Cytosine-phosphoguanine methylation of estrogen receptors in endometrial cancer

We hypothesize that estrogen receptors (ERs) are differentially expressed i n endometrial cancer. To test this hypothesis, we investigated the expressi on profile of ER alpha (ER alpha -A, ER alpha -B, ER alpha -C) and ER beta genes and CpG methylation status in endometrial cancer cell lines and tissu es using reverse transcription-PCR and methylation-specific PCR and direct DNA sequencing. The results demonstrated that ER alpha -A, ER alpha -B, and ER beta were normally expressed whereas ER alpha -C gene was inactivated i n all endometrial cancer cell lines. We further investigated the mechanisms of ER alpha -C gene inactivation through CpG methylation pathways, The tre atment with demethylating agent (5 ' -aza-2 ' -deoxycytidine) restored ER a lpha -C gene expression in all endometrial cancer cell lines. We further co nfirmed these findings with methylation-specific PCR and direct DNA sequenc ing and found that only ER alpha -C was methylated on all five different Cp G sites in ail cell Lines. We further analyzed 88 cancerous and 46 normal e ndometrial tissues. The results demonstrated that only ER alpha -C was inac tivated and methylated in 94% of cancer tissues. In 32 pairs of cancerous a nd normal endometrial tissues from the same patient, ER alpha -C was methyl ated in 29 of 32 cancer tissues but unmethylated in all normal endometrial tissues. This is the first report that demonstrates selective ER alpha -C g ene inactivation through CpG methylation pathway in uterine endometrial can cer.