F21 ESTABLISHMENT OF TYPE II 5a-REDUCTASE OVER-EXPRESSING CELL LINE
Kim CD1, Jang S1, Im M1, Hwang SL2, Chang SY2, Seo YJ1, Lee JH1, Parka JK1
1Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, Korea 2LG Household and Healthcare Research Park, Daejeon, Korea

Dihydrotestosterone (DHT) is the most potent male hormone, which is known for cause of androgenetic alopecia. The 5a-reductase type II is an enzyme that catalyzes the conversion of testosterone (T) to DHT, therefore it can be expected that specific inhibitor for type II 5a-reductase may improve the pathophysiologic status of androgenetic alopecia. So far, many researchers have tried to find 5a-reductase inhibitors using a prostate of experimental animals including mouse and rat as an enzyme source. However, 5a-reductase is known to have ~60 percent homology between human and rodent species. To overcome the defects in previous method, we established type II 5a-reductase over-expressing stable cell line. Type II 5a-reductase gene was cloned from human prostate cDNA library, and then used for construction of expression vector. After transfection into HEK293 cells, stable cells were selected by G418 treatment for 4 weeks. The expression of type II 5a-reductase gene was validated using RT-PCR and Western blot analysis. To measure the type II 5a-reductase activity, stable cells were lysed by sonication in citrate buffer (pH 5.5). Enzyme reaction was carried out in the presence of cellular extract, NADPH and [3H]testosterone as a substrate. After enzyme reaction, steroids were separated by thin layer chromatography and visualized by autoradiography. Ten µg of stable cell extract completely convert 1 µCi (0.01 nmol) of T into DHT. These results indicate that this cell line can be used as a good model to screen the specific inhibitors for type II 5a-reductase.