RREB1 Transcription Factor Splice Variants in Urologic Cancer
2011; Elsevier BV; Volume: 179; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.03.038
ISSN1525-2191
AutoresMatthew D. Nitz, Michael A. Harding, Steven C. Smith, Shibu Thomas, Dan Theodorescu,
Tópico(s)RNA modifications and cancer
ResumoRREB1 is an alternatively spliced transcription factor implicated in Ras signaling and cancer. Little is known about the expression of RREB1 isoforms in cell lines or human tumors, or about the clinical relevance of the latter. We have developed tools for IHC of RREB1 protein isoform-specific amplification of RREB1 mRNA and selective knockdown of RREB1 isoforms and use these to provide new information by characterizing RREB1 expression in bladder and prostate cancer cell lines and human tissue samples. Previously described splice variants RREB1α, RREB1β, RREB1γ, and RREB1δ were identified, as well as the novel variant RREB1ε. Total and isoform-specific mRNA expression was lower in most but not all tumors, compared with normal tissues. RREB1 IHC performed on a bladder cancer TMA did not indicate a relationship between total RREB1 expression and overall survival after radical cystectomy for invasive bladder cancer. In contrast, in vitro proliferation studies using the UMUC-3 bladder cancer cell line after selective isoform-specific knockdown of expression indicate that RREB1α is not necessary for proliferation, but that RREB1β may be required. These contributions should accelerate progress in the nascent RREB1 field by providing new reagents while also providing clues to the role of RREB1 isoforms in human cancer and raising the possibility of isoform-specific roles in human carcinogenesis and progression. RREB1 is an alternatively spliced transcription factor implicated in Ras signaling and cancer. Little is known about the expression of RREB1 isoforms in cell lines or human tumors, or about the clinical relevance of the latter. We have developed tools for IHC of RREB1 protein isoform-specific amplification of RREB1 mRNA and selective knockdown of RREB1 isoforms and use these to provide new information by characterizing RREB1 expression in bladder and prostate cancer cell lines and human tissue samples. Previously described splice variants RREB1α, RREB1β, RREB1γ, and RREB1δ were identified, as well as the novel variant RREB1ε. Total and isoform-specific mRNA expression was lower in most but not all tumors, compared with normal tissues. RREB1 IHC performed on a bladder cancer TMA did not indicate a relationship between total RREB1 expression and overall survival after radical cystectomy for invasive bladder cancer. In contrast, in vitro proliferation studies using the UMUC-3 bladder cancer cell line after selective isoform-specific knockdown of expression indicate that RREB1α is not necessary for proliferation, but that RREB1β may be required. These contributions should accelerate progress in the nascent RREB1 field by providing new reagents while also providing clues to the role of RREB1 isoforms in human cancer and raising the possibility of isoform-specific roles in human carcinogenesis and progression. The Ras family of GTPases, with their regulators and effectors, have been implicated in tumor progression.1Traynor P. McGlynn L.M. Mukhergee R. Grimsley S.J. Bartlett J.M. Edwards J. An increase in N-Ras expression is associated with development of hormone refractory prostate cancer in a subset of patients.Dis Markers. 