Results Ligands regulate ERa protein levels and transcription rates independently We first examined the kinetics of ERa protein turnover Baricitinib clinical in MCF 7 cells following treatment with estradiol, two SERMs and two SERDs. It has been proposed that ligand dependent ERa regulation may result from the presence a long aliphatic side chain on steroid core. Thus in this study we selected RU39 and RU58 which are derivatives of 17b estradiol but with different side chains. RU39 has a dimethyl amino ethoxy phenyl side chain similar to the one in tamoxifen, while RU58 has a bulky hydrophobic side chain similar to the one in Ful vestrant. ERa protein levels in E2, ICI and RU58 treated MCF 7 cells rapidly decreased.

Time course experiments showed that 1 h after E2 induction, the detected amount of ERa protein accounted for only 40% of ERa levels before treatment, after 4 h, ERa levels were as low as 20% of the quantity of ERa present in untreated cells, and after 16 h ERa protein remained at a level equivalent to the one observed 1 h after addition of E2. Treatment with SERDs resulted in 70% reduction of ERa protein levels after 1 h, 4 h and even 16 h reaching 95% after 1 h exposure to ICI. Treatment of MCF 7 cells with OHT or RU39, two com pounds classified as SERM, reduced from 40% to 50% of ERa protein levels at the initial 1 h time point and about 20% after 16 h and 4 h treatment with OHT and RU39, respectively. In addition, ERa protein levels were almost equivalent to the ones detected in untreated cells after 4 h or 16 h culture in the presence of OHT or RU39.

Hence, ERa protein levels are stabilized by SERMs. To assess whether changes in protein levels reflect variations of ERa protein stability or of transcription rates of the ESR1 gene, we quantified ERa mRNA accu mulated following 16 h treatment with the different compounds. ESR1 mRNA expression was greatly reduced after treatment with ERa ligands. In the presence of E2, only 40% of ESR1 mRNA could be recovered. Similarly, treatment with SERMs and SERDs repressed ESR1 mRNA transcription by 45% 60% rela tive to untreated MCF 7 cells. Despite the fact that a reduction in ERa protein levels was readily detectable after 1 h and significant after 16 h, we observed that E2 induced a 7 to 10 fold increase in mRNA levels of the ERa target gene GREB1 compared to mock treated cells. GREB1 transcription was inhibited by SERMs and SERDs.

These results were expected since E2 is known to activate this ERa target gene, while SERMs Drug_discovery and SERDs are antiestrogens and thus repress GREB1 transcription in ERa positive mammary tumour cells. Thus, in MCF 7 cells, variations in ERa protein levels do not necessarily correlate with ESR1 transcription in the presence of ligands. We note that the decrease in ERa protein levels is more pronounced after treatment with SERDs than after addition of E2, while the effect of hormone and SERDs on ESR1 mRNA accumulation was comparable.