Aibzip, a Novel Bzip Gene Located on Chromosome 1Q21.3 That Is

[CANCER RESEARCH 62, 721–733, February 1, 2002] AIbZIP, a Novel bZIP Gene Located on Chromosome 1q21.3 That Is Highly Expressed in Prostate Tumors and of Which the Expression Is Up-Regulated by Androgens in LNCaP Human Prostate Cancer Cells1

Heng Qi, Catherine Fillion, Yvan Labrie, Jose´e Grenier, Andre´a Fournier, Louise Berger, Mohamed El-Alfy, and Claude Labrie2 Molecular Endocrinology and Oncology Research Center, Centre Hospitalier Universite´Laval Research Center (CHUQ) and Laval University, Quebec G1V 4G2, Canada

ABSTRACT Although a number of cytokines and growth factors have been shown to modulate AR activity, the primary regulators of AR Androgens play an important role in the development and physiology of activity are the androgens testosterone and its active metabolite the normal prostate as well as in prostate cancer cell proliferation. Com- 5␣-dihydrotestosterone. These steroid hormones interact with the parison of the mRNA expression profiles of control and R1881-treated cultures of LNCaP human prostate cancer cells using cDNA subtraction ligand-binding domain of AR and induce conformational changes led to the identification of a novel transcription factor that we named that allow AR to interact with transcriptional coregulators of which Androgen-Induced bZIP (AIbZIP) protein. AIbZIP is a 395 aa protein the specific roles in AR-mediated gene regulation remain to be with homology to cyclic AMP-responsive element binding protein/activat- determined (4). AR can modulate gene expression directly by ing transcription factor transcription factors. It contains an NH2-terminal interacting with specific elements in the regulatory regions of activation domain, a central bZIP domain, and a COOH-terminal trans- target genes (5) or indirectly by activating various growth factor membrane domain. The AIbZIP gene is localized on chromosome 1q21.3 signaling pathways (6). and consists of 10 exons. A major 1.7-kb transcript was detected exclu- Androgens play a determining role in prostate differentiation sively in the prostate as well as in breast and prostate cancer cell lines. and development (7). Androgens have been shown to stimulate the Androgens up-regulate AIbZIP mRNA and protein levels in a dose- dependent manner. The kinetics of AIbZIP mRNA up-regulation and the growth of prostate cancer cells both in vitro and in vivo, and they results of experiments with cycloheximide suggest that AIbZIP may be a are recognized to play an important role in prostate tumor growth delayed response gene. Immunoreactive AIbZIP protein was primarily (8). Since the original discovery of Charles Huggins in 1941, detected in the cytoplasm of prostatic luminal epithelial cells. Similarly, blockade of androgen formation and action has evolved as the full-length AIbZIP-green fluorescent protein fusion proteins were local- cornerstone of the treatment of prostate cancer (9). Genetic alter- ized in the cytoplasm of LNCaP cells, whereas a truncated form of AIbZIP ations of AR consisting of AR gene amplification (10) and muta- lacking the putative transmembrane domain was exclusively nuclear. tions in the AR coding sequence (11) are frequently observed in Examination of AIbZIP protein and mRNA expression in a series of prostate cancer and are believed to contribute in some manner to transurethral resection of the prostate and needle biopsy specimens indi- cancer progression. In fact, AR mutations are found in several cated that AIbZIP is expressed at higher levels in cancerous prostate cells compared with noncancerous prostate cells. The highly tissue-specific commonly studied human prostate cancer cell lines including expression profile, androgen regulation, chromosomal localization, and LNCaP, CWR22, MDA PCa 2a, and MDA PCa 2b (12–15). Given expression profile of AIbZIP in prostate tumors suggest that AIbZIP may the importance of androgens and the AR in prostate development play an important role in prostate cancer and in androgen receptor and cancer, it is important to identify genes that are regulated by signaling in prostate cells. Future studies will confirm a possible relation- androgens in the human prostate. ship between AIbZIP and prostate cancer. Transcription factors of which the expression is regulated by androgen in prostate cells are a particularly interesting class of INTRODUCTION androgen-responsive gene because they control the expression of a specific subset of androgen-responsive genes. Relatively few an- The AR3 belongs to the superfamily of nuclear steroid hormone drogen-responsive transcription factors have been identified to receptors, a family of transcription factors that modulate hormone- date. One of these, NKX3.1, is a homeobox gene located on regulated gene expression (1). The AR gene is localized on the X chromosome 8p21 that is believed to play an important role in chromosome (2). The AR gene encodes two functionally equivalent prostate differentiation and the control of prostate cell proliferation proteins that are translated from different initiation codons (3). (16, 17). Disruption of NKX3.1 in mice results in dysplasia of the Like other nuclear receptors, AR contains distinct functional do- prostatic epithelium (18), and loss of NKX3.1 expression is ob- mains consisting of an NH -terminal domain, a central DNA- 2 served in a high proportion of hormone-refractory and metastatic binding domain, and a COOH-terminal ligand-binding domain. prostate tumors (19). Androgens also down-regulate the expression of the zinc finger transcription factor early growth response-␣ (20) Received 7/9/01; accepted 12/3/01. The costs of publication of this article were defrayed in part by the payment of page and induce the orphan nuclear receptor TR3 (21), c-myc, and nm23 charges. This article must therefore be hereby marked advertisement in accordance with (22) in LNCaP cells. The orphan nuclear receptor TR3 is particu- 18 U.S.C. Section 1734 solely to indicate this fact. larly interesting, because TR3 translocates from the nucleus to 1 Supported by a fellowship from Le Fonds de Recherche en Sante´ du Que´bec (to C. L.). Financial support for this work was provided by Endorecherche. mitochondria in response to proapoptotic signals and induces cy- 2 To whom requests for reprints should be addressed, at Molecular Endocrinology and tochrome c release and apoptosis (23). Down-regulation of EGR-␣ Oncology Research Center, Centre Hospitalier Universite´Laval Research Center, Quebec G1V 4G2, Canada. Phone: (418) 654-2296; Fax: (418) 654-2761; E-mail: Claude. by androgens in LNCaP cells is consistent with the growth-stim- [email protected]. ulatory effect of androgens in prostate cells, because the EGR-␣ 3 The abbreviations used are: AR, androgen receptor; AIbZIP, Androgen-Induced protein is nearly identical to transforming growth factor-␤-induc- bZIP; CREB, cAMP-responsive element binding protein; ATF, activating transcription factor; TURP, transurethral resection of the prostate; PSA, prostate-specific antigen; ible early gene that has been shown to inhibit cell proliferation FISH, fluorescence in situ hybridization; BAC, bacterial artificial chromosome; GAPDH, (24). In fact, EGR-␣ and transforming growth factor-␤-inducible glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslated region; EGFP, enhanced green fluorescent protein; aa, amino acid; JTB, jumping translocation breakpoint; PAR, early gene are expressed from alternative promoters of the same prostate androgen-regulated. gene through the use of alternative first exons (25). 721