2008; 24: 157-165Crossref PubMed Scopus (5) Google Scholar, 2Erlich S. Tal-Or P. Liebling R. Blum R. Karunagaran D. Kloog Y. Pinkas-Kramarski R. Ras inhibition results in growth arrest and death of androgen-dependent and androgen-independent prostate cancer cells.Biochem Pharmacol. 2006; 72: 427-436Crossref PubMed Scopus (38) Google Scholar, 3Weber M.J. Gioeli D. 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RREB-1 is a transcriptional repressor of HLA-G.J Immunol. 2009; 183: 6948-6959Crossref PubMed Scopus (57) Google Scholar hZIP1,18Milon B.C. Agyapong A. Bautista R. Costello L.C. Franklin R.B. Ras responsive element binding protein-1 (RREB-1) down-regulates hZIP1 expression in prostate cancer cells.Prostate. 2010; 70: 288-296PubMed Google Scholar p16,19Zhang S. Qian X. Redman C. Bliskovski V. Ramsay E.S. Lowy D.R. Mock B.A. p16 INK4a gene promoter variation and differential binding of a repressor, the ras-responsive zinc-finger transcription factor, RREB.Oncogene. 2003; 22: 2285-2295Crossref PubMed Scopus (68) Google Scholar and PSA.20Mukhopadhyay N.K. Cinar B. Mukhopadhyay L. Lutchman M. Ferdinand A.S. Kim J. Chung L.W. Adam R.M. Ray S.K. Leiter A.B. Richie J.P. Liu B.C. Freeman M.R. The zinc finger protein ras-responsive element binding protein-1 is a coregulator of the androgen receptor: implications for the role of the Ras pathway in enhancing androgenic signaling in prostate cancer.Mol Endocrinol. 2007; 21: 2056-2070Crossref PubMed Scopus (42) Google Scholar RREB1 has also been found to bind nuclear proteins, such as CtBP,21Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Coordinated histone modifications mediated by a CtBP co-repressor complex.Nature. 2003; 422: 735-738Crossref PubMed Scopus (636) Google Scholar NeuroD,15Ray S.K. Nishitani J. Petry M.W. Fessing M.Y. Leiter A.B. Novel transcriptional potentiation of BETA2/NeuroD on the secretin gene promoter by the DNA-binding protein Finb/RREB-1.Mol Cell Biol. 2003; 23: 259-271Crossref PubMed Scopus (42) Google Scholar and androgen receptor (AR).20Mukhopadhyay N.K. Cinar B. Mukhopadhyay L. Lutchman M. Ferdinand A.S. Kim J. Chung L.W. Adam R.M. Ray S.K. Leiter A.B. Richie J.P. Liu B.C. Freeman M.R. The zinc finger protein ras-responsive element binding protein-1 is a coregulator of the androgen receptor: implications for the role of the Ras pathway in enhancing androgenic signaling in prostate cancer.Mol Endocrinol. 2007; 21: 2056-2070Crossref PubMed Scopus (42) Google Scholar The initial study of RREB1 found that the gene product bound the calcitonin promoter in medullary thyroid carcinomas in response to Ras.11Thiagalingam A. De Bustros A. Borges M. Jasti R. Compton D. Diamond L. Mabry M. Ball D.W. Baylin S.B. Nelkin B.D. RREB-1, a novel zinc finger protein, is involved in the differentiation response to Ras in human medullary thyroid carcinomas.Mol Cell Biol. 1996; 16: 5335-5345Crossref PubMed Scopus (121) Google Scholar In bladder cancer, the tumor suppressor p16 is commonly lost as an early event in tumorigenesis, and RREB1 was found to bind and repress transcription of the Cdkn2a locus.19Zhang S. Qian X. Redman C. Bliskovski V. Ramsay E.S. Lowy D.R. Mock B.A. p16 INK4a gene promoter variation and differential binding of a repressor, the ras-responsive zinc-finger transcription factor, RREB.Oncogene. 