Activator protein 1 (AP-1)– and nuclear factor κB (NF-κB)–dependent transcriptional events in carcinogenesis

Free Radical Biology & Medicine, Vol. 28, No. 9, pp. 1338 –1348, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

PII S0891-5849(00)00220-3

Forum: Signal Transduction ACTIVATOR PROTEIN 1 (AP-1)– AND NUCLEAR FACTOR ␬B (NF-␬B)–DEPENDENT TRANSCRIPTIONAL EVENTS IN CARCINOGENESIS TIN-CHEN HSU,† MATTHEW R. YOUNG,* JOAN CMARIK,*

and

NANCY H. COLBURN*

†

*Gene Regulation Section, National Cancer Institute—FCRDC and IRSP, SAIC-Frederick, Frederick, MD, USA (Received 6 January 2000; Accepted 9 February 2000)

Abstract—Generation of reactive oxygen species (ROS) during metabolic conversion of molecular oxygen imposes a constant threat to aerobic organisms. Other than the cytotoxic effects, many ROS and oxidants are also potent tumor promoters linking oxidative stress to carcinogenesis. Clonal variants of mouse epidermal JB6 cells originally identified for their differential susceptibility to tumor promoters also show differential reduction-oxidation (redox) responses providing a unique model to study oxidative events in tumor promotion. AP-1 and NF-␬B, inducible by tumor promoters or oxidative stimuli, show differential protein levels or activation in response to tumor promoters in JB6 cells. We further demonstrated that AP-1 and NF-␬B are both required for maintaining the transformed phenotypes where inhibition of either activity suppresses transformation response in JB6 cells as well as human keratinocytes and transgenic mouse. NF-␬B proteins or extracellular signal–regulated kinase (ERK) but not AP-1 proteins are shown to be sufficient for conversion from transformation-resistant to transformation-susceptible phenotype. Insofar as oxidative events regulate AP-1 and NF-␬B transactivation, these oxidative events can be important molecular targets for cancer prevention. © 2000 Elsevier Science Inc. Keywords—AP-1, NF-␬B, JB6 cells, Tumor promotion, Free radical

INTRODUCTION

mation, hyperoxia, ultraviolet (UV) and ionizing irradiation, some heavy metals, and certain oxidant chemicals also increase the cellular production of ROS [3,6,7]. Excessive ROS react with and modify macromolecules resulting either in alterations of DNA structure such as point mutations, sister chromatid exchange, and chromosomal aberrations [1,8,9], or in functional modifications of reactive proteins. The biological consequences of ROS are changes in signal transduction, gene expression, and posttranscriptional or posttranslational modification that alter cell growth and differentiation and consequently carcinogenesis [7,10 –12]. Aerobic organisms constantly battle the adverse effects of ROS by increasing the production of biochemical antioxidants (glutathione, tocopherol, ascorbate, ␤-carotene, retinoic acid, or pyridine nucleotides) [10,13,14], or by inducing endogenous antioxidant enzymes including superoxide dismutase (SOD), catalase, thioredoxin, glutathione peroxidase (GPX) and reductase (GRS), and others [13,15– 22]. These scavenging antioxidant molecules and the endogenous antioxidant enzymes attenuate the ROS concentration to maintain an intracellular reduction and oxidation (redox) balance.