More recently, Xu et al. (26) identified a number of putative were cultured for 3 days in hormone-depleted 2% FCS before each experiment. androgen-responsive genes using serial analysis of gene expression to The medium was changed at 2–3 day intervals. At the start of each experiment, compare the expression profiles of control and R1881-treated LNCaP the medium was replaced with identical medium containing the indicated cells. Seven of these genes were transcription factors that were not concentrations of compounds. Each experiment was performed at least three known previously to be androgen-regulated, including KRAB-associ- times, and all of the experiments were performed in LNCaP cells between passage 27 and 37. ated protein 1, prostate-derived Ets factor, mitochondrial transcription The synthetic androgen R1881 was purchased from NEN Life Science factor 1, a sox-like transcription factor, and the orphan nuclear recep- Products Inc. (Boston, MA). The antiandrogen hydroxyflutamide was kindly tor ear-2, all of which were up-regulated by androgens. Two tran- provided by Schering-Plough Research Institute (Kenilworth, NJ). Actinomy- scription factors, the homeobox protein Hox B13 and the histone cin D and cycloheximide were from ICN Biomedicals, Inc. (Aurora, OH). acetylase/deacetylase CHD4/Mi-2␤, were down-regulated by R1881. Northern Blotting. The human multiple tissue blots (2 ␮g poly(A)ϩ RNA/ Whereas the androgen regulation of these genes remains to be con- lane) were obtained from Clontech Laboratories, whereas total RNA was firmed, it is of interest to note that the prostate-specific transcription isolated from cell lines using Tri-Reagent (Molecular Research Center, Inc., factor PDEF interacts with AR and enhances androgen-induced acti- Cincinnati, OH). Electrophoresis of cancer cell line RNA samples, Northern vation of the PSA promoter (27). blotting, hybridization, and autoradiography were performed as described (28). Our laboratory is interested in identifying androgen-regulated genes The human tissue and cell line blots were probed with the A25 cDNA probe in human prostate cancer cells. We hypothesize that these genes may that was used to screen the LNCaP cDNA library. The human tissue blots were also probed with the full-length AIbZIP cDNA. Radiolabeled cDNA probes for play an important role in androgen-induced prostate development and human ␤-actin (Clontech Laboratories) and human GAPDH were used as androgen-dependent prostatic diseases such as prostate cancer. We loading controls for the human multiple tissue blots and cell line blots, report here the cDNA cloning, gene structure, expression profile, respectively. A portion of the GAPDH cDNA (nucleotides 172–717 of Gen- androgen regulation, and partial characterization of a novel androgen- Bank accession no. M33197) was amplified by PCR using oligonucleotides regulated transcription factor that we have named AIbZIP. AIbZIP is 5Ј-ATTGACCTCAACTACATGGT-3Ј and 5Ј-CTTGCCCACAGCCTTG- a novel member of the CREB/ATF family of transcription factors that GCAG-3Ј and radiolabeled with [␣-32P]dCTP. is highly expressed in prostate tumors and of which the expression is RNase Protection Assays. To confirm that clone A25 corresponded to an up-regulated by androgen in LNCaP cells. androgen-responsive transcript, the plasmid containing the A25 cDNA fragment (pCRII-SSH2-A25/AS) was used to generate a cRNA probe. The plasmid was linearized with EcoRV, and a 434-bp probe (including 135 bp of vector/ MATERIALS AND METHODS linker sequences) was synthesized using Sp6 RNA polymerase. To confirm that AIbZIP corresponded to an androgen-responsive gene, we Cloning of AIbZIP. A 299-bp cDNA fragment corresponding to nucleo- constructed a plasmid (pBSKS-A25-3ЈUTR/AS) to generate a probe that tides 314–612 of the full-length AIbZIP cDNA was isolated from androgen- would hybridize to a region of the AIbZIP transcript that is not recognized by treated LNCaP human prostate cancer cells using the CLONTECH PCR-Select the A25 probe. A cDNA fragment containing the last 64 bp of the open reading cDNA Subtraction kit (Clontech Laboratories, Palo Alto, CA) as described frame and the first 173 bp of the 3ЈUTR of AIbZIP (nucleotides 1203–1439) previously (28). This fragment, designated A25, was excised as a 354-bp insert was cloned into pBluescript II KS (ϩ/Ϫ) by PCR using oligonucleotides from the pCRII plasmid by EcoRI digestion, radiolabeled with [␣-32P]dCTP 5Ј-GATGGGAGGGAAGCCAAGA-3Ј and 5Ј-CAAATGCAGACTAGGC- using the DECAprimer II DNA labeling kit (Ambion, Austin, TX), and used CTC-3Ј. The plasmid was linearized with BamHI, and the 318 bp probe to screen 4 ϫ 105 recombinant plaques of a LNCaP cDNA library. After three (including 81 bp of vector sequences), designated 3ЈUTR, was produced using rounds of screening, positive clones were excised in vivo directly into the T3 RNA polymerase. The 3ЈUTR probe was used for most of the experiments phagemid pBK-CMV and sequenced using the T7 sequencing kit (USB Cor- presented in this report. poration, Cleveland, OH) and Deaza G/A T7 sequencing mixes (Amersham A probe designated as E4-7 was designed for the purpose of distinguishing Pharmacia Biotech, Piscataway, NJ) as necessary. the full-length AIbZIP transcript from putative alternative transcripts that were FISH. To confirm that the BAC clone RP11-422P24 (GenBank accession isolated as cDNA clones. To generate the required probe, a cDNA fragment no. AL358472) contained the AIbZIP gene, we amplified two fragments by encompassing nucleotides 610–890 of the full-length AIbZIP cDNA was PCR using sense primers 5Ј-GATGGGAGGGAAGCCAAGA-3Ј and 5Ј-CT- cloned into pBluescript II KS (ϩ/Ϫ) by PCR. The 362 bp E4-7 probe GTTCTGCACAGAACCA-3Ј in combination with the reverse primer 5Ј- (including 81 bp of vector sequences) was produced using T3 RNA polymer- CAAATGCAGACTAGGCCTC-3Ј. The BAC clone was then biotinylated by ase after linearizing the plasmid (pBSKS-A25-E4-7/AS) with BamHI. nick translation and hybridized in situ to metaphase chromosomes from normal A cRNA probe for ␤-actin was used as an internal control in all of the human lymphocytes (29). Chromosomes were counterstained with propidium RNase protection experiments. A fragment of the ␤-actin cDNA (nucleotides iodide and 4Ј,6-diamidino-2-phenylindole. After hybridization, the biotiny- 704–947 of GenBank accession no. X00351) was amplified by PCR and lated probe was detected with avidin-FITC. Images of metaphase preparations cloned into the XbaI and KpnI sites of pBluescript SK. The plasmid was were captured digitally using a cooled CCD camera (Biomedical Photometrics, linearized with DdeI, and a 145-bp cRNA probe that protects a 130-bp Inc., Waterloo, ON). FISH was performed in Dr. S. Scherer’s laboratory in the fragment of the human ␤-actin mRNA (nucleotides 818–947 of the cDNA) Department of Genetics at The Hospital for Sick Children, Toronto, Ontario, was synthesized using T7 RNA polymerase. Canada. The cRNA probes were labeled with [␣-32P]UTP (800 Ci/mmol) using Cell Lines and Culture Conditions. All of the cell lines used in these the Riboprobe in vitro Transcription System (Promega Corp., Madison, experiments were obtained from American Type Culture Collection (Manas- WI). RNase protection assays were performed using 10 ␮g of total RNA sas, VA). The human breast and prostate cancer cell lines and corresponding using the RPA III kit (Ambion, Inc.) according to the manufacturer’s culture conditions were described previously (28). Human kidney 293 cells instructions. Protected fragments were separated by electrophoresis in 6% were cultured in MEM containing 10% FCS, 1% nonessential amino acids, 100 acrylamide 7 M urea gels. The gels were exposed to Hyperfilm MP ␮g/ml streptomycin, and 100 IU/ml penicillin. (Amersham Pharmacia Biotech, Aylesbury, United Kingdom) and X-ray Experiments designed to characterize the regulation of AIbZIP expression films were quantitated by scanning densitometry using the Bioimage 110 S by androgens in LNCaP cells were performed as described (28). In brief, the (Millipore Corp., Bedford, MA). AIbZIP mRNA levels were corrected for LNCaP cells used in the time course, R1881 dose-response and cycloheximide/ ␤-actin mRNA levels, and the AIbZIP:␤-actin ratios in the control cells of actinomycin D experiments (Fig. 7) were cultured in RPMI 1640 supplemented each experiment were assigned a value of 1. AIbZIP mRNA levels in with hormone-depleted 0.25% FCS for 6 days before each experiment. Under treated cells are expressed as AIbZIP:␤-actin ratios relative to those in the these conditions, LNCaP cells remain in G1 for 24 to 48 h after addition of 0.1 corresponding control cells. nM of R1881 (data not shown). The LNCaP cells used in the experiments Expression Vectors. To express AIbZIP as a fusion to the NH2 terminus of designed to assess the effects of antiandrogens on AIbZIP expression (Fig. 8) EGFP, the coding sequence of AIbZIP was amplified from the full-length 722

