Two Newly Characterized Germinal Center B-Cell-Associated Genes, GCET1 and GCET2, Have Differential Expression in Normal and Neoplastic B Cells
2003; Elsevier BV; Volume: 163; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63637-1
ISSN1525-2191
AutoresZenggang Pan, Yulei Shen, Cheng Du, Guimei Zhou, Andreas Rosenwald, Louis M. Staudt, Timothy C. Greiner, Timothy W. McKeithan, Wing C. Chan,
Tópico(s)Peptidase Inhibition and Analysis
ResumoA group of genes are highly expressed in normal germinal center (GC) B cells and GC B-cell-derived malignancies based on cDNA microarray analysis. Two new genes, GCET1 (germinal center B-cell expressed transcript 1) and GCET2, were cloned from selected expressed sequence tags (IMAGE clone 1334260 and 814622, respectively). GCET1 is located on chromosome 14q32 and has four splicing isoforms, of which the longest one is 1787 bp and encodes a 435-amino acid protein. GCET2 is located on 3q13.13, and the cloned fragment is 3270 bp, which encodes a protein of 178 amino acids. Blast search showed that GCET1 has a highly conserved serine proteinase inhibitor (SERPIN) domain and is located on a chromosomal locus containing seven other SERPIN family members. GCET2 is a likely homologue of the mouse gene M17, a GC-expressed transcript. Analysis of the GCET2 protein sequence indicated that it may be involved in signal transduction in the cytoplasm. Northern blot and real-time polymerase chain reaction analyses confirmed that GCET1 is highly restricted to normal GC B cells and GCB-cell-derived cell lines. Although GCET2 is also a useful marker for normal and neoplastic GC B cells, it has a wider range of expression including immature B and T cells. Real-time polymerase chain reaction assay showed that both GCET1 and GCET2 are preferentially expressed in follicular lymphoma and diffuse large B-cell lymphoma with GC B-cell differentiation, confirming previous microarray gene expression analysis, but neither one is entirely specific. Multiple markers are necessary to differentiate the GCB from the activated B-cell type of diffuse large B-cell lymphoma with a high degree of accuracy. A group of genes are highly expressed in normal germinal center (GC) B cells and GC B-cell-derived malignancies based on cDNA microarray analysis. Two new genes, GCET1 (germinal center B-cell expressed transcript 1) and GCET2, were cloned from selected expressed sequence tags (IMAGE clone 1334260 and 814622, respectively). GCET1 is located on chromosome 14q32 and has four splicing isoforms, of which the longest one is 1787 bp and encodes a 435-amino acid protein. GCET2 is located on 3q13.13, and the cloned fragment is 3270 bp, which encodes a protein of 178 amino acids. Blast search showed that GCET1 has a highly conserved serine proteinase inhibitor (SERPIN) domain and is located on a chromosomal locus containing seven other SERPIN family members. GCET2 is a likely homologue of the mouse gene M17, a GC-expressed transcript. Analysis of the GCET2 protein sequence indicated that it may be involved in signal transduction in the cytoplasm. Northern blot and real-time polymerase chain reaction analyses confirmed that GCET1 is highly restricted to normal GC B cells and GCB-cell-derived cell lines. Although GCET2 is also a useful marker for normal and neoplastic GC B cells, it has a wider range of expression including immature B and T cells. Real-time polymerase chain reaction assay showed that both GCET1 and GCET2 are preferentially expressed in follicular lymphoma and diffuse large B-cell lymphoma with GC B-cell differentiation, confirming previous microarray gene expression analysis, but neither one is entirely specific. Multiple markers are necessary to differentiate the GCB from the activated B-cell type of diffuse large B-cell lymphoma with a high degree of accuracy. In the T-cell regions of secondary lymphoid organs, naïve B lymphocytes have two alternative fates after activation. Some of them will differentiate into primary plasma cells whereas others will migrate to form germinal centers (GC) and undergo further development.1Ahmed R Gray D Immunological memory and protective immunity: understanding their relation.Science. 1996; 272: 54-60Crossref PubMed Scopus (1452) Google Scholar, 2Cariappa A Pillai S Antigen-dependent B-cell development.Curr Opin Immunol. 