2003; 22: 2285-2295Crossref PubMed Scopus (68) Google Scholar Depletion of RREB1 by siRNA slows cell migration and cell spreading in breast cell lines,22Melani M. Simpson K.J. Brugge J.S. Montell D. Regulation of cell adhesion and collective cell migration by hindsight and its human homolog RREB1.Curr Biol. 2008; 18: 532-537Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar whereas in breast cancer and osteosarcoma cells RREB1 binds the p53 promoter and transactivates p53 expression on DNA damage.14Liu H. Hew H.C. Lu Z.G. Yamaguchi T. Miki Y. Yoshida K. DNA damage signalling recruits RREB-1 to the p53 tumour suppressor promoter.Biochem J. 2009; 422: 543-551Crossref PubMed Scopus (32) Google Scholar The RREB1 gene is a locus of viral integration for hepatitis B virus in hepatocellular carcinoma.23Tamori A. Yamanishi Y. Kawashima S. Kanehisa M. Enomoto M. Tanaka H. Kubo S. Shiomi S. Nishiguchi S. Alteration of gene expression in human hepatocellular carcinoma with integrated hepatitis B virus DNA.Clin Cancer Res. 2005; 11: 5821-5826Crossref PubMed Scopus (62) Google Scholar Furthermore, RREB1 was identified as a potential oncogene in Moloney murine leukemia virus (MuLV) infected p19ARF and p53 knockout mice.24Uren A.G. Kool J. Matentzoglu K. de Ridder J. Mattison J. van Uitert M. Lagcher W. Sie D. Tanger E. Cox T. Reinders M. Hubbard T.J. Rogers J. Jonkers J. Wessels L. Adams D.J. van Lohuizen M. Berns A. Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks.Cell. 2008; 133: 727-741Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar Finally, the human RREB1 locus has been found to be amplified in melanoma and is currently an area of intense investigation for its potential in molecular diagnostic testing.25Morey A.L. Murali R. McCarthy S.W. Mann G.J. Scolyer R.A. Diagnosis of cutaneous melanocytic tumours by four-colour fluorescence in situ hybridisation.Pathology. 2009; 41: 383-387Crossref PubMed Scopus (88) Google Scholar, 26Gerami P. Wass A. Mafee M. Fang Y. Pulitzer M.P. Busam K.J. Fluorescence in situ hybridization for distinguishing nevoid melanomas from mitotically active nevi.Am J Surg Pathol. 2009; 33: 1783-1788Crossref PubMed Scopus (110) Google Scholar, 27Pouryazdanparast P. Newman M. Mafee M. Haghighat Z. Guitart J. Gerami P. 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Classifying ambiguous melanocytic lesions with FISH and correlation with clinical long-term follow up.Mod Pathol. 2010; 23: 413-419Crossref PubMed Scopus (112) Google Scholar In prostate cancer, RREB1 binds the PSA promoter only in association with AR to repress transcription.20Mukhopadhyay N.K. Cinar B. Mukhopadhyay L. Lutchman M. Ferdinand A.S. Kim J. Chung L.W. Adam R.M. Ray S.K. Leiter A.B. Richie J.P. Liu B.C. Freeman M.R. The zinc finger protein ras-responsive element binding protein-1 is a coregulator of the androgen receptor: implications for the role of the Ras pathway in enhancing androgenic signaling in prostate cancer.Mol Endocrinol. 2007; 21: 2056-2070Crossref PubMed Scopus (42) Google Scholar In summary, the current literature on RREB1 suggests context-dependent phenotypes that may suppress or promote carcinogenesis and tumor progression. RREB1 is a transcription factor containing between 13 and 15 zinc finger domains, depending on alternative splicing.16Date S. Nibu Y. Yanai K. Hirata J. Yagami K. Fukamizu A. Finb, a multiple zinc finger protein, represses transcription of the human angiotensinogen gene.Int J Mol Med. 2004; 13: 637-642PubMed Google Scholar It was initially described as a 755-amino-acid C2H2 zinc finger protein (RREB-1),11Thiagalingam A. De Bustros A. Borges M. Jasti R. Compton D. Diamond L. Mabry M. Ball D.W. Baylin S.B. Nelkin B.D. RREB-1, a novel zinc finger protein, is involved in the differentiation response to Ras in human medullary