on the other hand, some hope to eventually identify some p53 modulating compounds. What are the biological consequences of redox-induced changes in p53 activity? Rainwater et al. have recently used site-directed mutagenesis technique to change each of 12 cysteine residues in murine p53 to serine residue and studied these cysteine-mutants for p53 activities. 38 The results show that these cysteinemutants can be divided into three categories: (a) those with substitution at positions of 40, 179, 274, 293, or 308 had little or no effect on p53 function; (b) those

Redox regulation of transcriptional activators with substitution at positions of 121,132, 138, or 272, which are localized in the loop-sheet-helix region of the site-specific DNA-binding domain, caused partial loss of p53 function; and (c) those with substitution at positions of 173, 235, or 239, which have been implicated in the zinc binding region, 29 had completely lost p53 function as well as gained oncogenic function as measured by a transformation assay. 38 These results clearly demonstrate that abrogation of redox regulation of cysteine residues involved in DNA binding by mutation causes the loss of p53 activity and suggest an important role of cellular redox status in regulating p53 function. As discussed above, we have shown that OP, a metal chelating and DNA intercalating reagent, can enhance p53 activity. What is the status of p53 target genes such as Waf-14~ following p53 activation and what is the consequence of target gene activation, if any? Using two murine tumor cell lines in which OP has shown to activate p53 activity, we have found that Waf-1 expression is, indeed, dramatically induced following OP treatment. This activation of Waf-1 leads to apoptosis in both cell lines (Bian, Wang, Jacobs, and Sun, manuscript submitted). The results clearly demonstrate that drug-induced p53 activation upregulated p53 target gene expression and caused biological changes. REDOX REGULATION OF A P - 1 0 N C O G E N E S

AP- 1, activator protein 1, is a complex of oncogene proteins of the Jun and Fos families. Three mammalian Jun proteins (c-Jun, Jun B, and JunD) and four Fos family members (c-Fos, Fra-1, Fra-2, and F o s B ) h a v e been identified. 6'7 Both c-Jun and c-Fos are nuclear transcription factors, consisting of 340-amino acids and 381-amino acids, respectively. Jun proteins contain a transcriptional activation domain at the amino-terminus, a basic domain for DNA binding, and leucine zipper domain for dimerization at the carboxyl-terminus. Fos proteins have leucine-zipper and basic domains in the center portion of the molecules. 6'7 AP-1 proteins, the Jun-Jun homodimer or Jun-Fos heterodimer, bind to TPA-response elements (TRE) or AP-1 binding sites, 5'-TGAG/CTCA-3' to transcriptionally activate effector genes. The c-jun and c-fos genes are inducible by a broad range of extracellular stimuli and function as intermediary transcriptional regulators in signal transduction processes leading to proliferation and transformation. 6,7

In vitro studies In vitro redox regulation of AP-1 DNA binding occurs at a posttranslational level. Curran and co-workers

339

initially identified a nuclear factor that stimulates the DNA-binding activity of Jun and Fos in vitro. This activity was inhibited by oxidant reagents and was partially mimicked by reducing agents. 4s These results suggested that AP-1 DNA binding may be subject to redox regulation. To identify critical cysteine residues in Jun and Fos proteins that are sensitive to redox regulation, the same group mutated two cysteine residues localized at the leucine zipper and basic regions, which are highly conserved among members of the jun and fos gene families. A single cysteine residue (Cys 252 in chicken Jun) in the DNA-binding domains (basic region) of the two proteins was identified as participating in DNA binding and subject to redox regulation. 49 Reduction of this residue by reducing agents such as DTT, NADPH, GSH, or /3-mercaptoethanol (/3-ME) increased AP-1 DNA binding, whereas oxidation by diamide or GSSG, or alkylation by NEM inhibited DNA binding. 49-5z Inhibition of DNA binding was mediated by an intermolecular disulphide bridge formed between the cysteine residues of each basic region within a dimer under nonreducing conditions. 52 Other cysteine residues in the DNA-binding domain of Jun (Cys 272) and Fos (Cys154), have been recently found to be required for gold(l) thiolates and antioxidant selenite induced inhibition of AP-1 DNA binding and transactivation, 53 indicating a codon specificity in redox regulation. The nuclear factor initially identified to activate AP-1 DNA binding was later purified and cloned and designated Ref-1 for redox factor. 54'55Ref-1 was found to be a previously characterized DNA repair enzyme. 56'57The redox and DNA-repair activities of Ref1 are encoded by N-terminal and C-terminal domains, respectively. 58 Cys 65 in Ref-1 protein was identified as the redox active site 59 and the cysteine directly involved in interaction between Ref-1 and Jun. 58'~9

Cell culture studies In intact cells, redox regulation of AP- 1 activity was found to be occurring at both the transcriptional and posttranslational levels. 47 Both the oxidant H202 and the antioxidant PDTC strongly induce expression of the c-jun and c-fos genes (ref. 47 and references therein). 6° AP-1 activities (DNA binding and transactivation) were, however, only moderately activated by H202, but strongly activated by antioxidants, including PDTC, NAC, butylated hydroxyanisole (BHA), and thioredoxin (an 11.5 kDa protein, functioning as a physiological antioxidant). 47'61'62In contrast to the in vitro resuits, lowering the GSH levels with BSO, an inhibitor of y-glutamylcysteine synthetase, or diamide, a thioloxidizing agent, stimulates AP-1 DNA binding as well as transactivation of chloramphenicol acetyltransferase

340

Y. SUN and L.W. OBERLEY

(CAT) activity driven by a glutathiones S-transferase (GST) gene regulatory element (EpRE) in HepG2 hepatoma cells. 63 Induction of these activities is inhibited by thiol compounds NAC and G S H . 63 Although oxidized AP-1 can be reduced and, therefore, reactivated by reducing agents in vitro (see above), there is no evidence showing that AP- 1 can be reversibly oxidized and reduced in intact cells. 47 This may explain the difference seen between cell-free and in intact cell studies. The reason for discrepancy between strong induction of c-jun and c-fos expression and weak stimulation of AP-1 activity by H202 is not clear at present. The possibility of direct damage of the AP-1 molecules by H202 has been, however, excluded. 47 Since AP-1 activation by antioxidants is mediated by induction of cjun/c-fos expression, AP-1 has been classified as a secondary antioxidant responsive factor. 47'64 What is the biological significance of these redoxinduced biochemical changes in AP-1 activities? The fact that redox sensitive Cys 252 at the DNA binding domain of c-Jun is replaced by serine in the transforming viral oncogene v-jun 65 suggested that oncogenic potential of v-jun may contribute to the escape from this redox regulatory process. Indeed, in the case of c-Fos, compared to wild-type truncated protein, a truncated Fos protein (F118-211) with C y s 154 mutated to serine showed a threefold increase in AP-1 DNA binding activity and an increased transforming activity as demonstrated by increased number and size of transformed colonies. 66 The results suggest that redox regulation may limit the formation of a functional Jun-Fos complex in vivo, therefore controlling the oncogenic potential of AP-1 proteins. Furthermore, oxidants H202 and superoxide anion can induce c-jun and c-fos expression in a number of cell models (refs. 67, 68, and references therein) and overexpression of c-Jun or AP1 has been shown to cause cellular proliferation and transformation in those cells. 67'69On the other hand, the phenolic antioxidant tert-butylhydroquinone (BHQ) induces fra-1 expression. The Fra proteins heterodimerize with Jun protein to form stable AP- 1 complexes that have low transactivation activity. Furthermore, Fra-I suppresses AP- 1 activity induced by TPA or expression of c-Jun and c-Fos. The Fra-1 induced attenuation of oncogenic Jun-Fos AP1 activity may provide an explanation for the antitumor-promoting activity of phenolic antioxidants. 7° Thus, redox induced biochemical modifications have significant biological consequences. REDOX REGULATION OF NF4cB SIGNAL TRANSDUCER