2002; 14: 241-249Crossref PubMed Scopus (103) Google Scholar GC B cells proliferate rapidly and at the same time acquire the capacity to undergo somatic hypermutation and isotype switching. A stringent process of selection also occurs with survival only of cells expressing high-affinity antibody to the selecting antigen.3Steinman RM Dendritic cells and the control of immunity: enhancing the efficiency of antigen presentation.Mt Sinai J Med. 2001; 68: 106-166PubMed Google Scholar, 4Han S Zheng B Takahashi Y Kelsoe G Distinctive characteristics of germinal center B cells.Semin Immunol. 1997; 9: 255-260Crossref PubMed Scopus (85) Google Scholar, 5Kelsoe G Life and death in germinal centers (redux).Immunity. 1996; 4: 107-111Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar This complex maturation process is associated with the acquisition of a unique gene expression profile that distinguishes GC B cells from B cells at other stages of maturation. A number of known genes and uncharacterized genes that are represented as expressed sequence tags (ESTs) are highly expressed in normal and malignant GC B cells, but not in resting or activated peripheral blood B cells.6Alizadeh AA Eisen MB Davis RE Ma C Lossos IS Rosenwald A Boldrick JC Sabet H Tran T Yu X Powell JI Yang L Marti GE Moore T Hudson Jr, J Lu L Lewis DB Tibshirani R Sherlock G Chan WC Greiner TC Weisenburger DD Armitage JO Warnke R Lavy R Wilson W Grevor MR Byrd JC Botstein D Brown PD Staudt LM Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (8074) Google Scholar, 7Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3219) Google Scholar Gene expression profiling studies on a large number of diffuse large B-cell lymphomas (DLBCLs) have shown the presence of a unique subset with overexpression of genes in the GC B-cell signature.6Alizadeh AA Eisen MB Davis RE Ma C Lossos IS Rosenwald A Boldrick JC Sabet H Tran T Yu X Powell JI Yang L Marti GE Moore T Hudson Jr, J Lu L Lewis DB Tibshirani R Sherlock G Chan WC Greiner TC Weisenburger DD Armitage JO Warnke R Lavy R Wilson W Grevor MR Byrd JC Botstein D Brown PD Staudt LM Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (8074) Google Scholar This type of lymphoma has a better prognosis than other types that do not exhibit this expression signature. The gene expression profile is very heterogeneous even within individual subsets of DLBCL, but a limited number of genes in the GC B-cell signature have been identified to be most diagnostic of the GCB subtype of DLBCL.7Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3219) Google Scholar Two of the ESTs, which are important predictors of prognosis on DLBCL and have the highest P values in differentiating GCB-DLBCL from other subtypes,7Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3219) Google Scholar were selected for cloning. The characteristics of these two genes are described below. Two ESTs were selected from the NCI_CGAP_GCB1 library based on the expression profiling data of our previous cDNA microarray study of normal and neoplastic B cells.6Alizadeh AA Eisen MB Davis RE Ma C Lossos IS Rosenwald A Boldrick JC Sabet H Tran T Yu X Powell JI Yang L Marti GE Moore T Hudson Jr, J Lu L Lewis DB Tibshirani R Sherlock G Chan WC Greiner TC Weisenburger DD Armitage JO Warnke R Lavy R Wilson W Grevor MR Byrd JC Botstein D Brown PD Staudt LM Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (8074) Google Scholar, 7Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3219) Google Scholar These ESTs are highly expressed in normal GC B cells, GCB-derived DLBCLs, and follicular lymphomas (FLs) (Figure 1). Using NCBI Genomic BLAST, the chromosomal locations of the selected ESTs were determined. The 50-kb chromosomal regions flanking each of the ESTs were searched to find other human ESTs close to or overlapping with the selected ESTs. Polymerase chain reaction (PCR) primers were designed to determine the presence and orientation of these ESTs on the cDNA (from the lymphoma cell line DHL16; see below) containing the original EST. Also, primers were designed according to known EST sequences for rapid amplification of cDNA ends (RACE)8Schaefer BC Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends.Anal Biochem. 