- Short communication
- Open Access
Benzo[a]pyrene diol epoxide suppresses retinoic acid receptor-β2 expression by recruiting DNA (cytosine-5-)-methyltransferase 3A
© Ye and Xu; licensee BioMed Central Ltd. 2010
- Received: 14 January 2010
- Accepted: 28 April 2010
- Published: 28 April 2010
Tobacco smoke is an important risk factor for various human cancers, including esophageal cancer. How benzo [a]pyrene diol epoxide (BPDE), a carcinogen present in tobacco smoke as well as in environmental pollution, induces esophageal carcinogenesis has yet to be defined. In this study, we investigated the molecular mechanism responsible for BPDE-suppressed expression of retinoic acid receptor-beta2 (RAR-β2) in esophageal cancer cells. We treated esophageal cancer cells with BPDE before performing methylation-specific polymerase chain reaction (MSP) to find that BPDE induced methylation of the RAR-β2 gene promoter. We then performed chromatin immunoprecipitation (ChIP) assays to find that BPDE recruited genes of the methylation machinery into the RAR-β2 gene promoter. We found that BPDE recruited DNA (cytosine-5-)-methyltransferase 3 alpha (DNMT3A), but not beta (DNMT3B), in a time-dependent manner to methylate the RAR-β2 gene promoter, which we confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis of the reduced RAR-β2 expression in these BPDE-treated esophageal cancer cell lines. However, BPDE did not significantly change DNMT3A expression, but it slightly reduced DNMT3B expression. DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-Aza) and DNMT3A small hairpin RNA (shRNA) vector antagonized the effects of BPDE on RAR-β2 expressions. Transient transfection of the DNMT3A shRNA vector also antagonized BPDE's effects on expression of RAR-β2, c-Jun, phosphorylated extracellular signal-regulated protein kinases 1/2 (ERK1/2), and cyclooxygenase-2 (COX-2), suggesting a possible therapeutic effect. The results of this study form the link between the esophageal cancer risk factor BPDE and the reduced RAR-β2 expression.
- Esophageal Cancer
- Esophageal Cancer Cell
- Esophageal Epithelial Cell
- Esophageal Cancer Cell Line
- Tobacco Carcinogen
Tobacco smoke is an important cause of human cancers, as it contains more than 60 carcinogens [1–4], which are major risk factors for cancers of the head and neck, lung, esophagus, pancreas, and bladder [5–9]. Benzo [a]pyrene diol epoxide (BPDE), a carcinogen present in tobacco smoke and environmental pollution, has been shown to induce gene mutations (such as in p53 and KRAS genes) in vitro [10–13]. Previously, we identified and cloned several BPDE-binding genes (such as ATM and BRCA2) and the cytosine-phosphate-guanine (CpG) islands of various gene promoters . Cigarette smoke has been shown to cause morphologic changes and the loss of retinoic acid receptor-beta2 (RAR-β2) expression in the lung tissues of experimental animals . Cigarette smoke, specifically the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, has also been shown to induce the methylation of the RAR-β2 gene promoters in murine lung cancer models . We have also previously shown that RAR-β2 expression is suppressed in premalignant and malignant esophageal cells [17–19]. Consequently, expression of epidermal growth factor receptor (EGFR), extracellular signal-regulated protein kinases 1/2 (ERK1/2), activated protein-1 (AP-1), and cyclooxygenase-2 (COX-2) are induced by BPDE . Numerous studies have demonstrated that RAR-β2 expression is frequently and progressively lost in premalignant and malignant tissues of the head and neck, lung, esophagus, pancreas, mammary gland, prostate, and other sites [20–22]. Lost expression of RAR-β2 in these various human cancers has been shown to be due to hypermethylation of its gene promoter [23–27]. However, it is still unknown if, and if so, how BPDE suppresses the expression of RAR-β2 and induces methylation of its gene promoter.
