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Address correspondence to C. Huang, Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Rd., Tuxedo, NY 10987. Tel.: (845) 731-3519. Fax: (845) 351-2118. email: chuanshu{at}env.med.nyu.edu
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Abstract |
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Key Words: polycyclic aromatic hydrocarbons; signal transduction; protein kinases; transcription factor; tumor promotion
Abbreviations used in this paper: 5-MCDE, (±)-anti-5-methylchrysene-1,2-diol-3,4-epoxide; AP-1, activator protein-1; B[a]P, benzo[a]pyrene; B[a]PDE, (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide; ERK, extracellular signalregulated protein kinase; JNK, c-Jun NH2-terminal kinase; PAH, polycyclic aromatic hydrocarbon; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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Introduction |
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The tumor promotion phase is a long-term reversible process characterized as the activation of transcription factors and regulation of their target genes through signal transduction pathways (Slaga and Fischer, 1983; Pitot and Dragan, 1996). The mouse epidermal JB6 Cl41 cell line is a well-characterized and widely used cell culture model for tumor promotion studies (Bernstein and Colburn, 1989; Dong et al., 1994, 1995; Huang et al., 1997a, 1998; Li et al., 1997; Watts et al., 1998). In transformation-sensitive (P+) but not transformation-resistant (P) JB6 cell lines, tumor promoters, such as phorbol esters or growth factors, induce activator protein-1 (AP-1) activity and neoplastic transformation (Bernstein and Colburn, 1989). Inhibition of AP-1 induction by TAM67 (a transactivation domain deletion mutant of c-Jun; Brown et al., 1993) blocks 12-O-tetradecanoylphorbol-13-acetate (TPA) and EGF-induced AP-1 transactivation and cell transformation (Dong et al., 1997). Several other events are also found to be required for different chemical-induced tumor promotion, including activation of extracellular signalregulated protein kinases (ERKs), nuclear factor-B, and PI-3K (Huang et al., 1996, 1997b, 1998; Li et al., 1997; Watts et al., 1998; Hsu et al., 2000). Importantly, this cell line is derived from mouse skin; thus, the results generated from Cl41 cells will provide a strong basis for further investigation in mouse skin, which is an important bioassay system for the evaluation of tumor promotion in vivo (Slaga and Fischer, 1983; Pitot and Dragan, 1996). Therefore, Cl41 cells were used in this paper as the in vitro model to investigate the tumor-promoting activity of 5-MCDE, the metabolite of 5-methylchrysene. We found that 5-MCDE induces AP-1 transcriptional activity in the Cl41 cell line, and this induction occurs via a PI-3K/Aktdependent signaling pathway.
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Results |
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Discussion |
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AP-1 is an important transcription factor that governs the expression of genes involved in intercellular communication, amplification, and primary pathogenic signals spreading as well as initiation and acceleration of tumorigenesis (Shaulian and Karin, 2002). It recognizes both TPA response elements (5'-TGAG/CTCA-3') and cAMP response elements (5'-TGACGTCA-3'; Chinenov and Kerppola, 2001). The Cl41 cell line, which is used in this paper, proceeds to an anchorage-independent growth phenotype and tumorigenicity upon the induction by phobol ester (TPA), EGF, or TNF- (Huang et al., 1996, 1997b, 1999a; Watts et al., 1998). AP-1 activation is identified as one of the required events for the aforementioned transformations (Colburn and Smith, 1987; Bernstein and Colburn, 1989; Dong et al., 1994; Singh et al., 1995; Huang et al., 1997a). Both the AP-1 molecular inhibitor TAM67 (a transactivation domain deletion mutant of c-Jun) and AP-1 transrepressing retinoids can block TPA-induced AP-1 transactivation, cell transformation, and tumor induction (Dong et al., 1994; Huang et al., 1997a,b). Thus, the importance of AP-1 in tumor promotion is well established. The mechanisms of cell transformation by AP-1 are probably through its manipulation on the target genes expressions (Chinenov and Kerppola, 2001; Shaulian and Karin, 2001). For instance, c-Jun is a positive regulator on gene expression of cyclin D1 and FasL, and a negative regulator of p16ink4a, p53, and p21cip1/waf1 (Shaulian and Karin, 2001), whereas c-Fos is required for expression of many matrix metalloproteinases (Saez et al., 1995). In this paper, 5-MCDE was shown to induce AP-1 transactivation. Although it does not change the c-Fos protein level, 5-MCDE exposure increases the expressions of Jun B, Fos B, Fra-1, and Fra-2. More importantly, the phosphorylations of c-Jun protein at serines 63 and 73 are markedly induced after 5-MCDE treatment. Furthermore, 5-MCDE exposure also increases the expression of AP-1 target genes, such as cyclin D1 and collagenase type I, which are associated with cell cycle regulation and tumor induction, respectively (Schonthal et al., 1988; Saez et al., 1995; Shaulian and Karin, 2002). Because 5-MCDE is identified as a complete carcinogen in animal carcinogenesis models, our results strongly suggest that AP-1 activation is one of the events necessary for the tumor promotion by 5-MCDE.
