Methylation of CpG island is not a ubiquitous mechanism for the loss of oestrogen receptor in breast cancer cells

Abstract

Summary Methylation has been shown to play an important role in the down-regulation of oestrogen receptors (ER) in breast cancer cells. One critical question that remains unclear is whether methylation can account for the loss of ER expression in cells derived from an ERpositive cell line. This laboratory has established an in vitro cell system using long-term growth of human ER-positive breast cancer cell line T47D in oestrogen-free medium. A clonal cell line, T47D:C4:2 (C4:2), has been characterized. Unlike T47D:A18 (A18), which is a T47D line maintained in oestrogen medium, C4:2 has lost the expression of ER and hormone responsiveness. DNA fingerprinting and restriction fragment length polymorphism (RFLP) analysis results confirmed that C4:2 was of the same lineage as A18. These cell lines provide an invaluable system to study the mechanism of ER expression and regulatory pathways leading to hormone-independent growth. The results here clearly demonstrate that the ER CpG island in C4:2 cells remains unmethylated. The loss of ER in the cell line must be due to mechanisms other than methylation. We also evaluated the ER CpG island in the MDA-MB-231 :10A (10A) cell line, which is a clone from the MDA-MB-231 line obtained from ATCC and the DNA from the MDA-MB-231 cell line used in the original report. Unlike the cell line from the report, which showed a full methylation pattern in the island, the 1 OA line only showed a partial methylation pattern in the CpG island. Possible mechanisms pertaining to the heterogeneous methylation pattern of the ER CpG island in the breast cancer cells are discussed.

DNA methylation analysis at distal and proximal promoter regions of the oestrogen receptor gene in breast cancers

“…In other words, these results showed that hypermethylation at the promoter regions of the ER-α gene might be quite important for ER negativity accompanying tumour progression. Chen et al (1998) reported that the ER-α CpG island in C4:2 cells, a subclone of T47D cells without ER-α expression, remained unmethylated. This result shows that the loss of ER-α in these specific breast cancer cells must be due to a mechanism other than methylation.…”

A-432411, a novel indolinone compound that disrupts spindle pole formation and inhibits human cancer cell growth

“…Western Blot Analysis Western blot analyses were done using a modified version of previously described methods (10,16). Briefly, cells were seeded in 12-well plates and treated with the reagents for 24 hours.…”

Molecular and Cellular Determinants of Estrogen Receptor α Expression

“…If ER Ϫ cells were treated with either 5-aza-dC or TSA alone, however, 5-aza-dC only weakly activated ER expression, whereas TSA did not activate ER. Chen et al established a clonal cell line derived from T47D cells (T47D:C4:2) that exhibited a loss of ER expression and responsiveness to hormones due to its maintenance in estrogen-free medium (7). Importantly, the ER CpG island in C4:2 cells was not methylated, indicating that methylation was not responsible for ER silencing in these cells.…”

“…Importantly, the ER CpG island in C4:2 cells was not methylated, indicating that methylation was not responsible for ER silencing in these cells. It was thus hypothesized that other steps may precede methylation, including a loss of transcriptional activators or the presence of factors that block the activity of methyltransferases (7).…”