Reactive oxygen species (ROS) including superoxide anion, hydrogen peroxides, and hydroxyl anion are generated due to an incomplete reduction of molecular oxygen to water during aerobic metabolism [1–5]. InflamDr. Nancy Colburn is currently Chief of the Gene Regulation Section in the Basic Research Laboratory at the National Cancer Institute in Frederick, Maryland. Drs. Tin-Chen Hsu, Matthew Young, and Joan Cmarik are senior postdoctoral fellows in the Gene Regulation Section who received their Ph.D.s from Thomas Jefferson University, University of Maryland, and Vanderbilt University, respectively. Dr. Hsu has characterized activated forms of NF-␬B and AP-1 complexes required for tumor promotion. Dr. Young has identified ERK dependent events required for AP-1 activation. Dr. Cmarik has cloned a novel inhibitor of tumor promotion. The Colburn laboratory has pioneered the use of the mouse JB6 model to identify molecular events required for tumor promotion that may be targeted for cancer prevention. The JB6 model has proven to be predictive for in vivo mouse skin carcinogenesis and for human keratinocyte progression models. Address correspondence to: Dr. Nancy Colburn, Gene Regulation Section, National Cancer Institute, Building 560, Room 21-89, Frederick, MD 21702, USA; Tel: (301) 846-1342; Fax: (301) 846-6907; E-Mail: [email protected]. 1338

AP-1– and NF-␬B– dependent transformation

In addition to their cytotoxic consequences, many oxidants appear to be natural tumor promoters. In tumor promotion–sensitive (P⫹) mouse epidermal JB6 cells, ROS stimulate the cell growth in soft agar or in monolayer cultures [15,23,24]. Oxidative stress has been implicated in both initiation and promotion/progression phases of carcinogenesis [1]. Reduced levels of antioxidant enzymes, including manganese SOD (MnSOD), thioredoxin reductase or, in some cases, CuZnSOD, were implicated in transformation of MCF-7, HeLa, and Jurkat cells, respectively [1,17–20,25–32]. In addition, the levels of small molecule antioxidants, acting to antagonize oxygen free radicals were lowered in many transformed cell types [1,16,25,27,33–38]. Overexpression of manganese SOD (MnSOD), a natural antioxidant, was shown to revert transformation or tumor promotion response in human melanoma cells UACC-903, human breast cancer MCF-7 cells, mouse epidermal JB6 cells [29,32,40], and other cells [41,42]. MOUSE EPIDERMAL JB6 CELLS: A UNIQUE CELL MODEL TO STUDY OXIDATIVE STRESS AND TUMOR PROMOTION

Mouse epidermal JB6 cells and their clonal variants represent a unique model characterized by their differential responsiveness to tumor promoter–induced neoplastic transformation [24,43– 45]. Upon induction by phorbol ester (TPA), epidermal growth factor (EGF), or tumor necrosis factor-alpha (TNF-␣), the tumor promotion–sensitive (P⫹) JB6 cells proceed irreversibly to an anchorage-independent growth phenotype and tumorigenicity, whereas the tumor promotion–resistant (P⫺) variants of JB6 cells are inert to induced transformation [44 – 46]. The molecular responses contributing to the differential transformation response in JB6 clonal variants have been studied extensively [44,47]. Among the responses that distinguish P⫹ and P⫺ JB6 cells, some are required for neoplastic transformation of JB6 cells as listed in Table 1. In addition, as summarized in Table 2, there are other differential responses whose possible causal significance remains to be determined. These JB6 cell variants showing differential tumor promotion responses also show differential oxidative responses [15,24,35,48], and elevation of superoxide anion has been shown to be a required event in phorbol ester– induced tumor promotion [49,50]. The levels of antioxidant enzymes, CuZnSOD and catalase, in P⫹ JB6 cells are twice those in P⫺ cells resulting from increased concentrations of mRNA for these genes [15,23,35]. The cooperation between elevated CuZnSOD and catalase protects P⫹ JB6 cells from oxidative damage and the subsequent cytotoxicity by converting superoxide to hydrogen peroxide (CuZnSOD) then to water molecules

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Table 1. Events Required for Transformation Expression or activity is differential in P⫹ and P⫺ JB6 cells Higher in

Event Elevation of superoxide anion/antioxidant protection Activator protein-1 (AP-1) activation Extracellular related kinase (ERK1/2) expression Nuclear factor ␬B (NF-␬B) activation Phospho-inositol-3-kinase activation Tissue inhibitor of metalloproteinase-3 (TIMP-3) downregulation and transcriptional silencing A7-1/Pdcd4 downregulation

