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• W1988630685 abstract "cTAGE-1 is a cutaneous-T-cell-lymphoma-specific tumor antigen recently identified by serologic identification of antigens by recombinant expression cloning. This study was aimed at identifying and characterizing related genes. Rapid amplification of cDNA ends and DNA screening led to five new members of the cTAGE gene family belonging to four different genes, two of which were differentially spliced (cTAGE-1/2 and cTAGE-5). Expression analysis using reverse transcription polymerase chain reaction revealed that cTAGE-1, cTAGE-1B, and cTAGE-5A expression was restricted to testis and tumor tissues, whereas the other cTAGE members were found in two to eight other normal tissues (of 27 tissues tested). Tumor-specific protein expression of cTAGE-5 was confirmed by Western blotting. Sero-reactivity against cTAGE-1, cTAGE-4, cTAGE-5A, and cTAGE-5B was found only in tumor patients (cutaneous T cell lymphoma and melanoma). The immunogenic epitope of cTAGE-1 was determined by using epitope mapping and sera of two cutaneous T cell lymphoma patients. Moreover, cTAGE-1, cTAGE-4, cTAGE-5A, and cTAGE-5B could be detected in most types of tumor tissues and cell lines at variable frequencies, including those of cutaneous T cell lymphoma, melanoma, head and neck squamous cell carcinoma, breast carcinoma, and colon carcinoma. We conclude that cTAGE-1 and cTAGE-5 are new cancer germline antigens and that tumor-specific splicing of cTAGE genes may lead to further candidate proteins for specific immunotherapy of cutaneous T cell lymphoma and other malignancies. cTAGE-1 is a cutaneous-T-cell-lymphoma-specific tumor antigen recently identified by serologic identification of antigens by recombinant expression cloning. This study was aimed at identifying and characterizing related genes. Rapid amplification of cDNA ends and DNA screening led to five new members of the cTAGE gene family belonging to four different genes, two of which were differentially spliced (cTAGE-1/2 and cTAGE-5). Expression analysis using reverse transcription polymerase chain reaction revealed that cTAGE-1, cTAGE-1B, and cTAGE-5A expression was restricted to testis and tumor tissues, whereas the other cTAGE members were found in two to eight other normal tissues (of 27 tissues tested). Tumor-specific protein expression of cTAGE-5 was confirmed by Western blotting. Sero-reactivity against cTAGE-1, cTAGE-4, cTAGE-5A, and cTAGE-5B was found only in tumor patients (cutaneous T cell lymphoma and melanoma). The immunogenic epitope of cTAGE-1 was determined by using epitope mapping and sera of two cutaneous T cell lymphoma patients. Moreover, cTAGE-1, cTAGE-4, cTAGE-5A, and cTAGE-5B could be detected in most types of tumor tissues and cell lines at variable frequencies, including those of cutaneous T cell lymphoma, melanoma, head and neck squamous cell carcinoma, breast carcinoma, and colon carcinoma. We conclude that cTAGE-1 and cTAGE-5 are new cancer germline antigens and that tumor-specific splicing of cTAGE genes may lead to further candidate proteins for specific immunotherapy of cutaneous T cell lymphoma and other malignancies. cutaneous T cell lymphoma associated antigen rapid amplification of cDNA ends serologic identification of antigens by recombinant expression cloning Cutaneous T cell lymphoma (CTCL) summarizes a heterogeneous group of T cell malignancies characterized by primary manifestation in the skin, which originate in most cases from CD4-T lymphocytes (Burg et al., 1997Burg G. Kempf W. Haeffner A. Cutaneous lymphomas.Curr Probl Dermatol. 1997; 9: 137-204Crossref Scopus (80) Google Scholar;Willemze et al., 1997Willemze R. Kerl H. Sterry W. EORTC classification for primary cutaneous lymphomas: A proposal from the Cutaneous Lymphoma Study Group of the European Organization for Research and Treatment of Cancer.Blood. 