prostate cancer research in taiwan - Anticancer Research

ANTICANCER RESEARCH 28: 1967-2018 (2008)

PROSTATE CANCER RESEARCH IN TAIWAN

Edited by TAI C. CHEN Clinical Research Center Boston University School of Medicine Boston, MA, U.S.A. and YI-FEN LEE Department of Urology University of Rochester Medical Center Rochester, NY, U.S.A.


Prostate Specific Antigen Gene Expression in Androgen Insensitive Prostate Carcinoma Subculture Cell Line KE-HUNG TSUI1,2,3, TSUI-HSIA FENG4, LI-CHUAN CHUNG5, CHUN-HSIANG CHAO5, PHEI-LANG CHANG1,3 and HORNG-HENG JUANG2,3,5 1Department

of Urology, 2Molecular Image Center and 3Bioinformation Center, Chang Gung Memorial Hospital, Kwei-Shan, Tao-Yuan; 4School of Nursing and 5Department of Anatomy, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan, R.O.C.

Abstract. A novel prostate cancer cell line (PC-J) was isolated from an androgen independent non-prostate specific antigen (non-PSA) producing carcinoma cell line. The homologous correlation between PC-J and PC-3 was determined by short tandem repeat analysis. The PSA promoter activity was detected by transient expression assay in the PCJ and LNCaP cells but not in androgen insensitive PC-3 cells. When the PC-J cells were cotransfected with androgen receptor, androgen receptor coactivators and PSA reporter vector cells, the reporter assays indicated that nuclear receptor coactivator 4 (NCOA4) but not androgen receptor activator 24 (ARA24) increased the sensitivity and maximum stimulation of dihydrotestosterone (DHT)-inducing PSA promoter activity. The RT-PCR assays revealed that the expression of several tumor markers, including interleukin-6, prostate stem cell antigen (PSCA), prostate epithelium-specific Ets transcription factor (PDEF) and matriptase, was lower in the PC-J cells than in the PC-3 cells. This cell model elucidated the regulation of PSA expression and enabled comparison of the gene profile at different stages of metastasis in prostatic carcinoma. Prostate cancer is the most prevalent cancer and the second leading cause of cancer deaths among American males (1). The prostate specific antigen (PSA), one of the most important proteins secreted in prostate fluid and serum PSA, is a well-known marker for diagnosing and monitoring prostate cancer (2). However, in vitro study of PSA regulation in hormone refactory prostate cancer in vitro has been limited since PSA does not express in highly metastasic prostate carcinoma cells (3). Correspondence to: Dr. Horng-Heng Juang, Department of Anatomy, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan, R.O.C. Fax: +886 3 2118112, e-mail: [email protected] Key Words: Prostate, PSA, ARA24, NCOA4, IL-6, PSCA, PEDF, matriptase.

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The PC3M cells, originally derived from liver metastases produced in nude mice following intrasplenic injection of PC-3 cells (4), have been widely used in the study of prostate. However, PC3M and PC-3 do not express the PSA gene and PSA promoter activity (5). Changes in serum PSA concentration are associated with cancer metastasis, recurrence, response to treatment and survival (6). During the past decade, in vivo and in vitro studies have indicated that some androgen receptor coactivators affect the progression from androgen dependence to androgen independence. Additionally, several abnormalities in expression and function of AR coactivators have been found to be associated with progression of prostate cancer (7, 8). The expression of NCOA4/ARA70 is increased in highgrade prostate cancer tissues, as well as in hormonerefractory LNCaP xenografts and prostate cancer cell lines (9). Another AR N-terminal interacting protein, ARA24, interacts with the polyglutamine tract, a region within the Nterminus of the AR linked to Kennedy disease (10). In situ hybridization assays have indicated that both AR-positive and AR-negative prostatic cell lines and xenografts expressed ARA24 (11). Other studies employing in situ RNA hybridization to analyze primary prostate carcinomas with different degrees of differentiation indicate that NCOA4 and ARA24 are preferentially expressed in high-grade prostatic intraepithelial neoplasia suggesting their involvement in prostate tumorigenesis (12). With tumor progression almost continuous expression of AR was revealed with increased expression of ARA24, but decreased expression of ARA70. However, the changing levels of these coactivators show no correlation with Gleason score (12). Both NCOA4 and ARA24 can enhance AR transactivation in transient overexpression of AR in PC-3 cells using a reporter vector with MMTV promoter (10, 13). Other studies have suggested that relative levels of ARA24 and NCOA4 might differentially modulate AR transcriptional activity within the promoter/enhancer region of kallikrein 2 (KLK2) and PSA of breast cancer cells (14). However, whether or not AR 1969