1995; 227: 255-273Crossref PubMed Scopus (284) Google Scholar, 9Bertioli D Rapid amplification of cDNA ends.Methods Mol Biol. 1997; 67: 233-238PubMed Google Scholar (Table 1).Table 1Primers for 5′ RACE in Cloning GCET1Gene specific primer designed from EST AA805575CTGCAATAGAGGTGCCTAACSMART II A oligonucleotide sequenceAAGCAGTGGTATCAACGCAGAGTACGCGGGUniversal primer A Mix (UPM)Long CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGTShort CTAATACGACTCACTATAGGGCNested universal primer A (NUP)AAGCAGTGGTATCAACGCAGAGTPrimers for Cloning GCET2 Utilizing EST SequencesdbEST IDGenBank acc. no.CS0DG007YD15 (5′)AL560376Forward primerAGTCAGGAGTTGCCCTGTCAGReverse primerTAGGTCTGGTCAACATTGTCC814622 (5′)AA480985Forward primerTTAAGAAGAACGATCCTTGGAGReverse primerATCAGGACACATAGAGGAGTGC3064524AW576133Forward primerATTGGGTATCTTGAAGCACTCCReverse primerACTGGACCATTGTGGAAGTAG684286 (5′)AA236080Forward primerCTGCATCTTGGTATCTCTTCCTCReverse primerAGAAGCTGAGCCTCCAAGTAGPrimers and Probes Used for Real-Time PCRGCET1Forward primerTGTCAGTGAAGAGGGCACTGAReverse primerGACCATCCTTCGATCGGACTATProbeFAM-CCACAGCAGCTACCGCCACCAA-TAMRAGCET2Forward primerAATATGAACTTCTCATGCCTCACAGAReverse primerAATGCTAGTCCAGCCACTTCACTAProbeFAM-TCTGCAACAGCCACGTCCACTTATGG-TAMRAGAPDHForward primerGAAGGTGAAGGTCGGAGTCReverse primerGAAGATGGTGATGGGATTTCProbeJOE-CAAGCTTCCCGTTCTCAGCC-TAMRAACTBForward primerTGCCGACAGGATGCAGAAGReverse primerGCCGATCCACACGGAGTACTProbeFAM-TCAAGATCATTGCTCCTCCTGAGCGC-TAMRAB2MForward primerTGTGCTCGCGCTACTCTCTCTReverse primerTCTGCTGGATGACGTGAGTAAACProbeFAM-CCTGGAGGCTATCCAGCGTACTCCAAA-TAMRA Open table in a new tab To clone the corresponding genes from selected ESTs, cytoplasmic total RNA was isolated from a cultured GCB-derived lymphoma cell line, DHL16, using RNeasy Midi Kit (Qiagen, Valencia, CA). Cytoplasmic total RNA was further treated with DNase I (Promega, Madison, WI), and then mRNA was isolated using the Poly(A) Pure mRNA purification kit (Ambion, Austin, TX). cDNA was synthesized using SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA) following the manufacturer's protocol. A universal adaptor, SMART IIA oligonucleotide, hybridized to the three cytosine residues added to the 3′ end of the cDNA during reverse transcription (RT) and acted as a template for further extension of the cDNA strand, with incorporation of a universal SMART primer sequence. 5′ RACE was performed using the SMART RACE cDNA amplification kit. In this reaction, a universal primer mix containing a long and a short primer was applied (Table 1). The long primer, combining with the gene-specific primer, amplified the specific template in the initial cycles. With the accumulation of the gene-specific template, the higher concentration short primer acted as a main universal primer for further amplification. Thus, this PCR-suppression effect ensures that the gene-specific cDNA is preferentially amplified with dramatically reduced background.10Matz M Shagin D Bogdanova E Britanova O Lukyanov S Diatchenko L Chenchik A Amplification of cDNA ends based on template-switching effect and step-out PCR.Nucleic Acids Res. 1999; 27: 1558-1560Crossref PubMed Scopus (333) Google Scholar 5′ RACE was performed for 35 cycles at 94°C for 30 seconds, 62°C for 40 seconds, and 70°C for 5 minutes using a T-Personal thermocycler (Biometra, Horsham, PA). The amplified products were electrophoresed in a 1% agarose gel and the specific bands were purified using the QIAquick gel extraction kit (Qiagen) and cloned into pCR2.1 vector using the TA cloning kit (Invitrogen, Carlsbad, CA). Individual white clones were randomly picked from plates and cultured in 5 ml of Amp+ LB medium, and plasmids were extracted using Qiagen QIAprep Spin Miniprep Kit (Qiagen). DNA sequencing was performed using a Perkin-Elmer Applied Biosystems model 377 DNA sequencer (Perkin-Elmer, Emeryville, CA). The sequencing data were analyzed using the GCG program and BLAST search of the GenBank database. The expression of GCET1 and GCET2, relative to three housekeeping genes, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), ACTB (β-actin), and B2M (β-2-microglobulin), were measured using an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA). Primers and probes for GCET1, GCET2, ACTB, and B2M were designed using Primer Express software (Applied Biosystems), as shown in Table 1. Probe and primers for GAPDH were purchased from