thyroid carcinomas.Mol Cell Biol. 1996; 16: 5335-5345Crossref PubMed Scopus (121) Google Scholar although subsequent analyses in chicken and human cells indicated that RREB1 encodes a longer protein of 1656 (Finb) amino acids (AA) in humans.13Fujimoto-Nishiyama A. Ishii S. Matsuda S. Inoue J. Yamamoto T. A novel zinc finger protein, Finb, is a transcriptional activator and localized in nuclear bodies.Gene. 1997; 195: 267-275Crossref PubMed Scopus (16) Google Scholar, 31Miyake J.H. Szeto D.P. Stumph W.E. Analysis of the structure and expression of the chicken gene encoding a homolog of the human RREB-1 transcription factor.Gene. 1997; 202: 177-186Crossref PubMed Scopus (8) Google Scholar A second variant encoding 1397 amino acids [Finb (cl-32)] with a unique C-terminus was also identified.13Fujimoto-Nishiyama A. Ishii S. Matsuda S. Inoue J. Yamamoto T. A novel zinc finger protein, Finb, is a transcriptional activator and localized in nuclear bodies.Gene. 1997; 195: 267-275Crossref PubMed Scopus (16) Google Scholar Two additional RREB1 C-terminal isoforms exhibiting addition or removal of cassette exons were isolated and designated Finb188 (1742AA) and Finb159 (1476AA). These isoforms exhibit a translation start site 57 bp upstream of the earlier described Finb and Finb (cl-32) isoforms (Figure 1A).16Date S. Nibu Y. Yanai K. Hirata J. Yagami K. Fukamizu A. Finb, a multiple zinc finger protein, represses transcription of the human angiotensinogen gene.Int J Mol Med. 2004; 13: 637-642PubMed Google Scholar The authors also discovered that Finb contains regions without homology to consensus protein sequence of Finb188. cDNA sequence alignment of Finb and Finb188 reveals that the former does not conform to sequences for the human genome, potentially because of cloning artifacts.16Date S. Nibu Y. Yanai K. Hirata J. Yagami K. Fukamizu A. Finb, a multiple zinc finger protein, represses transcription of the human angiotensinogen gene.Int J Mol Med. 2004; 13: 637-642PubMed Google Scholar Thus, there is a critical need for consensus on the RREB1 proteins. Given the number and diversity of known targets of RREB1 signaling, and that splice variants encoding four protein isoforms have already been described, it is likely that RREB1 signaling is mechanistically and phenotypically complex and that expression of isoforms in various proportions could contribute to this complexity. Surprisingly, there has been no comprehensive evaluation of RREB1 isoforms in human cancer cell lines and normal or tumor tissues to date. Furthermore, no study has shown evidence suggestive of isoform-specific phenotypes. With the present study, we developed tools to evaluate RREB1 isoforms in human bladder and prostate cell lines and human tumors at the RNA and immunohistochemical levels. We then used these tools to evaluate the expression patterns and their clinical relevance in human samples. Here, we present evidence that isoforms have different functional effects on in vitro bladder cancer cell proliferation. Human cancer cell lines were grown as described previously.32Lee J.K. Havaleshko D.M. Cho H. Weinstein J.N. Kaldjian E.P. Karpovich J. Grimshaw A. Theodorescu D. A strategy for predicting the chemosensitivity of human cancers and its application to drug discovery.Proc Natl Acad Sci USA. 2007; 104: 13086-13091Crossref PubMed Scopus (258) Google Scholar TERT cells33Chapman E.J. Hurst C.D. Pitt E. Chambers P. Aveyard J.S. Knowles M.A. Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway.Oncogene. 