NF-KB, nuclear factor KB, is an inducible transcriptional activator. It is a heterodimer containing a 50- and a 65-kDa subunit (termed p50 and p65). Both p50 and

p65 contain a highly conserved N-terminal region that is responsible for DNA binding and dimerization. In addition, p65 contains a C-terminal region as the transactivation domain, allowing the protein to function as a transcriptional activator. 71-74 There are two forms of NF-~:B in the cell: an inactive form in the cytosol and an active form in the nucleus. In uninduced cells, NFKB is present in the cytosol, where it is bound to a specific inhibitor, IKB. 75 Upon induction by any one of a variety of agents including cytokines, mitogens, tumor promoters, and v i r u s , 4 IKB becomes phosphorylated and/or rapidly proteolysed in an apparently redox status-dependent manner and released from NFKB. 76-79 This dissociation allows the p50 and p65 to migrate to the nucleus, bind to a decameric DNA sequence, 5'GGGACTTTCC-3' and activate target genes, which include a wild variety of cellular and viral genes involved in inflammatory and immune responses. 4 The functions of NF-KB include reception/ integration of multiple stimuli, cytoplasmic/nuclear signalling, activation of immediate early and early genes and human immunodeficiency virus type 1 (HIV1) gene expression. Biologically, NF-xB is likely to be involved in the molecular processes of AIDS pathogenesis and carcinogenesis, as well as immunological and neurological disorders. 4'79-81

In vitro studies Specific DNA binding of NF-KB, as shown by in vitro electrophoretic mobility-shift assays (EMSAs), was inhibited by oxidants diamide and GSSG, SHmodifying agent NEM, but was stimulated by reductants, /3-mercaptoethanol (B-ME), DTT, NAC, GSH, and L- or I>cysteine and reducing proteins, thioredoxin and Ref-1.5°'51'82-86This redox regulation of NF-KB activity was mediated by the p50 subunit of NF-KB. s° A conserved cysteine residue ( C y s 6] o r Cys62)71'72 at the N-terminal DNA binding region of p50 was later found to play a critical role in DNA binding activity of NF-KB.82-84 Serine substitution of this cysteine residue by site-directed mutagenesis reduces NF-KB binding as well as rendering the protein insensitive to sulfhydrylmodifying agents, or reducing proteins. 82-84

Cell culture studies In analogy with p53 and AP-1, NF-KB, when redox regulated, showed an opposite effect in intact cells and in cell-free conditions. That is, oxidation, instead of reduction shown in vitro, activates DNA-binding activity of NF-•B in intact cells. In untreated Jurkat and HeLa cells where NF-KB is in an inactive form, oxidation by H202 induces NF-KB DNA binding as well

Redox regulation of transcriptional activators as transactivation activities. 47'64 H202 also synergistically potentiates TPA induced NF-KB activation. 47'64 The H202 induced activation was counteracted by antioxidants NAC, PDTC, BHA, and metal chelators desferal and OP. NAC and PDTC also blocked the activation of NF-KB by a variety of agents including TPA and TNF-o~.47'64"87'88 This effect was mediated by suppression of the release of IKB from NF-tcB. 47"64'87'88 Therefore, redox regulation by small molecules of NFKB in intact cells appears to be mediated by directly or indirectly releasing the IKB from NF-KB. 89 Because intracellular thiols regulate activation of NF-~:B, 9° and oxidants are known to alter tyrosine kinase and phosphatase activities, 91'92 it has been proposed that intracellular redox status controls NF-KB by regulating tyrosine phosphorylation event(s). 91-94 Thioredoxin regulation of NF-KB in intact cells has been reported to be cell line dependent. In HeLa cells, transient expression or exogenous addition of thioredoxin resulted in a dose-dependent inhibition of NF-KB DNA binding and transactivation activities. 62 In Cos- 1 or Jurkat cells, however, transient transfection of thioredoxin enhanced NF-KB transactivation activity. 84 These effects were probably mediated through the interference with IKB release 62 or through the reduction of Cys 61/62 in the p50 protein. ~ What are the biological consequences of reduction/ oxidation of NF-KB? In contrast to AP-1, which showed an enhanced transforming activity after escaping from redox regulation by mutation, 66 the mutant p50 protein (Rel) having a serine substitution at Cys 6u62, which abolished redox control, failed to efficiently transform chicken lymphoid cells assayed both in vitro and in vivo. 85 Once again, escape from redox control caused phenotypical change of cells, but in a transcriptional activator-specific manner. Furthermore, TNF-ce induced apoptosis, which was mediated by NFKB, was blocked by the antioxidant NAC. 95 These observations suggest the significance but complexity of redox regulation of transcription factors in physiological and pathologic conditions. REDOX REGULATION OF OTHER

TRANSCRIPTIONAL ACTIVATORS Several other transcriptional activators were also subject to redox regulation. The redox status influences their DNA binding, transactivation, and/or biological activities. We will give a brief review below to introduce the readers to this rapid moving field.

C-myb proto-oncogene The myb gene encodes a helix-turn-helix transcriptional activator that primarily regulates the proliferation

341

and differentiation of hematopoietic progenitor cells. 96 The c-Myb protein has a molecular weight of 75 kDa and contains a DNA binding domain in the N-terminus and a transactivation domain at the central part of the molecule. It binds specifically to the DNA consensus sequence PyAACT/GG (refs. 96, 97 and references therein). In vitro study showed that the DNA binding of purified c-Myb protein was strongly stimulated by the reductant DTT, while oxidation with diamide or alkylation with NEM inactivated Myb DNA binding. 97 Site-directed mutagenesis identified a single conserved cysteine residue, Cys 130, in the DNA binding domain of Myb protein that was responsive to redox regulation. Substitution of Cys 13° with serine almost completely abolished DNA binding, indicating that reduced Cys I3° is essential for Myb DNA binding. 97 In addition, another cysteine residue, Cys 43 in the DNA binding domain of Myb protein was also found to be sensitive to SH-alkylation regulation. Cys 43 was accessible to NEM alkylation in the free Myb protein. DNA binding was inactivated/abolished by NEM treatment in a dose-dependent manner. Mutation Cys 43 to Va143 rendered Myb insensitive to NEM. 98 These results suggest that Cys 43 and/or Cys 13° in the DNA binding domain of the c-Myb protein could function as a molecular sensor for a redox regulator to turn specific DNA binding on or off, thus controlling effector gene expression. Because compared to wild-type protein, a cysteine mutant in the DNA binding domain of v-Myb protein has lost both transactivation and transformation activities, 99 it is possible that redox regulation of cysteine residues at DNA binding domain may cause change of biological functions in vivo.

Ets transcription factor The ets gene family encodes a novel class of sequence specific DNA binding proteins. ETS-1, a member of the ets gene family, is a nuclear phosphoprotein that binds to purine-rich DNA sequences (sequences containing the common core trinucleotide GGA) and functions as a transcription factor. 1°°'~°1 Both v-Ets and c-Ets are subject to redox regulation. Oxidants diamide and SH-alkylating agent NEM inactivate DNA binding by Ets-1 proteins. The C-terminal (DNA binding domain) Cys 394 was identified as being important for redox regulation in vitro, but not in vivo. ~02The biological significance of this redox regulation is not clear at present. It will be of interest to know whether the tumor suppressor activity of Etsl, which is mediated by its transactivation activity, ~°3 is subject to redox regulation.