P⫹

References [40,148,149] [24,49,50] [43,150] [53,54]

P⫹ P⫹ P⫹ P⫹ P⫺/P⫹ vs. Tx P⫺

Hsu et al., submitted [151,152] [153,154] [130]

Expression or activity is not differential in P⫹ and P⫺ JB6 cells ODC, osteopontin, p107, JNK, and Bcl-2

[131, 134, 135, 155, 156]

(catalase). The P⫺ JB6 cells, however, are more susceptible to oxidation-mediated injury due to the lack of active machinery to counteract reactive oxygen. In P⫹ JB6 cell overexpressing only CuZnSOD, a potentiated NF-␬B response was observed due to elevated hydrogen peroxides converted from molecular oxygen by CuZnSOD. Overexpression of catalase, individually or in addition to CuZnSOD co-expression, efficiently converted the CuZnSOD-generated hydrogen peroxide to water and resulted to reduced NF-␬B and transformation responses in P⫹ JB6 cells [15,23,35,51] and other cells [17,19]. Overexpression of MnSOD, a mitochondria SOD, also Table 2. Events Differential in P⫹ and P⫺ JB6 Cells

Event c-jun expression Fra-1 related protein downregulation Phosphorylation of MARCKS protein (80 kD PKC substrate) Expression of FKBPRP (FK506BP58) TIS1 immediate early gene induction TIS21 gene induction Pleckstrin gene induction 6-ketoprostaglandin F1␣ release Prolonged PKC inactivation by benzoyl peroxide Synthesis of 15- and 16-kD nuclear proteins Higher levels and synthesis of phosphatidylethanolamine; increased activation of phosphatidylcholine-specific phospholipase D Pristane stimulated inducible binding to CRE; repression of phosphodiesterase activity Cyclins A, B1, D1 induction HMG-I(Y) protein expression

Higher in

References

P⫹ P⫺ P⫺

[111] [157] [158]

P⫺ P⫺ P⫺ P⫺ P⫺

[159] [160] [160] [168] [161] [162]

P⫹ P⫹

[163] [164]

P⫹

[165]

P⫹ P⫹

[166] [147]

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suppressed the transformation response in P⫹ JB6 cells [40] whereas overexpression of extracellular signal–regulated kinase (ERK1/2), a second messenger of oxidative and other stimuli, conferred transformation response on P⫺ JB6 cells [52–54]. Because of their differential gene expression responses to oxidative stress and tumor promoters, JB6 cells offer a useful model to study the contribution of oxidative stress to tumor promotion, and to identify the altered gene expression events that may transduce the stimuli [24]. REDOX REGULATION OF AP-1 AND NF-␬B TRANSCRIPTION FACTOR ACTIVITIES AND THE ROLE OF ERK

NF-␬B and AP-1 are two eukaryotic transcription factors that regulate genes implicated in ROS-induced responses, and both factors are targets of oxidative stimuli [56 – 63]. Exogenous expression of MnSOD in MCF-7 breast cancer cells altered their intracellular redox states and suppressed their transformation phenotypes [32,55]. The tumor suppression by MnSOD in MCF-7 cells was accompanied by inhibition of AP-1 and NF-␬B activities providing a link among tumor formation, reactive oxygen, and transcription factors, AP-1 and NF-␬B. Similarly, in P⫹ JB6 cells overexpressing MnSOD, the suppressed TPA-induced transformation response was shown to be associated with decreased expression of c-fos and c-jun suggesting a role for AP-1 and NF-␬B in oxidation-mediated transformation [32, 40]. Activation of NF-␬B by hydrogen peroxide was detected in various cell types [7,10,11,64 – 69]. The event was cell-type specific and appeared to be regulated primarily by glutathione and its converting enzymes [7,70 – 72], in that depletion of glutathione in Jurkat cells suppressed NF-␬B activation induced by hydrogen peroxides [67]. The antioxidant, pyrrolidinedithiocarbamate (PDTC), has been shown to inhibit TNF-␣– induced NF-␬B activation in transformed human endothelial cells [73] as well as TPA- or TNF-␣–induced activation of NF-␬B and neoplastic transformation in JB6 cells [74]. N-acetyl-L-cysteine (NAC), another antioxidant, was also reported to suppress NF-␬B activation induced by TNF-␣, LPS, or UV in Jurkat and HeLa cells [61,75]. Overexpression of MnSOD, catalase, or GPX reduced NF-␬B activation induced by TPA, LPS, TNF-␣, or hydrogen peroxide in human melanoma cells [62,76]. Expression of thioredoxin abolished TPA-induced NF-␬B activation in HeLa cells [77]. Although a series of studies supported the notion that NF-␬B is a target and mediator of oxidative stress, the failure of hydrogen peroxides to activate NF-␬B in some T cell lines [71–78] and of catalase to block NF-␬B induction