cDNA by PCR and cloned into the BglII and KpnI sites of pEGFP-N3 revealed using the Supersignal Ultra Chemiluminescent detection system (Clontech Laboratories). The fusion protein encoded by plasmid pEGFPN3- (Pierce, Rockford, IL). AIbZIP contains aa 1–395 of AIbZIP, 10 amino acids (GATGPGSIAT) en- AIbZIP Immunoblots. To assess AIbZIP protein levels in R1881-treated coded by vector polylinker DNA, and EGFP. Plasmid AIbZIP (1–290)-EGFP LNCaP cells, equal amounts of protein extracts (30 ␮g/lane) were electro- was made by amplifying the first 290 codons of AIbZIP by PCR and subclon- phoresed on 12% SDS-PAGE gels and transferred to nitrocellulose filters. The ing the DNA fragment in-frame with the EGFP open reading frame in plasmid blots were incubated overnight at 4°C with rabbit polyclonal antibody against pEGFP-N3. AIbZIP (diluted 1:1000 in 5% milk and 1 ϫ Tris-buffered saline; 0.9% NaCl To express AIbZIP as a fusion to the COOH-terminus of the DNA-binding and Tris-HCl 10 mM) followed by a 3-h incubation at 37°C with peroxidase- domain of GAL4, the coding sequence of AIbZIP was amplified from the conjugated AffiniPure goat antimouse IgG (Jackson ImmunoResearch Labo- full-length cDNA by PCR and cloned into the EcoRI and XbaI sites of pM ratories Inc.). AIbZIP protein levels were quantified using scanning densitom- (Clontech Laboratories). The fusion protein encoded by pM-AIbZIP contains etry. AIbZIP protein levels in R1881-treated cells are expressed relative to the aa 1–147 of GAL4, three amino acids (PEF) encoded by vector polylinker levels determined in control LNCaP cells, which were assigned a value of 1. DNA, aa 1–395 of AIbZIP, and two residues (SR) that are encoded by the XbaI The AIbZIP levels (relative to control) achieved with 0.01, 0.1, 1, and 10 nM site that precedes the termination codon. Plasmids expressing GAL4 fused to R1881 in two independent experiments were within 4% of the calculated various deletion mutants of AIbZIP were constructed using conveniently average values. placed restriction sites or PCR. The sequence of each AIbZIP expression Prostate Specimens. The prostate specimens used in our studies were plasmid used in our experiments was verified. kindly provided by Dr. Jean Emond (Hoˆtel-Dieu de Le´vis, Le´vis, Que´bec, AIbZIP Localization Experiments. For transient expression of EGFP and Canada), Dr. Bernard Teˆtu (Hoˆtel-Dieu de Que´bec, Que´bec City, Que´bec, EGFP fusion proteins, 1 ϫ 105 LNCaP cells were seeded in four-well chamber Canada), and by Drs. Martin Lemay and Michel Tremblay (Centre Hospi- slides (9 ϫ 18 mm; Nalge Nunc International, Naperville, IL) in RPMI 1640. talier de l’Universite´ Laval, Que´bec City, Que´bec, Canada). Thirty-seven The cells were transfected with pEGFP-N3, pEGFPN3-AIbZIP, or pEGFPN3- different prostate specimens were used to evaluate AIbZIP expression by AIbZIP (1–290) (1 ␮g DNA/well) 24 h after plating using Lipofectin (Life immunohistochemistry and/or in situ hybridization. These tissues consisted Technologies, Inc., Rockville, MD). After transfection (24 h), the cells were of 20 specimens from men undergoing TURP for symptomatic benign fixed with Formalin solution (Sigma Chemical Co. Diagnostics, St. Louis, prostatic hyperplasia and 17 paraffin blocks of formalin-fixed archival MO). Crystal/Mount (Biomeda Corp., Foster City, CA) was used to mount the prostatic needle core biopsies displaying infiltrating adenocarcinoma. The coverslips to the slides. Fluorescent proteins were detected by fluorescence TURP specimens were fixed by immersion in 2% glutaraldehyde, 4% microscopy, and photographs were taken using a digital camera (SPOP RT formaldehyde, and 3% dextran in 0.05 M phosphate buffer (pH 7.4) for in Slider; Diagnostic Instruments, Inc., Sterling Heights, MI) mounted directly on situ hybridization or in 10% neutral buffered formalin for immunohisto- a BX-60 microscope (Olympus, Melville, NY). The stability of AIbZIP-EGFP chemistry. They were then processed and embedded in paraffin blocks. fusion proteins was verified in human kidney 293 cells. The cells were cultured BT-474 and MDA-MB-231 breast cancer cells were used as controls for the in MEM, and the corresponding expression plasmids were transfected using immunohistochemical detection of AIbZIP. The cells were harvested, fixed ExGen 500 (MBI Fermentas, Amherst, NY). The cells were harvested 24 h in 10% neutral buffered formalin, centrifuged in 2% agarose, postfixed in after transfection. 10% neutral buffered formalin, and embedded in paraffin. GAL4-AIbZIP Experiments. Human kidney 293 cells were seeded in In Situ Hybridization. Paraffin sections (4 ␮m) were collected on MEM containing 10% FCS at 1.5 ϫ 105 cells/well in 12-well plates (21 Superfrost Plus (Fisher) glass slides. The sections were deparaffinized in mm/well; Becton Dickinson Labware, Bedford, MA) 24 h before transfec- toluene, rehydrated through graded alcohol, and then treated with protein- tion. Each well was transfected with 1.5 ␮g of plasmid DNA comprised of aseK[1␮g/ml in 100 mM Tris-HCl, 50 mM EDTA, (pH 8.0)] for 30 min 1.125 ␮g pM plasmid expressing either the GAL4 DNA-binding domain at 37°C. Sections were then immersed in prehybridization buffer [50% alone, GAL4 fused to AIbZIP, or GAL4 fused to VP16 (pM3-VP16), and formamide, 0.8 M NaCl, 120 mM Tris base (pH 8.0), 8 mM EDTA, 200 0.25 ␮g of a GAL4-responsive luciferase reporter plasmid (pGL3- ␮g/ml salmon sperm DNA, 200 ␮g/ml tRNA, and 4% dextran sulfate] for G5E1BTATA). pRLnull (0.125 ␮g; Promega) was used as an internal 2 h at room temperature. The [35S]UTP-labeled 318-bp antisense AIbZIP control for transfection efficiency. Transfections were performed using riboprobe was generated by in vitro transcription using T3 RNA polymer- Exgen 500 (MBI Fermentas). After 48 h, the cells were lysed in Passive ase from pBSKS-A25–3ЈUTR/AS linearized with BamHI. The [35S]UTP- Lysis Buffer (Promega), and Firefly and Renilla luciferase activities were labeled 303-bp sense (control) riboprobe was generated by in vitro tran- determined using a Dual-Luciferase Reporter Assay System (Promega) scription using T7 RNA polymerase from pBSKS-A25–3ЈUTR/AS according to the manufacturer’s instructions. Firefly luciferase activity linearized with EcoRI. Sections were incubated overnight at 53°Cin values were normalized using Renilla luciferase activity values. The sta- hybridization buffer (prehybridization buffer plus 10 mM DTT and 2 ϫ 106 bility of GAL4-AIbZIP fusion proteins was verified in T-47D cells. The cpm probe). To remove the excess probe, the sections were washed twice cells were transfected with the corresponding plasmids using FuGENE in 50% formamide, 2 ϫ SSC at 58°C for 90 min. The slides were (Roche, Indianapolis, IN) and harvested 48 h after transfection. dehydrated in graded alcohol, air-dried, and then dipped in NBT-2 emul- AIbZIP Antibody Preparation. A synthetic peptide corresponding to aa sion (Eastman Kodak, Rochester, NY; diluted 1:1 in distilled water) and 379–392 of AIbZIP (KPRPSGRIRSVLHA) was synthesized on an ABI 433A exposed for 14 days. Slides were developed and counterstained with H&E. peptide synthesizer using FastMoc chemistry. The peptide was purified on a The silver grains resulting from hybridization of sense and antisense cRNA Vydak 22 ϫ 250 mm C18 reverse-phase high-performance liquid chromatog- probes were counted manually by overlaying photomicrographs with ace- raphy column using a 0.1% trifluoroacetyl/acetonitrile gradient. Rabbits were tate sheets. immunized with a total of 0.5 mg of peptide per rabbit. Immunohistochemistry. Immunostaining was performed using Zymed SP Immunoblotting. For immunoblotting, cells were harvested in lysis buffer kits (San Francisco, CA). Paraffin sections (4 ␮m) were deparaffinized in [6 M urea, 20 mM Tris (pH 6.8), 1% SDS, Roche Complete Protease Inhibitor toluene and rehydrated through ethanol. Endogenous peroxidase activity was ␮ mixture, pepstatin, and DTT] and then sonicated. Protein extracts (30 or 40 g) eliminated by preincubation with 3% H2O2 in methanol for 20 min. A micro- were separated by SDS-PAGE on 12% or 13.5% acrylamide gels and trans- wave retrieval technique using citrate buffer was applied (30). After cooling ferred to nitrocellulose. EGFP fusion proteins, GAL4 fusion proteins, and the slides, nonspecific binding sites were blocked using 10% goat serum for 30 ␣-tubulin were detected using the following commercially available primary min. Sections were incubated with the rabbit polyclonal AIbZIP antibody antibodies: Living Colors full-length A.v. polyclonal antibody (Clontech Lab- diluted 1:400 for2hatroom temperature, washed in PBS buffer, and oratories), mouse monoclonal antibody RK5C1 raised against the DNA-bind- incubated with biotinylated antirabbit secondary antibody for 12 min and then ing domain of GAL4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and with streptavidin-peroxidase for another 12 min. Under microscope monitor- mouse IgM antitubulin antibody (Santa Cruz Biotechnology). The following ing, diaminobenzidine was used as the chromogen to visualize the biotin/ peroxidase-conjugated AffiniPure secondary antibodies were obtained from streptavidin-peroxidase complexes. Counterstaining was performed using #2 Jackson ImmunoResearch Laboratories Inc. (West Grove, PA): goat antirabbit Gill’s hematoxylin for 45 s. For the negative control, the preimmune serum IgG, goat antimouse IgM, and goat antimouse IgG. Immune complexes were was used at the same dilution as the primary antibody. 723