2006; 25: 5037-5045Crossref PubMed Scopus (77) Google Scholar were a gift from Margaret A. Knowles. LUL2 cells were isolated from lung tumors by successive passages of the UMUC-3 cell line by tail vein injection into nude mice (unpublished data). With strict observance of NIH and University of Virginia guidelines and with approval of the Institutional Review Board, deidentified flash-frozen and archival tissues of human bladder and prostate cancer and adjacent non-neoplastic epithelia, procured by the University of Virginia Biorepository and Tissue Research Facility, were obtained for analysis of RREB1 expression. The tumor tissue was macrodissected to approximately 80% purity, as previously described.34Titus B. Frierson Jr, H.F. Conaway M. Ching K. Guise T. Chirgwin J. Hampton G. Theodorescu D. Endothelin axis is a target of the lung metastasis suppressor gene RhoGDI2.Cancer Res. 2005; 65: 7320-7327Crossref PubMed Scopus (100) Google Scholar The bladder TMA described here has been reported previously.35Smith S.C. Nicholson B. Nitz M. Frierson Jr, H.F. Smolkin M. Hampton G. El-Rifai W. Theodorescu D. Profiling bladder cancer organ site-specific metastasis identifies LAMC2 as a novel biomarker of hematogenous dissemination.Am J Pathol. 2009; 174: 371-379Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar RNA isolation was performed using an RNeasy mini kit (Qiagen, Valencia, CA) and cDNA synthesis using iScript (Bio-Rad, Hercules, CA). Quantitative RT-PCR (qRT-PCR) was performed on cDNA using iQ SYBR Green Supermix (Bio-Rad). RNA quantification was performed as previously described.36Nitz M.D. Harding M.A. Theodorescu D. Invasion and metastasis models for studying RhoGDI2 in bladder cancer.Methods Enzymol. 2008; 439: 219-233Crossref PubMed Scopus (17) Google Scholar The following primers were used with annealing temperatures: RREB1 (total) forward 5′-CTTCCTATAACTGCCCCC-3′, reverse 5′-ATGAGTGGTCGGCTCCTCC-3′; RREB1α forward 5′-TGGATCCCATGATAGCACAGAC-3′, reverse 5′-TGCTCTCTGTCCCGTGAGG-3′; RREB1β forward 5′-CACATGCTCACACACACTGACA-3′, reverse 5′-CCGACGGCTGCTCTCTGT-3′; RREB1δ forward 5′-ACCAACTGCCTGCAGAAGATCA-3′, reverse 5′-GTATGGCCTTTCCCCAGTGTGT-3′; RREB1ε forward 5′-TACAGAACAACCCTTCAATTCCT-3′, reverse 5′-TATGGCCTTTCCCCTGAG-3′. The PCR for the 5′ and 3′ ends of RREB1 was performed using AccuPrime SuperMix II (Invitrogen, Carlsbad, CA) for 35 cycles with the following primers: RREB1-5′ forward 5′-TCGGATTGGCAGAAGGAA-3′, reverse 5′-CAGGCTCAGCAGGTTGGT-3′; RREB1 3′ forward 5′-CGGAACTCGTACACCAACTG-3′, reverse 5′-CGCTGTGGGTGGACTCATTC-3′; RREB1 full length forward 5′-GATCAAGCTTACGTCAAGTTCGCCCGCT-3′, reverse 5′-GATCCTCGAGTCACTCCATCCCCACGAG-3′. RREB1δ and RREB1ε were cloned out of UMUC-3 cells. Sequences were submitted to GenBank: RREB1δ (HM369361) and RREB1ε (HM369360). Total RNA was isolated as described above and cDNA synthesis using SuperScript III first-strand synthesis supermix (Invitrogen) with 50 μmol/L random hexamers and 1 μg total RNA. The PCR reaction also included 10× PfuUltra HF reaction buffer (Agilent Technologies, Foster City, CA), 100 mmol/L dNTP mix (Agilent), 5% dimethyl sulfoxide, 1 mol/L betaine solution (Sigma-Aldrich, St. Louis, MO). The primers used were as follows: RREB1 cloning forward 5′-GATCATCGATATGACGTCAAGTTCGCCC-3′, reverse 5′-GATCTCTAGACTCCATCCCCACGAGCTG-3′. PCR products were isolated in a 1% agarose gel and purified using a QIAquick gel extraction kit (Qiagen). Isolated products were digested at 37°C by the enzymes ClaI and XbaI (New England Biolabs, Ipswich, MA) and were ligated