Differential display RT PCR of total RNA from human foreskin fibroblasts for investigation of androgen-dependent gene expression

Abstract

Male sexual differentiation is a process that involves androgen action via the androgen receptor. Defects in the androgen receptor, many resulting from point mutations in the androgen receptor gene, lead to varying degrees of impaired masculinization in chromosomally male individuals. To date no specific androgen regulated morphogens involved in this process have been identified and no marker genes are known that would help to predict further virilization in infants with partial androgen insensitivity. In the present study we first show data on androgen regulated gene expression investigated by differential display reverse transcription PCR (dd RT PCR) on total RNA from human neonatal genital skin fibroblasts cultured in the presence or absence of 100 nM testosterone. Using three different primer combinations, 54 cDNAs appeared to be regulated by androgens. Most of these sequences show the characteristics of expressed mRNAs but showed no homology to sequences in the database. However 15 clones with significant homology to previously cloned sequence were identified. Seven cDNAs appear to be induced by androgen withdrawal. Of these, five are similar to ETS (expression tagged sequences) from unknown genes; the other two show significant homology to the cDNAs of ubiquitin and human guanylate binding protein 2 (GBP‐2). In addition, we have identified 8 cDNA clones which show homologies to other sequences in the database and appear to be upregulated in the presence of testosterone. Four of these clones again are similar to ETS from unknown genes. Three differential expressed sequences that appear to be upregulated in the presence of testosterone show significant homology to the cDNAs of L‐plastin and one to the cDNA of testican. This latter gene codes for a proteoglycan involved in cell social behavior and therefore of special interest in this context. The results of this study are of interest in further investigation of normal and disturbed androgen‐dependent gene expression. © 1996 Wiley‐Liss, Inc.

Androgen receptor amplification is reflected in the transcriptional responses of Vertebral-Cancer of the Prostate cells

“…2A,B). We next compared the expression of nine AR targets; FKBP51, C6orf81, TMPRSS2, PSA, S100P, ChGn, SLC45A3, SPOCK1, and NKX3.1 (Amler et al, 2000;Bolton et al, 2007;Lin et al, 1999;Young et al, 1991;Averboukh et al, 1996;Tomlins et al, 2007;Nitsche et al, 1996;Bieberich et al, 1996), in control siRNA (siSCR)-and siAR-transfected VCaP cells that were treated with vehicle or R1881 by measuring their mRNAs by RT-qPCR. As shown in Fig.…”

The human ovarian surface epithelium is an androgen responsive tissue

“…For the androgen receptor an exon spanning fragment stretching from nucleotide position 1653 in exon 1 to position 2845 in exon 4 (Lubhahn et al, 1989) was amplified using the sense primer 5'-TGG ATG GAT AGC TAC TCC GG-3' and the antisense primer 5'-ACT ACA CCT GGC TCA ATG GC-3' (Nitsche et al, 1996), to give a product of 479 base pairs. For GAP-DH expression an exon spanning fragment was amplified using the sense primer 5'-AAA TGA GCC CCA GCC TTC T-3', and the antisense primer 5'-AGT CAA CGG ATT TGG TCG TA, to give a product of 315 base pairs.…”

Die pathogenetische Bedeutung der 5α-Reduktase-Isoenzyme für die androgenetische Alopezie

“…Ein vielversprechender Ansatz für die weitere Forschung wäre die Identifikation solcher Gene und Genprodukte, um die paradoxe Wirkung von DHT auf das Haarwachstum aufzuklären und eine spezifische, nicht auf Hormon-oder Rezeptorebene wirkende Therapie androgenabhängiger Störungen des Haarwachstums zu entwickeln. Derartig androgenabhängi-ge Gene sind mit Hilfe differentieller Genanalysen ("differential display RT-PCR") in humanen Vorhautfibroblasten [47] und Glattmuskelzellinien aus der humanen Prostata [24] teilweise identifiziert worden. Bislang wurde nur die Protease Nexin-1 als ein mögliches DHT-abhängiges Gen in der dermalen Haarpapille beschrieben [63].…”