Fig. 1. Nucleotide and deduced amino acid sequence of human AIbZIP. The longest cDNA clone obtained in LNCaP cells is 1564-bp long and contains an open reading frame of 395 amino acids. The numbers on the left indicate the nucleotide number, whereas amino acids are numbered on the right. A stop codon upstream of and in-frame with the first putative methionine is double underlined. The leucine and cysteine residues that constitute the leucine zipper are shown as white and the ,ء letters in black boxes. The termination codon is indicated by polyadenylation signal is underlined. The cDNA fragment isolated by cDNA subtraction (clone A25) contained nucleotides 314–612. The cRNA probe used for RNase protection assays contained nucleotides 1203–1439.

Nucleotide Sequence Accession No. The GenBank accession no. of the signal (AAUAAA) ϳ20 bp from its 3Ј extremity. Searches of the AIbZIP clone sequence reported here is AF394167. expressed sequence tag database at National Center for Biotechnology Information revealed several matches to AIbZIP, and a matching RESULTS contig could be assembled from three partially overlapping ESTs (AL519763, BG699998, and AW270016). Cloning of AIbZIP. We used PCR-select cDNA subtraction to Functional Domains of AIbZIP. The putative initiating methio- identify new androgen-regulated genes in LNCaP human prostate nine of cDNA A25-15 is located in a purine-rich region with simi- cancer cells that were exposed to the synthetic androgen R1881 for larity to a Kozak consensus sequence (5Ј-AAGCATGG-3Ј) that would 24 h. The cDNA fragments that were isolated were sequenced to be predicted to favor translation initiation, whereas the second methi- determine their identity to known genes, and novel cDNAs were onine lies in a less favorable context 110 codons further downstream then used as probes in RNase protection assays to confirm that the (31). Assuming that translation initiates at the first ATG codon, the corresponding mRNAs were truly androgen-regulated. One of the A25-15 open reading frame encodes a 395 amino acid protein. A validated androgen-responsive transcripts identified in LNCaP 4 cells was detected using a probe derived from clone A25, a 299-bp search of the Conserved Domain Database using RPS-BLAST with cDNA that did not match any known human gene in the GenBank the amino acid sequence of cDNA A25-15 revealed that it contained database. a region with extensive similarity to the bZIP domain of CREB/ATF To obtain a full-length A25 cDNA we screened a LNCaP cDNA transcription factors (Fig. 2A). For this reason, the protein encoded by library using radiolabeled clone A25 as the probe. Twenty-five cDNA cDNA A25-15 was designated AIbZIP. clones were isolated and characterized. The longest cDNA, number The bZIP domain of AIbZIP is located between residues 219 and A25-15, was 1564 bp in length and contained a 1188-bp open reading 280 (Fig. 2B). It is comprised of a lysine- and arginine-rich basic frame preceded by a 78-bp 5Ј-UTR (Fig. 1). A termination codon region separated from the leucine zipper by a 5-amino acid spacer. A (TGA) is located 30 bp upstream of and in-frame with the first bipartite nuclear localization signal (KKVRRKIRNKQSAQDSR- methionine codon of cDNA A25-15, thereby confirming that this RRKKE) is contained within the bZIP domain (aa 223–240). The cDNA includes the complete protein coding sequence. The 3Ј-UTR of cDNA A25-15 is 298 bp in length and contains a polyadenylation 4 Internet address: http://www.ncbi.nlm.nih.gov/. 724

Fig. 2. Features of AIbZIP. A, schematic representation of AIbZIP protein. The relative positions of the basic region, putative transmembrane domains (TMD), and leucine (L) and cysteine (C) residues of the leucine zipper are indicated. B, alignment of the bZIP domain of AIbZIP with those of human CREB-H (AB050902), human CREB3 (AF211848), mouse OASIS (AB017614), Drosophila melanogaster BBF-2 (A42140), human ATF-6 (XM_018271), human ATF-4 (NM_001675), and human ATF-3 (L19871). The basic and leucine zipper domains are indicated. Invariant residues are shown in white on a black background. C, dendogram of representative CREB/ATF proteins, CREB-H, CREB3, and AIbZIP. The dendogram was derived from the complete open reading frames of AIbZIP, CREB-H, CREB3, ATF3, B-ATF (U15460), CRE-BP1 (NM_001880), CREB (M27691), ATF4, and ATF6 using the GCG com- puter program PILEUP.

bZIP domain of AIbZIP is most similar to that of human CREB-H shows little homology to either the COOH-terminal TMD of (69% amino acid identity), human CREB3 (62% amino acid identity), AIbZIP or that of CREB-H. and mouse OASIS (54% identity) proteins (32–34). In fact, 17 of 21 Gene Structure and Chromosomal Location of AIbZIP. To amino acids that constitute the basic domain as well as the 5 spacer determine the genomic organization of the AIbZIP gene we searched amino acids present in AIbZIP are identical to those in CREB-H. The the GenBank genomic sequence database and found that the complete leucine zipper of AIbZIP is similar to that of CREB-H. It contains five sequence of the AIbZIP cDNA is contained in human chromosome 1 repeats and consists of five leucine residues and one cysteine residue clone RP11-422P24 (GenBank accession no. AL358472). Compari- at the second heptad position (aa 258) of the leucine zipper. CREB3 son of the genomic and cDNA sequences allowed us to determine the and OASIS contain tyrosine substitutions at the same position. Twen- exon-intron junctions of the AIbZIP gene (Table 1). The AIbZIP gene ty-two of the 36 amino acids present between the first and last consists of 10 exons ranging from 74 to 493 bp in size. The first exon leucines are identical in AIbZIP and CREB-H. The bZIP domain of codes for the 5Ј UTR of the mRNA, whereas the AIbZIP open reading AIbZIP shows less homology (26–35% identity) to the bZIP domains frame is encoded by exons 2–10. As shown in Fig. 3A, the AIbZIP of human ATF3, ATF4, and ATF6 proteins. gene lies in close proximity to the JTB/PAR gene (36, 37). The two On the basis of the similarities between the bZIP domains of genes are arranged in a head-to-head orientation, and the last 94 bp of AIbZIP, CREB-H, and CREB3, we have assigned AIbZIP to the AIbZIP exon 10 correspond to the last 94 bp of JTB/PAR exon 5. We CREB/ATF subfamily of bZIP proteins. In a recent review, Hai used FISH to confirm the chromosomal location of the gene encoding and Hartman (35) classified mammalian ATF/CREB proteins into AIbZIP. Hybridization of biotinylated BAC clone RP11-422P24 to six subgroups that are represented by CREB, CRE-BP1, ATF3, metaphase chromosomes confirmed that this BAC maps to 1q21.3 ATF4, ATF6, and B-ATF. Proteins between subgroups have little (data not shown). in common except for the bZIP domain, whereas the structural similarity between proteins within subgroups extends to regions a outside the bZIP domain. A dendogram analysis performed using Table 1 Exon-intron structure of AIbZIP gene the amino acid sequences of AIbZIP, CREB-H, CREB3, and those cDNA Exon positionb Size (bp) 3Ј splice junction 5Ј splice junction of representative ATF/CREB proteins revealed that AIbZIP, 11–74 74 . . . AGAAAGA AGAACAG/gtaatgc CREB-H, and CREB3 segregate into a distinct subgroup of ATF/ 275–252 178 cctgcag/AAGCATG TGGCTGT/gtgagtg CREB proteins (Fig. 2C). 3 253–499 247 actgtag/GGCCTTC CAGCTAG/gtcagtg Analysis of the protein sequence of AIbZIP (PSORTII server)5 4 500–621 122 actccag/ATCAGTG AACCCTG/gtgagtc 5 622–714 93 cccccag/CTGCCCT CACCAAG/gtaacat also revealed the presence of two putative transmembrane domains 6 715–821 107 ctccaag/GCAGAGG AGAGCAG/gtacgcc (aa 169–191 and 294–314) flanking the bZIP domain. The COOH- 7 822–890 69 attctag/GGTGGCA ACAACAT/gtgagtg terminal TMD of AIbZIP (TSTCVLILLFSLALIILPSFS) bears 8 891–975 85 ttcccag/CTCCTTC TGTTTTG/gtaccat 9 976–1072 97 ctcccag/ATTCTTC CACGGAG/gtgagag significant homology to the TMD of CREB-H (CVAVLLLSFA- 10 1073–1564 492 ctcacag/TGACTTC AAATTGT/poly A LIILPSI, aa 323–339). The putative TMD located in the NH2- a The AIbZIP gene is located in BAC clone no. RP11-422P24 (accession no. terminal half of AIbZIP (RAGTVAPVPCTTLLPCQTLFLTD) AL358472), which consists of four unordered pieces of genomic sequences of 1q21.3. The exon and intron junction sequences are shown as capital and small letters, respectively. b Nucleotide position numbers are indicated in Fig. 1 (GenBank accession no. 5 Internet address: http://psort.ims.u-tokyo.ac.jp. AF394167). 725

