Role of Estrogen Receptor Gene Demethylation and DNA Methyltransferase·DNA Adduct Formation in 5-Aza-2′deoxycytidine-induced Cytotoxicity In Human Breast Cancer Cells

Abstract

The cytosine analog 5-aza-2-deoxycytidine is a potent inhibitor of DNA methyltransferase. Its cytotoxicity has been attributed to several possible mechanisms including reexpression of growth suppressor genes and formation of covalent adducts between DNA methyltransferase and 5-aza-2-deoxycytidine-substituted DNA which may lead to steric inhibition of DNA function. In this study, we use a panel of human breast cancer cell lines as a model system to examine the relative contribution of two mechanisms, gene reactivation and adduct formation. Estrogen receptor-negative cells, which have a hypermethylated estrogen receptor gene promoter, are more sensitive than estrogen receptor-positive cells and underwent apoptosis in response to 5-aza-2-deoxycytidine. For the first time, we show that reactivation of a gene silenced by methylation, estrogen receptor, plays a major role in this toxicity in one estrogen receptor-negative cell line as treatment of the cells with anti-estrogen-blocked cell death. However, drug sensitivity of other tumor cell lines correlated best with increased levels of DNA methyltransferase activity and formation DNA⅐DNA methyltransferase adducts as analyzed in situ. Therefore, both reexpression of genes like estrogen receptor and formation of covalent enzyme⅐ DNA adducts can play a role in 5-aza-2-deoxycytidine toxicity in cancer cells.Current studies suggest that DNA methyltransferase (DNA MTase), 1 the enzyme that methylates cytosines that are 5Ј to guanosines, plays a role in human carcinogenesis. In general, the level of DNA MTase activity is elevated significantly in neoplastic cells compared with normal cells (1). Moreover, increased enzyme activity is characteristic of the progression of both colon and lung cancer (2, 3). Studies demonstrate that overexpression of DNA MTase leads to the tumorigenic conversion of NIH3T3 cells (4), whereas decreasing the levels of DNA MTase through a combination of genetic and pharmacologic means drastically reduces the incidence of colonic adenomas in the Apc min mouse model of colon carcinogenesis (5). These studies provide substantial evidence for the involvement of DNA MTase in oncogenesis.There are at least two potential mechanisms by which DNA MTase may influence oncogenicity. Elevated levels of DNA methylation may lead to increased frequency of C to T transition mutations derived from deamination of methylcytosine (6). Alternatively, increased DNA MTase may play a role in the establishment of altered patterns of methylation at CpG island sequences found in the 5Ј region of genes involved in growth control and tumor progression (7). For example, aberrant hypermethylation of CpG islands in cancer cells has been implicated in the transcriptional inactivation of the Rb, p16, estrogen receptor (ER), E-cadherin, and glutathione S-transferase Pi genes (8 -12).These studies have sparked a renewed interest in the use of DNA MTase inhibitors such as the cytosine analogs 5-azacytidine and 5-aza-2Ј-deoxycytidine (5-aza-dC) in the treatment of human cancers. In vitro...

Methylation status of breast cancer resistance protein detected by methylation‐specific polymerase chain reaction analysis is correlated inversely with its expression in drug‐resistant lung cancer cells

“…This evidence was confirmed by bisulfite sequencing analysis and the treatment of non-BCRP-expressing PC-6 cells with 5-aza-dC, which acts as a DNA methyltransferase inhibitor only after its incorporation into DNA and leads to irreversible binding of methyltransferases to analog bases and to their depletion. 25,36 Moreover, BCRP was re-expressed by 5-aza-dC in a dose-dependent manner. These findings suggest that promoter methylation is responsible for transcriptional silencing of BCRP in PC-6 cells and that demethylation of at least 1 allele is necessary for BCRP re-expression and for BCRP-mediated resistance to SN-38 in PC-6/SN2-5H cells.…”

Expression of DNA methyl-transferase (DMT) and the cell cycle in human breast cancer cells

“…It had been previously demonstrated with six breast cancer cell lines that DMT mRNA, protein, and enzyme activity (Ottaviano et al, 1994;Ferguson et al, 1997), were elevated in ER-negative breast cancer cells compared to ER-positive cells. Because DMT protein is known to be expressed primarily during the S phase of the cell cycle in normal cells (Szyf et al, 1985(Szyf et al, , 1991, we ®rst determined whether the elevated DMT protein level in ER-negative cell lines was simply due to a large S phase fraction in those cells.…”

“…Increased expression of DMT is an early event in two experimental models of cancer (Belinsky et al, 1996;Miyoshi et al, 1993), and overexpression of DMT can promote a transformed phenotype in NIH3T3 cells (Wu et al, 1993). In ER-negative breast cancer cell lines, DMT RNA and protein levels are signi®cantly elevated compared to ER-positve cell lines (Ottaviano et al, 1994;Ferguson et al, 1997), but little is known about the regulation of DMT expression and activity in breast cancer. If deregula-tion of DMT is necessary to initiate or maintain aberrant methylation of the ER CpG island, then examining the regulation of DMT expression in breast cancer may greatly improve our understanding of breast cancer progression.…”

Epigenetic Information and Estrogen Receptor Alpha Expression in Breast Cancer

“…The genome contains two types of information: genetic and epigenetic; the epigenetic information provides instructions on how, where, and when the genetic information should be used. The most important known form of epigenetic information in mammalian cells is DNA methylation, the covalent addition of a methyl group to the 5ʹ position of cytosine, predominantly within the CpG dinucleotide [9,12,13]. CpG islands are GpC-and CpG-rich regions of 1 kilobase (kb) that are usually associated with the promoter Figure 1.…”