1997; 90: 354-371PubMed Google Scholar). The most frequent forms of CTCL are Mycosis fungoides and the leukemic varient sézary syndrome (Weinstock and Horn, 1988Weinstock M. Horn J. Mycosis fungoides in the United States. Increasing incidence and descriptive epidemiology.JAMA. 1988; 260: 42-46Crossref PubMed Scopus (272) Google Scholar). Therapeutic approaches vary between the different types of CTCL, but are generally successful in early stages but often not in the aggressive, later stages of the disease. Immunologic therapies such as vaccination with peptides or peptide-loaded dendritic cells (Nestle et al., 1998Nestle F.O. Alijagic S. Gilliet M. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells.Nat Med. 1998; 4: 328-332Crossref PubMed Scopus (2640) Google Scholar;Nestle, 2000Nestle F.O. Dendritic cell vaccination for cancer therapy.Oncogene. 2000; 19: 6673-6679Crossref PubMed Scopus (67) Google Scholar;Rosenberg, 2001Rosenberg S.A. Progress in human tumour immunology and immunotherapy.Nature. 2001; 411: 380-384Crossref PubMed Scopus (1143) Google Scholar) could be a promising alternative, but validated tumor-specific antigens are still lacking for the treatment of CTCL, whereas for melanoma a variety of tumor antigens and immune responses are known (Renkvist et al., 2001Renkvist N. Castelli C. Robbins P.F. Parmiani G. A listing of human tumor antigens recognized by T cells.Cancer Immunol Immunother. 2001; 50: 3-15Crossref PubMed Scopus (384) Google Scholar). The T cell receptor (TCR) has been proposed as a tumor-specific target (Berger et al., 1998Berger C.L. Longley B.J. Imaeda S. Christensen I. Heald P. Edelson R.L. Tumor-specific peptides in cutaneous T cell lymphoma. Association with class I major histocompatibility complex and possible derivation from the clonotypic T cell receptor.Int J Cancer. 1998; 76: 304-311Crossref PubMed Scopus (52) Google Scholar) and recently an adenovirus-mediated vaccination with TCR-α and TCR-β chains fused to an immunogenic protein has been demonstrated to protect against tumor challenge in a mouse model (Wong and Levy, 2000Wong C.P. Levy R. Recombinant adenovirus vaccine encoding a chimeric T cell antigen receptor induces protective immunity against a T cell lymphoma.Cancer Res. 2000; 60: 2689-2695PubMed Google Scholar). The disadvantage of this kind of treatment is the high grade of individualization needed, and the fact that the proof of principle is still lacking in humans. Two interesting cell surface proteins have recently been discovered for CTCL (Bagot et al., 2001aBagot M. Moretta A. Sivori S. CD4(+) cutaneous T cell lymphoma cells express the p140-killer cell immunoglobulin-like receptor.Blood. 2001; 97: 1388-1391Crossref PubMed Scopus (97) Google Scholar;Nikolova et al., 2001Nikolova M. Tawab A. Marie-Cardine A. Bagot M. Boumsell L. Bensussan A. Increased expression of a novel early activation surface membrane receptor in cutaneous T cell lymphoma cells.J Invest Dermatol. 2001; 116: 731-738Crossref PubMed Scopus (24) Google Scholar), although their immunotherapeutic usage has still to be proven. CD8+ reactive infiltrates have been shown to be CTCL specific and lytic (Berger et al., 1996Berger C.L. Wang N. Christensen I. Longley J. Heald P. Edelson R.L. The immune response to class I-associated tumor-specific cutaneous T cell lymphoma antigens.J Invest Dermatol. 1996; 107: 392-397Crossref PubMed Scopus (94) Google Scholar;Bagot et al., 1998Bagot M. Echchakir H. Mami Chouaib F. Isolation of tumor-specific cytotoxic CD4+ and CD4+CD8dim+ T cell clones infiltrating a cutaneous T cell lymphoma.Blood. 1998; 91: 4331-4341PubMed Google Scholar). In a recent study,Vermeer et al., 2001Vermeer M.H. van Doorn R. Dukers D. Bekkenk M.W. Meijer C.J. Willemze R. CD8+ T cells in cutaneous T cell lymphoma: Expression of cytotoxic proteins, Fas ligand, and killing inhibitory receptors and their relationship with clinical behavior.J Clin Oncol. 