Methylation of conserved CpG sites neighboring the beta retinoic acid response element may mediate retinoic acid receptor beta gene silencing in MCF-7 breast cancer cells

Abstract

We investigated the mechanism of retinoic acid receptor (RAR) b2 gene silencing in breast cancer cells. Transfection experiments indicated that MCF-7 cells transactivate an exogenous b2 promoter (71470/+156) to the same extent as MTSV1.7 breast epithelial cells, which express endogenous RARb2. This was true even in the context of replicated chromatin, suggesting a cisacting rather than a trans-acting defect. Cytosine methylation, a cis-acting DNA modi®cation, has been implicated in RARb2 silencing in cancer cells. Upon bisul®te genomic sequencing, we found that 3 CpG sites in the b2 RARE region were variably methylated in MCF-7 cells but were not methylated in MTSV1.7 cells or in 2 MDA-MB-231 subclones that di ered in RARb2 expression (high in clone A2, low in clone A4). However, the 5'-UTR region was hypermethylated in clone A4 relative to clone A2 cells. Following 5-azacytidine treatment, RA and trichostatin A markedly induced RARb2 expression in MCF-7 cells but not in MDA-MB-231 clone A4 cells. A b2 RARE reporter construct in which the methylation-susceptible cytosines in the sense strand were replaced by thymine displayed marked loss of activity in a replicated chromatin-dependent manner. We conclude that cytosine methylation contributes to RARb2 gene silencing in MCF-7 cells and that methylation of the RARE region may be particularly important.

Eicosapentaenoic Acid Demethylates a Single CpG That Mediates Expression of Tumor Suppressor CCAAT/Enhancer-binding Protein δ in U937 Leukemia Cells

“…Our results are consistent with the site-specific CpG demethylation of C/EBP␦ proximal promoter in primary breast cancer (59). Moreover, evidence demonstrating gene promoter site-specific methylation as a mechanism of tumor suppressor gene silencing in various types of cancer cells has been reported (60,61). A single CpG demethylation appears to be the molecular event associated with a putative antitumor gene expression in prostate carcinogenesis (62).…”

Histone deacetylases inhibitors as anti-angiogenic agents altering vascular endothelial growth factor signaling

“…The blocking of HDAC activity by a variety of inhibitors, including trichostatin-A (TSA) (Yoshida et al, 1990), suberoylanilide hydroxamic acid (SAHA) (Richon et al, 1998) and trapoxin (TPX) (Kijima et al, 1993), modulates the di erentiation of normal and malignant cells. TSA suppresses Ras-induced neurite outgrowth of PC12 cells (Futamura et al, 1995), induces di erentiation of AML1-ETO leukemia cells (Wang et al, 1999), enhances beta retinoic acid receptor expression in epithelial cells (Arapshian et al, 2000) and prevents myo®broblastic di erentiation of rat hepatic stellate cells (Niki et al, 1999). Inhibition of HDAC also forces cancer cells to undergo apoptosis (Glick et al, 1999) and causes signi®cant suppression of human prostatic tumor growth in nude mouse (Butler et al, 2000).…”

Hs.137007 is a novel epigenetic marker hypermethylated and up-regulated in breast cancer

“…For example, individual tumor suppressors or proto-oncogenes that have CpG islands in their promoter were examined for changes in methylation in normal breast and cancer cells and tissues (17,18). In addition, malignant cells cultured in the presence of 5-aza-2'-deoxycytidine and genes that showed altered expression before and after treatment were selected as candidates.…”