Figure 2. Formation of androgen induced DSB requires AR and TOP2B but not cell cycle progression.

(A) Representative western blot shows robust depletion of TOP2B and AR in si TOP2B and AR transfected cells. (B) LNCaP cells were transiently transfected with siRNAs targeting TOP2B, AR or scrambled control. Seventy-two hours after transfection and concomitant androgen deprivation, cells were treated with 100 nM DHT or vehicle control for 6 h and subjected to neutral comet assay. Shown is the distribution of COMET tail moments across 50 randomly selected cells for each condition. (C) Stimulation of LNCaP cells with the androgen receptor agonist DHT (100 nM for 6 h) induced DSBs as measured by neutral comet assay, whereas treatment with the androgen receptor antagonist enzalutamide (10 μM for 6 h) did not induce DSBs. (D) To evaluate the role of cell cycle progression in androgen induced DSB formation, LAPC4 cells were either treated with solvent control or 50 μM lovastatin for 36 h prior to stimulation with 100 nM DHT for 6 h. Note that androgen induced γH2A.X foci formation was not different between control and lovastatin pre-treated cells. (E) S-phase cell fraction, as measured by the percentage of EdU positive cells, was not different during the early phase of androgen stimulation. (F) Treatment with DHT and enzalutamide for 6 h did not significantly change cell cycle distribution of LNCaP cells grown in charcoal stripped FBS.