Applied Biosystems. The probes for GCET1, GCET2, ACTB, and B2M were labeled with FAM (carboxyfluorescein) at the 5′ ends as the reporter dye and TAMRA (carboxytetramethylrhodamine) at the 3′ ends as the quencher dye. The GAPDH probe was labeled with JOE (carboxy-4,5-dichloro-2,7-dimethoxyfluorescein) as the reporter dye and TAMRA as the quencher dye. Total RNA or mRNA was extracted from cell lines, clinical samples, and normal tissues: DHL16, SUDHL 6 (FL cell lines);11Epstein AL Levy R Kim H Henle W Henle G Kaplan HS Biology of the human malignant lymphomas IV: functional characterization of ten diffuse histiocytic lymphoma cell lines.Cancer. 1978; 42: 2379-2391Crossref PubMed Scopus (133) Google Scholar OCI-LY7, OCI-LY19 (GCB-DLBCL cell lines), OCI-LY3, OCI-LY10 (ABC-DLBCL cell lines);12Davis RE Brown KD Siebenlist U Staudt LM Constitutive nuclear factor kappaB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells.J Exp Med. 2001; 194: 1861-1874Crossref PubMed Scopus (895) Google Scholar BLCL (B-lymphoblastoid cell line derived in the authors’ laboratory); U266 (myeloma cell line);13Nilsson K Bennich H Johansson SG Ponten J Established immunoglobulin producing myeloma (IgE) and lymphoblastoid (IgG) cell lines from an IgE myeloma patient.Clin Exp Immunol. 1970; 7: 477-489PubMed Google Scholar Nalm6 (pre-B-cell line);14Han T Dadey B Minowada J Unique leukemic non-T/non-B lymphoid cell lines (REH and KM-3): absence of MLR-S and presence of suppressor cell activity for normal T-cell response.J Clin Lab Immunol. 1978; 1: 237-243PubMed Google Scholar L428 (Hodgkin lymphoma cell line);15Schaadt M Diehl V Stein H Fonatsch C Kirchner HH Two neoplastic cell lines with unique features derived from Hodgkin's disease.Int J Cancer. 1980; 26: 723-731Crossref PubMed Scopus (113) Google Scholar NK92 (NK cell line)16Gong JH Maki G Klingemann HG Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells.Leukemia. 1994; 8: 652-658PubMed Google Scholar and Jurkat (T-leukemia cell line);17Schneider U Schwenk HU Bornkamm G Characterization of EBV-genome negative “null” and “T” cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma.Int J Cancer. 1977; 19: 621-626Crossref PubMed Scopus (542) Google Scholar lymphoma samples, 11 DLBCL, 10 FL, 6 chronic lymphocytic leukemia (CLL); nonneoplastic tissues, spleen, tonsil, thymus, and peripheral blood B cells. All these RNA samples were treated with DNase I, and then 500 ng to 3 μg of RNA was transcribed into cDNA using Superscript II-reverse transcriptase (Life Technologies, Inc., Grand Island, NY) following the manufacturer's directions. A corresponding PCR on RNA from each sample (non-RT control) was performed to verify the absence of genomic DNA contamination. Real-time PCR was performed with the TaqMan Universal PCR Master Mix (Applied Biosystems) using 5 μl of cDNA with or without dilution in a 25-μl reaction mixture with a final concentration of 200 nmol/L of probe and 200 nmol/L of primers. After incubation at 50°C for 2 minutes, AmpliTaq Gold was activated by incubation at 95°C for 10 minutes. Forty PCR cycles were performed with denaturation at 95°C for 15 seconds, and combined annealing and extension at 60°C for 1 minute. Serial dilutions of cDNA from DHL16 were used to construct standard curves for the target genes (GCET1 and GCET2) and the endogenous reference genes (GAPDH, ACTB, and B2M). For each unknown sample, the relative amount of target cDNAs and reference cDNAs applied to the PCR reaction system were calculated using linear regression analysis from the corresponding standard curves. Then the normalized expression level of the target gene in each sample was calculated by dividing the quantity of the target transcript with the quantity of corresponding reference transcript. The normalized values of the target transcript were used to compare its relative expression levels in different samples. The three B-cell compartments (GC, mantle zone, and marginal zone) in 5-μm-thick frozen section of reactive tonsils or spleens were isolated using laser-captured microdissection with Arcturus PixCell II system (Arcturus Engineering, Mountain View, CA). The sections on plain glass slides were fixed with 70% ethanol for 30 seconds, washed in diethyl pyrocarbonate-treated water, stained with Mayer's hematoxylin for 30 