Real‐time measurement of E2: ERα transcriptional activity in living cells

Abstract

Kinetic analyses of diverse physiological processes have the potential to unveil new aspects of the molecular regulation of cell biology at temporal levels.17β-estradiol (E2) regulates diverse physiological effects by binding to the estrogen receptor α (ERα), which primarily works as a transcription factor.Although many molecular details of the modulation of ERα transcriptional activity have been discovered including the impact of receptor plasma membrane localization and its relative E2-evoked signaling, the knowledge of real-time ERα

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“…According to the previous results, we observed in Y537SNLuc cells that both Clo and Fenti reduced in a dose-dependent manner the ERE-promoter activity ( Figure 5 e). As expected [ 13 , 22 ], also ICI prevented Y537S ERα mutant ERE-promoter activity ( Figure 5 e).…”

“…Next, we employed these 21 drugs in the in-cell assays described in [ 20 , 21 , 22 ] using one cell line modeling primary tumors (i.e., MCF-7 cells) and one cell line modeling MBC expressing the Y537S mutated transcriptionally hyperactive ERα (i.e., Y537S). In particular, we treated each cell line with each drug at a final treatment concentration of 2 μM for 3 and 7 days and measured both the ERα levels and cell proliferation as described in [ 21 ] as well as the wild type (wt) and Y537S mutant ERα transcriptional activity at 1 or 3 days after drug administration as described in [ 20 , 22 ] and searched for those molecules blocking the different aspects of ERα signaling. Figure 1 c shows in red or green squares the positive hits in each assay for each cell line.…”

“…Remarkably, Clo and Fenti also significantly reduced the basal promoter activity in the absence of E2 ( Figure 5 d). To further strengthen the notion that Clo and Fenti inhibit the E2-induced ERα-dependent transcriptional activity, we next employed the Y537SNLuc cells that stably express the same ERE-containing promoter activity [ 22 ], which is constitutively activated by the Y537S ERα mutant present in this CRISPR-CAS9 engineered cell line [ 13 ]. According to the previous results, we observed in Y537SNLuc cells that both Clo and Fenti reduced in a dose-dependent manner the ERE-promoter activity ( Figure 5 e).…”

“…These methods not only speed up the research process through an unbiased approach but also help to screen a high number of candidates in a very limited amount of time. In this respect, we have developed and characterized several assays to directly measure in cell lines different aspects of the E2:ERα proliferative signaling including either ERα-dependent transcriptional activity both controlled by wild type and Y537S mutant receptor, cell proliferation or ERα levels, in a high-throughput format [ 20 , 21 , 22 ].…”

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“…For growth curves ( Figure 1 A,B and Figure S2 ) and drug synergy studies, the xCELLigence DP system (ACEA Biosciences, Inc., San Diego, CA) Multi-E-Plate station was used to measure the time-dependent response to the indicated drugs, using real-time cell analysis (RTCA), as previously reported [ 23 , 24 ]. Briefly, the number of cells (i.e., normalized cell index) is directly proportional to the measured electric impedance of the cells on the well surface.…”