In the process of identifying the upstream-regulating signal transduction pathway, we found that the PI-3K pathway is involved in 5-MCDEinduced AP-1 activation. Using both PI-3K inhibitor (wortmannin and LY294002) and the dominant-negative mutant PI-3K (p85) can substantially inhibit AP-1 transactivation induced by 5-MCDE. This result is consistent with our previous findings that PI-3K activation is the upstream kinase event responsible for mediation of AP-1 transactivation induced by TPA or EGF (Huang et al., 1996, 1997b). PI-3K also plays an important role in skin tumor progression phase. It is suggested by the observations that wortmannin and dominant-negative p85 subunit of PI-3K inhibit the TPA- and EGF-induced cell transformation and prevent the invasion of MDA-MB-435 cells, whereas a constitutively active p110 subunit of PI-3K increases their invasion (Huang et al., 1996, 1997b; Adelsman et al., 1999). Together, we speculate that the PI-3K signal pathway is involved in carcinogenic effects of 5-MCDE.
Among the downstream kinases of PI-3K, Akt has been identified as the main signal transmitter. Akt, which is also known as PKB, is the cellular homologue of the retroviral oncogene v-Akt (Datta et al., 1999; Scheid and Woodgett, 2001). The activation of Akt has been identified in many cancers (Cheng et al., 1996; Bellacosa et al., 1998; Ruggeri et al., 1998; Shayesteh et al., 1999). For instance, amplification of Akt was observed in cancers of the breast, ovaries, and pancreas (Cheng et al., 1996; Bellacosa et al., 1998; Ruggeri et al., 1998; Shayesteh et al., 1999). The loss of function of tumor suppressor gene PTEN, which converts PIP3 back to PIP2 and thus mitigates the effects of Akt activation caused by PI-3K, is also seen in a wide spectrum of human cancers (Ali et al., 1999). Besides the PI-3Kdependent pathway, increased calcium can also activate the calcium/calmodulin-dependent kinase kinase, which then activates Akt by directly phosphorylating Akt at the Thr308 (Chen et al., 2002). In this work, Akt was found to be involved in 5-MCDEinduced AP-1 transactivation and activated in a PI-3Kdependent manner. Overexpression of a dominant-negative subunit of PI-3K blocked the 5-MCDEinduced Akt phosphorylations at Ser473 and Thr308. Introduction of dominant negative mutant Akt specifically impairs 5-MCDEinduced activation of AP-1. These results indicate that 5-MCDE induces AP-1 activation through the PI-3KAkt pathway.
The role of another possible PI-3K downstream signaling pathway, mTORp70S6K, in 5-MCDEinduced AP-1 activation was also investigated. Although the exact relationship between mTOR and PI-3K remains to be determined, mTOR has been found to be phosphorylated and activated by Akt in mitogen-simulated cells and PTEN-deficient cells (Brunet et al., 1999; Scheid and Woodgett, 2001). Moreover, application of an mTOR inhibitor had antiproliferative and anticancer effects in PTEN-deficient cells (White, 1998; Laine et al., 2000). mTOR controls the mammalian translation machinery by activating p70S6K, which enhances the translation of mRNA with 5' polypyrimidine tracts, as well as by inhibiting 4E-BP1 (also known as PHAS-1), which is a translation inhibitor binding to the CAP structure present at the 5' termini of mRNAs (Thomas and Hall, 1997; Hara et al., 1998). Currently, the mTOR derivative CCI-779 has entered a clinical trial for cancer treatment (Mills et al., 2001; Owa et al., 2001). However, we found mTOR is not involved in the 5-MCDEinduced AP-1 activation pathway in Cl41 cells. This notion was supported by the following evidence: (a) rapamycin can't decrease the 5-MCDEinduced AP-1 activation; (b) dominant-negative mutant p85 impaired 5-MCDEinduced AP-1 activation of all time points tested, and it only inhibited the early phase but not the late phase of p70S6K phosphorylation; and (c) overexpression of dominant-negative mutant Akt impairs AP-1 activation, but not p70S6K phosphorylation by 5-MCDE.