2001; 19: 4322-4329PubMed Google Scholar could demonstrate that the percentage of infiltrating CD8+ T cells within biopsy samples of mycosis fungoides correlated significantly with prognosis. This suggests an important role for the CD8+ cytotoxic T lymphocytes in antitumor response in CTCL and further supports the idea of an immunologic therapy for this disease (Bagot et al., 2001bBagot M. Nikolova M. Schirm-Chabanette F. Wechsler J. Boumsell L. Bensussan A. Crosstalk between tumor T lymphocytes and reactive T lymphocytes in cutaneous T cell lymphomas.Ann N Y Acad Sci. 2001; 941: 31-38Crossref PubMed Scopus (40) Google Scholar;Edelson, 2001Edelson R.L. Cutaneous T cell lymphoma. The helping hand of dendritic cells.Ann N Y Acad Sci. 2001; 941: 1-11Crossref PubMed Scopus (90) Google Scholar). Yet, a set of specific targets is still needed in order to compose such a strategy without the drawback of immune escape from a singular antigen. By a SEREX (serologic identification of antigens by recombinant expression cloning) approach using a testis phage library and sera of patients suffering from CTCL, we have recently identified a new potential target named cTAGE-1 (CTCL associated antigen 1, AF177229), which is expressed in around 35% of the tested CTCL tumor specimens (Eichmüller et al., 2001Eichmüller S. Usener D. Dummer R. Stein A. Thiel D. Schadendorf D. Serological detection of cutaneous T cell lymphoma-associated antigens.PNAS. 2001; 98: 629-634Crossref PubMed Scopus (140) Google Scholar). The expression of cTAGE-1 within normal tissues is restricted to testis, which makes it very attractive for immune therapy as the testis is immune privileged with low or even absent expression of human leukocyte antigens (Chen and Old, 1999Chen Y.T. Old L.J. Cancer-testis antigens: Targets for cancer immunotherapy.Cancer J Sci Am. 1999; 5: 16-17Crossref PubMed Scopus (4) Google Scholar) and adds it to the still increasing family of so-called cancer-testis antigens (Old, 2001Old L.J. Cancer/testis (CT) antigens – A new link between gametogenesis and cancer.Cancer Immunity. 2001; 1: 1PubMed Google Scholar). Sequence analysis, database searches, and Northern blot analysis suggested that cTAGE-1 is an alternative splicing product of a larger gene and may belong to a gene family (Eichmüller et al., 2001Eichmüller S. Usener D. Dummer R. Stein A. Thiel D. Schadendorf D. Serological detection of cutaneous T cell lymphoma-associated antigens.PNAS. 2001; 98: 629-634Crossref PubMed Scopus (140) Google Scholar). Thus this study aimed at the identification of the cTAGE family and the evaluation of its members as putative tumor-specific targets. We have used rapid amplification of cDNA ends (RACE) elongation, library screening by DNA probes, and database analyses for unraveling homologous genes, as well as reverse transcription polymerase chain reaction (RT-PCR), Northern, Southern, and Western blotting, and SEREX for further characterization of the new genes. Tissue specimens were obtained from tumor tissues during routine surgical treatment with the permission of the local ethical board. Specimens were obtained from 19 CTCL including 14 mycosis fungoides (up to four cases stage I, five cases stage II, and five cases stage IV), three Sézary syndromes, one T-zone lymphoma, and one CD30+ lymphoma, as well as 31 colon tumors, 28 head and neck squamous cell carcinomas, and 23 breast carcinomas. Additionally, four CTCL cell lines were used for RNA isolation including the mycosis fungoides derived MyLa (Kaltoft et al., 1992Kaltoft K. Bisballe S. Dyrberg T. Boel E. Rasmussen P.B. Thestrup Pedersen K. Establishment of two continuous T cell strains from a single plaque of a patient with mycosis fungoides.In Vitro Cell Dev Biol. 1992; 28a: 161-167Crossref PubMed Scopus (110) Google Scholar), the Sézary syndrome cell line SeAx (Kaltoft et al., 1988Kaltoft K. Bisballe S. Rasmussen H.F. Thestrup Pedersen K. Boehncke W.H. Volker H. Sterry W. C-type particles are inducible in Se-Ax, a continuous T cell line from a patient with Sezary's syndrome.Arch Derm Res. 1988; 280: 264-267Crossref PubMed Scopus (13) Google Scholar), HH (lymphomatoide papulosis, ATCC number CRL-2105), and HuT-78 (Sézary syndrome, ATCC number TIB-161). As non-CTCL cell lines we used 28 melanoma cell lines, six head and neck squamous cell carcinoma cell lines, and eight colon carcinoma cell lines. A large number of control cDNAs each derived from several donor samples were used to analyze expression specificity of the new genes: human multiple fetal tissue cDNA