Methylation-mediated regulation of the glutathione S-transferase P1 gene in human breast cancer cells

Abstract

Understanding the mechanisms that regulate the human pi class GST (GSTP1) gene expression in breast cancer cells is of particular importance to the study of breast cancer biology. In cultured human breast cancer cell lines, GSTP1 is exclusively expressed in estrogen receptor-negative (ER−) cells but is undetectable in receptor-positive (ER+) cells. Previously, we examined transiently transfected GSTP1 promoter activities, in vitro GSTP1 promoter–DNA interactions, and GSTP1 mRNA stability. These studies indicated that transiently transfected GSTP1 promoter elements and GSTP1 mRNA stability could only partially explain cell line-specific expression of endogenous GSTP1. In the present study, we examined whether the methylation status of the GSTP1 CpG island plays an important role in GSTP1 regulation. Southern blot analysis revealed that the GSTP1 CpG island is hypermethlyated in ER+, GSTP1 non-expressing cell lines but is undermethylated in ER−, GSTP1 expressing cell lines. Moreover, partial demethylation of the GSTP1 CpG island by treatment with 5-aza-2′-deoxycytidine resulted in de novo gene expression in ER+ cell lines, as detected by RT-PCR, Northern blot and Western blot analyses. Our data strongly indicate that methylation status of the promoter contributes significantly to the levels of GSTP1 expressed in ER− and ER+ breast cancer cell lines.

Introduction

Glutathione S-transferases (GST) are a multigene family of enzymes that catalyse the conjugation of glutathione with electrophilic, hydrophobic compounds, including carcinogens, natural toxins and exogenous drugs (Coles and Ketterer, 1990; Hayes and Pulford, 1995; Mannervik and Danielson, 1988; Tew, 1994). The resulting complex formed by this reaction is usually less toxic than the parent xenobiotic and is eventually metabolized and exported via glutathione-dependent transport systems (Ishikawa, 1992). Because of these conjugating activities, GSTs are believed to be vital in mediating a variety of normal cellular detoxification reactions.

The pi class GST (GSTP1-1) is of particular interest to the study of breast cancer biology. In breast cancer cells, increased GSTP1 expression is associated with other altered biological properties. In cultured breast cancer cells, GSTP1 is exclusively expressed in estrogen receptor-negative (ER−) cells but not in receptor-positive (ER+) cells (Moscow et al., 1988). In addition, GSTP1 is coordinately expressed with other genes, including epidermal growth factor receptor, protein kinase C and glutathione peroxidase (Borner et al., 1987; Davidson et al., 1987; Townsend et al., 1991), and inversely related to cytochrome P4501A1 expression (Vickers et al., 1989). In some ER+ cultured breast cancer cells, repeated exposure of these cells to certain anticancer agents results in a multidrug resistant phenotype associated with increased GSTP1 expression and other altered biochemical properties, including the loss of ER expression (Batist et al., 1986). The coordinated expression of these genes suggests that GSTP1 and these other genes may share common regulatory mechanisims in breast cancer cells. Consequently, determination of processes that regulate GSTP1 expression is of considerable importance to breast cancer biology.

To investigate the regulatory mechanisms involved in differential GSTP1 expression, we employed model breast cancer cell lines that either express (ER−) or do not express (ER+) GSTP1. Previously, we examined transiently transfected GSTP1 promoter activities, in vitro GSTP1 promoter–DNA interactions and GSTP1 mRNA stability. These studies indicated that efficiency of expression from transiently transfected GSTP1 promoter elements and GSTP1 mRNA stability could not fully explain cell line-specific expression of endogenous GSTP1 (Jhaveri and Morrow, 1998; Jhaveri et al., 1997; Morrow et al., 1992). In the present study, we evaluate whether DNA methylation is involved in modulating GSTP1 promoter activity. In cancer cells, methylation of genes within a GC-rich region is frequently associated with diminished transcriptional activity (Graff et al., 1995; Herman et al., 1996; Ottaviano et al., 1994). The 5′ region of GSTP1 contains GC rich regions, including a CpG island (Cowell et al., 1988; Morrow et al., 1989). Methylation of these sequences could potentially contribute to GSTP1 silencing in non-expressing cells. Indeed, in prostate cancer, loss of GSTP1 expression is associated with increased methylation of GSTP1 GC-rich sequences (Lee et al., 1994). Here we present evidence that DNA methylation plays a significant role in modulating GSTP1 expression in breast cancer cell lines. GSTP1 is hypermethylated in non-expressing cells but undermethylated in expressing cells. Furthermore, treatment of non-expressing cell lines with a demethylating agent activates GSTP1 expression and this expression is associated with decreased methylation of the GSTP1 promoter.