342

Y. SuN and L. W. OBERLEY

Sp-1 transcription factor The zinc-finger transcription factor Sp- 1 binds to the " G C b o x " (5'G/TG/AGGCGGFFG/AG/AC/T 3') via three zinc fingers of the Cys2His2 type. Jc~ The zinc ion is an essential cofactor for specific DNA binding of Sp1. l°5 This feature provides a structural basis for redox regulation of Sp-1. Indeed, in vitro Sp-1 DNA binding was abolished by the oxidants H202 and diamide as well as the SH-alkylating agent NEM and iodacetamide. Oxidant-induced inactivation of DNA binding can be quantitatively reversed by reductants, /3-ME, DTT, and GSH. 1°6 The metal depletion of Sp-1 protein dramatically increased the sensitivity of Sp-1 DNA binding to NEM, H202, and physiological concentration of GSSG, indicating the role played by zinc in the protection of Sp-1 transcription activity. J06 Sp-1 DNA binding was surprisingly related to the aging process. The Sp-1 DNA binding efficiency was remarkedly reduced using nuclear extract from old animals compared to young animals. 1°7 Because reactive oxygen intermediates are known to increase during aging, it is logical to examine the possible redox regulation of Sp-1 activity. Indeed, in vitro binding assays as measured by gel retardation and DNase I footprintings, showed that high concentration of DTT, if added to the aged nuclear extract, fully restored the Sp-1 DNA binding activity, while addition of H202 tO young nuclear extract strongly decreased binding, l°s The similar observation has been made when purified Sp-1 proteins were used for the assay. 1°8 The results clearly demonstrated that Sp-1 transcription activator is subjected to redox regulation both in vitro and in vivo situation. Because Sp1 binding sites have been found in many genes, redox regulation of this transcription activator is expected to have a broad influence on expressions of many target genes, which in turn, leads to many biological changes.

redoxin or DTT activates this binding.112 In vitro study showed that the binding of GR to DNA was also inhibited by sulfhydryl-modifying reagents such as methyl-methanethiosulfonate and oxidant H202, and this inhibition can be reversed by reductant DTT. JJ3.~14 The experiment performed in intact cells also showed that the DNA binding activity of GR is sensitive to redox modulation. 115 GR-DNA binding and transactivation were dramatically decreased in cell extracts treated with DEM or BSO (both are GSH depleting agents) or oxidant H202. These inhibitions can be reversed by antioxidant NAC.115 In analogy to Spl, glucocorticoid responsiveness is impaired during aging, and this aging-related alteration is not always paralleled by a reduction in receptor number.ll6 It is possible that increased oxygen radicals during aging could play a role in modulating GR activity in vivo.

Early growth response- 1 (egr-1) transcription factor Egr-1 is a zinc finger transcription factor rapidly induced by growth factors and other extracellular stimuli. 117 It binds to its specific DNA-binding sequence GCGGGGGCG through the interaction of three zinc finger motifs. This binding, however, is subject to redox regulation in vitro through the zinc finger domain. Reductants, DTT, /3-ME, and Ref-1 protein activate binding, while oxidants diamide and SH-alkylating agent NEM inactivate binding in a dose-dependent manner. 118 The possible biological significance of redox regulation of Egr- 1 was suggested by an intact cell study. Both H202 and DEM (a glutathione-depleting agent) were shown to inhibit TPA-induced Egr- 1 DNA binding. Treatment of myeloid lines HL-60 and KG1 with DEM prevented TPA-induced differentiation. ~19 This apparent association implies that abrogation of TPA-induced activation of Egr-I may lead to failure of cellular differentiation in these cells.

Glucocorticoid receptor The glucocorticoid receptor (GR) belongs to a family of ligand-inducible nuclear transcription factors. It exists in the cytosol in an inactive form. Upon binding to its ligand, the glucocorticoid-receptor complex is translocated into the nucleus where the GR dissociates with its ligand and binds to specific glucocorticoid response elements (GRE, 5' AGAA/TCA(G)A/T 3'). l°9 The DNA binding is mediated through the zinc-finger domain of the DNA binding region, consisting of two groups of four cysteines tetrahedrically coordinated with two zinc ions. 11° It has been long known that activation of the GR is subject to redox modulation. Sulfhydryl-modifying reagents such as NEM prevent the binding of cytosol GR to glucocorticoid, 111 while thio-

Thyroid transcription factors Thyroid transcription factors (TTF) I and II are part of a superfamily with retinoic acid receptor and GR. They are implicated in the control of thyroid- and lungspecific gene expression. They bind to the promoter of thyroglobulin and thyroperoxidase genes and transactivate their expressions, which are involved in thyroid tissue differentiation. The DNA binding activity of both factors is subject to redox regulation. 12°'121 In the case of TTF-1, oxidants diamide or GSSG decrease TTF-I DNA binding activity. This inhibited activity can be fully restored by reducing agents, DTT and GSH. The oxidants-induced decrease in DNA binding was found to be due to the formation of disulfide bonds

Redox regulation of transcriptional activators

between two specific cysteine residues located outside the TTF-1 homodomain, rather than direct hinderance of TTF-1/DNA contacts.12° Redox regulation of TTF2 is probably mediated through the zinc finger domain.121 In vitro experiment showed that TTF-2 bound DNA as a dimer that required zinc. The oxidant diamide inactivated TTF-2 DNA binding activity, whereas the reductant DTT abolished this inhibition.121

Upstream stimulatory transcription factor ( USF) Transcription factor USF is a helix-loop-helix/leucine repeat protein that binds to the upstream stimulatory core sequence, CACGTG. It transactivates both adenoviral and several cellular gene expressions (ref. 122 and references therein). Purified USF is composed of 43- and 44-kDa polypeptides. USF43, a transcriptionally active component of USF, was found to be strongly affected in its DNA binding activity by redox changes. Dimerization and DNA binding of purified 43-kDa USF was greatly favored by reducing conditions and was dramatically lowered under nonreducing conditions. ~22 This redox regulation was later found to be due to the redox modulation of two cysteine residues, Cys 229 and C y s 248, both present in the helix-loophelix domain of the protein] 23 Oxidation resulted in both intra- and intermolecular covalent crosslinks through disulfide bond formation that inhibit DNA binding, presumably by blocking structural changes required for DNA interaction. Compared to wild-type form, mutant USF43 with serines substituting for cysteines was no longer sensitive to the reducing agent DTT in its DNA binding as well as transactivation potentials] 23 indicating a critical role of these cysteine residues in redox regulation.

Aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) The AhR is an intracellular protein that binds to a variety of environmentally important carcinogens, including polycyclic aromatic hydrocarbons and certain halogenated hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). 124 Upon binding to its ligand, AhR is activated and found in nuclear fraction where it forms heterodimer with AhR nuclear translocator protein, A R N T ] 25 Both AhR and ARNT are basic helix-loop-helix containing transcription factors. The AhR-ARNT heterodimer binds to xenobiotic responsive elements (XRE, 5'-T/GNGCGTGA/CG/CA-3'), 126 and transactivate the genes containing this consensus sequence in their promoter regions. Most of the downstream target/effector genes are genes encoding many antioxidant and drug metablism enzymes, such as two

343

cytochrome P-450 IA1 (CYPIA1 and CYPIA2) genes, and glutathione S-transferase Ya subunit geneJ 27 AhR/ ARNT, therefore, play an important role in carcinogenesis and drug metabolism. It has been recently found that the AhR/ARNT complex is also subject to redox regulation. 12~ Exposure of purified receptor to reductant DTT markedly increased its DNA-binding activity, while exposure to diamide substantially decreased its binding to XRE oligo. The diamide-induced inhibition of AhR DNA-binding activity could be reversed by the addition of excess DTT or by reduced thioredoxin in the presence of cytoplasmic and nuclear proteins. 128 Because there are no cysteine residues in the basic region of AhR and ARNT heterodimers, the redox regulation of the DNA binding of these proteins may involve a novel mechanism. 128 Whether AhR/ARNT are subject to redox modulation in vivo is not known at present. Given the fact that they transcriptionally regulate the expression of many genes involved in drug/carcinogen metabolism, redox regulation of this transcriptional activator in vivo could produce significantly biological consequences.

C O N C L U S I O N S AND P E R S P E C T I V E S

As reviewed above, many transcriptional activators are subject to redox modulation. These data are summarized and listed in Table 3. Apparent discrepancy in redox regulation of p53, AP-1, and NF-•B between in vitro (cell-free system) and in vivo (intact cells) experiments could mainly result from three factors: (1) in in vitro assay, redox compounds interact directly and solely with purified transcriptional activators, while in vivo experiment, the compounds must enter the cells, travel to the nucleus, and then interact with the target molecules. In vivo experiments involve how many Table 3. Redox Regulation of Transcriptional Activators

In Vitro DNA Binding

Transcriptional Activator p53 AP-I NF-KB c-myb Ets Sp-I GR Egr- 1 TTF USF AhR

Reduction

Oxidation

t T ~ T

1

T T T ~' T T

In lntact Cells D N A B i n d i n g and Transactivation Reduction

Oxidation

~T It

T ~T

T l l

l

T

l

344

Y. SUN and L.W. OBERLEY

molecules entered cells, whether they have been metabolized/modified before reaching and interacting with the targets, and what is the final concentration of unmetabolized compounds, if any, left to interact with the target molecules. Because the compounds are also interacting with other cellular proteins, the results we see could be the outcome of many protein-protein interactions triggered by redox treatment; (2) related to #1, antioxidants that scavenge radicals 39 are present in vivo but not in vitro; and (3) in the case of NF-KB, the redoxsensitive component of the signaling pathway is upstream, and regulation does not seem to involve the cysteine residues in v i v o . 89 Changes in cellular redox status alter DNA binding and transactivation activities of many transcriptional activators, which in turn, lead to changes in expression of many target genes with ultimate changes in cellular functions. Redox regulation, therefore, appears to be a broad regulatory system that allows cells to adapt to environmental changes. The advantages of these redoxmediated changes are that they are quick and reversible through the change of sulfhydryl groups and transitional metals and they do not require new protein synthesis. Conceptually, the big disadvantage is, however, nonspecificity. Many transcriptional activators can be regulated by redox reagents simultaneously and the biological outcome will depend upon the net results of crosstalk among these redox-regulated transcriptional activators. This makes the in vivo study of redox regulation of individual transcriptional activators extremely difficult. Moreover, many different oxidants or reductants can affect the same transcriptional activator. Obviously, nonspecificity in the cell is not a big problem or the cell would not work. The problem is our understanding. Cellular compartmentalization may be the reason for specificity. Particular signals may interact with particular activators in a localized part of the cell. It is expected in this field that (1) more transcriptional activators, particularly those with conserved cysteine residue(s) in DNA binding domain and zinc-figure transcription factors, such as WT-1 (Wilm's tumor) tumor suppressor gene ~29 will be found to be regulated by redox; (2) because cysteine mutations in the p53 gene have been found in many human tumors (see Tables 1 and 2) and some cysteine mutants have altered biochemical as well as biological properties, 3s'~3° extensive studies of redox regulation of human tumor cell lines harboring these p53 mutations will emerge; (3) crosstalk among transcriptional a c t i v a t o r s 64'131-133 after redox treatment will draw much attention; and (4) most importantly, redox regulation has to be extended from in vitro to in vivo models. Because reductants/oxidants are small molecules and are easily given as oral drugs,

human beings will greatly benefit frombreakthroughs in this field. A c k n o w l e d g e m e n t s - We thank Dr. Nancy H. Colburn at the National Cancer Institute for her critical reading of the manuscript and Drs. Dongzhou Liao and Ellen M. G. De Vries at the Mayo Clinic for releasing their database of p53 mutations in human cancers prior to publication. This article is dedicated to and in memory of Yi Sun's dear grandmother, Shen Wan-Zhen, who passed away on February 12, 1994.

REFERENCES

1. Wingender, E. Transcription regulating proteins and their recognition sequences. Eukaryotic Gene Exp. 1:11-48; 1990. 2. Buratowski, S. The basics of basal transcription by RNA polymerase II. Cell 77:1-3; 1994. 3. Zawel, L.; Reinberg, D. Initiation of transcription by RNA polymerase II: A multi-step process. Prog. Nucleic Acid Res. Mol. Biol. 44:67-108; 1993. 4. Baeuerle, P. A. The inducible transcription activator NF-KB: Regulation by distinct protein subunits. Biochim. Biophys. Acta 1072:63-80; 1991. 5. Jackson, S. P. Regulating transcription factor activity by phosphorylation. Trends Cell Biol. 2:104-108; 1992. 6. Vogt, P. K.; Bos, T. J. Jun: Oncogene and transcription factor. Adv. Cancer Res. 55:1-35; 1990. 7. Angel, P.; Karin, M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim. Biophys. Acta 1072:129-157; 1991. 8. Maltzman, W.; Czyzyk, L. UV irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells. Mol. Cell. Biol. 4:1689-1694; 1984. 9. Kastan, M. B.; Onyekwere, O.; Sidransky, D.; Vogelstein, B.; Craig, R. W. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 51:6340~311 ; 1991. 10. Binetruy, B.; Smeal, T.; Karin, M. Ha-Ras augments c-Jun activity and stimulates phophorylation of its activation domain. Nature 351:122-127; 1991. 11. Smeal, T.; Binetruy, B.; Mercola, D. A.; Birrer, M.; Karin, M. Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serine 63 and 73. Nature 354:494496; 1991. 12. Lees, J. A.; Buchkovich, K. J.; Marshak, D. R.; Anderson, C. W.; Harlow, E. The retinoblastoma protein is phosphorylated on multiple sites by human cdc2. EMBO J. 10:42794290; 1991. 13. Maxwell, S. A.; Roth, J. A. Postranslational regulation of p53 tumor suppressor protein function. Crit. Rev. Oncog. 5:23-57; 1994. 14. Storz, G.; Tartaglia, L. A.; Ames, B. N. Transcriptional regulator of oxidative stress-inducible genes: Direct activation by oxidation. Science 248:189-194; 1990. 15. Allen, F. Redox control of transcription: Sensors, response regulators, activators and repressors. FEBS Lett. 332:203-207; 1993. 16. Hidalgo, E.; Demple, B. An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein. EMBO J. 13:138-146; 1994. 17. Demple, B.; Am~ibile-Cuevas, C. F. Redox redux: The control of oxidative stress responses. Cell 67:837-839; 1991. 18. Ostrovsky de Spicer, P.; Maloy, S. PutA protein, a membraneassociated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator. Proc. Natl. Acad. Sci. USA 90:42954298; 1993. 19. Halliwell, B.; Gutteridge, J. M. C. eds. Free Radicals in Biology and Medicine. 2nd ed. Oxford: Clarendon Press; 1989; 1989. 20. Lane, D. P. P53 and human cancers. Br. Med. Bull. 50:582599; 1994.

