Estrogen receptor (ER)-negative breast cancer cells display extensive methylation of the ER gene CpG island and elevated DNA methyltransferase (DMT) expression compared to ER-positive cells. The present study demonstrates that DMT protein levels tightly correlate with S phase fraction in ER-positive cells, whereas ER-negative cells express DMT throughout the cell cycle. In addition, levels of p21(CIP1), which disrupts DMT binding to PCNA, are inversely correlated with DMT levels. Therefore increased DMT expression in ER-negative cells is not simply due to elevated S-phase fraction, but rather to more complex changes that allow cells to escape normal cell cycle-dependent controls on DMT expression. Because ER-negative breast tumors often have activated growth factor pathways, the impact of these pathways on DMT expression was examined in ER-positive cells. Stable transfection with fibroblast growth factors (FGFs) 1 and 4 led to increased DMT expression that could not be accounted for by a shift in S phase fraction. Elevated DMT protein expression in FGF-transfectants was accompanied by a significant decrease in p21, again suggesting a reciprocal relationship between these two proteins. However, acquisition of an estrogen-independent phenotype, even in conjunction with elevated DMT levels, was not sufficient to promote ER gene silencing via methylation. These results indicate that multiple steps are required for de novo methylation of the ER CpG island.
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We are grateful to Ravi Veeraswami and Xiaohui Lin for preparing and characterizing the DMT antibody. We also thank Robert Clarke of the Lombardi Cancer Center and Francis Kern from Southern Research Institute for generously providing the cell lines used in this study. The DMT antisense expression vector was generously provided by Paula Vertino at the Emory University School of Medicine. Finally, we are grateful to Jim Fluke of the Johns Hopkins Oncology Center Flow Cytometry Facility for performing the FACS analysis. This work was supported by grants from the American Cancer Society (NE Davidson, AT Ferguson), Susan G Komen Foundation (NE Davidson), the National Cancer Institute (NE Davidson, SJ Nass), and the United States Army Research and Materiel Command (SJ Nass). In addition, an AFLAC Travel Fellowship to SJ Nass made it possible to present part of this work at the 90th Annual Meeting of the American Association for Cancer Research.
- Breast cancer
- DNA methylation
- Estrogen receptor
- S phase