into the p3XFLAG-CMV-14 expression vector (Sigma-Aldrich). RREB1α and RREB1β,16Date S. Nibu Y. Yanai K. Hirata J. Yagami K. Fukamizu A. Finb, a multiple zinc finger protein, represses transcription of the human angiotensinogen gene.Int J Mol Med. 2004; 13: 637-642PubMed Google Scholar kindly provided by Dr. Akiyoshi Fukamizu (University of Tsukuba, Tsukuba, Japan), were subcloned into the p3XFLAG-CMV-14. The following antibodies were used in detection of RREB1: anti-RREB1 from GenWay Biotech (cat. no. 18-732-2922332; Biotech, San Diego, CA), from Cosmo Bio (cat. no: CBX-CBX00717; Carlsbad, CA; Tokyo, Japan), and from Sigma-Aldrich (cat. no. HPA001756), as well as anti-FLAG (cat. no: F1804; Sigma-Aldrich), anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-TBP (TFIID) (Santa Cruz Biotechnology). Nuclear and cytoplasmic isolates were made with an NE-PER extraction kit (Thermo Scientific, Rockford, IL). Immunoblotting and detection were performed as described previously.8Oxford G. Owens C.R. Titus B.J. Foreman T.L. Herlevsen M.C. Smith S.C. Theodorescu D. RalA and RalB: antagonistic relatives in cancer cell migration.Cancer Res. 2005; 65: 7111-7120Crossref PubMed Scopus (107) Google Scholar Immunohistochemistry (IHC) was performed on a Dako Autostainer instrument (Dako, Glostrup, Denmark) with the following protocol: antigen retrieval (125°C, Dako TRS9 buffer, 30 seconds), Dako dual endogenous enzyme block (10 minutes), RREB1 antibody (Sigma-Aldrich; 1:100 in Dako antibody diluent, 30 minutes), detection (Dako Envision dual link anti-rabbit, 30 minutes), chromogen (Dako diaminobenzidine Dab+ substrate, 10 minutes), and counterstain (hematoxylin, 5 minutes). Staining of RREB1 was scored semiquantitatively as negative (absence of staining), low/focal (a blush or positivity of cells 50% of cells). Transient vector transfection was performed using FuGENE 6 transfection reagent (Roche, Basel, Switzerland) according to the manufacturer's instructions. Stable expression was achieved by cutting RREB1-expressing p3XFLAG-CMV-14 vector with the ScaI restriction enzyme (New England Biolabs) and transfected with FuGENE 6. Selection was performed for 14 days in 800 μg/mL of G418 (Geneticin, Invitrogen). Oligofectamine (Invitrogen) was used to transiently transfect siRNA according to the manufacturer's instructions. All siRNA was transfected at a final concentration of 25 nmol/L: RREB1 total 5′-GGAGUUUGUUUGCAAGUAU-3′ and 5′-GUUCAGACCUAUCUUCCAU-3′ (used in combination at 12.5 nmol/L), GL2 5′-CGUACGCGGAAUACUUCGAdTdT-3′, RREB1 exon 8-1 5′-CCUGAGAAGAAACGGGCUUUU-3′, RREB1 exon 8-2 5′-CGCAAACACGGAGUUACCACCUGUU-3′, RREB1 exon 8-3 5′-GAUGUUGGAUCCCAUGAUAUU-3′, RREB1 exon 9-1 5′-CAGAGAAGAGCGACGAUGAdTdT-3′, RREB1 9-2 5′-CCACCAAGCUCAUGGACUUUU-3′, and RREB1 exon 9-3 5′-GGAAGAAGGUCUGCAGCGUdTdT-3′. In vitro cell growth assays were performed by using Alamar Blue (Invitrogen) fluorescence emission as described previously.37Havaleshko D.M. Cho H. Conaway M. Owens C.R. Hampton G. Lee J.K. Theodorescu D. Prediction of drug combination chemosensitivity in human bladder cancer.Mol Cancer Ther. 2007; 6: 578-586Crossref PubMed Scopus (40) Google Scholar Briefly, Alamar Blue fluorescence was measured at 96 hours after siRNA depletion in UMUC-3 cells. Using bladder and prostate cancer cell lines as models, primers were designed to interrogate expression of variants in the last four coding exons of RREB1, a region in alternative splicing has been described (Figure 1A).13Fujimoto-Nishiyama A. Ishii S. Matsuda S. Inoue J. Yamamoto