Redox regulation of transcriptional activators 21. El-Deity, W. S.; Kern, S.; Pietenpol, J. A.; Kinzler, K. W.; Vogelstein, B. Definition of a consensus binding site for p53. Nat. Genet. 1:45-49; 1992. 22. Baker, S. J.; Markowitz, S.; Fearon, E. R.; Willson, J. K.; Vogelstein, B. Suppression of human colorectal carcinoma cell growth by wildtype p53. Science 249:912-915; 1990. 23. Land, D. P. P53, guardian of the genome. Nature 358:15-16; 1992. 24. Hartwell, L. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell 71:543546; 1992. 25. Greenblatt, M. S.; Bennett, W. P.; Hollstein, M.; Harris, C. C. Mutations in the p53 suppressor gene: Clues to cancer etiology and molecular pathogenesis. Cancer Res. 54:4855-4878; 1994. 26. Zakut-Houri, R.; Bienz-Tadmor, B.; Givol, D.; Oren, M. Human p53 cellular tumor antigen: cDNA sequence and expression in COS cells. EMBO J. 4:1251-1255; 1985. 27. Hollstein, M.; Sidransky, D.; Vogelstein, B.; Harris, C. C. P53 mutations in human cancers. Science 253:49-53; 1991. 28. De Vries, E. M.; Ricke, D. O.; De Vries, T. N.; Hartmann, A.; Blaszyk, H.; Liao, D.; Soussi, T.; Kovach, J. S.; Sommer, S. Database of mutations in the p53 and APC tumor suppressor genes designed to facilitate molecular epidemiological analyses. Hum. Mutat. 7:202-213; 1996. 29. Cho, Y.; Gorina, S.; Jeffrey, P. D.; Pavletich, N. P. Crystal structure of a p53 tumor suppressor-DNA complex: Understanding tumorigenic mutations. Science 265:346-355; 1994. 30. Hainaut, P.; Milner, J. Redox modulation of p53 conformation and sequence-specific DNA binding in vitro. Cancer Res. 53:4469--4473; 1993. 31. Hainant, P.; Rolley, N.; Davies, M.; Milner, J. Modulation by copper of p53 conformation and sequence-specific DNA binding: Role for Cu(II)/Cu(I) redox mechanism. Oncogene 10:2732; 1995. 32. Hainaut, P.; Milner, J. A structural role of metal ions in the "wild-type" conformation of the tumor suppressor protein p53. Cancer Res. 53:1739-1742; 1993. 33. Butcher, S.; Hainaut, P.; Milner, J. Increased salt concentration reversibly destabilizes p53 quaternary structure and sequencespecific DNA binding. Biochem. J. 298:513-516; 1994. 34. Hainaut, P.; Butcher, S.; Milner, J. Temperature sensitivity for conformation is an intrinsic property of wild type p53. Br. J. Cancer 71:227-231 ; 1995. 35. Hupp, T. R.; Meek, D. W.; Midgley, C. A.; Lane, D. P. Activation of the cryptic DNA binding function of mutant form of p53. Nucleic Acids Res. 21:3167-3174; 1993. 36. Srinivasan, R.; Roth, J. A.; Maxwell, S. A. Sequence-specific interaction of a conformational domain of p53 with DNA. Cancer Res. 53:5361-5364; 1993. 37. Pavletich, N. P.; Chambers, K. A.; Pabo, C. O. The DNA-binding domain of p53 contains the four conserved regions and the major mutation hot spot. Genes Dev. 7:2556-2564; 1993. 38. Rainwater, R.; Parks, D.; Anderson, M. E.; Tegtmeyer, P.; Mann, K. Role of cysteine residues in regulation of p53 function. MoL Cell. Biol. 15:3892-3903; 1995. 39. Sun, Y. Free radicals, antioxidant enzymes and carcinogenesis. Free Radic. Biol. Med. 8:583-599; 1990. 40. Juven, T.; Barak, Y.; Zauberman, A.; George, D. L.; Oren, M. Wild type p53 can mediate sequence-specific transactivation of an internal promoter within the mdm2 gene. Oncogene 8:34113416; 1993. 41. E1-Deiry, W. S.; Tokino, T.; Velculescu, V. E.; Levy, D. B.; Parsons, R.; Trent, J. M.; Lin, D.; Mercer, W. E.; Kinzler, K. W.; Vogelstein, B. WAF- 1, a potential mediator of p53 tumor suppression. Cell 75:817-825; 1993. 42. Kern, S. E.; Kinzler, K. W.; Bruskin, A.; Jarosz, D.; Friedman, P.; Prives, C.; Vogelstein, B. Identification of p53 as a sequence-specific DNA binding protein. Science 252:1708-1711 ; 1991. 43. Sun, Y.; Nakamura, K.; Hegamyer, G. A.; Dong, Z.; Colbum, N. H. No point mutation of Ha-ras or p53 genes expressed in

44.

45.

46. 47. 48.

49. 50. 51. 52. 53.

54. 55. 56.

57.

58. 59.

60.

61. 62.

63. 64.

