Both hypomethylation and hypermethylation of DNA associated with arsenite exposure in cultures of human cells identified by methylation-sensitive arbitrarily-primed PCR.
In a previous study we reported that methylation within the promoter region of p53 was altered in human lung A549 cells exposed to arsenite over a 2-week period in culture. In the present study the methylation status of the 5' control region of the tumor suppressor gene, von Hippel Lindau syndrome (VHL), a gene known to be silenced transcriptionally by CpG methylation was assessed. No changes in DNA methylation in VHL in human kidney UOK cell lines exposed to arsenite were seen after 4 weeks in culture, assessed by simple HpaII digestion followed by PCR amplification. Using methylation-sensitive arbitrarily-primed PCR we identified eight differentially methylated regions of genomic DNA of approximately 300--500 bp from three UOK cell lines and from human lung A549 cells after arsenite exposure in culture. Six fragments were hypermethylated, and two were hypomethylated, relative to untreated controls. Sequence analysis revealed two DNA fragments contained repeat sequences of mammalian-apparent LTR retrotransposons, five contained promoter-like sequences, and 13 CpG islands were identified. Three fragments had 99-100% homology to regions on human chromosomes 6, 9, and 15 but these genes have not yet been identified. Our findings are consistent with a potential role for both hypermethylation and hypomethylation of DNA that coexist after exposure to arsenite. The results, in total, could support the existence of a state of DNA methylation imbalance that could conceivably disrupt appropriate gene expression in arsenite exposed cells.
Zhong
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Environmental Carcinogenesis Division (MD-68), National Health and Environmental Effects Research Laboratory, ORD, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA.
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D000818:Animals; D018053:Arsenites / Q000633:toxicity*; D002460:Cell Line; D002884:Chromosomes, Human, Pair 15; D002896:Chromosomes, Human, Pair 6; D002899:Chromosomes, Human, Pair 9; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects*; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000187:drug effects; D008025:Ligases; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000187:drug effects / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D018053:Arsenites; D011506:Proteins; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D019003:Deoxyribonuclease HpaII; D008025:Ligases; C497446:VHL protein, human; C015001:arsenite