a plasmid that expresses the fusion protein AIbZIP (1–290)-EGFP, which lacks the last 105 amino acids of AIbZIP (Fig. 4A). Plasmids expressing EGFP alone, AIbZIP-EGFP, or AIbZIP (1–290)-EGFP were transiently transfected into LNCaP cells. EGFP alone was distributed evenly in the nuclei and cytoplasm of cells, whereas AIbZIP-EGFP was predominantly localized in the cytoplasm in structures that could correspond to the Golgi apparatus (Fig. 4B). Interestingly, deletion of the COOH-terminal portion of AIbZIP that includes the putative transmembrane domain produced a fusion protein that localized exclusively in the nuclei of transfected cells. These results indicate that a domain responsible for the cytoplasmic localization of AIbZIP resides within its COOH-

terminal region. The putative NH2-terminal transmembrane domain does not appear to play an essential role in the cytoplasmic localization of AIbZIP. Immunoblots performed using an antibody against EGFP confirmed that the expression plasmids produced fusion proteins of the expected sizes (Fig. 4C). Transcriptional Activity of AIbZIP. To determine whether AIbZIP had transcriptional activity we constructed a plasmid expressing full-length AIbZIP as a fusion protein to the COOH Fig. 3. Gene structure of AIbZIP. A, schematic representation of the AIbZIP and JTB/PAR genes on chromosome 1q21. The 10 exons of the AIbZIP gene are shown as terminus of the DNA-binding domain of GAL4. The GAL4- Ⅺ, whereas the 5 exons of the JTB/PAR gene are shown as u. The arrows indicate the AIbZIP-expressing plasmid was cotransfected with a luciferase direction in which the genes are transcribed, and the f indicates the shared portion of reporter plasmid containing GAL4 DNA-response elements into a common exon. B, schematic representation of the full-length AIbZIP cDNA and of two other cDNAs isolated in LNCaP cells. The exons are numbered from 1 to 10, and human kidney 293 cells. Preliminary experiments confirmed that the coding region of each cDNA is shown in gray. The form 3 cDNA contains intron GAL4-AIbZIP could indeed activate a GAL4-responsive promoter 5(Int5). in a dose-dependent manner (data not shown). To map the activation domain of AIbZIP we then constructed plasmids expressing

Alternative Forms of AIbZIP. ATF/CREB genes frequently pro- GAL4-AIbZIP fusion proteins containing various NH2- or COOH- duce alternatively spliced transcripts that encode functional pro- terminal deletions within AIbZIP and tested their ability to activate teins (38). The full-length AIbZIP cDNA represents the most the GAL4-responsive promoter (Fig. 5A). Deletion of the first 48 common form of AIbZIP transcript in LNCaP cells. Nineteen other or 98 amino acids of AIbZIP completely abrogated GAL4-AIbZIP- cDNAs we characterized were identical in sequence to cDNA induced transactivation of the GAL4-responsive luciferase re- A25-15 except that their 5Ј UTRs varied in length from 32 to 78 bp porter. These results suggested that the activation domain of ϭ (median 44 bp). Two sequence variants of cDNA A25-15 were AIbZIP resides within its NH2-terminal portion. In fact, experi- represented in five other cDNA clones. One variant, form 2 (found ments performed using fusion proteins that express the first 71 or in two cDNA clones), had a deletion of nucleotides 764-1043, 100 amino acids of AIbZIP confirmed that the NH2-terminal region which corresponds to a complete deletion of exons 7 and 8, as well of AIbZIP is sufficient for activation. It is interesting to note that as partial deletions of exons 6 and 9 (Fig. 3B). The second variant, these fusion proteins were 30 times more active than the full-length designated form 3 (found in three cDNA clones), contained intron GAL4-AIbZIP fusion protein. Moreover, fusion proteins contain- 5 sequences. Both variants result in frameshifts that would produce ing the first 150–280 amino acids of AIbZIP were less active than truncated proteins lacking all (form 3) or most (form 2) of the bZIP the 71- and 100-residue fusion proteins. Immunoblots performed domain. To determine whether these transcripts were expressed at using a GAL4-specific antibody confirmed that the proteins that appreciable levels in LNCaP cells we designed a cRNA probe displayed less transcriptional activity were not expressed at lower (E4–7) that would discriminate between the wild-type AIbZIP levels (Fig. 5B). Therefore, we conclude that the transcriptional transcript and the two variants. Neither variant was detected in activity of the AIbZIP activation domain is negatively regulated by LNCaP cells (data not shown), and these two clones were not the COOH-terminal portion of AIbZIP. additionally studied. Tissue Expression Profile of AIbZIP. We performed Northern In addition to these cDNAs, some ESTs contained in the GenBank blots to confirm the size of the AIbZIP mRNA and to determine database (BI087317, BG763732, BF347981, and AL528505) are whether other AIbZIP-related transcripts are expressed in LNCaP identical to AIbZIP exon 2 except that they contain an additional cells. Total RNA was isolated from LNCaP cells after exposure to 0.1 241–277 bp DNA at their 5Ј end, which suggests that alternative nM of R1881 for 24 h, and AIbZIP mRNA was detected using transcripts could be expressed from the AIbZIP gene. On the basis of radiolabeled A25 cDNA as the probe. As shown in Fig. 6A, the the available genomic data, this alternative exon is predicted to lie 72 AIbZIP probe detected a major 1.7-kb band in control LNCaP cells. bp 5Ј to exon 1. We did not isolate a matching cDNA in LNCaP cells, The size of this transcript is in agreement with the length of the but this alternative transcript would not be predicted to alter the open longest AIbZIP cDNA we isolated (1564 bp). A less abundant tran- reading frame of AIbZIP unless other exons were also alternatively script that migrated slightly above the major band and which could spliced. correspond to an alternatively spliced AIbZIP transcript was also AIbZIP Localization. To assess the subcellular localization of detected. Exposure of LNCaP cells to R1881 caused a marked in- AIbZIP, the complete open reading frame of AIbZIP was cloned crease in the amount of 1.7-kb mRNA as well as in that of the less in-frame with sequences encoding EGFP to produce a plasmid abundant longer transcript. expressing AIbZIP as a fusion protein to the NH2 terminus of To determine the tissue expression profile of AIbZIP we performed EGFP (AIbZIP-EGFP). To evaluate the potential role of the Northern blot analysis using polyadenylated RNA purified from 16 COOH-terminal transmembrane domain of AIbZIP we constructed human tissues. CLONTECH Human and Human IV multiple tissue 726