345

preneoplastic-to-neoplastic progression as modeled in mouse JB6 cell variants. Mol. Carcinog. 8:49-57; 1993. Sun, Y.; Hegamyer, G.; Nakamura, K.; Kim, H.; Oberley, L. W.; Colburn, N. H. Alterations of the p53 tumor suppressor gene in transformed mouse liver cells. Int. J. Cancer 55:952-956; 1993. Sun, Y.; Nakamura, K.; Wendel, E.; Colburn, N. H. Progression toward tumor cell phenotype is enhanced by overexpression of a mutant p53 tumor suppressor gene isolated from nasopharyngeal carcinoma. Proc. Natl. Acad. Sci. USA 90:2827-2831; 1993. Sun, Y.; Dong, Z.; Nakarnura, K.; Colburn, N. H. Dosage dependent dominace over wild type p53 of a mutant p53 isolated from nasopharyngeal carcinoma. FASEB J. 7:944-950; •993. Meyer, M.; Pahl, H. L.; Baeuerle, P. A. Regulation of the transcription factors NF-KB and AP-I by redox changes. Chem. BioL Interact. 91:91-100; 1994. Abate, C.; Luk, D.; Gentz, R.; Ranscher, F. J.,, III; Curran, T. Expression and purification of the leucine zipper and DNAbinding domains of Fos and Jun: both Fos and Jun contact DNA directly. Proc. Natl. Acad. Sci. USA 87:1032-1036; 1990. Abate, C.; Patel, L.; Rauscher, F. J., III; Curran, T. Redox regulation of Fos and Jun DNA-binding activity in vitro. Science 249:1157-1161; 1990. Toledano, M. B.; Leonard, W. J. Modulation of transcription factor NF-KB binding activity by oxidation-reduction in vitro. Proc. Natl. Acad. Sci. USA 88:4328-4332; 1991. Galter, D.; Mihm, S.; DrOge, W. Distinct effects of glutathione disulphide on the nuclear transcripton factor ~cB and the activator protein-1. Eur. J. Biochem. 221:639-648; 1994. Bannister, A. J.; Cook, A.; Kouzarides, T. In vitro DNA binding activity of Fos/Jun and BZLFI but not C/EBP is affected by redox changes. Onogene 6:1243-1250; 1991. Handel, M. L.; Watts, C. K. W.; DeFazio, A.; Day, R. O.; Sutherland, R. L. Inhibition of AP-1 binding and transcription by gold and selenium involving conseved cysteine residues in Jun and Fos. Proc. Natl. Acad. Sci. USA 92:4497-4501; 1995. Xanthoudakis, S.; Curran, T. Identification and characterization of Ref-1, nuclear protein that facilitates AP-1 DNA-binding activity. EMBO J. 11:653~565; 1992. Xanthoudakis, S.; Miao, G.; Wang, F.; Pan, Y. E.; Curran, T. Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO ./. 11:3323-3335; 1992. Robson, C. N.; Hickson, I. D. Isolation of cDNA clones encoding a human apurinic/apyrimidinic endonuclease that corrects DNA repair and mutagenesis defects in E. Coli xth (exonuclease III) mutants. Nucleic Acids Res. 19:5519-5523; 1991. Demple, B.; Herman, T.; Chen, D. S. Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: Definition of a family of DNA repair enzymes. Proc. Natl. Acad. Sci. USA 88:11450-11454; 1991. Xanthoudakis, S.; Miao, G. G.; Curran, T. The redox and DNArepair activities of Ref-1 are encoded by nonoverlapping domains. Proc. Natl. Acad. Sci. USA 91:23-27; 1994. Walker, L. J.; Robson, C. N.; Black, E.; Gillespie, D.; Hickson, I. D. Identification of residues in the human DNA repair enzyme HAP1 (Ref-l) that are essential for redox regulation of Jun DNA binding. Mol. Cell. Biol. 13:5370-5376; 1993. Li, W. C.; Wang, G. M.; Wang, R. R.; Spector, A. The redox active components of H202 and N-acetyl-L-cysteine regulate expression of c-jun and c-fos in lens systems. Exp. Eye Res. 59:179-190; 1994. Holmgren, A. Thioredoxin and glutaredoxin systems. J. Biol. Chem. 264:13963-13966; 1989. Schenk, H.; Klein, M.; Erdbrugger, W.; DrOge, W; SchulzeOsthoff, K. Distinct effects of thioredoxin and antioxidants on the activation of transcription factors NF-tcB and AP-1. Proc. Natl. Acad. Sci. USA 91:1672-1676; 1994. Bergelson, S.; Pinkus, R.; Daniel, V. Intracellular glutathione levels regulate Fos/Jun induction and activation of glutathione S-transferase gene expression. Cancer Res. 54:36-40; 1994. Meyer, M.; Schreck, R.; Baeuerle, P.A. H~O2 and antioxidants

346

65. 66. 67.

68.

Y. Sun and L. W. OBERLEY have opposite effects on activation of NF-KB and AP- 1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J. 12:2005-2015; 1993. Maki, Y.; Bos, T. J.; Davis, M.; Starbuck, M.; Vogt, P. K. Avian sarcoma virus 17 carries the Jun oncogene. Proc. Natl. Acad. Sci. USA 84:2828-2832; 1987. Okuno, H.; Akahori, A.; Sato, H.; Xanthoudakis, S.; Curran, T.; Iba, H. Escape from redox regulation enhances the transforming activity of Fos. Oncogene 8:695-701; 1993. Timblin, C. R.; Janssen, Y. W. M.; Mossman, B. T. Transcriptional activation of the proto-oncogene c-jun by asbestos and H202 is directly related to increased proliferation and transformation of tracheal epithelial cells. Cancer Res. 55:2723-2726; 1995. Colburn, N. H. Gene regulation by active oxygen and other stress inducers: Role in tumor promotion and progression. In: Spatz, L.; Bloom, A. D, eds. Biological consequences of oxidative stress: Implications f o r cardiovascular disease and carcinogenesis. London: Oxford University Press; 1992;

1992:121-137. 69. Watts, R. G.; Ben-Aft, E. T.; Bernstein, L. R.; Birrer, M. J.; Winterstein, D.; Wendel, E.; Colburn, N. H. C-jun and multistage carcinogenesis: Association of overexpression of introduced c-jun with progression toward a neoplastic endpoint in mouse JB6 cells sensitive to tumor promoter-induced transformation. Mol. Carcinog. 13:27-36; 1995. 70. Yoshioka, K.; Deng, T.; Cavigelli, M.; Karin, M. Antitumor promotion by phenolic antioxidants: Inhibition of AP-1 activity through induction of Fra expression. Proc. Natl. Acad. Sci. USA 92:4972-4976; 1995. 71. Bours, V.; Villalobos, J.; Burd, P. R.; Kelly, K.; Siebenlist, U. Cloning of a mitogen-inducible gene encoding a KB DNA-binding protein with homology to the rel oncogene and to cell-cycle motifs. Nature 348:76-80; 1990. 72. Kieran, M.; Blank, V.; Logeat, F.; Vandekerckhove, J.; Lottspeich, F.; Le Bail, O.; Urban, M. B.; Kouftlsky, P.; Baeuerle, P. A.; Israel, A. The DNA binding subunit of NF-KB is identical to factor KBF1 and homologous to the rel oncogene product. Cell 62:1007-1018; 1990. 73. Ghosh, S.; Gifford, A. M.; Riviere, L. R.; Tempst, P.; Nolan, G. P.; Baltimore, D. Cloning of the p50 DNA binding subunit of NF-KB: Homology to rel and dorsal.. Cell 62:1019-1029; 1990. 74. Bull, P.; Morley, K. L.; Hoekstra, M. F.; Hunter, T.; Verma, I. M. The mouse c-rel protein has an N-terminal regulatory domain and a C-terminal transcriptional transactivation domain. Mol. Cell. BioL 10:5473-5485; 1990. 75. Baeuerle, P. A.; Baltimore, D. IKB: A specific inhibitor of the NF-KB transcription factor. Science 242:540-546; 1988. 76. Naumann, M.; Scheidereit, C. Activation of NF-KB in vivo is regulated by multiple phosphorylations. EMBO J. 13:45974607; 1994. 77. Hayashi, T.; Sekine, T.; Okamoto, T. Identification of a new serine kinase that activates NF-KB by direct phosphorylation. J. Biol. Chem. 268:26790-26795; 1993. 78. Henkel, T.; Machleidt, T.; Alkalay, I.; Kronke, M.; Ben-Neftah, Y.; Baeuerle, P. A. Rapid proteolysis of IKB-a is necessary for activation of transcription factor NF-KB. Nature 365:182-185; 1993. 79. Baeuerle, P. A.; Henkel, T. Function and activation of NFkappa B in the immune system. Annu. Rev. Immunol. 12:141179; 1994. 80. Higgins, K. A.; Perez, J. R.; Coleman, T. A.; Dorshkind, K.; McComas, W. A.; Sarmiento, U. M.; Rosen, C. A.; Narayanan, R. Antisense inhibition of the p65 subunit of NF-KB blocks tumorigenicity and causes tumor regression. Proc. Natl. Acad. Sci. USA 90:9901-9905; 1993. 81. Kaltschmidt, B.; Baeuerle, P. A.; Kaltschmidt, C. Potential involvement of the transcription factor NF-kappa B in neurological disorders. Mol. Aspects Med. 14:171-190; 1993. 82. Matthews, J. R.; Wakasugi, N.; Virelizier, J. L; Yodoi, J.; Hay, R. T. Thioredoxin regulates the DNA binding activity of NF-

xB by reduction of a disulphide bond involving cysteine 62. Nucleic Acids Res. 20:3821-3830; 1992.