seconds, followed by another water wash. The slide was then dehydrated with 70%, 95%, and 100% ethanol for 10 seconds each. Finally, the slide was passed through xylene twice, each for 30 seconds. A consecutive section was immunostained for CD3 to guide the dissection. Only well-defined GC, mantle zone, and marginal zone were dissected to avoid contamination with other populations. Cells were captured at the 15-μm laser setting with the laser pulse at 60 mW for 200 ms. DNA-free RNA was extracted using the Absolutely RNA MicroRNA isolation kit (Stratagene, La Jolla, CA) according to the manufacturer's instruction. After DNase I digestion, the column-adsorbed RNA was eluted into 30 μl of 10 mmol/L Tris-HCl (pH 7.5) and stored at −80°C. Total RNAs from cell lines were extracted with Trizol reagent (Invitrogen) according to the manufacturer's instruction. RNAs were separated by agarose gel containing formaldehyde and transferred to positively charged nylon membrane (Immobilon; Millipore Corp., Billerica, MA) with a vacuum transfer apparatus (Oncor Corp., Gaithersburg, MD). Plasmid clones with full-length open reading frame were labeled by random hexamer with Prime-a-Gene labeling kit (Promega, Madison, WI). Twenty-five ng of the PCR product were heated at 100°C for 3 minutes and then chilled on ice. Reaction mix and 32P-dCTP (3000 Ci/mmol, 10 mCi/ml; ICN Biomedicals, Irvine, CA) were added using the manufacturer's protocol. After a 1-hour incubation at room temperature, the unincorporated 32P-dCTP was removed by a Bio-P6 spin column (Bio-Rad, Hercules, CA). Hybridization was performed in a buffer containing 0.125 mol/L NaPO4, pH 6.8, 0.25 mol/L NaCl, 7% sodium dodecyl sulfate, 1 mmol/L ethylenediaminetetraacetic acid, 10% PEG 8000, and 50% formamide at 50°C overnight. The membrane then was washed three times with a 0.1× standard saline citrate and 0.4% sodium dodecyl sulfate solution at 70°C, and exposed to a Kodak X-OMAT film (Kodak, Rochester, NY) at −70°C in a cassette with intensifying screen. Diffuse large B-cell lymphoma (DLBCL) accounts for ∼40% of non-Hodgkin's lymphoma cases, and cDNA microarray assays have identified three gene expression subgroups of DLBCL—GC B cell-like (GCB), activated B cell-like (ABC), and type 3 DLBCL.7Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3219) Google Scholar A group of genes are preferentially expressed in the GCB-DLBCL subtype, including many known markers of GC differentiation, such as BCL-6, CD10, CD38, nuclear factor A-myb, and OGG1, as well as a number of uncharacterized genes represented as ESTs.6Alizadeh AA Eisen MB Davis RE Ma C Lossos IS Rosenwald A Boldrick JC Sabet H Tran T Yu X Powell JI Yang L Marti GE Moore T Hudson Jr, J Lu L Lewis DB Tibshirani R Sherlock G Chan WC Greiner TC Weisenburger DD Armitage JO Warnke R Lavy R Wilson W Grevor MR Byrd JC Botstein D Brown PD Staudt LM Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (8074) Google Scholar, 7Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3219) Google Scholar, 18Chan WC Huang JZ Gene expression analysis in aggressive NHL.Ann Hematol. 2001; 80: B38-B41PubMed Google Scholar Based on the cDNA microarray findings, two uncharacterized genes (IMAGE 1334260 and 814622) were selected from the GCB signature for our study. According to the cDNA microarray data,6Alizadeh AA Eisen MB Davis RE Ma C Lossos IS Rosenwald A Boldrick JC Sabet H Tran T Yu X Powell JI Yang L Marti GE Moore T Hudson Jr, J Lu L Lewis DB Tibshirani R Sherlock G Chan WC Greiner TC Weisenburger DD Armitage JO Warnke R Lavy R Wilson W Grevor MR Byrd JC Botstein D Brown PD Staudt LM Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (8074) Google Scholar, 7Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3219) Google Scholar these two genes have similar expression patterns in the selected normal and malignant lymphoid tissues studied. They are highly expressed in normal GC B cells and GCB-derived lymphomas, including FL and the GCB-DLBCL; and they are poorly expressed in both activated and resting peripheral blood B cells, resting and activated T cells, B-CLL, and the ABC-DLBCL (Figure 1). BLAST search of the GenBank database did not reveal any known genes matched to our selected EST clones. Searching the 50-kb genomic DNA region flanking either end of the selected ESTs revealed many additional clones in the human EST database, and the majority of these clones are from libraries constructed either from normal GC B cells or GCB-derived lymphoma cell lines. Two new genes were cloned from the selected EST clones (IMAGE 1334260 and 814622) and named GCET1 and GCET2, respectively. 