Stable expression of human 5α-reductase type II in COS1 cells due to chromosomal gene integration: a novel tool for inhibitor identification

Abstract

Inhibitors of human 5α-reductase type II are promising drug candidates for the treatment of benign prostatic hyperplasia which is associated with high prostatic DHT levels. In this study we describe the evaluation of potential inhibitors in a new cell assay. First a plasmid (pRcCMV-5αII) for the expression of human 5α-reductase type II was constructed by the use of the vector pRcCMV and transfected into the African green monkey fibroblast-like cell line COS1. By selection with G418 sulfate, ten COS1 single cell clones were obtained of which three stably exhibited high 5α-reductase activity. One single cell clone (COS1-5αIIST) was selected for further investigations. By Southern blot analysis, fluorescence in situ hybridization (FISH) and comparative PCR experiments it turned out that the expression plasmid pRcCMV-5αII has been integrated into the chromosome, resulting in a long-term stable expression of the foreign 5α-reductase gene. The newly established cell line was used for testing novel compounds on their inhibitory effect on human 5α-reductase type II. Using this whole cell assay, inhibitors with IC 50 values in the nanomolar range could be identified.