cofactors contribute to the regulatory mechanisms of PSA gene expression in androgen-insensitive prostate carcinoma cells remains unclear. In addition to isolating, PC-J, a subclone cell line, from the androgen insensitive prostate carcinoma cells, PC-3, this study attempted to elucidate how ARA24 and NCOA4 modulate the regulation of the PSA gene by androgen. The difference in gene profile of PC-3 and PC-J cells was also examined.

Materials and Methods

Cell culture and materials. The LNCaP, PC-3, and DU145 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The dihydrotestosterone (DHT; 5α-androstan17β-ol-3one) was purchased from Sigma (St. Louis, MO, USA). Fetal calf serum (FCS) was purchased from HyClone (Logan, UT, USA) and RPMI 1640 medium and RPMI 1640 phenol red free (RPMI-PRF) medium were purchased from Life Technologies (Rockville, MD, USA).

PC-3 subcloning. The PC-3 cells were plated in a 96-well plate with a limiting dilution. Individual colonies were examined to evaluate the expression of PSA using the reporter assay with the reporter vector (pPSABH). Reverse transcription-polymerase chain reaction. The total RNA was isolated with Trizol reagent and the cDNA was synthesized by using superscript III preamplification as described previously (15). Table I lists the primers used for amplifying specific genes. The quality of the cDNA was verified by performing controlled reactions using primers derived from β-actin-P and β-actin-R. The PCR reaction was carried out in a thermal cycler (T3000 Thermocycler, Biometra, Goettingen, Germany) under the following parameters: 30 cycles of 94˚C for 0.5 min, anneal temperature for 1 min and 72˚C for 1 min. The anneal temperature was 60˚C for β-actin, 50˚C for MGAT5 and matriptase, 52˚C for maspin and LXRβ and 55˚C for IL-6, PSCA and PDEF. The PCR products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining.

Immunoblot and Northern blot assays. The LNCaP, PC-3 and PC-J cells were incubated with RPMI 1640 medium with 10% FCS. Cells were lysed using a lysing buffer [62.5 mM Tris (pH 6.8), 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 7 M urea, 5 μg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride]. Equal amount of protein (40 μg) were loaded onto a 12% SDS-polyacrylamide gel and analyzed by the electrochemiluminescence (ECL) detection system (Amersham Biosciences, New Territories, Hong Kong), as described previously (16). The blotting membranes were probed with 1:200 diluted polyclonal PSA antiserum (Dako, Glostrup, Denmark) or 1:1000 diluted β-actin antiserum (C11, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The double strand DNA fragment of PSA was radioactively labeled with [α- 32P]dCTP by nick translation using the multiprimer DNA labeling system (Amersham Biosciences). Approximately 20 μg of total RNA from each cell was hybridized with the 32P-labeled βactin and PSA probes, respectively, as described previously (15).

Reporter vector constructs. The PSA gene promoter was isolated from the P1-derived artificial chromosome (PAC) clone (LLNLF214C7; Human Genome Mapping Project Resource Centre, UK).

