Saturday, October 29, 2011
Hall 1-2 (San Jose Convention Center)
4- aminobiphenyl (4-ABP) is a carcinogenic compound released in cigarette smoke that may contribute to breast cancer. Cytochrome b5 (b5) and NADH cytochrome b5 reductase (b5R) detoxify the oxidized form of 4-ABP, 4-ABP-NHOH. Previous studies have shown wide variability in b5 and b5R expression and activity in breast. Screening for single nucleotide polymorphisms (SNPs) in the genes encoding b5 (CYB5A) and b5R (CYB5R3) does not account for this phenotypic variability. DNA methylation can cause downregulation of gene expression. The objective of this study is to determine the methylation status of promoter regions of the CYB5A and CYB5R3 genes, in individual human breast and liver samples with outlier expression of b5 and b5R. We hypothesized that individual variability in the expression of CYB5A and CYB5R3 in human breast could be associated with the extent of DNA methylation. In order to prove our hypothesis breast and liver tissues were obtained from the Cooperative Human Tissue Network, DNA samples extracted and bisulfite treated using the MethylSEQr kit. Regions in the CpG islands in both genes were amplified using PCR. PCR purified products were cycle sequenced using Big Dye reagents. Finally, methylation sites were identified using Staden Package software. Preliminary data for the promoter region of both genes shows high methylation at distal CpG islands and low methylation of proximal CpG dinucleotides. Currently we are investigating whether the extent of methylation is related to the wide varibaility in the expression of these genes that has been previously observed in human liver and breast.