Key words: chemoprevention, detoxication, antioxidant responsive element, ARE, NRF1, CYP, GST, MAP kinase, carcinogenesis, protein kinase C

Abstract
This article provides an overview of the mechanisms by which cancer chemopreventive blocking agents increase the expression of detoxication and antioxidant genes. These agents all appear capable of transcriptionally activating a gene battery that includes NAD(P)H:quinone oxidoreductase, aldo-keto reductases, glutathione S-transferases, gamma-glutamylcysteine synthetase, glutathione synthetase and heme oxygenase. Gene induction occurs through the antioxidant responsive element (ARE), a process that is dependent on the NF-E2-related factors Nrf1 amd Nrf2. Under basal conditions, these basic region leucine zipper (bZIP) transcription factors are located in the cytoplasm of the cell bound to Keap1, and upon challenge with inducing agents they are released from Keap1 and translocate to the nucleus. Within the nucleus, Nrf1 and Nrf2 are recruited to the ARE as heterodimers with either small Maf proteins, FosB, c-Jun, JunD, ATF2 or ATF4. The role of protein kinases in transducing chemical stress signals to the bZIP factors that affect gene induction through the ARE is discussed.

Introduction
A wide range of chemicals, both naturally-occurring and synthetic, can protect against the development of cancer. These include coumarins, diterpenes, dithiolethiones, indoles, isothiocyanates, lactones, organosulphides and phenols (for a review, see reference [1]). Compounds with cancer chemopreventive properties have been subdivided into blocking agents and suppressing agents on the basis of the stage during carcinogenesis at which they act [2]. Blocking agents prevent carcinogens from modifying DNA and causing mutations. This is usually achieved by increasing the expression of detoxication and antioxidant enzymes in target tissues, though alterations in the pharmacokinetics of xenobiotics may also serve to protect against tumourigenesis. Such responses are thought to represent a form of cellular adaptation to chemical and oxidative stress [1]. By contrast, suppressing agents inhibit the later promotion and progression stages of neoplastic disease. Their actions include antagonism of oncogenes, activation of tumour suppressor proteins, inhibition of angiogenesis, stimulation of apoptosis or terminal differentiation, and modulation of arachidonic acid cascades.

Induction of detoxication and antioxidant genes as mechanisms of cancer chemoprevention
It was first shown over twenty years ago that chemopreventive blocking agents increase the hepatic levels of enzymes involved in drug metabolism [2,3]. Cytochrome P450 (CYP), NAD(P)H:quinone oxidoreductase (NQO), aldehyde dehydrogenase, aldo-keto reductase, glutathione S-transferase (GST), UDP-glucuronosyl transferase (UGT), and microsomal epoxide hydrolase represent some of the enzymes that are now known to be induced by chemopreventive agents [3-6]. These findings suggest that enhanced detoxification capacity represents a mechanism by which compounds with anticancer properties confer at least some of their beneficial effects.

Besides influencing drug metabolism, it is becoming recognised that blocking agents also induce antioxidant proteins and enzymes involved in the inflammatory response. Such proteins include the catalytic heavy subunit and the regulatory light subunit of -glutamylcysteine synthetase (GCS_h and GCSl), glutathione synthetase, -glutamyl transpeptidase, heme oxygenase (HO), the heavy and light subunits of ferritin, peroxiredoxin MSP23, and leukotriene B₄ 12-hydroxydehydrogenase [4,6,7]. Thus, increases in antioxidant status, scavenging of free radicals, and detoxification of both reactive oxygen species and the cytotoxic metabolites they generate through damaging and degrading macromolecules also represent mechanisms by which anticancer agents may protect against neoplasia [8].

Recognition that certain blocking agents serve to increase NQO and GST activities but not arylhydrocarbon hydroxylase activity (CYP1A1 and CYP1A2), whereas others induce all three activities, led Prochaska and Talalay [9] to refer to the former compounds as monofunctional inducers and the latter compounds as bifunctional inducers. Subsequent characterization of the 5-flanking regions of the rat NQO1, GSTA2 and CYP1A1 genes has revealed that monofunctional inducers transcriptionally activate the expression of these genes through the antioxidant responsive element (ARE) whilst bifunctional inducers act through the ARE as well as the xenobiotic responsive element (XRE) [1]. The fact that both monofunctional and bifunctional inducers stimulate the ARE suggests that this enhancer plays a central role in the anticancer actions of such agents.