83. Mitomo, K.; Nakayama, K.; Fujimoto, K.; Sun, X.; Seki, S.; Yamamoto, K. Two different cellular redox systems regulate the DNA-binding activity of the p50 subunit of NF-KB in vitro. Gene 145:197-203; 1994. 84. Hayashi, T.; Ueno, Y.; Okamoto, T. Oxidoreductive regulation of nuclear factor kappa B. Involvement of a cellular reducing catalyst thioredoxin. J. Biol. Chem. 268:11380-11388; 1993. 85. Kumar, S.; Rabson, A. B.; Gelinas, C. The RxxRxRxxC motif conserved in all Rel/KB proteins is essential for the DNA binding activity and redox regulation of the v-Rel oncoprotein. Mol. Cell. Biol. 12:3094--3106; 1992. 86. Okamoto, T.; Ogiwara, H.; Hayashi, T.; Mitsui, A.; Kawabe, T.; Yodoi, J. Human thioredoxin/adult T cell leukemia-derived factor activates the enhancer binding protein of human immunodeficiency virus type 1 by thiol redox control mechanism. Int. Immunol. 4:811-819; 1992. 87. Schreck, R.; Meier, B.; Mannel, D. N.; Droge, W.; Baeuerle, P. A. Dithiocarbamates as potent inhibitors of nuclear factor •B activation in intact cells. J. Exp. Med. 175:1181-1194; 1992. 88. Israel, N.; Gougerot-Pocidalo, M. A.; Aillet, F.; Virelizier, J. L. Redox status of cells influences constitutive or induced NFkappa B translocation and HIV long terminal activity in human T and monocytic cell lines. J. Immunol. 149:3386-3393; 1992. 89. Schreck, F.; Rieber, P.; Baeuerle, P. A. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-KB transcription factor and HIV-1. EMBO J. 10:2247-2258; 1991. 90. Staal, F. J. T.; Roederer, M.; Herzenberg, L. A.; Herzenberg, L. A. Intracellular thiols regulate activation of nuclear factor KB and transcription of human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 87:9943-9947; 1990. 91. Nakamura, K.; Hori, T.; Sato, N.; Sugie, K.; Kawakami, T.; Yodoi, J. Redox regulation of a src family protein tyrosine kinase p56 LcK in T cells. Oncogene 8:3133-3139; 1993. 92. Anderson, M. T.; Staal, F. J. T.; Gitler, C.; Hersenberg, L. A.; Hersenberg, L. A. Separation of oxidant-initiated and redoxregulated steps in the NF-KB signal transduction pathway. Proc. Natl. Acad. Sci. USA 91:11527-11531; 1994. 93. Menon, S. D.; Qin, S.; Guy, G. R.; Tan, T. H. Differential induction of nuclear NF-KB by protein phosphatase inhibitors in primary and transformed human cells: Requirement for both oxidation and phosphorylation in nuclear translocation. J. Biol. Chem. 268:26805-26812; 1993. 94. Staal, F. J. T.; Anderson, M. T.; Staal, G. E. J.; Hersenberg, L. A.; Gitler, C.; Hersenberg, L. A. Redox regulation of signal transduction: Tyrosine phosphorylation and calcium influx. Proc. Natl. Acad. Sci. USA 91:3619-3622; 1994. 95. Talley, A. K.; Dewhurst, S.; Perry, S.; Dollard, S. C.; Gummuluru, S.; Fine, S. M.; New, D.; Epstein, L. G.; Gendelman, H. E.; Gelbard, H. A. Tumor necrosis factor alpha-induced apoptosis in human neuronal cells: Protection by the antioxidant N-acetylcysteine and the genes bcl-2 and crmA. Mol. Cell. Biol. 15:2359-2366; 1995. 96. Shen-Ong, G. L. C. The myb oncogene. Biochim. Biophys. Acta 1032:39-52; 1990. 97. Guehmann, S.; Vorbrueggen, G.; Kalkbrenner, F.; Moelling, K. Reduction of a conserved Cys is essential for Myb DNA-binding. Nucleic Acids Res. 20:2279-2286; 1992. 98. Myrset, A. H.; Bostad, A.; Jamin, N.; Lirsac, P. N.; Toma, F.; Gabrielsen, O. S. DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein. EMBO J. 12:4625-4633; 1993. 99. Grasser, F. A.; LaMontagne, K.; Whittaker, L.; Stohr, S.; Lipsick, J. S. A highly conserved cysteine in the v-Myb DNA binding domain is essential for transformation and transcriptional trans-activation. Oncogene 7:1005-1009; 1992. 100. Wasylyk, B.; Hahn, S. L.; Giovane, A. The Ets family of transcription factors. Eur. J. Biochem. 211:7-18; 1993. 101. Wasylyk, B.; Wasylyk, C.; Flores, P.; Begue, A.; Leprince, D.;

Redox regulation of transcriptional activators

102. 103. 104.

105.

106.

107. 108. 109.

110.

111.

112. 113.

114.

115.

116. 117.

118.

119.