panel (Becton Dickinson Clontech, Palo Alto, CA; content: fetal brain, heart, kidney, liver, lung, skeletal muscle, spleen, thymus) and cDNA made from RNA panels or individual RNAs (Becton Dickinson Clontech; content: bone marrow, brain, colon, heart, kidney, liver, lung, breast, ovary, placenta, peripheral blood lymphocytes, prostate, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus, trachea). All experiments handling human tissue were carried out in accordance to Helsinki principles. Total RNA was isolated using a variant of the guanidinium thiocyanate/phenol method (peqGold TriFast, Peqlab, Erlangen, Germany) and treated with DNase. cDNA was obtained using a first strand cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany). RT-PCR was done as described previously (Eichmüller et al., 2002Eichmüller S. Usener D. Jochim A. Schadendorf D. mRNA expression of tumor-associated antigens in melanoma tissues and cell lines.Exp Dermatol. 2002; 11: 292-301Crossref PubMed Scopus (36) Google Scholar). The quality of the cDNA was tested by RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer (forward, gtttacatgt tccaatatga ttccac; reverse, tcatatttgg caggtttttc tagac; 57°C annealing temperature; product size 638 bp; all primers are given in 5′3′ notation). The following primer pairs were used to distinguish all different cTAGE members, whereby primer setsIII were used for the analysis of cTAGE-1/2 splicing variants (cf. Figure 1): primer set I (forward, ctcctgactt ctttcccaac ctttacc; reverse, tgcaattccc acctaacttc cattctg; 60°C, 714 bp), primer set II (forward, ctctccccgc tgaaaatgaa gcaac; reverse, gcttagggaa gacgaaggga aggaa; 63°C, 1002 bp), primer sets I-III (forward, tagggctaac cctcagccat ac; reverse, ctccttctca aagctggcat tc; 63°C, 364 bp), cTAGE-3 (forward, aacagaaagt taatgactga gttatatc; reverse, cactctatct tcagaatgtg ggggtggg; 60°C, 1195 bp), cTAGE-4 (forward, gccataaaca gctttttcag caag; reverse, ttttaaactt ctggttctgc tggtc; 60°C, 1270 bp), cTAGE-5A (forward, ccagcacttg gcactttatt cg; reverse, tatttcatcc tcaagttcag aatt; 55°C, 578 bp), cTAGE-5B (forward, ctctccctgc tgaaaatgaa gccac; reverse, tcgttagtct catccgccaa tgcttc; 60°C, 308 bp), cTAGE-5C/MGEA-6 (forward, gtgtcatcat tttgagcttt gg; reverse, tgcttcactg cctttccttc; 50°C, 107 bp, published byHeckel et al., 1997Heckel D. Brass N. Fischer U. cDNA cloning and chromosomal mapping of a predicted coiled-coil proline-rich protein immunogenic in meningioma patients.Hum Mol Genet. 1997; 6: 2031-2041Crossref PubMed Scopus (27) Google Scholar), and cTAGE-5D/MGEA-11 (forward, agcatttggc agaggctcac; reverse, tggaatcaaa cctgagggga ac; 60°C, 706 bp). The primers had to be designed in consideration of sequence homologies between cTAGE members negotiating primer sensitivity. In order to control specificity of the RT-PCR detection, all PCR products were separated, blotted onto nitrocellulose membranes, and hybridized using digoxigenin-labeled plasmid-PCR products as probes according to the manufacturer's protocol (Roche Diagnostics). RACE-PCR (rapid amplification of cDNA ends) was done using primers located within cTAGE-1 both in 5′ and 3′ directions to get the full-length gene (5′ primer, tccttcccct tgccacagca agagaaaagc agaac; 3′ primer, tgcaagctgg gccaccaagc cattgcttc), and starting within cTAGE-2 in the 3′ direction (primer, tttcccccca cctcctccag gaaccgtg). As target, RNA from testis and CTCL cell lines was amplified using a RACE kit (SmartTM cDNA amplification kit; Becton Dickinson Clontech). Similar to the SEREX approach, a testis cDNA library was screened using cTAGE-1- and cTAGE-2-specific probes to identify homologous genes (Usener et al., 2003Usener D. Gerhardt A. Schadendorf D. Eichmüller S. Sero-reactivity against MAGE-A and LAGE-1 proteins in melanoma patients.Br J Dermatol. 