Biotransformation of blocking agents is often essential for them to affect gene induction. Evidence indicates that activation of ARE-driven gene expression requires a metabolic stimulus. Blocking agents that work through the ARE either possess a thiol-active moiety or are converted within the body to a metabolite bearing such a group [1,10]. For example, beta-naphthoflavone (beta-NF) requires to be oxidised by CYP1A1 to a putative quinone- or hydroquinone-containing metabolite [11]. Similarly, butylated hydroxyanisole (BHA) requires to be converted, through O-demethylation catalysed by another CYP isoenzyme, to tert-butylhydroquinone (t-BHQ) and ethoxyquin is oxidised to the alpha,beta-unsaturated carbonyl-containing compound 2,2,4-trimethyl-6-quinolone [3,12].

In addition, a significant number of blocking agents exist, including isothiocyanates, that stimulate the ARE directly [13]. Figure 1 shows the cis-acting elements and metabolic events by which anticarcinogens regulate gene expression. It should be noted that as indoles only appear to stimulate XRE-driven gene expression, and not ARE-driven gene expression (at least in LS-174 and Caco-2 human colon cells), they ought not to be classed as bifunctional inducers [14].

Identification of the antioxidant responsive element
The ARE was first characterized by Pickett and his colleagues as an enhancer within a 41 base pair region from the 5'-flanking region of the rat GSTA2 gene that was responsive to beta-NF [15]. Initially, the enhancer was referred to as a beta-NF responsive element [11,15], but was re-designated the ARE once it was recognised to respond not only to beta-NF but also to phenolic antioxidants that undergo redox cycling [16]. Furthermore, the ARE responds to H₂O₂ and hypoxia, as well as to alpha,beta-unsaturated carbonyls and hydroperoxides that are typically generated during oxidative stress [16,19]. This enhancer is therefore now thought to play an important role in the regulation of antioxidant genes in response to exposure to pro-oxidants [1,8].

Deletion and mutational analyses of the rat GSTA2 promoter identified the core sequence required for basal and/or inducible activity of the ARE as 5'-TGACNNNGC-3' [16]. Subsequent functional analysis has shown that the T nucleotide in the 5' part of the ARE is essential for both its basal and inducible activity, whereas the adjacent G is only essential for induction [20]. Mutation of the other more proximal nucleotides attenuates, but does not abolish, ARE activity [20].

Within the promoter of the murine Gsta1 gene a 41 base pair region exists that shares 95% sequence identity with the ARE-containing region in rat GSTA2 [1,21]. The enhancer flanking mGsta1 was originally called an electrophile responsive element (EpRE) because it can be stimulated by dimethyl fumarate and trans-4-phenyl-3-buten-2-one as well as by t-BHQ and beta-NF [21].

ARE enhancers have been found in the promoters of the rat and human NQO1 genes [22,23], and in the genes encoding the human GCS_h and GCS₁ subunits [24,25]. Also, the ARE has been demonstrated to be present in the 5-flanking region of the mouse and human HO-1 genes [26,27], where it has been called a stress responsive element (StRE) because it responds to heme, heavy metals, arsenite and 12-O-tetradecanoylphorbol-13-acetate. This enhancer has similarly been identified in the promoters of the murine metallothionein 1 gene [28], the rat inducible nitric oxide synthase gene [29] and the rat gamma-glutamyl transpeptidase gene [30]. The human spermidine/spermine N¹-acetyltransferase gene may also contain an ARE, but in this instance it has been called a polyamine-responsive element [31].

Comparison between the ARE enhancers in the promoters of rGSTA2, mGsta1, rNQO1, hNQO1 and rGSTP1 allowed a consensus sequence to be proposed, namely, 5'-T^A/_CANN^A/_GTGA^C/_TNNNGC^A/_G-3' [17]. Based on this consensus sequence, Wasserman and Fahl [17] employed a bioinformatics approach to identify ARE-like motifs in the promoters of rodent genes encoding the GSH transporter, ferritin-L, tyrosinase and interleukin 6. The significance of these putative enhancers has yet to be established.