Our current findings provide further evidence that BPDE may play a role in esophageal cancer development and progression by suppressing RAR-β2 expression. As mentioned above, numerous studies have demonstrated that RAR-β2 expression is frequently and progressively lost in premalignant and malignant tissues and cells [20–22]. These studies clearly indicate that RAR-β2 functions as a tumor suppressor gene. RAR-β2 gene promoter methylation is believed to be responsible for the lost expression of RAR-β2 in various human cancers, including esophageal cancer [20–27]. Lost expression of RAR-β2 and methylation of the RAR-β2 gene promoter have been used as diagnostic markers of tumorigenesis . For example, methylation of the RAR-β2 gene promoter has been found in early-stage breast cancer  and has been detected in the bronchial aspirates of lung cancer patients at a much higher frequency than in patients with benign lung disease . Furthermore, the RAR-β2 gene promoter has been detected at a high level of methylation in esophageal cancer and was found to be associated with RAR-β2 gene silencing in this disease . Cigarette smoke has been shown to downregulate RAR-β2 expression, but not that of RAR-α or RAR-γ in the lungs of ferrets . However, the cause of this lost RAR-β2 expression or RAR-β2 gene promoter methylation is not fully understood.
The current study mechanistically links the esophageal cancer risk factor BPDE to suppressed RAR-β2 expression and RAR-β2 gene promoter methylation, which may help in the development of novel strategies against this deadly disease by using chemopreventive agents to antagonize the effects of BPDE on esophageal epithelial cells. Recent studies have shown that cigarette smoke, specifically the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, reduced RAR-β2 expression or induced RAR-β2 gene promoter methylation in experimental animals . Epigallocatechin gallate induced a concentration- and time-dependent reversal of RAR-β2 gene promoter methylation in esophageal cancer cell lines, resulting in the restoration of RAR-β2 expression . The factors known to be associated with aberrant CpG island methylation include local DNA structure changes, carcinogen exposure, increased DNA-methyltransferase activity, and microsatellite instability [31, 32]. Tobacco carcinogens are also known factors in the methylation of various tumor suppressor genes, including the RAR-β2 gene promoter [33–35]. However, the defined causes of CpG island methylation in cancer are largely unknown. In the current study, we found that BPDE recruited DNMT3A to methylate the RAR-β2 gene promoter and thus silence its gene expression, which in turn, may contribute to the malignant transformation of esophageal epithelial cells. Taken together, the results of this study form the link between the esophageal cancer risk factor BPDE and the reduced RAR-β2 expression, which may help in the development of novel strategies against this now deadly disease by antagonizing the effects of BPDE on esophageal epithelial cells with anti-methylation agents.
Cell culture and drug treatment
Esophageal squamous cancer cell lines TE-3 and TE-12 and adenocarcinoma cell line SKGT4 were grown and maintained as described elsewhere [17–19]. BPDE was purchased from Midwest Research Institute (Kansas City, MO) and 5-Aza from Sigma (St. Louis, MO). The treatment schedules were the same as those used in our previous studies [17–19].
RNA isolation and Northern blotting
TRIzol reagent (Invitrogen, Carlsbad, CA) was used to extract RNA from monolayer cultures, and the plasmid pRC/CMV (Invitrogen, San Diego, CA), which contains human RAR-β2 cDNA, was prepared for using as the Northern blotting probe as previously described .
MSP and DNA sequencing
DNA isolated from these cells was subjected to MSP using an MSP kit (Zymed, South San Francisco, CA) according to the manufacturer's instructions. The primers used to amplify the methylated RAR-β2 genes were 5'-TCGAGAACGCGAGCGATTCG-3' and 5'-GACCAATCCAACCGAAACGA-3'. The primers used to amplify the unmethylated RAR-β2 genes were 5'-TTGAGAATGTGAGTGATTTGA-3' and 5'-AACCAATCCAACCAAAACAA-3'. The PCR conditions used were the same as those described previously . The PCR products were cloned into the pGEM-T easy vector (Promega, Madison, WI), amplified, and sequenced in our institutional DNA sequencing facility with T7 primer.
The ChIP assay was performed with a kit from Millipore (Billerica, MA), according to the manufacturer's protocol, with two clones (#5D11 and 8E11) of anti-BPDE antibodies (Trevigen, Gaithersburg, MD).
Protein extraction and Western blotting
Total cellular protein was isolated for Western blotting as previously described [17–19]. The antibodies used were anti-c-Jun/AP-1, anti-DNMT3A (Santa Cruz Biotechnology, Santa Cruz, CA), anti-COX-2 (DB Transduction Laboratories, Lexington, KY), anti-phosphorylated-Erk1/2, anti-DNMT3B (Cell Signaling Technology, Beverly, MA), and anti-β-actin antibody (Sigma).
