Benzo[a]pyrene diol epoxide suppresses retinoic acid receptor-beta2 expression by recruiting DNA (cytosine-5-)-methyltransferase 3A.
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-beta2) 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-beta2 gene promoter. We then performed chromatin immunoprecipitation (ChIP) assays to find that BPDE recruited genes of the methylation machinery into the RAR-beta2 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-beta2 gene promoter, which we confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis of the reduced RAR-beta2 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-beta2 expressions. Transient transfection of the DNMT3A shRNA vector also antagonized BPDE's effects on expression of RAR-beta2, 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-beta2 expression.
Ye
Fei
F
Department of Clinical Cancer Prevention, Unit 1360, The University of Texas M, D, Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Xu
Xiao-Chun
XC
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity*; D015152:Blotting, Northern; D015153:Blotting, Western; D002273:Carcinogens / Q000633:toxicity*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D011401:Promoter Regions, Genetic; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D000097584:DNA Methyltransferase 3B
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D002273:Carcinogens; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D018168:Receptors, Retinoic Acid; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C082925:retinoic acid receptor beta