Role of basic region leucine zipper transcription factors in regulation of detoxication/antioxidant genes and sensitivity to carcinogenesis
Similarity exists between the ARE and the DNA-binding motifs of transcription factors within the basic region leucine zipper (bZIP) superfamily. Friling et al [21] highlighted the fact that part of the core ARE bears some resemblance to the 5-TGA^C/_GT^A/_CA-3 DNA recognition motif for AP-1. Although the ARE consensus sequence is clearly distinct from the classic AP-1 binding site, it is nevertheless related to the DNA-binding sites of other bZIP transcription factors such as nuclear factor-erythroid 2 (NF-E2), 5'-^A/_GTGA^C/_GTCAGC^A/_G-3' [32], and the viral oncoprotein v-Maf, 5'-TGCTGACTCAGCA-3' [33].

Conclusions
NF-E2 is the founding member of a family of cap ‘n’ collar (CNC) bZIP transcription factors that includes NF-E2-related factors 1, 2 and 3 (Nrf1, Nrf2 and Nrf3) [32,34-36] and the more distantly related Bach1 and Bach2 [37]; Figure 2 shows a cartoon depicting the conserved Neh2, CNC and bZIP domains of p45 NF-E2, Nrf1, Nrf2 and Nrf3; the Nrf2-ECH homology domain 2 (Neh2) has been defined by Itoh et al [38]. The possibility that any of the CNC bZIP factors might mediate gene regulation through the ARE was first tested by Venugopal and Jaiswal [39]. These investigators demonstrated, by transient co-transfection of expression vectors for various bZIP proteins and reporter gene constructs into HepG2 cells, that overexpression of both Nrf1 and Nrf2 significantly increases ARE-driven gene expression. Furthermore, electrophoretic mobility shift assays demonstrated that hNrf1 was capable of binding the ARE found flanking hNQO1 [39], and that rNrf2 can bind the ARE in the promoters of rGSTA2 and rNQO1 [20].

The physiological role of Nrf1 has been the subject of several investigations. Nrf1 clearly performs a critical biological function during development since disruption of the gene is embryonically lethal [40,41]. Transient transfection of an expression construct for Nrf1 into COS-1 cells can both trans-activate transcription from the promoter of the gene for GCS_h and cause a concomitant increase in intracellular glutathione [42]. Evidence that Nrf1 contributes in vivo to protection against oxidants has been obtained from the finding that fibroblasts derived from embryos of mice bearing a targeted disruption of Nrf1 have a diminished expression of both GCS₁ and glutathione synthetase, and this is accompanied by an increased sensitivity to paraquat and CdCl₂ [43].

The contribution of Nrf2 to the regulation in vivo of detoxication and antioxidant genes has been evaluated not only by examining the phenotype of cells that overexpress this bZIP protein, but also by examining cells and tissues that lack the factor. Transient transfection of wild-type Nrf2 into HepG2 cells results in increased reporter activity from GCS_h and GCS₁ promoter transgenes [44]. Stable transfection of L929 cells with a regulatable dominant negative mutant form of Nrf2 was shown to diminish substantially induction of HO-1 by CdCl₂, ZnSO₄, sodium arsenite and t-BHQ [45]. Fibroblasts from Nrf2 (-/-) mice were demonstrated to express only about 15% of mRNA for GCS_h and GCS₁ when compared with fibroblasts from Nrf2 (+/+) mice [46]. The decreased expression of GCS subunits in Nrf2 (-/-) fibroblasts was also reflected by the level of GSH which was only about 50% of that in fibroblasts expressing the factor [46]. Macrophages from Nrf2 (-/-) mice have been shown to exhibit impaired expression of HO-1, peroxiredoxin MSP23, the 60 kDa stress protein A170, and the cystine membrane transporter (system X_c^-) [47]. The liver, forestomach and small intestine from Nrf2 (-/-) mice have been found to exhibit reduced constitutive and/or inducible expression of Nqo1, aflatoxin B₁ aldehyde reductase, Gsta1/2, Gsta3, Gsta4, Gstm1, Gstm5, Gstp1/2, Gstp1/2, Ugt1a6, microsomal epoxide hydrolase, GCS_h, manganese superoxide dismutase and catalase [12,48-50].

