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Testosterone Modulates Mitochondrial Aconitase in the Full-Length Human Androgen Receptor-Transfected PC-3 Prostatic Carcinoma Cells

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Testosterone Modulates Mitochondrial Aconitase in the Full-Length Human Androgen Receptor-Transfected PC-3 Prostatic Carcinoma Cells 121 Testosterone modulates mitochondrial aconitase in the full-length human androgen receptor-transfected PC-3 prostatic carcinoma cells H-H Juang, M-L Hsieh1 and K-H Tsui1 Department of Anatomy, Chang Gung University, 259 Wen-Hua 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan, Republic of China 1Department of Urology, Chang Gung Memorial Hospital, Kwei-Shan, Tao-Yuan 333, Taiwan, Republic of China (Requests for offprints should be addressed to H-H Juang; Email: [email protected]) Abstract In vitro studies indicated that dihydrotestosterone (DHT) stimulates the enzymatic activity of the mitochondrial aconitase (mACON) in androgen-sensitive prostatic carcinoma cells, LNCaP. Cell proliferation assay determined that DHT doubles the optimal proliferation response of LNCaP cells. The androgen-insensitive human prostatic carcinoma cells, PC-3, were overexpressed in the human androgen receptor to assess the involvement of the native androgen receptor in the regulation by DHT of mACON gene expression. A stable-transfected clone that expresses the full-length androgen receptor was selected and termed PCAR9. The results revealed that DHT-treated PCAR9 cells paradoxically not only reduced the enzymatic activity of mACON but also blocked the biosynthesis of intracellular ATP attenuating cell proliferation. Transient gene expression assay indicated that DHT divergently regulates the promoter activity of the mACON gene in LNCaP and PCAR9 cells. This study suggested that DHT regulates mACON gene expression and the proliferation of cells in a receptor-dependent model through modulation by unidentified non-receptor factors. Journal of Molecular Endocrinology (2004) 33, 121–132 Introduction sensitive prostatic carcinoma (LNCaP) cells; how- ever, early study has indicated that DHT treatment The differentiation, development and maintenance inhibits the proliferation of LNCaP cells which of the prostate gland are under the direct control were long-term cultured in a medium without of androgen (Cunha et al. 1987). The epithelial steroids (Liao et al. 1995). Other studies have also compartment of the prostate gland in humans and shown that DHT suppresses malignancy and other animals has the unique function of accumu- triggers the cell cycle arrest of the androgen lating and secreting extremely high levels of citrate receptor-transfected bone metastatic prostate cells, (Franklin et al. 1986). The net citrate secretion rate PC-3 (Yuan et al. 1993, Heisler et al. 1997). Hence, is determined by the rates of citrate synthesis and the function of androgen and androgen receptor in citrate oxidation; testosterone controls the balanc- the prostate has been suspected to vary with the ing mechanism in the rat ventral prostate (Costello state of malignancy. & Franklin 1991). Evidence is emerging that the Aconitase (aconitase hydratase, EC4·2·1·3) con- enzymatic activity of many enzymes involved in taining a [4Fe–4S] cluster is regarded as the key metabolizing citrate in the prostate also depends on enzyme responsible for the interconversion of dihydrotestosterone (DHT) (Costello & Franklin citrate and isocitrate in the citric acid cycle 2002). Mutation of the androgen receptor is not a (Emptage et al. 1983). Citrate oxidation via the major mechanism in the process of prostate cells Krebs cycle is the major pathway for energy that become androgen growth independent (Taplin production from carbohydrate and fat metabolism. et al. 1995). It is well known that DHT has a From previous reports, the citrate that is synthe- positive effect on the proliferation of androgen- sized by prostate cells is accumulated and secreted Journal of Molecular Endocrinology (2004) 33, 121–132 Online version via http://www.endocrinology.org 0952–5041/04/033–121 © 2004 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 02:47:30AM via free access 122 H-H JUANG and others · Androgen regulates mitochondrial aconitase rather than oxidized. The limiting mitochondrial Construction of the androgen receptor aconitase (mACON) activity prevents citrate from overexpression vector entering the Krebs cycle that induces accumulation The full-length human androgen receptor cDNA and secretion of citrate from the prostate epithelial clone was kindly provided by Dr S Liao (Chang cells (Costello & Franklin, 1991). A bioenergetic et al. 1988). Two fragments of human androgen hypothesis of prostate malignancy suggests that a receptor cDNA were digested: one was digested normal citrate-producing prostatic epithelial cell with EcoRI and HindIII, and the other was becomes a citrate-oxidizing cell following a digested with HindIII and XbaI resulting in 2·3 transformation in which mACON is not limiting and 1·3 kb fragments respectively. The overexpres- (Costello & Franklin 1994). Our previous in vitro sion vector, pcDNA3 (Invitrogen), was digested study demonstrated that mACON-antisense stable- with EcoRI and