Abstract
Differential display was used to identify novel androgen-regulated genes in the human prostatic adenocarcinoma cell line LNCaP. A 322-base pair cDNA fragment that was consistently induced by the synthetic androgen R1881 revealed 100% homology with the human phosphatidic acid phosphatase type 2a isozyme very recently reported by Kai et al. (PAP-2a; Kai., M., Wada, I., Imai, S.-i., Sakane, F., and Kanoh, H. (1997) J. Biol. Chem. 272, 24572-24578). The fragment was used to clone the corresponding cDNA from a human prostate library. The deduced amino acid sequence confirmed the identity with human PAP-2a. The inducibility of PAP-2a mRNA by androgens was confirmed by Northern blot hybridization. The effect was time- and dose-dependent with a maximal stimulation (4-fold) after 24 h of treatment with 10(-9) M R1881. The steroid specificity of PAP-2a mRNA regulation was found to be in agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells, supporting the involvement of the androgen receptor in the induction process. Furthermore, low basal levels of PAP-2a mRNA and absence of androgen inducibility were observed in the poorly differentiated and androgen receptor-negative cell lines PC-3 and DU-145. Induction of PAP-2a mRNA was not affected by the protein synthesis inhibitor cycloheximide and was accompanied by a marked increase in PAP-2 activity as measured by the conversion of phosphatidic acid into diacylglycerol in membrane fractions of LNCaP. Comparison of the expression of PAP-2a mRNA in 50 different human tissues revealed ubiquitous expression. The highest levels, however, were observed in the prostate. Since PAP-2 plays a pivotal role in the control of signal transduction by lipid mediators such as phosphatidate, lysophosphatidate, and ceramide-1-phosphate, the ability of androgens to stimulate the expression and activity of this enzyme in prostatic cells may provide an important opportunity for cross-talk between signaling pathways involving lipid mediators and androgens.
Highlights
The nucleotide sequence reported in this paper has been submitted to EMBL, GenBank, and DDBJ data bases with accession number Y14436
Use of differential display polymerase chain reaction (DD-PCR) Analysis to Identify Androgen-regulated Genes in LNCaP Cells: Identification of One of the Androgeninduced Genes as PAP-2a—To identify mRNAs that are regulated by androgens, DD-PCR was performed on total RNA derived from LNCaP cells treated with control medium or R1881
The synthetic androgen R1881 was selected for these studies to circumvent the rapid metabolization of natural androgens such as 5␣-dihydrotestosterone and testosterone reported in LNCaP cells [24]
Summary
Cell Culture and Androgen Treatment—Human prostatic adenocarcinoma cell lines were purchased from the American Type Culture Collection (Rockville, MD) and maintained in a humidified atmosphere of 5% CO2 in air in RPMI 1640 medium (LNCaP) or in Dulbecco’s modified Eagle’s medium (PC-3 and DU-145) supplemented with 10% fetal calf serum (FCS), 3 mM L-glutamine, 100 g/ml streptomycin, and 100 units/ml penicillin (all from Life Technologies Inc., Paisley, Scotland). PCR reactions were performed in a total volume of 20 l containing 2.5 M downstream primer (dT11MN), 0.5 M arbitrary 10-mer primer, 2 M dNTPs, 2 Ci of [␣-33P]dATP (Amersham Int.), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 1.1 l of cDNA solution, and 0.5 unit of AmpliTaq DNA Polymerase (Perkin Elmer, Norwalk, CT). For the determination of PAP activity, 1.5 g of protein of LNCaP cell membranes were incubated at 37 °C for 20 min, in a solution containing 100 M [14C]1-␣-dipalmitoyl phosphatidic acid (NEN Life Science Products), 50 mM Hepes/NaOH (pH 7.0), and 3 mM Triton X-100, in a final volume of 100 l as described by Hoer et al [23]. Equal results were obtained using 32P-labeled phosphatidic acid as substrate [23]
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