Fig. 4. Localization of AIbZIP in LNCaP cells. A, schematic representation of AIbZIP-EGFP and AIbZIP (1–290)-EGFP fusion proteins. The puta- N tive NH2-terminal (TMD ) and COOH-terminal (TMDC) transmembrane domains of AIbZIP are depicted as u. B, fluorescence micrographs of LNCaP cells transiently transfected with plasmids expressing EGFP, AIbZIP-EGFP, and AIbZIP (1– 290)-EGFP (ϫ40 magnification). The arrow in the middle panel points to cytoplasmic AIbZIP-EGFP (arrowhead indicates the Golgi apparatus) whereas the arrows in the right panel point to nuclear AIbZIP (1–290)-EGFP. C, immunoblot of whole- cell extracts from human kidney 293 cells (Lane 1) and 293 cells transiently transfected with plasmids expressing EGFP (Lane 2), AIbZIP-EGFP (Lane 3), and AIbZIP (1–290)-EGFP (Lane 4). EGFP and AIbZIP-EGFP fusion proteins were detected using a polyclonal antibody against EGFP. The positions of molecular size markers (in kDa) are shown on the left. The black arrowheads on the right indicate the positions of the respective fusion proteins and EGFP. A Mr 48,000 fusion protein was detected in cells that were transfected with the plasmid expressing AIbZIP (1–290)-EGFP (Lane 4). This protein did not interfere with the evaluation of the subcellular localization of AIbZIP. The levels of ␣-tubulin were determined as a loading control (bottom panel).

blots were hybridized to the AIbZIP A25 cDNA probe. A 1.7-kb band lines in which AIbZIP mRNA was most abundant (data not was detected only in human prostate, whereas a shorter 700-bp band shown). We did not verify whether AIbZIP is expressed in normal was detected in the colon (Fig. 6B). AIbZIP or AIbZIP-related tran- mammary cells. However, based on these data, it is possible that scripts were not detected in any other human tissue even after pro- AIbZIP expression is restricted to hormone-sensitive tissues, i.e., longed exposure (6 days) of the blots to autoradiography film. An the prostate and mammary gland. identical tissue distribution pattern was observed using the full-length Androgen Regulation of AIbZIP Expression in LNCaP Cells. AIbZIP cDNA as the probe (data not shown). Although some ESTs To additionally characterize the regulation of AIbZIP expression containing portions of the AIbZIP cDNA have been isolated in non- by androgens we assessed the effect of R1881 on AIbZIP mRNA prostate cDNA libraries, the absence of detectable full-length AIbZIP levels as a function of time and dose. AIbZIP mRNA levels were transcript in tissues other than the prostate suggested that AIbZIP determined in LNCaP cell cultures between 1 and 72 h after the might be a prostate-specific gene. addition of 0.1 nM of R1881. This dose of R1881 was selected To additionally test this hypothesis we performed RNase pro- because it is the same dose that was used to treat LNCaP cultures tection assays using the 3ЈUTR cRNA probe to determine the that served for the cDNA subtraction. As shown in Fig. 7A, AIbZIP expression profile of AIbZIP mRNA in human breast and prostate mRNA levels (expressed as AIbZIP:␤-actin ratios) did not change cancer cell lines. In addition to LNCaP cells, AIbZIP mRNA was markedly up to 12 h after the addition of 0.1 nM of R1881. detected in the androgen-insensitive prostate cancer cell lines DU However, 2–3-fold changes in AIbZIP mRNA levels were ob- 145 and PC-3 (Fig. 6C). This finding indicates that AIbZIP ex- served starting 24 h after the addition of R1881. Similar results pression is not exclusively regulated by androgens, because DU were observed in two other experiments. 145 and PC-3 cells do not express AR. Interestingly, AIbZIP The induction of AIbZIP expression by R1881 was less rapid mRNA was also detected in all of the human breast cancer cell than that of other androgen-responsive genes such as NKX3.1 (17) lines tested except for MDA-MB-231 cells. As in prostate cancer and PSA (28), which suggests that AIbZIP is not an early response cells, AIbZIP expression in breast cancer cells was not strictly gene. Therefore, we examined the effect of the protein synthesis dependent on AR expression, because some of the cell lines that inhibitor cycloheximide and the RNA synthesis inhibitor actino- express AIbZIP do not express AR. The full-length 1.7-kb AIbZIP mycin D on R1881-induced AIbZIP expression in LNCaP cells. transcript was also detected by Northern blot analysis in those cell The effects of actinomycin D and cycloheximide were compared 727

Fig. 5. Mapping the transcriptional activation domain of AIbZIP. A, full-length AIbZIP and a series of deletion mutants expressed as fusion proteins to the COOH-terminus of the GAL4 DNA-binding domain were tested for their ability to activate a GAL4-responsive luciferase reporter plasmid (pGL3-G5E1BTATA) in transient transfection experiments in human kidney 293 cells. The GAL4-AIbZIP constructs were tested in parallel in three [two for GAL4-AIbZIP(49–395)] sep- arate transfection experiments. Each transfection was performed in triplicate, and luciferase activity was quantified 48 h after transfection and corrected for transfection efficiency. The relative luciferase activity obtained with each construct is expressed as the mean Ϯ SE of three [two for GAL4-AIbZIP(49–395)] experiments. B, expression of GAL4-AIbZIP fusion proteins in transiently transfected T-47D cells. Fusion proteins were detected using an antibody against the DNA-binding domain of GAL4. The letters at the bottom of each lane correspond to the GAL4-AIbZIP constructs shown in A. Fusion proteins D, E, and F are barely visible because of low expression (D and E)or the proximity of a cross-reacting polypeptide (F). Their Tubulin was used as a-␣ .ء positions are indicated by loading control (bottom panel).

with that of Casodex, an AR antagonist. The cell culture conditions and the dose of R1881 used in this experiment were identical to those used for the cDNA subtraction experiment and the time course experiment. In the experiment shown in Fig. 7B, R1881 caused a 2-fold increase in AIbZIP mRNA levels at 24 h. This effect was completely blocked by actinomycin D (1 ␮g/ml), cy- Ϫ6 cloheximide (10 ␮g/ml), and Casodex (3 ϫ 10 M), which suggests that androgens regulate AIbZIP expression indirectly. The dose of R1881 that we used to isolate androgen-responsive transcripts by cDNA subtraction (0.1 nM) reproducibly caused a 2–3-fold increase in AIbZIP mRNA levels in several independent experiments (Figs. 6A, and 7, A and B). To determine whether higher doses of R1881 could produce a correspondingly larger increase in AIbZIP mRNA levels, LNCaP cells were cultured for 24 h in the presence of 0.001–10 nM R1881. As shown in Fig. 7C, 1nM of R1881 and 10 nM of R1881 caused ϳ5- and 6-fold increases in AIbZIP mRNA levels, respectively, compared with a 2.3-fold increase in cells exposed to 0.1 nM of R1881. We performed two additional experiments to examine the effect of R1881 on AIbZIP protein levels in LNCaP cells. The cells were grown under the same culture conditions that were used for the RNA experiments presented in Fig. 7C. Protein extracts were prepared from cells that were exposed to increasing doses of R1881 for 24 h, and AIbZIP protein levels were determined using a rabbit polyclonal antibody raised against a COOH-terminal peptide of AIbZIP (aa 379–392, KPRPSGRIRSVLHA). This polyclonal antibody is specific for AIbZIP, because it recognizes a protein of the expected size (Mr ϳ43,000) in BT-474 cells, which express high levels of AIbZIP Fig. 6. Expression profile of AIbZIP in human tissues and cell lines. A, Northern blot analysis (10 ␮g of total RNA) of AIbZIP mRNA in LNCaP cultures grown in the presence mRNA, whereas no protein was detected in MDA-MB-231 cells that or absence of 0.1 nM R1881 for 24 h. The blot was hybridized to the A25 and GAPDH express low levels of AIbZIP mRNA (see Fig. 6C). Preimmune serum cDNA probes. The positions of the AIbZIP mRNA and RNA size markers are indicated. did not detect AIbZIP (data not shown). R1881 elicited nearly iden- B, Northern blot analysis of AIbZIP mRNA in human tissues. CLONTECH human multiple tissue blots (2 ␮g polyadenylated RNA) were hybridized to the A25 cDNA probe tical dose-related increases in AIbZIP protein levels in two independ- and exposed to film for 2 days after hybridization. The blots were also hybridized to a ent experiments (Fig. 7D). A 2.9-fold (average of two experiments) ␤-actin cDNA probe. C, determination of the AIbZIP mRNA expression profile in human breast and prostate cancer cell lines by RNase protection assay. Total RNA (10 ␮g) from increase in AIbZIP protein levels was observed at the dose of 1 nM of each of the indicated cell lines was hybridized to the AIbZIP 3ЈUTR and ␤-actin cRNA R1881. probes. 728

