RAD001 and letrozole were synthesized in the laboratories of Novartis Pharma AG, Basel, Switzerland. All chemicals, unless otherwise stated, were molecular grade and purchased from Sigma, Poole, Dorset UK. All tissue culture grade plastics were purchased from Thermo Fisher Scientific Nunc, Leices tershire UK. Tissue culture MCF7 AROM1 and BT474 AROM3 were derived from parental cell lines to stably express CYP19. These modified cell lines were given the suffix AROM to distinguish them from the parental cells. AROM cells were maintained in phenol red containing RPMI 1640 medium containing 2 mM glutamine, 10 ug/ml insulin, and 10% fetal bovine serum supplemented with 1 mg/ml G418.

MCF7 cells that had adapted to long term estrogen deprivation were maintained in phenol red free RPMI 1640 medium containing 2 mM glutamine, 10 ug/ml insulin supplemented with 10% dextran coated charcoal stripped FBS , referred to as DCC. For all experiments, cells lines were stripped of steroids for 3 days before seeding by cul turing in DCC in the absence of insulin. Cell proliferation assays Cell lines were seeded into 12 well plates at densities between 1 and 4 104 cells per well. Cell monolayers were left to acclimatize for 24 hours before treatment with the drug combinations indicated for 6 days, with daily changes. Cell number was determined by using a Z1 Coulter Counter. The com bination effects between RAD001 and 4 OH tamoxifen or letrozole were analyzed by using isobolograms.

To determine the nature of the interaction between RAD001 and letrozole or 4 OH tamoxifen, combination studies were performed by using Chou and Talalays constant ratio combination design and quantified by using Calcusyn software. The combination indices for 50%, 75%, and 90% growth inhibition were obtained by using mutually nonexclusive Monte Carlo simulations, and statistical tests were applied to determine whether the CI values at multiple effect levels were significantly different from CI 1. In this analysis, CI scores significantly lower than 1 were defined as synergistic. CI 1, as antagonis tic. and a CI 1, as additive. Experiments were set up in triplicate. Transcription assay Cell lines were seeded in Anacetrapib 24 well plates at 7 104 cells per well in DCC medium for all cell lines except BT474, which was seeded at 1 105 cells per well. Twenty four hours later, monolayers were transfected with Fugene with 0.

1 ug of EREIItkluc and 0. 1 ug of pCH110 overnight, before treatment with the drugs indicated. After 24 hours, luciferase and b galactosidase activ ities were measured by using a luminometer. Western blotting Cell monolayers were extracted as described previously. Protein concentrations were quantified by using BioRad protein assay kit. Proteins were resolved with SDS PAGE and transferred to nitro cellulose filters. Filters were probed with specific antibodies as indicated.