5?-reductase in human embryonic kidney cell line HEK293: Evidence for type II enzyme expression and activity

Abstract

Human 5alpha-reductase catalyses the last step in androgen biosynthesis, namely the reduction of testosterone (T) to the more potent androgen dihydrotestosterone (DHT). The enzyme is therefore considered to be an important drug target for androgen related diseases such as benign prostatic hyperplasia and prostate cancer. The present study displays evidence that the human embryonic kidney cell line HEK293 which is frequently used in recombinant target protein expression contains an endogenous 5alpha-reductase type II activity. After an incubation of 24 h 1 x 10(6) HEK293 cells converted 23% of the substrate 4-androstene-3,17-dione (7.5 nM) to the product 5alpha-androstane-3,17-dione. Reverse transcription polymerase chain reaction was carried out to identify the mRNA of the isoform responsible for the 5alpha-reductase activity. Only with type II specific primers a fragment with the predicted size was amplified, while with type I specific primers no band could be observed. An antiserum against human 5alpha-reductase type II was raised by immunizing a rabbit with a hemocyanin-conjugated peptide corresponding to amino acid 29 to 44 of the type II enzyme. Western blot analysis of different fractions of a HEK293 homogenate performed with this antiserum detected a band at 45 kDa in the nuclear and microsomal fraction corresponding to 5alpha-reductase type II protein.

Subcellular localization of the five members of the human steroid 5α-reductase family

“…Some authors have reported that human SRD5A1 is associated with nuclear fractions upon expression in insect Sf9 cells [28] , [29] but later papers [30] have instead reported retrieval of enzyme activity from the microsomal fraction. Expression of SRD5A2 and SRD5A1 in HEK293 and in LNGK-9 PCa cells yielded activity in the nuclear and microsomal fractions [31] , [32] . Upon heterologous expression and purification in yeast, SRD5A1 appears to be associated to nuclei, whereas SRD5A2 is in the microsomal fraction [33] .…”

C11-hydroxy and C11-oxo C19 and C21 Steroids: Pre-Receptor Regulation and Interaction with Androgen and Progesterone Steroid Receptors

“…Although classed as non-steroidogenic, previous studies have shown the endogenous expression of some enzymes in the HEK293 cell line. While not observed in the present study, one such enzyme includes SRD5A which catalyses the reduction of the C4/C5 double bond [ 17 , 51 , 52 ]. However, conversion of 11OH5αdione by 11βHSD2 in the present study yielded both 11K5αdione and 11KDHT, suggesting the expression of endogenous 17βHSD isozyme/s, which was also reported in a previous study by our group [ 32 ].…”

Effects ofLeuzea carthamoideson Human Breast Adenocarcinoma MCF-7 Cells Determined by Gene Expression Profiling and Functional Assays

“…The inhibitory activity of Leuzea root extract and 20-hydroxyecdysone towards 5α-reductase, type II was determined using HEK-II cell homogenates and substrate 1β[ 3 H]-androstenedione (505 nM) as well as the well known 5α-reductase inhibitor finasteride as positive control (IC 50 = 25 nM) as previously described [18], [19].…”