2003Google Scholar). Briefly, specific DNA probes were obtained by PCR using cTAGE-1- and cTAGE-2-specific primers and the appropriate plasmids as targets. Twenty-five micrograms DNA of each PCR was digoxigenin labeled (Roche Diagnostics), pooled, and diluted in 50 ml Dig Easy Hyb solution. The testis phage library that was used for the SEREX screening and cTAGE-1 isolation (Eichmüller et al., 2001Eichmüller S. Usener D. Dummer R. Stein A. Thiel D. Schadendorf D. Serological detection of cutaneous T cell lymphoma-associated antigens.PNAS. 2001; 98: 629-634Crossref PubMed Scopus (140) Google Scholar) was recombinantly expressed and RNA was blotted to nitrocellulose membranes. Membranes were prehybridized (Dig Easy Hyb; 2 h; 45°C), and hybridized with the probes (overnight, 45°C). Positive clones were identified with a digoxigenin-specific antibody coupled to an alkaline phosphatase, which catalyzed BCIP and NBT to a violet color. Clones were isolated to monoclonality, plasmid was excised (in vivo excision according to the manufacturer's protocol; Stratagene), and the insert was sequenced. Moreover, serum reactivity to the newly identified phagemid clones was analyzed by a secondary SEREX screen as described previously (Eichmüller et al., 2001Eichmüller S. Usener D. Dummer R. Stein A. Thiel D. Schadendorf D. Serological detection of cutaneous T cell lymphoma-associated antigens.PNAS. 2001; 98: 629-634Crossref PubMed Scopus (140) Google Scholar;Usener et al., 2003Usener D. Gerhardt A. Schadendorf D. Eichmüller S. Sero-reactivity against MAGE-A and LAGE-1 proteins in melanoma patients.Br J Dermatol. 2003Google Scholar). Sera from up to 30 CTCL patients, 15 melanoma patients, and 17 healthy donors were used to screen each of the phage clones in at least two independent experiments. Using epitope mapping as previously described (Kneissel et al., 1999Kneissel S. Queitsch I. Petersen G. Behrsing O. Micheel B. Dübel S. Epitope structures recognised by antibodies against the major coat protein (g8p) of filamentous bacteriophage fd (Inoviridae).J Mol Biol. 1999; 288: 21-28Crossref PubMed Scopus (32) Google Scholar) we determined the immunogenic epitope of cTAGE-1 as recognized by patients' antibodies. Briefly, 15mer peptides with an offset by one amino acid covering all possible open reading frames on cTAGE-1 were synthesized on polyethylene-glycol-activated cellulose membrane. The membranes were blocked with 10% milk, 5% sucrose, and 1×casein block (Sigma-Genosys, Cambridge, U.K.) in TBS-T (Tris-buffered saline containing 0.05% Tween-20). Membranes were incubated with CTCL patients' or normal sera diluted 1:500 in incubation buffer (TBS-T containing 0.05% milk, 5% sucrose, 1×casein block) for 2 h followed by incubation with a peroxidase-coupled goat antihuman IgG (H+L) (Dianova) diluted 1:10,000 for 1 h at room temperature. Antibody binding was visualized by enhanced chemiluminescence staining (Amersham Bioscience). RNA from normal tissues (Becton Dickinson Clontech) as well as RNA from cell lines from different tumor entities (CTCL, melanoma, and colon carcinoma) was used for Northern blotting. RNA was blotted onto nylon membranes (Hybond N, Amersham Bioscience) and fixed by ultraviolet crosslinking. The probes were obtained by gene-specific PCR using a cTAGE-2 plasmid and were labeled by means of a Redi Prime II kit and 32P-dCTP (Amersham Bioscience). Membranes were prehybridized (2 h at 55°C), incubated with the probe overnight at 55°C, and subsequently washed in progressively higher stringency (2×sodium citrate/chloride buffer (SSC)/0.1 sodium dodecyl sulfate (SDS) at 55°C for 15 min and 0.2×SSC/0.1 SDS at 65°C for 5 min). Autoradiography was conducted at –80°C for up to 5 d. Filters were rehybridized with GAPDH probe to prove RNA integrity and to compare RNA amount. For Southern blotting, genomic DNA was isolated from the CTCL cell lines HH, HuT-78, MyLa, and SeAx (blood and cell culture DNA mini kit; Qiagen, Hilden, Germany), cut by HindIII, separated, and blotted onto positively charged nylon membranes. PCR products of cTAGE-1 and cTAGE-2 plasmids were used as probes for labeling. After prehybridization (6×SSC and 2×Denhardt; 2 h at 63°C) membranes were incubated with the specific probe (3×SSC, 1×Denhardt, overnight at 60°C) and washed (2×SSC at 55°C and 0.2×SSC at 60°C, 2×30 min each). As negative control we used a radioactive-labeled empty plasmid (pUC18). Two polyclonal rabbit antibodies (K64 and K65) were raised