XbaI, and the linearized plasmid transfected PC-3 cells have lower citrate utility, DNA was ligated with the 2·3 kb EcoRI–HindIII intracellular ATP biosynthesis and cell proliferation and the 1·3 kb HindIII–XbaI human androgen than the mock-transfected cells (Juang, 2004a). cDNA fragments, resulting in the insertion of the Although early studies have demonstrated that full-length androgen receptor cDNA in the poly- androgen treatment promotes citrate oxidation and adenlyate region that was controlled by the human citrate secretion in LNCaP cells, in transient rat cytomegalovirus immediate-early (CMV) promoter androgen receptor-transfected PC-3 cells, in the rat (pcDNA3AR). Proper ligation was confirmed by ventral prostate and in the pig prostate (Costello extensive restriction mapping and sequencing. et al. 1995, 1996, Franklin et al. 1995, Juang et al. 1995), the regulation of mACON in the human prostate is not well understood. Stable transfection of androgen receptor into PC-3 cells PC-3 cells were transfected with pcDNA3AR Materials and methods overexpression vector using lipofectin as described Cell culture and chemicals by the manufacturer (Life Technologies). Briefly, the transfected cells were incubated for 48 h in PC-3 and LNCaP cell lines were obtained from the RPMI 1640 supplemented with 10% fetal bovine American Type Culture Collection. LNCaP cells serum (FBS). Then, the cells were incubated for 2 are the unique tumor cell line derived from the weeks in the same medium supplemented with supraclavicular lymph node metastasis from the Geniticin (G418; PAA laboratories, Linz, Austria) human prostatic carcinoma which exhibits in- to a final concentration of 800 µg/ml. The resulting creased proliferation in response to androgens Geniticin-resistant cells were plated in a 96-well in vitro (Horoszeqicz et al. 1983). The PC-3 cell line plate with a limiting dilution. One selected single, was isolated from the bone metastasis of a resistant colony was then examined to evaluate the 62-year-old patient with stage IV prostatic cancer expression of the androgen receptor using immuno- (Kaighn et al. 1979). 5-Androstan-17-ol-3 one blotting assay and cell immunocytochemical stain- (DHT) and charcoal (dextran-coated) were pur- ing. One clone that expressed the highest chased from Sigma and the BCA protein concentration of the full-length androgen receptor concentration assay kit was purchased from Pierce (110 kDa) in the nuclei was selected and termed (Rockford, IL, USA). Fetal calf serum (FCS) was the PCAR9. The mock-transfected PC-3 cells purchased from HyClone (Logan, UT, USA) and (PCDNA) were transfected with controlled RPMI 1640 medium and RPMI 1640 phenol pcDNA3 expression vector that lacked the andro- red-free (RPMI-PRF) medium were purchased gen receptor cDNA insert. The PCDNA cells were from Life Technologies). The FCS, which was clonally selected in the same way as the PCAR9 treated with charcoal (1 g/500 ml FCS) for 24 h to cells. remove the steroids, was considered to be the charcoal–dextran-treated FCS (CD-FCS) in this study. PC-3 and LNCaP cells were maintained in Immunocytochemical staining of cells an RPMI 1640 medium that contained 10% FCS LNCaP, PCAR9 and PCDNA cells were grown on and the medium was changed twice a week. the surface of the cover slides for 48 h. Cells were Journal of Molecular Endocrinology (2004) 33, 121–132 www.endocrinology.org Downloaded from Bioscientifica.com at 09/29/2021 02:47:30AM via free access Androgen regulates mitochondrial aconitase · H-H JUANG and others 123 Table 1 Primers required by RT-PCR to synthesize the human androgen receptor (AR) and -actin cDNAs Sequence Location Primer AR1sense 58-CCAAGACCTACCGAGGAGCT-38 +47 to +66 AR1antisense 58-GCTGTGAAGGTTGCTGTTCC-38 +355 to +336 AR2sense 58-CGGACGAGGATGACTCAG-38 +458 to +475 AR2antisense 58-TCTTCAGTGCTCTTGCCTGC-38 +914 to +895 AR3sense 58-CTGGCTTCCGCAACTTACAC-38 +2172 to +2191 AR3antisense 5-8TGGTAGAAGCGTCTTGAGCA-38 +2576 to +2577 -Actin-sense 58-GAAGATCAAGATCATTGCTCCTCC-38 +975 to +998 -Actin-antisense 58-CTGGTCTCAAGTCAGTGTACAGG-38 +1698 to +1676 washed with PBS and then fixed with cold acetone promoter as a mammalian positive control vector for 15 min. Cells were immunocytochemically for the glucocorticoid inducible expression of CAT. stained using a VECTASTAIN ABC kit accor- After the transfected cells were grown for 12 h in ding to the manufacturer’s instructions (Vector; RPMI-PRF medium without FCS, the cells were Burlingame, CA, USA). Cells were detected using cultured in the same medium with 2% CD-FCS, 1:500 of anti-bovine mitochondrial aconitase with or without 10 nM DHT, for an additional antiserum (kindly donated by Dr R B Franklin) and 48 h. Cells were harvested and resuspended in rabbit anti-human androgen receptor N-terminal 100 µl 0·25 M Tris buffer (pH 7·5) for CAT assay (N-20) and C-terminal (C-19) polyclonal antibodies as described elsewhere (Juang et al. 1995). (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Mitochondrial aconitase activity assay Cells were suspended in 100 µl 0·25 M sucrose Reverse transcription (RT)-PCR buffered with 50 mM HEPES and 0·007% Total RNA was isolated using Trizol reagent and digitonin on ice for 5 min.
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