Besides the PI-3K pathway, MAPKs (ERKs, p38K, and JNKs) are also found to involve in AP-1 activation by many other stimuli. Serum and growth factors activate AP-1 transcriptional activity mainly through ERKs, whereas the induction of AP-1 by proinflammatory cytokines and genotoxic stress is mostly through activating JNKs and p38K cascades (Gruda et al., 1994; Hill et al., 1994; Chang and Karin, 2001). In this work, we demonstrated that PI-3KAkt pathway mediated AP-1 activation specifically through activation of ERKs and JNKs. Although our most current data have shown that p38K are activated and involved in 5-MCDEinduced AP-1 activation (unpublished data), the signaling pathways leading to activation of p38K are still under investigation in our laboratory.
In summary, the present work demonstrates that 5-MCDE induces AP-1 activity in a dose- and time-dependent manner. AP-1 activation by 5-MCDE is mediated through a PI-3K/Akt/ERKs, JNKs-dependent, and mTOR-independent pathway. This conclusion is based on the results provided from using chemical inhibitors, including wortmannin, Ly294002, and rapamycin, or overexpression of dominant-negative mutants, such as p85, Akt-T308A/S473A, DN-ERK2, and DN-JNK1. This paper is highly relevant to the understanding of the molecular mechanisms involved in tumor promotion effects by 5-MCDE, but may also provide some useful information for cancer chemoprevention by enabling manipulation of the upstream regulatory signal pathways leading to AP-1 activation. Although mechanisms underlying the initiating signaling pathways by 5-MCDE are not well understood, we anticipate that oxidative stress, such reactive oxygen species, may be involved. This notion is supported by findings that 5-MCDE is capable of inducing oxidative DNA damage. Investigations are underway to determine if this is the case.
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Materials and methods |
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Cell culture
The JB6 P+ mouse epidermal Cl41 cell line and its transfectants were cultured in monolayers at 37°C, 5% CO2, using MEM that contained 5% FBS, 2 mM L-glutamine, and 25 µg gentamicin per milliliter as described previously (Huang et al., 1997a, 1998; Watts et al., 1998). The cultures were dissociated with trypsin and transferred into new 75-cm2 culture flasks (Fisher Scientific) from one to three times per week.
Generation of stable cotransfectants
Cl41 cells were cultured in a 6-well plate until they reached 8590% confluence. 1 µg of CMV-neo vector and 20 µl of LipofectAMINE reagent, with 2 µg of AP-1-luciferase reporter plasmid DNA, mixed with 12 µg of dominant-negative mutant (SR-Akt-T308A/S473A) or vector control were used to transfect each well in the absence of serum. After 1012 h, the medium was replaced with 5% FBS MEM. Approximately 3036 h after the beginning of the transfection, the cells were digested with 0.033% trypsin, and cell suspensions were plated into 75-ml culture flasks and cultured for 2428 d with G418 selection (400 µg/ml). The all stable clones (stable pool) in a flask were designed as a mass culture. The stable transfectants were identified by measuring both the basal level of luciferase activity and the blocking Akt activation. Stable transfectants, Cl41 AP-1 mass4 and Cl41 AP-1 Akt-T308A/S473A mass1, were established and cultured in G418-free MEM for at least two passages before each experiment.
AP-1 activity assay
Confluent monolayers of P+1-1 cells or Cl41 cell stable transfectants were trypsinized, and 8 x 103 viable cells suspended in 100 µl MEM supplemented with 5% FBS were added to each well of 96-well plates. Plates were incubated at 37°C in a humidified atmosphere with 5% CO2 in air. After the cell density reached 8090%, cells were exposed to 5-MCDE at a final concentration as indicated in the figures for AP-1 induction. At different periods after treatment, the cells were extracted with lysis buffer (Promega) and their luciferase activity was determined by the Luciferase assay using a luminometer (Wallac 1420 Victor 2 multilable counter system; PerkinElmer) after the addition of 100 µl of lysis buffer for 30 min at 4°C. The results are expressed as AP-1 activity relative to control medium containing the same concentration of DMSO only (relative AP-1 activity).
Northern blot
Whole RNA was extracted from monolayer cells with TRIzol® reagent according to the manufacturer's instructions (Invitrogen). 15 µg RNA was run on a 1% agarose/formaldehyde gel at 120 V for 1.5 h. The RNA was transferred to a nylon membrane (Ambion) by vacuum for 1.5 h and cross-linked by UV wave. The murine collagenase I (MMP13) probe was PCR-amplified from mouse mRNA (Yu et al., 2002). The probe was labeled with -[32P]dCTP using Prime-a-Gene Labeling System (Promega). The radioactivity was detected by phosphoImager using a scanner (model StormTM 860; Molecular Dynamics).