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Table I. Primers used in this study. Name

Actin-P Actin-R IL-6P IL-6R Maspin-P Maspin-R LXRβ-P LXRβ-R PDEF-P PDEF-R PSCA-P

Sequence

PCR size (bp)

5’-GAAGATCAAGATCATTGCTCCTCC-3’ 5’-CTGGTCTCAAGTCAGTGTACAGG-3’ 5’-AAAGAGGCACTGGCAGAAAA-3’ 5’-CATGCTACATTTGCCGAAGA-3’ 5’-AGATGGCCACTTTGAGAACATT-3’ 5’-GAGGACTTAACATGGGCTATGC-3’ 5’-CGCTACAACCACGAGACAGA-3’ 5’-CCACCCTAGGAAGAGGAAGG-3’ 5’-GACCAGTGAGGAGAGCTGGACCGA-3’ 5’-TGACCTTGGGCTCTGGAAGGTCAG-3’ 5’-ATGAAGGCTGTGCTGCTTGCCCTGT TGATG-3’ PSCA-R 5’-CTAGAGCTGGCCGGGTCCCCAGAGC AGCAG-3’ Matiptase-P 5’-GCTCATCACACTGATAACCAACAC-3’ Matriptase-R 5’-TAGAAGAATTTGAAGCGCACCTTCAC-3’ MGAT5-P 5’-GACCTGCAGTTCCTTCTTCG-3’ MGAT5-R 5’- AGAGCAGGAGGTCACCTTG-3’ RANP 5’-CGATGGCTGCGCAGGGAGAG-3’ RANR 5’-TTCATTCTCACAGGTCATCATCC-3’

723 399 733 514

451 371 221

631 660

The PAC clone was digested with Hind III and one 6-kbp DNA fragment was subcloned into the pGEM3 vector (Promega Bioscience, San Luis Obispo, CA, USA). The reporter vectors (pPSAH, -41 to -5874; pPSABHE, -4801 to -3933 with -41 to -589; pPSAKH, -41 to -1557; pPSABH, -41 to -589; pPSAPCR1, -41 to -465; pPSAPCR2, -41 to -310; pPSAPCR3, -41 to 193, pPSAPCR4 -41 to -123) containing 5’-flanking region of the human PSA gene were cloned into the luciferase reporter vector (pbGL3; promega Bioscience) as described previously (3, 16). The mouse mammary tumor virus (MMTV) reporter vector was constructed by insertion of MMTV promoter into the promoterless lucifease reporter vector as described previously (3). The enhanced green fluorescent proteins (EGFP) reporter vectors containing the different DNA fragments of the 5’-flanking region of PSA gene were constructed by digesting the pPSABH with Bgl II and Hind III, pPSAH with Hind III, and pPSABHE with Sac I and Hind III and were then cloned into the pEGFP-1, a promoterless EGFP vector (BD Biosciences, Bedford, MA, USA).

Expression vector constructs. The human NCOA4 overexpression vector was constructed by cloning NCOA4 cDNA vectors (DKFZp762E1112; RZPD, Berlin, Germany) after digestion with Eco RI and Xho I into the pcDNA3 overexpression vector controlled by the cytomegalovirus (CMV) promoter. The cDNA fragment of human ARA24 was synthesized by RT-PCR with two primers (RANP and RANR; Table I) from the total RNA extracted from the LNCaP cells. The synthesized cDNA fragment (–2 to +772) was cloned into the pGEM-T vector (Promega Bioscience) and then ligated into the overexpression vector, pcDNA3 (Invitrogen, Carlsbad, CA, USA) after digestion with Eco I. The correct ligation and orientation was confirmed by extensive restriction mapping and sequencing. The androgen receptor (AR) overexpression vector was cloned described previously (17).

Tsui et al: PSA Regulation in Androgen Insensitive Prostate Cells

Figure 1. Transient gene expression assay of PSA promoter/enhancer reporter vectors transfected into PC-J cells. (A). Various PSA promoterluciferase gene constructs transfected into PC-J cells. (B). PC-J cells were transfected with PSA promoter/enhancer constructs in the presence or absence of 10 nM DHT. Luciferase activity was examined 48 h after transfection. White boxes: DHT treated group and black boxes: mock-treatment control group. Data are mean-percentage (±S.E., n=6) of the luciferase activity relative to that of control reporter vector (pGL3). (C). PC-J cells cotransfected with PSA promoter/enhancer constructs and androgen receptor overexpression vector in the presence of different concentrations of DHT. Luciferase activity was determined 48 h after transfection. Data are the mean-percentage stimulation (±S.E., n=6) of the luciferase activity induced by DHT treatments relative to that of the mock-treatment control sample (*p