Quantifying ER Function Using High-Throughput Imaging in Breast and Other Cancer Cells

摘要

We are testing the hypothesis that PRL-array-containing cell lines can be used effectively to identify novel molecular players in ERalpha transcriptional activation and repression, and in determining temporal patterns of function of all known ERalpha-coregulators. We are using special cell lines containing chromosomally integrated arrays of estrogen-responsive reporters combined with automated and other types of single cell microscopic, imaged based assays. This systems-biology level approach will integrate functional data from multiple readouts at a single cell level for ERalpha and CoRs including 1) nuclear targeting, 2) promoter array occupancy, 3) large scale chromatin modeling, 4) histone modifications, and 5) mRNA synthesis. ERalpha is differentially recruited to the PRL-array in HeLa cells by estrogen or epidermal growth factor, two critical signaling modalities in breast cancer. Transcription and chromosomal condensation mediated by ERalpha can be uncoupled. ERalpha mediated accumulation of reporter transcripts has differential periodicity in response to E2 or EGF. E2- induces a novel 24-hour cyclical pattern of accumulation of mRNA. Significant progress in technology and analytical techniques and modification of planned reporter constructions will facilitate achieving our proposed objectives.