Figure 2

Oestradiol inhibits the growth of MCF-7:5C cells and induces apoptosis. MCF-7:5C cells were cloned from wild-type MCF-7:WS8 cells following long-term growth (∼1 year) in oestrogen-free RPMI medium containing 10% (v v⁻¹) dextran charcoal-stripped (DCC) foetal bovine serum (FBS), 2 mM glutamine, 100 U ml⁻¹ penicillin-streptomycin, 6 ng ml⁻¹ bovine insulin, and 1 × nonessential amino acids. (A) For DNA assays, MCF-7:5C cells were seeded into 12-well plates at a density of ∼20 000 cells per well in RPMI medium. The cells were left for 24 h to acclimatise, and then treated with 0.1, 1, or 10 nM oestradiol (E₂) for a total of 6 days, with the control cells receiving <0.1% ethanol vehicle. Cells were re-fed on days 3 and 5. Total DNA (μg) per well was used to measure cell growth. The data represent the average of five separate experiments. (B) Apoptotic cells were identified/quantified by double staining with recombinant FITC-conjugated annexin V and propidium iodide (PI), using the Annexin V-FITC kit (Immunotech, Beckman Coulter). For experiments, MCF-7:WS8 and MCF-7:5C cells were seeded in 100 mm plates at a density of 1 × 10⁶ per plate in either oestrogen-free RPMI medium containing 10% DCC stripped fetal bovine serum (SFS) or MEM containing 5% DCC stripped calf serum (SCS). The cells were left for 24 h to acclimatise and then treated with either 1 nM E₂ or less than 0.1% ethanol vehicle (control) for 72 h. Data shown represent three separate experiments. It should be noted that oestradiol treatment of MCF-7:WS8 cells in oestrogen-free MEM media containing 5% SCS did not have any significant effect on apoptosis (data not shown).