by TPA or TNF-␣ in COS-1 cells [79], suggests that ROS species other than hydrogen peroxides may be operative in some systems. Reactive oxygen has been shown to regulate AP-1 activation [56,60,61,63]. Expression of c-jun and c-fos is transiently induced by TPA [80,81] and other mitogens [82,83]. Furthermore, hydrogen peroxide, UV-C and UV-A, ionizing radiation, asbestos, and dioxin have been reported to induce AP-1 activity through transcriptional, post-transcriptional, and post-translational mechanisms [84 – 88]. It was suggested that activation of the mitogenactivated protein kinase (MAPK) pathway may be responsible for AP-1 activation by oxidative stimuli. Oxidative stimuli was shown to induce phosphorylation of Elk-1 by ERK1/2 resulting in a potentiated interaction with serum response factor (SRF) and enhanced binding to serum response element (SRE) on the promoter region of c-fos for elevated expression [65,66,89]. Hydrogen peroxide not only induced the activation of ERK1/2 [90], it also induced NF-␬B activation [58], suggesting that NF-␬B, like AP-1, may act as a downstream target of ERK1/2 in response to oxidation. In contrast, thioredoxin or reduced oxygen pressure (hypoxia) also activated AP-1 where de novo protein synthesis of c-Fos and, to a lesser extent, c-Jun was required since addition of cycloheximide abolished the AP-1 induction [91,92]. The mechanism for AP-1 activation by antioxidant needs further characterization. Exposure of HeLa cells to UV-C and hydrogen peroxides, however, causes a rapid increase in AP-1 binding independently of new protein synthesis [93,94] suggesting that Fos and Jun activation occur as a result of post-translational modifications involving changes in phosphorylation states known to modulate AP-1 activity [7]. ERK has been reported to phosphorylate the Ser-243 at C-terminal of c-Jun while dephosphorylation of several C-terminal Ser/Thr appears to be necessary to unmask the DNA-binding domain of c-Jun [7,95,96]. Activation of c-Jun requires a phosphorylation at Ser-63 and Ser-73 at the N-terminal transactivation domain by the JUN kinase (JNK) induced directly by UV-C or indirectly by hydrogen peroxides and nitric oxide through the induction of p21ras [97–100]. Redox Factor 1 (Ref-1), a class II hydrolytic apurinic/ apyrimidinic endonuclease, has been shown to stimulate the DNA-binding activity of AP-1 proteins by a redoxdependent mechanism in cooperation with thioredoxin [56,102]. Thioredoxin was shown to increase DNA-binding activity of AP-1 or NF-␬B by reducing Cys-154 in c-Fos and Cys-272 in c-Jun or Cys-62 in p50 of NF-␬B, all located in the DNA-binding domains [92,103–105]. Ref-1 also stimulated DNA-binding activity of AP-1 and NF-␬B by maintaining the above cysteine residues in reduced states in which Ref-1 regulated the redox state in