PCR analysis of the RAR-β2 gene promoter
The DNA-protein complex from the ChIP assay was then subjected to PCR analysis. The primers used for the RAR-β2 gene promoter were 5'-TCATTTGAAGGTTAGCAGCCCGGGTA-3' and 5'-GGAGGCAAATGGCATAGAAA-3', which generated a 502-bp PCR product after 35 cycles.
DNMT3A shRNA and transient gene transfection
DNMT3A shRNAs were purchased from OriGene Technologies (Rockville, MD). They were used for knocking down DNMT3A expression using Lipofectamine 2000 (Invitrogen) and treated with 0.5 μg/mL of puromycin for 48 h. The total cellular protein from these cells was subjected to Western blotting analysis of DNMT3A expression, and RNA from duplicate experiments was subjected to RT-PCR analysis of RAR-β2 expression as previously described .
5-Aza, 5-aza-2'-deoxycytidine; MSP, methylation-specific polymerase chain reaction; BPDE, benzo [a]pyrene diolepoxide; ChIP, chromatin immunoprecipitation; CpG, cytosine-phosphate-guanine; DNMT-3A, DNA (cytosine-5-)-methyltransferase 3 alpha; RT-PCR, reverse transcription-polymerase chain reaction; RAR-beta2, retinoic acid receptor-β2.
This work was supported in part by National Cancer Institute (NCI) grant R01 CA117895, a Cancer Center Supporting Grant (CA16672) for our core DNA facility, and a seed grant from Duncan Family Institute for Cancer Prevention and Risk Assessment. We thank the Department of Scientific Publications at The University of Texas M. D. Anderson Cancer Center for editing the manuscript.
- Osborne MR, Crosby NT: Benzopyrenes. 1987, Cambridge, UK: Cambridge University Press.Google Scholar
- Hoffmann D, Hecht SS: Advances in tobacco carcinogenesis. Chemical Carcinogenesis and Mutagenesis I. Handbook of Experimental Pharmacology. Edited by: Cooper CS, Grover PL. 1990, 94: 63-102. Berlin, Germany: Springer-Verlag.View ArticleGoogle Scholar
- Harvey RG: Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenesis. 1991, Cambridge, UK: Cambridge University Press.Google Scholar
- Hecht SS: Cigarette smoking: cancer risks, carcinogens, and mechanisms. Langenbecks Arch Surg. 2006, 391: 603-613. 10.1007/s00423-006-0111-zView ArticlePubMedGoogle Scholar
- Smoking, Tobacco, Cancer Program (U.S.): Smoking, Tobacco, and Cancer Program: 1985-1989 Status Report. 1990, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, National Cancer Institute. NIH Publication No. 90-3107. Bethesda, MD.Google Scholar
- Zhang ZF, Kurtz RC, Sun M, Karpeh M, Yu GP, Gargon N, Fein JS, Georgopoulos SK, Harlap S: Adenocarcinomas of the esophagus and gastric cardia: medical conditions, tobacco, alcohol, and socioeconomic factors. Cancer Epidemiol Biomarkers Prev. 1996, 5: 761-768.PubMedGoogle Scholar
- Gammon MD, Schoenberg JB, Ahsan H, Risch HA, Vaughan TL, Chow WH, Rotterdam H, West AB, Dubrow R, Stanford JL, Mayne ST, Farrow DC, Niwa S, Blot WJ, Fraumeni JF: Tobacco, alcohol, and socioeconomic status and adenocarcinomas of the esophagus and gastric cardia. J Natl Cancer Inst. 1997, 89: 1277-1284. 10.1093/jnci/89.17.1277View ArticlePubMedGoogle Scholar
- Tran GD, Sun XD, Abnet CC, Fan JH, Dawsey SM, Dong ZW, Mark SD, Qiao YL, Taylor PR: Prospective study of risk factors for esophageal and gastric cancers in the Linxian general population trial cohort in China. Int J Cancer. 2005, 113: 456-463. 10.1002/ijc.20616View ArticlePubMedGoogle Scholar
- Xu X-C: Risk factors and gene expression in esophageal cancer. Cancer Epidemiology, Host Susceptibility Factors. Edited by: Verma M. 2009, I: 335-360. New York: Humana Press.Google Scholar
