Figure 3.

UGT2B15 and UGT2B17 double deletion permits sustained levels of free and active androgens. A and B, CRISPR-Cas knockout ablates expression of UGT2B15 and UGT2B17 mRNA (A) and (B) protein. Transcript expression by qRT-PCR was assessed in triplicate and normalized to RPLP0 and expression in Ctrl cells. Experiments were repeated 3 times and error bars represent the S.D. C, UGT2B15 and UGT2B17 KO in LNCaP reverses rapid loss of free DHT and testosterone that otherwise occurs in Ctrl LNCaP and is comparable with persistence of free androgens in C4-2 cells. Cells were treated with [³H]DHT (10 nm) or [³H]testosterone (100 nm) for the indicated incubation times, steroids were extracted from media and analyzed by HPLC in triplicate. D, intracellular free [³H]DHT is sustained in glucuronidation-deficient cells. Ctrl and KO cells were treated with [³H]DHT (10 nm) and the radioactive signal was assessed in the organic extract from treated cells in triplicate. E and F, UGT2B15 and UGT2B17 ablation allows for sustained free intracellular DHT with direct treatment (10 nm) (E) and sustains free intracellular DHT and testosterone (T) when treated with the adrenal precursor steroid DHEA (100 nm) (F). Cells were treated for the indicated incubation time in triplicate and intracellular steroids were quantitated by MS. Experiments were repeated three times in E and twice in F. Error bars represent the S.D. in C–F.