Breast cancer cells can recognize and respond to different levels of progestins to achieve different phenotypic outputs [RNA-seq]

The steroid hormone progesterone, acting through its nuclear progesterone receptor (PR), has complex physiologic activities with different levels of hormones manifesting distinct and sometimes opposing phenotypic responses in target tissues. However, most of what is currently known about the transcriptional activity of PR comes from studies performed using progestins at levels that are in the high physiologic range (>10nM), relevant only in the luteal phase of the reproductive cycle and pregnancy in humans. These studies do not consider the non-linearity of responses to progestins that exist in physiology and are not informative as to the mechanisms by which low levels of progestins, as occurs during menopause (0.1-0.3nM), exert their biological activities. Thus, we undertook to define the mechanisms which enable cells to recognize and respond to different levels of progestins. Overall design: T47D cells were plated 2.5x10^5 cells per well in 6-well culture plates. Cells were allowed to attach overnight in phenol red-free RPMI 1640 supplemented with 8% charcoal-stripped FBS (CFS), NEAA, and NaPyr. Synchronization was achieved by 24-48hr serum starvation in phenol red-free RPMI 1640 supplemented with 0.1% CFS, NEAA, and NaPyr. Synchronized T47D cells were treated with the relevant assay compounds. Cell plates were incubated at 37*C in the presence of 5% CO2 for the noted amount of time. Upon treatment completion, media was removed by aspiration, and cells were gently washed with sterile PBS. Isolation of total RNA was performed using the Aurum™ Total RNA Mini-Kit according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Total RNA concentration and quality were determined using a Nanodrop 1000 (ThermoFisher).