Stehelin, D. The c-ets proto-oncogenes encode transcription factors that cooperate with c-Fos and c-Jun for transcriptional activation. Nature 346:191-193; 1990. Wasylyk, C.; Wasylyk, B. Oncogenic conversion of Ets affects redox regulation in vivo and in vitro. Nucleic Acids Res. 21:523-529; 1993. Suzuki, H.; Romano-Spica, V.; Papas, T.; Bhat, N. ETS1 suppresses tumorigenicity of human colon cancer cells. Proc. Natl. Acad. Sci. USA 92:44424446; 1995. Kadonaga, J. T.; Carner, K. R.; Masiarz, F. R.; Tjian, R. Isolation of cDNA encoding transcription factor Spl and functional analysis of the DNA binding domain. Cell 51:10791090; 1987. Westin, G.; Schaffner, W. Heavy metal ions in transcription factors from HeLa cells:Spl, but not octamer transcription factor requires zinc for DNA binding and for activator function. Nucleic Acids Res. 16:5771-5781 ; 1988. Knoepfel, L.; Steinkiihler, C.; Can5, M.-T.; Rotilio, G. Role of zinc-coordination and of the glutathione redox couple in the redox susceptibility of human transcription factor SP1. Biochem. Biophys. Res. Commun. 201:871-877; 1994. Ammendola, R.; Mesuraca, M.; Russo, T.; Cimino, F. Spl DNA binding efficiency is highly reduced in nuclear extracts from aged rat tissues. J. Biol. Chem. 267:17944-17948; 1992. Ammendola, R.; Mesuraca, M.; Russo, T.; Cimino, F. The DNA-binding efficiency of Spl is affected by redox changes. Eur. J. Biochem. 225:483-489; 1994. Payvar, F.; DeFranco, D.; Firestone, G. L.; Edgar, B.; Wrange, O.; Okret, S.; Gustafsson, J.-A.; Yamamoto, K. R. Sequencespecific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region. Cell 35:381-392; 1983. Freedman, L. F.; Luisi, B. F.; Korszun, Z. R.; Basavappa, R.; Sigler, P. B.; Yamamoto, K. R. The function and structure of the metal coordination sites within the glucocorticoid receptor DNA binding domain. Nature 334:543-546; 1988. Schaumburg, B. P. Investigations on the glucocorticoid-binding protein from rat thymocytes: II. Stability, kinetics and specificity of binding of steroids. Biochim. Biophys. Acta 261:219-235; 1972. Grippo, J. F.; Holmgren, A.; Pratt, W. B. Proof that the endogenous, heat-stable glucocorticoid receptor-activating factor is thioredoxin. J. Biol. Chem. 260:93-97; 1985. Bodwell, J. E.; Holbrook, N. J.; Munck, A. Evidence for distinct sulfhydryl groups associated with steroid- and DNA-binding domains of rat thymus glucocorticoid receptors. Biochemistry 23:4237-4242; 1984. Tienrungroj, W.; Meshinchi, S.; Sanchez, E. R.; Pratt, S. E.; Grippo, J. F.; Holmgren, A.; Pratt, W. B. The role of sulfhydryl groups in permitting transformation and DNA binding of the glucocorticoid receptor. J. Biol. Chem. 262:6992-7000; 1987. Esposio, F.; Cuccovillo, F.; Morra, F.; Russo, T.; Cimino, F. DNA binding activity of the glucocorticoid receptor is sensitive to redox changes in intact cells. Biochim. Biophys. Acta 1260:308-314; 1995. Roth, G. S.; Livingston, J. N. Dissociation of glucocorticoid inhibition of glucose transport and metabolism by increase in adipocyte age and/or size. Endocrinology 104:423-428; 1979. Sukhatme, V. P.; Cao, X.-M.; Change, L. C.; Tsaimorris, C.-H.; Stamenkovich, D.; Ferreira, P. C. P.; Cohen, D. R.; Edwards, S. A.; Shows, T. B.; Curran, T.; LeBeau, M. M.; Adamson, E. D. A zinc-finger encoding gene co-regulated with c~fos during growth and differentiation and after cellular depolarization. Cell 53:37-43; 1988. Huang, R.-P.; Adamson, E. D. Characterization of the DNAbinding properties of the early growth response-1 (Egr-1) transcription factor: Evidence for modulation by a redox mechanism. DNA Cell Biol. 12:265-273; 1993. Esposito, F.; Agosti, V.; Morrone, G.; Morra, F.; Cuomo, C.; Russo, T.; Venuta, S.; Cimino, F. Inhibition of the differentiation of human myeloid cell lines by redox changes induced through glutathione depletion. Biochem. J. 301:649-653; 1994.

347

120. Amone, M. I.; Zannini, M.; Di Lauro, R. The DNA binding activity and the dimerization ability of the thyroid transcription factor I are redox regulated. J. Biol. Chem. 270:12048-12055; 1995. 121. Civitareale, D.; Saiardi, A.; Falasca, P. Purification and characterization of thyroid transcription factor 2. Biochem. J. 304:981-985; 1994. 122. Pognonec, P.; Roeder, R. G. Recombinant 43-kDa USF binds to DNA and activates transcription in a manner indistinguishable from that of natural 43144-kDa USF. Mol. Cell. Biol. 11:5125-5136; 1991. 123. Pognonec, P.; Kato, H.; Roeder, R. G. The Helix-Loop-Helix/ Leucine repeat transcription fractor USF can be functionally regulated in a redox-dependent manner. J. Biol. Chem. 267:24563-24567; 1992. 124. Nebert, D. W.; Puga, A.; Vasiliou, S. Role of the Ah receptor and the dioxin-inducible lAb] gene battery in toxicity, cancer, and signal transduction. Ann. N Y Acad. Sci. 685:624640; 1993. 125. Reyes, H.; Reisz-Porszasz, S.; Hankinson, O. Identification of the Ah receptor nuclear translocator protein (Arm) as a component of the DNA binding form of the Ah receptor. Science 256:1193-1195; 1992. 126. Lusska, A.; Shen, E.; Whitlock, J. P., Jr. Analysis of proteinDNA interactions at a dioxin-responsive enhancer. J. Biol. Chem. 268:680-685; 1993. 127. Nebert, D. W. The Ah locus: Genetic differences in toxicity, cancer, mutation, and birth defects. Crit. Rev. Toxicol. 20:153174; 1989. 128. Ireland, R. C.; Li, S.-Y.; Dougherty, J. J. The DNA binding of purified Ah receptor heterodimer is regulated by redox conditions. Arch. Biochem. Biophys. 319:470-480; 1995. 129. Call, K. M.; Glaser, T.; Ito, C. Y.; Buckler, A. J.; Pelletier, J.; Haber, D. A.; Rose, E. A.; Kral, A.; Yeger, H.; Lewis, W. H.; Jones, C.; Housman, D. E. Isolation and characterization of a zinc-finger polypeptide gene at the human chromosome 11 Wilms' tumor locus. Cell 60:509-520; 1990. 130. Ory, K.; Legros, Y.; Auguin, C.; Soussi, T. Analysis of the most representative tumour-derived p53 mutants reveals that changes in protein conformation are not correlated with loss of transactivation or inhibition of cell proliferation. EMBO J. 13:34963504; 1994. 131. Stein, B.; Baldwin, A. S., Jr.; Ballard, D. W.; Greene, W. C.; Angel, P.; Herrlich, P. Cross-coupling of the NF-KB p65 and Fos/Jun transcription factors produces potentiated biological function. EMBO J. 12:3879-3891; 1993. 132. Goldberg, Y.; Treier, M.; Ghysdael, J.; Bohmann, D. Repression of AP- l-stimulated transcription by c-Ets- 1. J. BioL Chem. 269:16566-16573; 1994. 133. Masquilier, D.; Sassone-Corsi, P. Transcriptional cross-talk: Nuclear factor CREM and CREB bind to AP-1 sites and inhibit activation by Jun. J. Biol. Chem. 267:22460-22466; 1992.

ABBREVIATIONS

AhR--aryl hydrocarbon recepotor AP- 1-- activator protein 1 ARNT--AhR nuclear translocator B HA-- butyl-hydroxyanisole BHQ--butylhydroquinone BSO--buthionine sulfoximine CATIchloramphenicol acetyltransferase DEM-- diethylmaleate D T T - - 1 , 4 - d i t h i o - 1- t h r e i t o l Egr- 1 --early growth response- 1 EMSAIelectrophoretic mobility-shift assay GR--glucocorticoid receptor

348

Y. SUN and L. W. OSERLEY

GRE--glucocorticoid response element GSH--glutathione (reduced form) GSSG--glutathione (oxidized form) GST--glutathione S-transferase H202--hydrogen peroxide /3-ME--~-mercaptoethanol NAC--N-acetyl-L-cysteine NADPH--nicotinamide adenine dinucleotide phosphate (reduced form) NEM--N-ethyl-maleimide NF-xB--nuclear factor KB O P - - 1,10-phenanthroline

PDTC--pyrrolidine dithiocarbamate TCDD - - 2,3,7,8 -tetrachlorodibenzo-p-dioxin Ref-1--nuclear redox factor-1 TPA-- 12- O-tetradecanoylphorbol-13-acetate TRE--TPA-response element TF--transcription factors TTF--thyroid transcription factor USF--upstream sfimulatory transcription factor VHL--von Hippel-Lindau disease tumor suppressor gene WT-1--Wilm's tumor suppressor gene-1 XRE--xenobiotic responsive element