- Venkatachalam S, Denissenko MF, Alvi N, Wani AA: Rapid activation of apoptosis in human promyelocytic leukemic cells by (+/-)-anti-benzo[a]pyrene diol epoxide induced DNA damage. Biochem Biophys Res Commun. 1993, 197: 722-729. 10.1006/bbrc.1993.2539View ArticlePubMedGoogle Scholar
- Denissenko MF, Pao A, Tang M, Pfeifer GP: Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in p53. Science. 1996, 274: 430-432. 10.1126/science.274.5286.430View ArticlePubMedGoogle Scholar
- Wang D, You L, Sneddon J, Cheng SL, Jamasbi R, Stoner GD: Frameshift mutation in codon 176 of the p53 gene in rat esophageal epithelial cells transformed by benzo[a]pyrene dihydrodiol. Mol Carcinog. 1995, 14: 84-93. 10.1002/mc.2940140204View ArticlePubMedGoogle Scholar
- Mass MJ, Jeffers AJ, Ross JA, Nelson G, Galati AJ, Stoner GD, Nesnow S: Ki-ras oncogene mutations in tumors and DNA adducts formed by benz[j]aceanthrylene and benzo[a]pyrene in the lungs of strain A/J mice. Mol Carcinog. 1993, 8: 186-192. 10.1002/mc.2940080309View ArticlePubMedGoogle Scholar
- Liang Z, Lippman SM, Kawabe A, Shimada Y, Xu X-C: Identification of benzo[a]pyrene diol epoxide-binding DNA fragments using DNA-immunoprecipitation technique. Cancer Res. 2003, 63: 1470-1474.PubMedGoogle Scholar
- Wang XD, Liu C, Bronson RT, Smith DE, Krinsky NI, Russell M: Retinoid signaling and activator protein-1 expression in ferrets given beta-carotene supplements and exposed to tobacco smoke. J Natl Cancer Inst. 1999, 91: 60-66. 10.1093/jnci/91.1.60View ArticlePubMedGoogle Scholar
- Vuillemenot BR, Pulling LC, Palmisano WA, Hutt JA, Belinsky SA: Carcinogen exposure differentially modulates RAR-beta promoter hypermethylation, an early and frequent event in mouse lung carcinogenesis. Carcinogenesis. 2004, 25: 623-629. 10.1093/carcin/bgh038View ArticlePubMedGoogle Scholar
- Song S, Xu XC: Effect of benzo[a]pyrene diol epoxide on expression of retinoic acid receptor-beta in immortalized esophageal epithelial cells and esophageal cancer cells. Biochem Biophys Res Commun. 2001, 281: 872-877. 10.1006/bbrc.2001.4433View ArticlePubMedGoogle Scholar
- Li M, Song S, Lippman SM, Zhang XK, Liu X, Lotan R, Xu XC: Induction of retinoic acid receptor-β suppresses cyclooxygenase-2 expression in esophageal cancer cells. Oncogene. 2002, 21: 411-418. 10.1038/sj.onc.1205106View ArticlePubMedGoogle Scholar
- Song S, Lippman SM, Zou Y, Ye X, Ajani JA, Xu X-C: Induction of cyclooxygenase-2 by benzo[a]pyrene diol epoxide through inhibition of retinoic acid receptor-β2 expression. Oncogene. 2005, 24: 8268-8276. 10.1038/sj.onc.1208992View ArticlePubMedGoogle Scholar
- Soprano DR, Qin P, Soprano KJ: Retinoic acid receptors and cancers. Annu Rev Nutr. 2004, 24: 201-221. 10.1146/annurev.nutr.24.012003.132407View ArticlePubMedGoogle Scholar
- Xu X-C: Tumor-suppressive activity of RAR-β2 in cancer. Cancer Lett. 2007, 253: 14-24. 10.1016/j.canlet.2006.11.019PubMed CentralView ArticlePubMedGoogle Scholar
- Mongan NP, Gudas LJ: Diverse actions of retinoid receptors in cancer prevention and treatment. Differentiation. 2007, 75: 853-870. 10.1111/j.1432-0436.2007.00206.xView ArticlePubMedGoogle Scholar
- Bean GR, Scott V, Yee L, Ratliff-Daniel B, Troch MM, Seo P, Bowie ML, Marcom PK, Slade J, Kimler BF, Fabian CJ, Zalles CM, Broadwater G, Baker JC, Wilke LG, Seewaldt VL: Retinoic acid receptor-β2 promoter methylation in random periareolar fine needle aspiration. Cancer Epidemiol Biomarkers Prev. 2005, 14: 790-798. 10.1158/1055-9965.EPI-04-0580View ArticlePubMedGoogle Scholar