5′ RACE for GCET1 revealed two major products of ∼1.5 and 1.8 kb (Figure 2A). After cloning and sequencing, we identified four splicing variants of 1787 bp, 1451 bp, 1488 bp, and 1547 bp, which contain an open reading frame of 435 amino acids, 335 amino acids, 337 amino acids, and 286 amino acids, respectively (Figure 2D). GCET1 contains the serine proteinase inhibitor (SERPIN) motif VSFNRTFLMMI. A Blast search of the human and animal protein databases using GCET1 protein as a query indicated that GCET1 contains the conserved SERPIN domain and has the highest homology (50 to 63%) to thyroxine-binding globulin from a variety of species, including human, sheep, rat, and mouse. The longest isoform, GCET1A (Figure 2E), was found to be identical to a previously reported gene, centerin,19Frazer JK Jackson DG Gaillard JP Lutter M Liu YJ Banchereau J Capra JD Pascual V Identification of centerin: a novel human germinal center B cell-restricted serpin.Eur J Immunol. 2000; 30: 3039-3048Crossref PubMed Scopus (30) Google Scholar which is a member of the SERPIN family, but no nucleotide sequence and protein sequence had been submitted to GenBank previously. GCET1 is located on chromosome 14q32 and spans ∼14 kb of genomic DNA. The longest isoform, GCET1A, has 5 exons (Figure 2B). On 14q32, close to GCET1 are seven other SERPIN members, including SERPIN A1, A2, A3, A4, A5, A6, and A10 (Figure 2B). Taken together, this indicated that GCET1 belongs to the SERPIN family. EST clone CS0DG007YD15 and 3064524 were confirmed to be co-localized with clone 814622 and 684286 on the same cDNA by PCR. The PCR products were cloned and sequenced. Multiple attempts at extending the sequence by 5′ or 3′ RACE beyond the most 5′ or 3′ EST clone failed to yield any additional sequence, indicating that we had or were very close to having the full-length sequence of the GCET2 transcript. The GCET2 cDNA we cloned is 3270 bp long, with 6 exons, encoding a 178-amino acid protein. It is located on chromosome 3q13.13 spanning ∼15 kb of genomic DNA (Figure 3A). Using GCET2 protein as a query to various databases showed a 34% homology identity to a human protein (accession no. AAH24174) (Figure 4A) encoded by a predicted gene located on 1q44, and 57% homology to the mouse M17 protein (germinal center expressed transcript) (Figure 4B), whose function has not been defined.20Christoph T Rickert R Rajewsky K M17: a novel gene expressed in germinal centers.Int Immunol. 1994; 6: 1203-1211Crossref PubMed Scopus (30) Google Scholar (Note: GenBank accession numbers: GCET1A, AY220118; GCET1B, AY220119; GCET1C, AY220120; GCET1D, AY220121; GCET2, AY212246.)Figure 4A: Sequence alignment between human GCET2 and AAH24174. The homology is 34% (46 of 135) (does not include positively charged amino acid residues). B: Homology between GCET2 and mouse M17 is 57% (89 of 157) (does not include positively charged amino acid residues). Bolded amino acid sequences are the possible conserved tyrosine phosphorylation sites.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Initial Northern blot hybridization of GCET1 showed a dominant band of ∼1.8 kb only in the cell line DHL-16 (Figure 2C). GCET2 has a dominant transcript of ∼1.5 kb and another transcript around 3.3 kb. Both transcripts were detected in DHL16 and the pre-B cell line Nalm-6 (Figure 3B). Faint signals were also seen with the NK-cell line, NK92. Analysis of GCET2 gene sequence showed five AATAAA polyadenylation signals located after 1540 bp (Figure 3C). According to the Northern blot results and sequence data, one or more of the proximal AAUAAA elements are likely to be functional, yielding the shorter transcript. Representative real-time PCR standard curves for ACTB, GCET1, and GCET2 are shown in Figure 5. Although the assays were very accurate and reproducible, the expression of a housekeeping gene was quite variable in different cells and tissues. Normalization using any single housek
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