Decreased Expression of Manganese Superoxide Dismutase in Transformed Cells Is Associated with Increased Cytosine Methylation of the SOD2 Gene

Abstract

Tumor cells express lower levels of manganese superoxide dismutase (MnSOD) than their normal counterparts. Enforced expression of MnSOD reverses the malignant phenotype of many transformed cells, suggesting that SOD2 is a tumor suppressor. The SOD2 gene contains a large CpG island spanning > 3.5 kb that starts near the 5' edge of the promoter and extends into intron 2. We hypothesized that the difference in SOD2 expression between tumor cells and their normal cell counterparts might be secondary to differences in their cytosine methylation patterns in this CpG island. To test this hypothesis, we analyzed the methylation status of the SOD2 gene in two cell line models that show differential MnSOD expression between normal and SV40-transformed cells: WI38 and MRC5 and their SV40-transformed variants, WI38-VA and MRC5-VA. We subdivided the SOD2 gene CpG island into 10 individual regions for analysis by bisulfite genomic sequencing. A region located in intron 2 displayed a significant increase in cytosine methylation in both transformed cell lines that expressed low levels of MnSOD mRNA compared with their normal cell counterparts. Recent studies by others have shown that SOD2 intron 2 is a potent transcriptional enhancer. The association between increased cytosine methylation of the SOD2 intron 2 region and decreased MnSOD expression in transformed cells compared with their normal counterparts suggests that an epigenetic mechanism contributes to the differential SOD2 gene expression between these normal and SV40-transformed cells.

Epigenetic Regulation of Manganese Superoxide Dismutase Expression in Human Breast Cancer Cells

“…[21][22][23] The expansive CpG island at the 5' end of the SOD2 gene makes it a candidate for control by DNA methylation. 10 Therefore, we tested the hypothesis that decreased MnSOD expression in human breast cancer cells was associated with increased SOD2 promoter cytosine methylation. To test this hypothesis we used bisulfite sequencing to analyze eight CpG sites located between -1422 and -1164 upstream from the transcription initiation start site of MnSOD (Fig.…”

“…For example, hypermethylation within the second intron of SOD2 was associated with decreased MnSOD levels in immortalized fibroblasts. 10 More recently, additional studies have shown that methylation of the SOD2 promoter decreases the expression of MnSOD in multiple myeloma. 26 Epigenetic silencing by DNA methylation is heritable, but is dependent upon the activity of DNA methyltransferases.…”

“…Five micrograms of genomic DNA was modified with sodium bisulfite under conditions previously described. 10 We designed primers specific to bisulfite modified DNA to analyze CpG methylation at the SOD2 promoter -1422 to -1164 upstream from the transcription initiation site. To achieve specificity, we amplified this region in two successive rounds of nested PCR.…”

“…9 SOD2, the gene that encodes MnSOD, is located on chromosome 6q25 and is characterized by an expansive GC-rich promoter extending from approximately 1500 bp upstream through the second exon. 10 Many studies have identified transcriptional elements in this region that regulate SOD2 expression. SOD2 transcription is mediated trough a TATA-less proximal promoter containing several transcriptional cis-elements.…”

“…Currently a role for DNA methylation in controlling SOD2 expression is emerging. Hypermethylation of CpG islands at SOD2 decreases MnSOD expression in immortalized fibroblasts 10 and multiple myeloma cells. 25,26 This is facilitated by DNA methylation inhibiting transcription factor binding at the SOD2 promoter.…”