AP-1– and NF-␬B– dependent transformation

the presence of reducing agents like thioredoxin and thioredoxin reductase [56,106,107]. In cells treated with TPA or ionizing radiation, thioredoxin is imported into the nucleus in response to oxidative stimuli and interacts with Ref-1, implying that Ref-1 participates in relaying a redox signal from the cytoplasm to the nucleus to activate the nuclear transcription factors [108 –110]. TRANSACTIVATION OF AP-1 AND NF-␬B IS REQUIRED FOR TUMOR PROMOTER–INDUCED TRANSFORMATION

Clonal variants of JB6 cells having differential transformation responses to TPA, EGF, or TNF-␣ also show differential activation of AP-1 with elevated AP-1 response detected only in P⫹ JB6 cells [43,46,111]. Expression of a transactivation-deficient mutant of c-Jun (TAM67) or exposure to AP-1 transrepressing retinoids suppressed AP-1 activation and the inducible transformation responses in P⫹ JB6 cells demonstrating that AP-1 activation is required in tumor promoter–induced transformation [112]. Overexpression of c-jun, the only differentially expressed AP-1 protein, failed to alter the AP-1 nonresponsive or the transformation-inert phenotypes of P⫺ JB6 cells suggesting that although AP-1 activity is required for transformation response induced by TPA, EGF, or TNF-␣, AP-1 protein expression is not limiting in P⫺ cells and is insufficient to confer transformation susceptibility [113]. ERK proteins are, however, limiting in P⫺ JB6 cells, and restoration of ERK levels confers both AP-1 response and transformation response [53,114]. Similar observations regarding AP-1 and NF-␬B, discussed later, are applicable to mouse and human keratinocyte models as well as to a transgenic mouse model where TAM67 expression also suppressed or reversed their respective progression phenotypes or prevented tumor promotion [115,116]. An increase in c-fos mRNA expression and protein synthesis was observed in both the P⫹ and P⫺ JB6 cells upon TPA induction, indicating that mitogen-induced expression of c-fos is not the limiting factor for reduced AP-1 activation in the P⫺ JB6 cells [111]. Induced expression of c-jun is also not limiting for AP-1 activation in P⫺ cells as discussed above. Electrophoretic mobility shift assays (EMSA) showed that JunD and possibly JunB are the major Jun family members present in TPA-induced AP-1 DNA– binding complexes. The c-Jun protein was not detected in AP-1 complexes in untreated P⫹ JB6 cells or in those transiently induced upon TPA exposure [167] (Young and Colburn, unpublished). The presence of JunB in AP-1 complexes appears to be induced by TPA in the P⫹ JB6 cells to a greater extent than in the P⫺ variants, whereas the level of JunD, although higher in P⫹ than in P⫺, appears constant with or without TPA induction. Based on these