RT-PCR
The RNA was extracted by the Trizol® method mentioned in the previous paragraph. The cDNA was synthesized from 2 µg RNA using First-Strand Synthesis System for RT-PCR according to manufacturer's instructions (Invitrogen). 1 µl of synthesized cDNA was used for following multiplex PCR. The murine cyclin D1 (Klein et al., 2003) and ß-actin (sense, 5'-CCT GTG GCA TCC ATG AAA CT-3'; antisense, 5'-GTG CTA GGA GCC AGA GCA GT-3') primers were added together and PCR-amplified for 21 cycles. The PCR products were separated on 2% agarose gel.
PI-3K assay
PI-3K activities were assayed as described previously (Huang et al., 1996, 1997b). In brief, cells were cultured in monolayers in 100-mm dishes using normal culture medium. The media were replaced with 0.1% FBS MEM that contained 2 mM L-glutamine and 25 µg of gentamicin per milliliter after the cell density reached 7080%. 45 h later, the cells were incubated with fresh serum-free MEM medium for 34 h at 37°C. 5-MCDE was added to cell cultures for PI-3K induction. Cells were washed once with ice-cold PBS and lysed in 400 µl of lysis buffer per plate (20 mM Tris, pH 8, 137 mM NaCl, 1 mM MgCl2, 10% glycerol, 1% NP-40, 1 mM dithiothreitol, 0.4 mM sodium orthovanadate, and 1 mM PMSF). The lysates were centrifuged and the supernatants incubated at 4°C with 40 µl of agarose beads (previously conjugated with the monoclonal anti-phosphotyrosine antibody Py20) overnight. Beads were washed twice with each of the following buffers: (1) PBS with 1% NP-40 and 1 mM dithiothreitol; (2) 0.1 M Tris, pH 7.6, 0.5 M LiCl, and 1 mM dithiothreitol; and (3) 10 mM Tris, pH 7.6, 0.1 M NaCl, and 1 mM dithiothreitol. Beads were incubated for 5 min on ice in 20 µl of buffer 3, and 20 µl of 0.5 mg/ml phosphatidylinositol (previously sonicated in 50 mM Hepes, pH 7.6, 1 mM EGTA, and 1 mM NaH2PO4) were added. After 5 min at RT, 10 µl of the reaction buffer were added (50 mM MgCl2, 100 mM Hepes, pH 7.6, and 250 µM ATP containing 5 µCi of -[32P]ATP), and beads were incubated for an additional 15 min. Reactions were stopped by adding 15 µl 4 N HCl and 130 µl of chloroform/methanol (1:1). After vortexing for 30 s, 30 µl from the phospholipid-containing chloroform phase were spotted onto TLC plates coated with silica gel H containing 1.3% potassium oxalate and 2 mM EDTA applied in H2O/methanol (3:2). Plates were heated at 110°C for at least 3 h before use. Plates were placed in tanks containing chloroform/methanol/NH4OH/H2O (600:470:20:113) for 4050 min until the solvent reached the top of the plates. Plates were dried at RT and autoradiographed (Huang et al., 1996, 1997b).
Western blot
3 x 104 Cl41 transfectants were cultured in each well of 6-well plates to 7080% confluence with normal culture medium. The cell culture medium was replaced with 0.1% FBS MEM supplemented with 2 mM L-glutamine and 25 µg gentamicin per milliliter and cultured for 33 h. Cells were incubated in serum-free MEM for 34 h at 37°C, and were then exposed to 5-MCDE for various lengths of time. Cells were washed once with ice-cold PBS and extracted with SDS-sample buffer. The cell extracts were separated on polyacrylamideSDS gels, transferred, and probed with one of the specific antibodies as indicated. The protein bands specifically bound to primary antibodies were detected using an antirabbit IgG-AP-linked and an ECF Western blotting system (Huang et al., 2002; Amersham Biosciences).
Statistical analysis
t test was used to determine the significance of differences in AP-1 activities between 5-MCDE treated and DMSO control. The differences were considered significant at a P < 0.05.
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Acknowledgments |
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This work was supported in part by grants from National Institutes of Health (NIH)/National Cancer Institute (CA094964, CA103180, and CA112557) and NIH/National Institute of Environmental Health Sciences (ES012451).
Submitted: 5 January 2004
Accepted: 29 January 2004
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