T. A novel zinc finger protein, Finb, is a transcriptional activator and localized in nuclear bodies.Gene. 1997; 195: 267-275Crossref PubMed Scopus (16) Google Scholar, 16Date S. Nibu Y. Yanai K. Hirata J. Yagami K. Fukamizu A. Finb, a multiple zinc finger protein, represses transcription of the human angiotensinogen gene.Int J Mol Med. 2004; 13: 637-642PubMed Google Scholar Predicted PCR product sizes for the four RREB1 isoforms characterized thus far were 1237 bp for Finb188, 1072 bp for Finb182 (RREB-1, Finb), 439 bp for Finb159, and 348 bp for Finb (cl-32). In nine cell lines examined, bands corresponding to all four splice variants were identified (Figure 1B). Each band was sequenced to confirm the PCR products corresponded to the predicted variant. To examine whether the first six identified coding exons of RREB1 transcripts underwent alternative splicing, primers spanning from the first to the seventh coding exon were tested on seven cell lines. Each cell line showed a single band, leading us to conclude that alternative splicing of RREB1 occurs predominantly in the exon 6 to exon 10 region of the RNA (Figure 1C). To search for novel splice variants, primers were designed to interrogate all 10 coding exons (Figure 1B). PCR products for Finb188/182 and Finb (cl-32) were identified, as well as a novel variant migrating at a much smaller size (Figure 1D). Sequencing of this band revealed it to be a novel isoform of RREB1, lacking coding exons 7, 8, and 9 and causing a frame shift in exon 10, resulting in a loss of a C2H2 zinc finger and a new translation stop site. Given these findings, we propose the following nomenclature for the RREB1 splice variants: RREB1α, RREB1β, RREB1γ, RREB1δ, and RREB1ε (Figure 1A). To evaluate RREB1 isoform expression, we developed primers for qRT-PCR that allow specific amplification of each isoform. These primers were used in conjunction with primers spanning the second and third exons, the area we found not to be involved in differential splicing, to detect total RREB1 mRNA expression. We examined one telomerase (TERT) immortalized urothelial cell line,33Chapman E.J. Hurst C.D. Pitt E. Chambers P. Aveyard J.S. Knowles M.A. Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway.Oncogene. 2006; 25: 5037-5045Crossref PubMed Scopus (77) Google Scholar five urothelial bladder cancer cell lines, and two prostate cancer cell lines. The data presented in Figure 1B, as well as the use of isoform-specific primers to estimate relative abundance of isoforms, demonstrated that RREB1α and RREB1β comprised the vast majority of total RREB1 mRNA (see Supplemental Figure S1 at http://ajp.amjpathol.org). Expression of RREB1γ was so low as to not be reliably quantifiable. Expression of RREB1δ typically was only 1% to 4%, and RREB1ε expression was <1% of total RREB1. For further studies, therefore, we focused on total RREB1, RREB1α, and RREB1β. The expression levels for total RREB1, RREB1α, and RREB1β in cell lines were variable (Figure 2A). Notably, the total RREB1 expression is higher in the nontransformed TERT urothelial cell line than in all of the urothelial (bladder) cancer cell lines. Next we examined paired human normal and cancerous bladder (n = 10) and prostate (n = 4) tissues for total RREB1 (Figure 2B), RREB1α (Figure 2C), and RREB1β (Figure 2D) mRNA expression. Of 10 bladder cancers, 7 samples had lower total RREB1 expression, as well as lower expression of the specific α and β isoforms, than their no
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