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observations, Bernstein and colleagues suggested that JunD is a limiting factor in AP-1 complexes in P⫺ JB6 cells, contributing to reduced AP-1 response. Although c-Fos protein was expressed in both P⫹ and P⫺ JB6 cells upon TPA induction, it was present only in the AP-1 DNA– binding complexes of only the P⫺ JB6 cells, suggesting that c-Fos does not participate in the formation of an active AP-1 complex and may function as an inhibitory factor [167]. Fra-1 and Fra-2 were present in DNA-binding complexes of both P⫹ and P⫺ cells even at the basal condition without induction. Fra-1, although constitutively expressed, was induced by TPA to a greater level in DNA-binding complexes of P⫹ than P⫺ JB6 cells and remained at an elevated level for greater than 18 h. Thus, Fra-1 appears to be the dominant Fos family protein in transcriptionally active AP-1 complexes in JB6 cells and its major partner in the AP-1 heterodimer appears to be JunD and/or JunB. Inhibition of NF-␬B activation in P⫹ JB6 cells using a chemical antioxidant, PDTC, was shown to be accompanied by inhibition of AP-1 activation when the transformation response was also suppressed [74]. Inhibition of AP-1 by dominant negative Jun (TAM67) in human keratinocytes demonstrated the associated inhibition of NF-␬B during suppression of their transformed phenotype [116]. Because the reduction of AP-1 and NF-␬B activities occur concurrently when the activity of either one is suppressed, NF-␬B may contribute instead of or in addition to AP-1 in neoplastic transformation of JB6 cells. Interaction between the p65 component of NF-␬B and c-Fos/c-Jun components of AP-1 was previously reported [117] providing a possible mechanism explaining coordinated regulation of AP-1 and NF-␬B signaling in JB6 cells. Previous studies suggested the requirement of NF-␬B for maintenance of tumor phenotype and/or for inhibition of apoptosis [118 –125]. Inhibition of NF-␬B by a nondegradable mutant of I␬B␣ (I␬B␣mut), an antisense RNA, or a transgenic null mutation results in tumor regression [120,126,127]. However, none of the previous reports showed that a NF-␬B component is sufficient to confer inducible transformation response. The fact that inhibition of AP-1 is invariably accompanied by inhibition of NF-␬B when transformation is suppressed in human keratinocytes suggests that NF-␬B regulated gene expression may also be required for neoplastic transformation. Our recent findings (Hsu et al., submitted) indicate that P⫹ and P⫺ JB6 cells are variants for activation of NF-␬B upon induction by TPA or TNF-␣. Inhibition of NF-␬B using a nondegradable mutant of I␬B␣ suppresses tumor promoter–induced transformation in P⫹ JB6 cells. Elevated levels of nuclear NF-␬B proteins and increased NF-␬B–specific DNA binding appear to contribute to the enhanced NF-␬B activity in P⫹ JB6 cells.

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It is yet unclear whether the redox states of the NF-␬B proteins, especially p50, also play a role in transformation responses of JB6 cells. Overexpression of NF-␬B proteins in P⫺ JB6 cells substantially increases both NF-␬B and AP-1 activities and confers transformation response to tumor promoters (Hsu et al., submitted). Our findings suggest that NF-␬B not only is required for tumor promoter–induced transformation response in JB6 cells but also plays a causal role in maintaining expression of transformed phenotype in human keratinocytes. THE AP-1 AND NF-␬B ACTIVATION AND INDUCED TRANSFORMATION RESPONSES ARE REGULATED BY ERK

Elevated levels of ERK1/2 proteins and their activities were previously observed in P⫹ JB6 cells leading to enhanced activation of AP-1 [53,54]. In P⫺ JB6 cells with reduced ERK protein levels, overexpression of ERK2 restored the cellular ERK activity and subsequently conferred transformation susceptibility. Elevated AP-1 and transformation responses to TPA or EGF were observed in P⫺ JB6 cells expressing ERK2. Conversely, a dominant negative mutant of ERK inhibited ERK activity in the P⫹ JB6 cells and blocked their TPA-induced AP-1 and transformation responses [53]. Therefore, an increased level of ERK2, protein concentration or activation, appears to be not only necessary but also sufficient to induce both AP-1 activation and neoplastic transformation in P⫺ JB6 cells. Identification of ERKdependent events needed to activate AP-1 will be important. Elevated NF-␬B response was also observed in P⫺ JB6 cells expressing ERK2 while a reduced level of NF-␬B activation was detected in P⫹ JB6 cells expressing dominant negative ERK2 (Hsu and Colburn, unpublished) suggesting that NF-␬B, in addition to AP-1, is a target of ERK signaling in JB6 cells. However, since NF-␬B is able to restore transformation response in P⫺ JB6 cells independent of ERK activation, NF-␬B also appears to be an essential and causal factor for neoplastic transformation of JB6 cells. It is important to ascertain whether ERK and NF-␬B act independently on separate pathways or interactively to produce transformation susceptibility. Modulation of ERK response coordinately altered the activation of AP-1 and NF-␬B suggesting that a combinatory role of AP-1, NF-␬B, and ERK is required for neoplastic transformation of JB6 cells. The differential responses of AP-1, NF-␬B, and ERK activation in P⫹ and P⫺ JB6 cells produced by tumor promoters or changes in redox states make JB6 cells a unique model to study oxidative stress and its resulting biological consequences as well as to investigate oxidative induction of carcinogenesis. Many events in Table 1 occur differen-