A major in vivo consequence of failure to express Nrf2 is increased sensitivity to xenobiotics. In many instances this phenotype is probably due to an impaired ability to resynthesize GSH following depletion by reactive chemicals. The Nrf2 (-/-) mice more readily develop liver damage caused by acetaminophen than do wild-type mice [51,52]. Also, the Nrf2 (-/-) mice readily develop pulmonary injury following treatment with butylated hydroxytoluene [53]. Like the liver and gastrointestinal tract, the lungs of the knockout mice have been found to contain diminished levels of mRNA for Nqo1, Ugt1a6, GCS₁, HO-1, superoxide dismutase and catalase. In the context of cancer chemoprevention, the most important finding in Nrf2 (-/-) mice is that the dithiolethione chemopreventive agent oltipraz fails to protect against benzo(a)pyrene-initiated cancer of the forestomach. Furthermore, the mutant mouse develops a larger number of tumours than do wild-type mice exposed to benzo(a)pyrene that have not received treatment with oltipraz [52]. This suggests that Nrf2 controls constitutive protective mechanisms against xenobiotics.

To our knowledge, nothing has been published about the contribution of other CNC bZIP transcription factors to the regulation of the ARE gene battery. However, red blood cells from p45 NF-E2 deficient mice are sensitive to oxidative stress and show loss of expression of Nqo1, Gsta3 and catalase [55]. Further work is required to determine whether Nrf3, Bach1 or Bach2 contribute to the regulation of detoxication and antioxidant genes.

Transcription factors recruited to the antioxidant responsive element

Both Nrf1 and Nrf2 bind target DNA sequences as heterodimers with other bZIP proteins. Initial characterization of p45 NF-E2 demonstrated that it bound DNA as a heterodimer with p18 NF-E2, also called MafK [32,56]. Using p45 NF-E2 as a paradigm, it seems likely that Nrf1 and Nrf2 heterodimerize with members of the small Maf protein family, MafF, MafG and MafK. Evidence that Nrf1 and Nrf2 can associate with small Maf proteins has been provided by electrophoretic mobility shift assays and immuno-precipitation experiments [20,48,57-59].

In addition to being able to form heterodimers with small Maf proteins, Nrf1 has been reported to dimerize with c-Jun, ATF2 and ATF4 [60,61], and Nrf2 has been reported to dimerize with c-Jun, ATF4, PMF and PPAR [62-65]. The physiological significance of Nrf1 and Nrf2 dimerizing with different bZIP proteins has received little attention to date. Transfection of MafG and MafK into cell lines negatively regulates ARE-driven gene expression [20,58], and it is therefore reasonable to suppose that Nrf1- and Nrf2-containing complexes will differ in the magnitude of transactivation depending on the relative amount of small Maf proteins present. Significantly, the bZIP factors MafG, ATF3, ATF4, c-Jun, JunB, Fra1and Fra2 are themselves inducible by H₂O₂ [66] CdCl₂ [63] and t-BHQ [67] and this will presumably introduce a temporal dimension into transcriptional activation through the ARE. It is anticipated that following drug exposure the complexes recruited to the ARE, and related enhancers, will change with time.

Sequences that flank the ARE consensus can influence its function [16,17]. For example, a number of gene promoters contain an ARE-related motif located close to the core enhancer that regulates basal expression. In the case of rGSTA2 and mGsta1 these two sequences are tandemly arrayed, whereas in rNQO1 and hNQO1 they are in reverse orientation and can form a 13 base pair palindrome [1,3,23]. It is possible that the sequence context of different AREs will influence the bZIP dimers that are recruited to the promoter. Certainly, Nguyen et al [20] have shown that rNfr2 binds the ARE in the rNQO1 gene promoter more avidly than the ARE in the rGSTA2 promoter. The binding specificity of different bZIP dimers towards palindrome-type and tandemly arrayed AREs ought to be investigated.

Intracellular sensors of chemopreventive blocking agents and transduction of a chemical stress signal to the ARE

The actin-binding protein Keap1 has been identified as a docking site for Nrf2 that is responsible for sequestering the bZIP protein in the cytoplasmic compartment of unstressed cells [68]. The association between the two proteins is between the C-terminal DGR domain of Keap1 and the N-terminal Neh2 domain of Nrf2. It is worth noting that Nrf1 also contains a Neh2 domain, and therefore it too probably interacts with Keap1 in vivo. A model depicting the movement of Nrf2 between compartments in non-stressed and chemically-stressed cells in shown in Figure 3.