Casodex did not completely block the effect of 10 nM of R1881. These results provide additional confirmation that R1881 regulation of AIbZIP is mediated by the AR. It is of interest to note that the AR expressed in LNCaP cells contains a mutation in its hormone-binding domain that alters the ligand-binding specificity of the AR and which imparts “androgen-like” activity to antiandrogens such as hydroxyflutamide (12). Because such aberrant behavior can result from specific ligand-induced alterations in receptor conformation, it is of interest to identify genes that may be differentially regulated by androgens and antiandrogens to gain some insight into AR structure- function relationships, AR-coactivator interactions, and target genes. Therefore, LNCaP cells were cultured in the presence of increasing concentrations of either R1881 (0.001–10 nM), hydroxyflutamide (0.001–500 nM), or Casodex (0.001–500 nM) for 48 h. The cells were harvested after 48 h, and RNA was isolated and processed for determination of AIbZIP mRNA levels by RNase protection assay. As expected, R1881 up-regulated AIbZIP mRNA levels, whereas Caso- dex did not (Fig. 8B). Surprisingly, hydroxyflutamide, which up- regulated PSA mRNA levels under these conditions (data not shown), did not markedly increase AIbZIP mRNA levels. AIbZIP mRNA Expression in Human Prostate. To evaluate AIbZIP mRNA localization and expression in the prostate we performed in situ hybridization. AIbZIP mRNA was not detected in noncancerous human prostate (Fig. 9, A and B). In fact, in normal prostate, the hybridization pattern obtained with the AIbZIP antisense probe was similar to that obtained with the control sense probe. In both cases, scattered silver grains were equally distributed throughout the epithelium, stroma, and lumen. The number of silver grains overlying epithelial cells that were generated by the antisense probe was similar to that obtained with the control sense probe (75 versus 80 in the specimen presented in Fig. 9). In contrast, hybridization of the antisense probe to sections of prostate adenocarcinoma revealed a more intense and epithelial-specific labeling (Fig. 9C). The number of silver grains overlying epithelial cells obtained with the antisense probe was approximately twice that obtained with the control sense probe (574 versus 236 in the specimen presented in Fig. 9). The results presented in Fig. 9 are representative of experiments performed on 10 sections of normal prostate and 11 sections of cancerous prostate. These results suggested that AIbZIP could be expressed at higher levels in prostate cancer cells compared with normal prostate.

Fig. 7. Androgen regulation of AIbZIP expression. A, time course of R1881 induction of AIbZIP in LNCaP cells. RNA was isolated from control and androgen-treated cultures between 1 and 72 h after addition of 0.1 nM R1881. B, effect of actinomycin D (1 ␮g/ml), Ϫ6 cycloheximide (10 ␮g/ml), and Casodex (3 ϫ 10 M) on induction of AIbZIP by 0.1 nM R1881. Actinomycin D and cycloheximide were added to the cultures 15 min before addition of R1881. The cells were harvested 24 h after addition of R1881. C, effect of increasing doses of R1881 on AIbZIP mRNA levels in LNCaP cells. LNCaP cells were cultured in medium containing 0.001–10 nM R1881 for 24 h. In each experiment, total RNA (10 ␮g) was hybridized to the AIbZIP 3ЈUTR and ␤-actin cRNA probes. AIbZIP mRNA levels are expressed as AIbZIP:␤-actin ratios (AIbZIP:␤-actin ratios in control cells were assigned a value of 1). The results presented here are representative of three independent experiments performed between passages 27 and 37. D, effect of increasing doses of R1881 on AIbZIP protein levels in LNCaP cells. LNCaP cells were cultured as in C, and AIbZIP protein was detected using a rabbit polyclonal antibody raised against a COOH-terminal peptide of AIbZIP. BT-474 and MDA-MB-231 cells were used as positive and negative controls, respectively. AIbZIP protein levels in LNCaP cells were quantitated by scanning densitometry and expressed relative to control LNCaP cells. The experiment was performed twice, and the average AIbZIP protein levels in R1881-treated cells are shown below the immunoblot. Fig. 8. Modulation of AIbZIP expression by antiandrogens. A, effect of Casodex Ϫ6 (3 ϫ 10 M) on induction of AIbZIP by 0.001, 0.05, 0.1, and 10 nM R1881 in LNCaP cells. The cells were exposed to the indicated compounds for 48 h. B, effect of increasing doses of R1881, hydroxyflutamide (OH-Flu), or Casodex on AIbZIP mRNA levels in Modulation of AIbZIP Expression by Antiandrogens. As LNCaP cells. The cells were cultured in the presence of the indicated compounds for 48 h. Ϫ6 ␮ Ј ␤ shown in Fig. 8A, 3 ϫ 10 M Casodex completely reversed up- In each experiment, total RNA (10 g) was hybridized to the AIbZIP 3 UTR and -actin cRNA probes. AIbZIP mRNA levels are expressed as AIbZIP:␤-actin ratios (AIbZIP:␤- regulation of AIbZIP mRNA levels induced by doses of R1881 actin ratios in control cells were assigned a value of 1). The results presented here are ranging from 0.001 to 0.1 nM. On the other hand, the same dose of representative of three independent experiments performed between passages 27 and 37. 729

Fig. 9. Determination of AIbZIP mRNA localization and expression in noncancerous (A and B, ϫ600 magnification) and cancerous (C and D, ϫ950 magnification) prostate tissue by in situ hybridization. A, section of noncancerous human prostate hybridized with the antisense AIbZIP cRNA probe. A background of scattered silver grains can be seen over the lumen (L), epithelial cells (E), and stromal cells (S). B, an adjacent section of the prostate specimen shown in A, hybridized with the control sense AIbZIP cRNA probe. C, section of cancerous human prostate hybridized with the antisense AIbZIP cRNA probe. Strong autoradio- graphic signals were detected in the cancerous (labeled “C”) cells. D, an adjacent section of the prostate specimen shown in C, hybridized with the control sense AIbZIP cRNA probe. Scattered silver grains are observed, and cancerous cells are not labeled. Quantita- tion of the silver grains is described in the text.

AIbZIP Protein Expression in Prostate Carcinoma. We used a specimens exhibited low or undetectable expression of AIbZIP similar rabbit polyclonal antibody raised against aa 379–392 of AIbZIP to to that presented in Fig. 11, A and C. All 15 of the tumor-containing assess AIbZIP protein expression by immunostaining. As shown in specimens exhibited an expression pattern similar to that presented in Fig. 10, this polyclonal antibody is specific for AIbZIP, because it Fig. 11, A and D. produced a strong reaction in BT-474 cells, which express high levels of AIbZIP protein, whereas no reaction was detected in MDA-MB- DISCUSSION 231 cells that do not express AIbZIP protein (see Fig. 7D). As expected, the preimmune serum did not produce a detectable reaction AIbZIP is a new gene that we discovered using cDNA subtraction (Fig. 10B). to identify androgen-induced transcripts in LNCaP human prostate We then assessed AIbZIP protein expression in prostate needle cancer cells. The full-length human AIbZIP cDNA encodes a 395- biopsy specimens that contained both noncancerous and cancerous amino acid protein of which the outstanding feature is the presence of prostate cells. Immunoreactive AIbZIP expression was low (Fig. 11A) a bZIP domain, hence the name AIbZIP. BZIP domains are comprised or undetectable (Fig. 11C) in noncancerous prostate tissue. In contrast, of a region of basic amino acids (bZIP) that is located immediately

