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

Z Chen,J Yang,A Ko,Vc Jordan

doi:10.1038/bjc.1998.31

Abstract

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 ER-positive 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 10A 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.

Highlights

The results showed that estrogen receptor (ER) CpG island is not methylated in either C4:2 or A18 cell lines
Restriction digests of ER-positive MCF-7 DNA resulted in complete digestion, indicating that the CpG island was not methylated
This study demonstrates clearly that the ER CpG island in C4:2 remains unmethylated after the loss of ER expression in T47D cells, and the results demonstrate that the loss of ER expression in breast cancer cells does not require methylation of the CpG island

Summary

Methods

Cell lines and cell cultureMDA-MB-231 and T47D were obtained from ATCC. MCF-7 cells were obtained from Dr Dean Edwards, in the Department of Southern blot analysis and RFLP analysisDNA was isolated from the cells with the QIAGEN Blood & Cell Culture Kits (QIAGEN, Chatsworth, CA, USA) according to the manufacturer's protocol. MCF-7 cells were obtained from Dr Dean Edwards, in the Department of Southern blot analysis and RFLP analysis. DNA was isolated from the cells with the QIAGEN Blood & Cell Culture Kits (QIAGEN, Chatsworth, CA, USA) according to the manufacturer's protocol. Genomic DNA from the other MDAMB-231 cell line was generously provided by Dr NE Davidson (The Johns Hopkins Oncology Center, Baltimore, MD, USA). The restriction enzymes were obtained from New England BioLabs (Beverly, MA, USA). Each restriction reaction contained 15 jig of the genomic DNA and 200 units of either SaclI or HhaI in the appropriate buffer at 37°C overnight. The digested DNA samples were precipitated with ethanol and digested again with 50 units of BsmI at 65'C for 6 h. Southern blot analysis was performed according to standard protocols (Ausubelt et al, 1994).

Results

Discussion

Conclusion