Induction and characterization of de novo methylation by benzo[A]pyrene in the cancer cell lines MCF-7 and MDA-MB-231

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Kozina, Vladimir Joseph
University of the Pacific Dissertations;Breast Cancer;DNA Methylation;Carcinogenesis Molecular aspects
thesis / dissertation description
The experiments presented were designed to test the hypothesis that the well-known carcinogen, benzo[a]pyrene has epigenetic effects, specifically the ability to alter cytosine methylation patterns. MCF-7 and MDA-MB-231 human breast cancer cells were treated for a period of sixty days with 100 nM benzo[a]pyrene. The methylation status of two genes, Ecadherin and GSTP 1 were examined using methyl-specific PCR and Southern blot analysis. After sixty days, no detectable change in methylation was observed. Evidence exists that de novo methylation is a consequence of transcriptional inactivity. Benzo[a]pyrene can contribute to transcriptional repression by sequestering the transcription factor, Spl. To test this hypothesis in our system, MCF-7 cells were transiently transfected with a reporter construct containing Sp 1 sites. These experiments demonstrated an 8.4 fold increase in reporter gene activity over a promoterless control plasmid; however, a difference could not be established between benzo[a]pyrene-treated and untreated cells.