Figure 6. Rapid estrogen stimulation of increased BRCA1 and BARD1 protein levels requires AKT activation but not new protein synthesis.

(A) MCF7 cells cultured in 10% CSS medium were stimulated with 10 nM E2 or pretreated with 10 μM LY294002 for 1 hour before stimulation with E2 for indicated amount of time (in hours). Immunoblot for BRCA1 and BARD1 proteins demonstrates that pre-incubation with LY294002 blocks the increase in total protein levels observed with E2 treatment alone. Samples treated with LY294002 alone showed similar levels of BRCA1 and BARD1 expression as the E2 + LY294002 groups (data not shown). pS473-AKT was detected to monitor AKT activation. Numbers below each panel represent relative densitometry values compared to the vehicle control.

(B) Immunoblots for indicated proteins from MCF7 cells cultured for 48 hours in CSS medium and then stimulated with 10 nM estrogen (E2) for the indicated amount of time (in hours) or with ethanol as vehicle control (V). Cells were pretreated with either 25 μM cycloheximide (CHX) or 1 μM ICI 182780 for 1 hour prior to addition of E2. Control groups were treated with inhibitor alone and matched to the 0.5 hour time point. Cyclin D1 was analyzed only in the E2 and E2+CHX treated samples as a control for the effectiveness of CHX treatment. Lamin immunoblot indicates equal loading. Numbers below each panel represent relative densitometry values compared to vehicle control.