AIbZIP protein levels were appreciably higher in prostatic carcinoma NH2-terminal to a “leucine zipper” (bZIP), i.e., repeats of leucine cells compared with the adjacent noncancerous tissue (Fig. 11A, and residues that are separated by six amino acids (39). These domains, compare Fig. 11C to Fig. 11D). The immunostaining reaction was which mediate both DNA binding and protein-protein interactions, are primarily localized in the cytoplasm of luminal epithelial cells. In present in proteins of the bZIP superfamily, which includes the noncancerous cells, the reaction was detected in structures that could ATF/CREB family of transcription factors. correspond to the Golgi apparatus (see inset in Fig. 11A). In cancerous Several characteristics of AIbZIP are unique. First, the structure of cells, a more intense reaction was detected in the cytoplasm, espe- AIbZIP differs from that of most known ATF/CREB proteins. The cially in the apical portions of these cells. A total of 20 noncancerous sequence of the bZIP domain of AIbZIP is most closely related to TURP specimens and 15 adenocarcinoma-containing needle biopsy those of CREB-H (32) and CREB3 (33), two recently identified and specimens were examined. The noncancerous cells present in these relatively understudied members of the ATF/CREB family. The sim- 730

manner by the COOH-terminal region of the protein. Omori et al. (32) also observed that the COOH-terminus of CREB-H inhibited the

transcriptional activity of its NH2-terminal activation domain. Immunohistochemistry and GFP fusion protein localization experiments revealed that AIbZIP is primarily a cytoplasmic protein. The protein appears to be localized in structures related to the secretory pathway such as the Golgi apparatus. Whereas this finding will need to be confirmed using more precise localization techniques such as electron microscopy, this characteristic of AIbZIP is particularly interesting considering that the closely related CREB-H and CREB3 proteins, as well as ATF6, are associated with the endoplasmic reticulum. Like AIbZIP, all three of the proteins contain COOH-terminal transmembrane domains, which, when deleted, cause the recombinant proteins to localize to the nucleus (32, 40, 41). Despite their highly homologous bZIP and transmembrane domains, a few structural features distinguish AIbZIP from CREB-H. First, CREB-H contains a second COOH-terminal leucine zipper (aa 411–432) anchored by four leucine residues that is not present in AIbZIP. Second, CREB-H contains a COOH-terminal KDEL-like sequence (GDEL; aa 458– 461) that can function as an endoplasmic reticulum retrieval sequence (42). A similar motif is not present in AIbZIP. Given the fact that AIbZIP possesses the functional domains and activity of a transcription factor, it is highly possible that AIbZIP translocates to the nucleus to regulate gene expression in response to specific though as yet undetermined stimuli. In support of this hypothesis, during the endoplasmic reticulum stress response (unfolded protein response), ATF6 translocates from the endoplasmic reticulum to the nucleus where it activates genes that encode molecular chap- erones (43–45). Haze et al. (40) found that this process involves

cleavage of ATF6 from a mature Mr 90,000 polypeptide to a nuclear- localized Mr 50,000 form of ATF6 that includes the bZIP and activation domains. Thus, it is conceivable that AIbZIP could exist in a transcriptionally inactive form in cytoplasmic compartments and that removal of the COOH-terminal “anchor” would activate AIbZIP. Another unique feature of AIbZIP is that androgens induce its expression in LNCaP cells. To our knowledge, AIbZIP is the first example of a human CREB/ATF protein of which the expression is regulated by androgen. Most CREB/ATF proteins are regulated post- translationally, primarily through phosphorylation (38) or, as demon- Fig. 10. AIbZIP protein expression in BT-474 (A and B) and MDA-MB-231 (C) cells strated recently for ATF6, by proteolysis (40). Contrary to CREB, (ϫ600 magnification). A, immunohistochemical detection of AIbZIP in a paraffin section of BT-474 cells using a rabbit polyclonal antibody raised against a COOH-terminal AIbZIP does not contain protein kinase A phosphorylation sites nor peptide of AIbZIP. A strong staining reaction can be detected in the cytoplasm of most does it contain a kinase-inducible domain (38). CREB/ATF proteins cells. B, paraffin section of BT-474 cells incubated with the preimmune serum. No staining reaction can be detected. C, the antibody used in A was used to detect AIbZIP in may also mediate the effects of androgens in other androgen-sensitive a paraffin section of MDA-MB-231 cells. No immunostaining reaction was detected. tissues. Kim et al. (46) reported recently that CREB mRNA levels increase in the submandibular glands of rats treated with testosterone. Induction of bZIP proteins by steroid hormones has also been ob- ilarity among AIbZIP, CREB-H, and CREB3 also extends to regions served in mammary epithelial cells. hXBP-1, a bZIP transcription outside the bZIP domain. In fact, a dendogram analysis performed factor more closely related to jun proteins, was found to be up- using the amino acid sequences of AIbZIP, CREB-H, CREB3, and regulated by estrogen in MCF-7 human breast cancer cells (47). Thus, those of representative ATF/CREB proteins revealed that AIbZIP, bZIP transcription factors may play an important role in steroid CREB-H, and CREB3 constitute a distinct subgroup of ATF/CREB hormone-dependent cellular events in hormone-sensitive tissues. proteins. One particularly interesting characteristic of AIbZIP is its highly

AIbZIP contains an NH2-terminal transcriptional activation do- restricted expression profile. On the basis of Northern blot analyses, main. Experiments performed using GAL4-AIbZIP fusion proteins full-length AIbZIP mRNA is only expressed in the human prostate as revealed that the first 71 amino acids of AIbZIP are sufficient to well as in breast and prostate cancer cell lines. Indeed, the shorter activate transcription of a GAL4-responsive promoter. This portion of transcript (0.7 kb) expressed in the colon is unlikely to encode AIbZIP is relatively rich (14 of 71 amino acids; 20%) in acidic amino full-length AIbZIP. This highly tissue-specific expression profile ad- acids such as aspartic acid and glutamic acid. Similarly, the activation ditionally supports the hypothesis that AIbZIP may mediate steroid- domains of CREB-H and CREB3 have been mapped to the first 141 responsive events in these tissues. However, AIbZIP expression is not and 38 amino acids, respectively (32, 33). Interestingly, the NH2- exclusively responsive to androgen in LNCaP cells, because AIbZIP terminal domain of AIbZIP was more transcriptionally active than the mRNA was detected in the absence of androgenic stimulation. In full-length AIbZIP protein. These results suggest that the activation preliminary experiments conducted to date, AIbZIP expression was domain of AIbZIP could be physically masked or regulated in some not modulated by steroid hormones in human breast cancer cell lines 731

Fig. 11. AIbZIP protein expression in two prostate needle biopsy specimens displaying infiltrating adenocarcinoma (ϫ600 magnification). A and B are two consecutive serial sections from one specimen, whereas C and D are two different areas from a single section of a second specimen. Noncancerous tissue is labeled “N,” whereas cancerous tissue is labeled “C.” A, the cytoplasm of noncancerous epithelial cells is weakly labeled, whereas a strong immunostaining reaction is detected in all cancerous cells. The two noncancerous cells identified by arrows are additionally magnified in the inset (top left hand corner of A). The staining reaction is detected in the Golgi apparatus. B, no reaction was detected when the preimmune serum was used. C, AIbZIP protein was not detected in the noncancerous cells of this specimen. D, a strong immunostaining reaction was detected in the cancerous cells present on an area adjacent to that shown in C.

(data not shown). The highly tissue-specific expression pattern of ACKNOWLEDGMENTS AIbZIP is reminiscent of that of CREB-H, which is expressed exclu- We thank David Turgeon for kindly providing the LNCaP cell library, Serge sively in the liver (32). Rivest for assistance with the fluorescence microscopy, and Catherine Trem- Probably the most interesting observation is that AIbZIP appears to blay for the in situ hybridization. We also thank Drs. Jean Emond, Martin be expressed at higher levels in prostate cancer cells compared with Lemay, Bernard Teˆtu, and Michel Tremblay for providing prostate tissue noncancerous prostate cells. The immunostaining data presented here samples, and the Centre Hospitalier Universite´ Laval Research Center art clearly show that AIbZIP protein levels are low or undetectable in department for the figures. noncancerous prostate cells compared with adjacent cancerous cells in which AIbZIP is uniformly and highly expressed. 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Heng Qi, Catherine Fillion, Yvan Labrie, et al.

Cancer Res 2002;62:721-733.

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