tially in P⫹ and P⫺ JB6 cells and are required for tumor promotion. Some of these events appear to associate with ROS signaling pathways, while the relationship of others to oxidative signaling remains to be identified.

DOWNREGULATION OF A NOVEL, DIFFERENTIALLY EXPRESSED PROTEIN PDCD4 IS REQUIRED FOR TUMOR PROMOTER–INDUCED TRANSFORMATION

A differential display of mRNA comparison of JB6 P⫹ and P⫺ cells identified seven differentially expressed genes, five of which were preferentially expressed in transformation-resistant P⫺ cells [138] The function of one of the genes preferentially expressed in P⫺ cells, pdcd4, has been previously unknown [128, 129]. To test the hypothesis that pdcd4 functions as an inhibitor of tumor promoter–induced transformation, whose expression must be downregulated if transformation is to occur, we expressed a pdcd4 antisense construct in P⫺ cells [130]. Clonal transfectants expressing antisense pdcd4 showed reduced expression of endogenous pdcd4 and a gain of transformation response, an effect that was reversed by expressing sense constructs complementary to the antisense sequence [130]. Thus, pdcd4 is a novel inhibitor of induced transformation whose molecular mechanism will be of interest to elucidate.

OTHER EVENTS THAT ARE REQUIRED FOR TRANSFORMATION OF JB6 CELLS

Other events shown in Table 1, although required for tumor promotion, show similar regulatory response in P⫹ and P⫺ JB6 cells; that is, genetic variants for these responses have not been identified among JB6 clonal lines. Such an example is ornithine decarboxylase (ODC) whose activation is necessary for TPA-induced transformation response in JB6 cells [131]. Inhibition of ODC by ␣-difluoromethylornithine attenuated TPA-induced ODC activity and transformation response while TPA-induced AP-1 activation was not altered. AP-1 inhibition by TAM67 did not affect ODC induction, suggesting that while both events are required they occur on separate pathways. It is not known if NF-␬B activation is involved in ODC-mediated transformation of JB6 cells but it appears unlikely because TAM67 also inhibits NF-␬B activation when ODC is unaffected. The roles of ROS in ODC induction and signaling need further characterization. Another TPA-inducible gene, osteopontin, was identified by subtractive hybridization using TPA-treated P⫹ and P⫺ cells [132,133] but turned out to be similarly expressed in P⫹ and P⫺ cells. The question of whether osteopontin induction is required for induced transfor-

AP-1– and NF-␬B– dependent transformation

mation was raised [134] but has not been definitively answered. Studies of Chang and coworkers [135–137] have characterized both expression and phosphorylation of osteopontin during JB6 cell transformation. Finally, Thurston et al. have found that one of the Rb family of pocket proteins, p107, is downregulated as a late event following TPA exposure of JB6 cells (Thurston et al., submitted). Antisense p107 expression speeds up and enhances the transformation response, suggesting that downregulation of p107 expression must occur for transformation to occur. OTHER EVENTS OCCURRING DIFFERENTIALLY IN Pⴙ AND Pⴚ JB6 CELLS