The mechanisms by which cells recognise the presence of chemopreventive blocking agents remain to be elucidated. However, a key feature of these chemicals is that they interact with thiol groups, and would therefore be expected to modify cysteine residues within proteins [1,10]. It is therefore possible that modification of cysteine residues within Keap1 or Nrf2 could serve to trigger the release of the bZIP protein from its tethering site. An alternative possibility discussed previously [8,69] is that the active site cysteine residue of protein kinase phosphatases could be modified by blocking agents, and their resulting inactivation allow protein kinase signalling to proceed unchecked. An example of this is provided by receptor-directed tyrosine phosphatases being inactivated by UV irradiation, oxidants and alkylating agents [70,71]. Similarly, certain class Mu and class Pi GST subunits have been reported to act as endogenous inhibitors of stress-activated protein kinases [72,73]. It can therefore be postulated that in the presence of chemopreventive agents, GSTM1 and GSTP1 relax their inhibition of protein kinase activity, possibly through either covalent or non-covalent binding of the xenobiotic by the transferase.

Three of the mitogen activated protein (MAP) kinases, extracellular-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), have been implicated in ARE activation [74-76]. Two lines of evidence link MAP kinases to the ARE response. Firstly, many of the xenobiotics that stimulate ARE-driven gene expression also activate protein kinase activity. Secondly, modulation of MAP kinase activity by pharmacological inhibition, knockout by dominant negative mutants, or overexpression of constitutively active mutants modifies ARE-driven reporter activity. The emerging picture is complex, and suggests that the involvement of specific MAP kinases is variable and may be ARE-specific, inducer-specific and tissue-specific. Such variation is well illustrated by reference to the p38 kinase. Kong and his colleagues demonstrated that in serum-starved human HepG2 cells and murine Hepa1c1c7 cells, both t-BHQ and -NF, but not the isothiocyanate sulforaphane, induced p38 activity [75]. These workers found induction by t-BHQ of transcription from the mGsta1 ARE was augmented in these liver cell lines by pre-treatment with the p38 inhibitor SB203580, suggesting p38 negatively regulates the ARE [75]. By contrast, activation of the human HO-1 gene ARE by CdCl₂ in human mammary MCF-7 cells is diminished by pre-treatment with SB203580 [27]. Finally, in the rat hepatoma H4IIE cell line, both t-BHQ and sulphur amino acid deprivation (SAAD) activated p38 [77,78]. Nonetheless, the resulting induction of rGSTA2 mRNA by SAAD, but not by t-BHQ, was attenuated by SB203580.

At present, it is unclear how MAP kinases impinge upon ARE activation, and whether PI3K is essential for the response [74-79]. Of the proteins with clearly defined roles in ARE activation, none can be described definitively as MAP kinase substrates. However, Nrf1, Nrf2 and Keap1 contain potential proline-directed serine/threonine residues, and therefore cannot be disregarded as possible substrates. Similarly, certain small Maf proteins also contain proline-directed serine and threonine residues and therefore might be phosphorylated by MAP kinases. Both c-Jun and ATF2 are substrates for JNK [80-82]. Thus, it is reasonable to ascribe some of the variable results noted above to tissue-specific differences in signalling pathways, and differences in the composition of bZIP heterodimers recruited to the ARE enhancers in the promoters of different genes.

Whilst MAP kinase signalling to the ARE may be mediated by the dimerization partners of Nrf1 or Nrf2, it should be recognised that these latter CNC bZIP factors are phosphorylated in vivo. It has recently been demonstrated that treatment of HepG2 cells with t-BHQ results in increased phosphorylation of Nrf2, and that this is associated with appearance of the transcription factor in the nucleus [83]. Evidence suggests that the phosphorylation in HepG2 cells is largely mediated by protein kinase C (PKC). In vitro, Nrf2 is an excellent substrate for PKC [83]. Furthermore, PKC activity can be augmented in HepG2 cells by t-BHQ treatment. Crucially, pre-treatment of HepG2 cells with staurosporine, a pharmacological inhibitor of PKC, has been reported to abolish both phosphorylation and nuclear translocation of Nrf2 in response to t-BHQ [83].