- Grote HJ, Schmiemann V, Geddert H, Rohr UP, Kappes R, Gabbert HE, Bocking A: Aberrant promoter methylation of p16(INK4a), RAR-β2 and SEMA3B in bronchial aspirates from patients with suspected lung cancer. Int J Cancer. 2005, 116: 720-725. 10.1002/ijc.21090View ArticlePubMedGoogle Scholar
- Wang Y, Fang MZ, Liao J, Yang GY, Nie Y, Song Y, So C, Xu X-C, Wang LD, Yang CS: Hypermethylation-associated inactivation of retinoic acid receptor beta in human esophageal squamous cell carcinoma. Clin Cancer Res. 2003, 9: 5257-5263.PubMedGoogle Scholar
- Li R, Saito T, Tanaka R, Satohisa S, Adachi K, Horie M, Akashi Y, Kudo R: Hypermethylation in promoter region of retinoic acid receptor-beta gene and immunohistochemical findings on retinoic acid receptors in carcinogenesis of endometrium. Cancer Lett. 2005, 219: 33-40. 10.1016/j.canlet.2004.06.044View ArticlePubMedGoogle Scholar
- Olasz J, Juhasz A, Remenar E, Engi H, Bak M, Csuka O, Kasler M: RAR beta2 suppression in head and neck squamous cell carcinoma correlates with site, histology and age. Oncol Report. 2007, 18: 105-112.Google Scholar
- Jiang Y, Liang ZD, Wu TT, Cao L, Zhang H, Xu X-C: ATM expression is associated with tobacco smoke exposure in esophageal cancer tissues and benzo[a]pyrene diol epoxide in cell lines. Int J Cancer. 2007, 120: 91-95. 10.1002/ijc.22121View ArticlePubMedGoogle Scholar
- Liu H, Zhou Y, Boggs SE, Belinsky SA, Liu J: Cigarette smoke induces demethylation of prometastatic oncogene synuclein-c in lung cancer cells by downregulation of DNMT3B. Oncogene. 2007, 26: 5900-5910. 10.1038/sj.onc.1210400View ArticlePubMedGoogle Scholar
- Fang MZ, Wang Y, Ai N, Hou Z, Sun Y, Lu H, Yang CS: Tea polyphenol (-)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines. Cancer Res. 2003, 63: 7563-7570.PubMedGoogle Scholar
- Issa JP: CpG island methylator phenotype in cancer. Nat Rev Cancer. 2004, 4: 988-993. 10.1038/nrc1507View ArticlePubMedGoogle Scholar
- Di Croce L, Raker VA, Corsaro M, Fazi F, Fanelli M, Faretta M, Fuks F, Lo Coco F, Kouzarides T, Nervi C, Minucci S, Pelicci PG: Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science. 2002, 295: 1079-1082. 10.1126/science.1065173View ArticlePubMedGoogle Scholar
- Lin RK, Hsieh YS, Lin P, Hsu HS, Chen CY, Tang YA, Lee CF, Wang YC: The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation and tumor suppressor gene hypermethylation in mice and lung cancer patients. J Clin Invest. 2010, 120: 521-532.PubMed CentralView ArticlePubMedGoogle Scholar
- Breton CV, Byun HM, Wenten M, Pan F, Yang A, Gilliland FD: Prenatal tobacco smoke exposure affects global and gene-specific DNA methylation. Am J Respirat Crit Care Med. 2009, 180: 462-467. 10.1164/rccm.200901-0135OC.View ArticleGoogle Scholar
- Oka D, Yamashita S, Tomioka T, Nakanishi Y, Kato H, Kaminishi M, Ushijima T: The presence of aberrant DNA methylation in noncancerous esophageal mucosae in association with smoking history: a target for risk diagnosis and prevention of esophageal cancers. Cancer. 2009, 115: 3412-3426. 10.1002/cncr.24394View ArticlePubMedGoogle Scholar
- Xu XC, Liu X, Tahara E, Lippman SM, Lotan R: Expression and up-regulation of retinoic acid receptor-beta is associated with retinoid sensitivity and colony formation in esophageal cancer cell lines. Cancer Res. 1999, 59: 2477-2483.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.