Because JB6 cell variants provide a unique model for studying the oxidative response, studies of events that are differentially regulated in cell variants with high or low oxidation states may contribute to the understanding of signaling by oxidative stress. These differentially occurring events are listed in Table 2. In addition, three uncharacterized sequences preferentially expressed in P⫺ cells and three in P⫹ cells (one with some sequence similarity to mRNA for vasoactive intestinal polypeptide precursor) have been identified by differential display [138,168]. The establishment of a causal or protective function of any of these events in neoplastic transformation in the JB6 model awaits the results of further experiments. However, it is interesting to speculate on the role of certain of these events in neoplastic transformation as well as oxidative signaling. Events occurring preferentially in P⫺ cells may protect the cells against transformation. 6-ketoprostaglandin F1␣ is a stable metabolite of PGI2, an agonist of adenylate cyclase. P⫺ cells released more 6-ketoprostaglandin F1␣ in response to the calcium ionophore A23187 than did P⫹ cells [139]. The cAMP elevator forskolin and the PGI2 analog carba-prostacyclin both inhibit TPA-induced anchorage-independent transformation of P⫹ cells [139,140]. These data suggest that enhanced levels of certain prostaglandin metabolites that regulate adenylate cyclase may inhibit neoplastic transformation in P⫺ cells. Pleckstrin was recently identified as a mRNA preferentially induced in P⫺ cells by TPA, and the mouse cDNA sequence was subsequently determined [168]. Pleckstrin contains two pleckstrin homology domains, protein motifs shown to interact with phosphoinositides and, in some cases, other proteins [141]. Proteins containing pleckstrin homology domains are thought to play a role in signal transduction by affecting the subcellular localization of other proteins with which they interact. Pleckstrin is the major PKC substrate found in platelets [142]. As PKC activity is stimulated by TPA, it is inter-

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esting to consider the possibility that the induction and possibly the phosphorylation of pleckstrin in response to TPA treatment of P⫺ cells may stimulate a signal transduction pathway with a protective outcome that is lacking in P⫹ cells. The HMG-I and -Y proteins are nonhistone chromosomal proteins. They are alternatively spliced transcripts of the same gene, and differ by only 11 amino acids present in I but lacking in Y [143]. Elevated levels of HMG-I(Y) proteins have been associated with neoplastic vs. normal cell lines, and with benign and malignant tumor tissue vs. normal tissue [144]. HMG-I(Y) proteins function as architectural transcription factors by bending DNA and also by directly interacting with transcription factors, including members of the AP-1 and NF-␬B families [145,146]. Thus, HMG-I(Y) might influence the expression of a large number of genes downstream of multiple transcription factors, and the finding that HMGI(Y) proteins are induced to greater levels and are more persistent in P⫹ than in P⫺ cells [147] suggests that these proteins may play a pivotal role in mediating tumor promoter–induced neoplastic transformation of P⫹ cells. Furthermore, HMG-Y protein was uniquely induced in the P⫹ cells, suggesting the possibility that HMG-Y may have a function distinct from that of HMG-I in neoplastic transformation of JB6 cells. No differences in activity or role of the 11 amino acids region unique to I are currently known. In conclusion, we have presented here findings and opportunities using a unique cell model, mouse epidermal JB6 cells. The clonal variants of JB6 cells show differential responses to tumor promoter–induced transformation and display differential redox states in response to oxidative stress. Since many tumor promoters are also generators of reactive oxygen, JB6 cells provide an ideal model to study the consequences of ROS generation during carcinogenesis. Many of the gene regulation events demonstrated to play essential roles in transformation may also respond to ROS signaling. Tables 1 and 2 list a group of events that are differentially regulated in clonal variants of JB6 cells. We believe that studies on JB6 cells may contribute to understanding of ROS response and other biological consequences yet to be characterized.

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ABBREVIATIONS

ROS—reactive oxygen species AP-1—activator protein 1 NF-␬B—nuclear factor ␬B ERK— extracellular signal–regulated kinase ref-1—redox factor 1 TPA—12-O-tetradecanoylphorbol-13-acetate TNF—tumor necrosis factor HMG— high-mobility group ODC— ornithine decarboxylase JNK—Jun N-terminal kinase Tx—transformation MAPK—mitogen-activated protein kinase