Concluding comments
In this review we have highlighted recent advances in our understanding of how cancer chemopreventive blocking agents transcriptionally activate gene expression. This response to blocking agents appears to represent cellular adapation to chemical stress. Although it is clear that Nrf1 and Nrf2 play pivotal roles in transcriptional activation of cytoprotective genes, much remains to be learnt about how their dimerizing partners might influence this process. It seems likely that a large number of bZIP heterodimers can bind the ARE core enhancer, and therefore there is potential for both subtlety and specificity in the response to chemopreventive agents that is at present poorly understood. Relatively little is known about the number of genes that can be included in the ARE gene battery. This is an area that warrents investigation since the existance of inducible transcription factors that appear to be regulated through ARE-like elements may result in distinct batteries of genes being indirectly inducible by chemopreventive blocking agents.

Acknowledgments
We thank the Association for International Cancer Research for funding our research into cancer chemoprevention. Dr Cecil B. Pickett and Professor Masayuki Yamamoto are thanked for invaluable discussions about the ARE.

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Figure legends

Figure 1
Regulation of XRE- and ARE-driven gene expression by chemopreventive blocking agents. This diagram illustrates the various metabolic pathways, transcription factors and cis-acting elements that are involved in transcriptional activation of genes by monofunctional and bifunctional inducers. Some of the chemicals that act directly as monofunctional inducers are listed, as are others that require biotransformation before they can stimulate the ARE (see right hand side of the figure). In addition, the ARE can be stimulated by certain Ah receptor ligands such as sudan III and beta-NF once they have been oxidised by CYP isoenzymes; these are referred to as bifunctional inducers [9]. Indoles are considered to be chemopreventive agents and although they cannot activate the ARE directly [14] it is unclear whether their metabolites are capable of stimulating this enhancer. Thus, the hatched arrow from indoles on the left hand side of the figure to the ARE only represents a tentative assignment. The consensus sequence for the ARE, shown at the bottom, is described elsewhere [15-17]. The consensus sequence for the XRE is taken from reference 18. Since AREs recruit either Nrf1 or Nrf2 as heterodimers with small Maf proteins, FosB, c-Jun, JunD, ATF2 or ATF4, the complex above ARE in the diagram is simply Nrf-bZIP (see below for further details).

Figure 2
Location of structural domains in the p45 subunit of Nuclear Factor-Erythroid 2 and related factors Molecular cloning of p45 NF-E2, Nrf1, Nrf2 and Nrf3 revealed that the C-terminal part of each protein contains a cap ‘n’ collar (CNC) domain (shown in black) immediately adjacent to a basic region leucine zipper (bZIP) domain (shown in diagonal lines, from top right to bottom left). Together, the CNC and bZIP domains have been referred to as Neh1 by Yamamoto and his colleagues [38]. The Neh2 domain can be identified as conserved region of about 90 amino acids within the N-terminal third of Nrf1 and the N-terminus of Nrf2 that is responsible for the negative regulation of at least Nrf2 by Keap1 (represented by boxed area containing diagonal lines, from top left to bottom right) [38]. For comparison, the small Maf proteins are show as bZIP factors that lack both the CNC and Neh2 domains.

Figure 3
Model of signalling pathways involved in activation of Nrf2 by chemopreventive blocking agents The left hand panel shows that under basal conditions Nrf2 resides in the cytoplasm tethered to Keap1 whereas small Maf proteins are constitutively nuclear [. The right hand panel shows that upon treatment with blocking agents, the activities of MAP kinases, PKC and PI3K are increased, presumably in response to thiol-dependent stimulation of “sensors” that recognise the presence of specific classes of xenobiotic [69-82]. Activation of protein kinases leads to release of Nrf2 from Keap1, and translocation of the transcription factor to the nucleus where it may dimerize with a small Maf protein or another bZIP protein, before binding ARE enhancers in the promoters of inducible genes [68]. The signalling pathways responsible for release of Nrf2 from Keap1 have not been defined (shown by hatched lines) and may involve phosphorylation of either protein. Equally, it could entail phosphorylation of the dimerizing partner for Nrf2. Evidence has been presented that PKC can phosphorylate Nrf2 [83], and this is therefore shown in a solid line.

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