against synthetic peptides derived from the open reading frame of cTAGE-5A (amino acids 756–770: GLIPPSNEPATEHPE). This epitope can be found in cTAGE-5A, cTAGE-5C, and cTAGE-5D, but not in cTAGE-5B. The synthetic peptide was coupled to KLH and two rabbits were immunized and boosted for two times each. A part of each serum was purified by an affinity purification carried out on a CnBr-Sepharose column loaded with synthetic peptide (custom antibodies; synthesis of the peptides, immunization, and purification by Biogenes, Berlin, Germany). Proteins for Western blotting were isolated with the peqGold TriFast System (Peqlab) or obtained commercially (normal tissues: bone marrow, brain, colon, placenta, prostate, spleen, skeletal muscle, small intestine, trachea, stomach, testis; BioChain Institute, Hayward, CA, and Becton Dickinson Clontech). Equal amounts of protein were separated by 12% SDS polyacrylamide gel electrophoresis and blotted to a Hybond nitrocellulose membrane (Amersham Bioscience). Membranes were blocked with 5% dry milk in phosphate-buffered saline/0.1% Tween (PBS-T) for at least 1 h and washed three times in PBS-T. Membranes were incubated with the cTAGE-5A antibody (1:10,000 in PBS-T containing 0.5% milk and 0.5% bovine serum albumin BSA) overnight at 4°C and subsequently washed three times in PBS-T. A horseradish-peroxidase-conjugated antirabbit antibody was used as secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:10,000 in PBS-T containing 0.5% milk and 0.5% BSA; 1 h at room temperature). After another wash in PBS-T (three times), antibody binding was visualized by enhanced chemoluminescence (Western blot analysis system; Amersham Bioscience). After stripping (2% SDS, 62.5 mM Tris at pH 6.7, and 100 mM β-mercaptoethanol; 56°C for 30 min) equivalent loading was confirmed by an additional hybridization using a monoclonal mouse anti-β-actin antibody (1:5000; Santa Cruz). Analysis of cTAGE-1 indicated that this mRNA might be an alternative splicing variant at the 3′ end of a longer gene (Eichmüller et al., 2001Eichmüller S. Usener D. Dummer R. Stein A. Thiel D. Schadendorf D. Serological detection of cutaneous T cell lymphoma-associated antigens.PNAS. 2001; 98: 629-634Crossref PubMed Scopus (140) Google Scholar). This could be confirmed by a 5′ RACE using a testis RACE-cDNA unraveling a 4.4 kb sequence in agreement with the size depicted by Northern blot analysis (Eichmüller et al., 2001Eichmüller S. Usener D. Dummer R. Stein A. Thiel D. Schadendorf D. Serological detection of cutaneous T cell lymphoma-associated antigens.PNAS. 2001; 98: 629-634Crossref PubMed Scopus (140) Google Scholar). This gene shows a clear open reading frame from bp 113 to bp 2377 representing an 86 kDa protein and was named cTAGE-2 (Accession number AF273058). We had already identified chromosome 18p11.2 as the genomic localization of cTAGE-1 (Eichmüller et al., 2001Eichmüller S. Usener D. Dummer R. Stein A. Thiel D. Schadendorf D. Serological detection of cutaneous T cell lymphoma-associated antigens.PNAS. 2001; 98: 629-634Crossref PubMed Scopus (140) Google Scholar) and cTAGE-2 fits to the same chromosomal region as an unspliced sequence with 99.6% similarity (Figure 1). Two homologous expressed sequence tags derived from a testis library (Soares testis NHT), which were named IMAGp998F133560 (Accession number AA868597) and IMAGp998P031789 (Accession number AA399523), were obtained from the Resource Center of the German Human Genome Project (Berlin and Heidelberg) and sequenced. They fitted also to chromosome 18p11.2, but spanned over different parts of cTAGE-1 and partly cTAGE-2, with an additional gap within IMAGp998F133560, and were renamed cTAGE-1B and cTAGE-1C (Figure 1). We used gene-finding programs (GCG: Genscan, HMMGene) to search for possible promoter sequences and exons on chromosome 18p11.2. In fact, Genscan could identify a promoter 2.5 kb upstream of cTAGE-2, an initializing exon, and one terminating exon (Figure 1). As Genscan does not predict untranslated regions, the differences in the 3′ end are not relevant. The coding regions predicted by HMMGene were shorter, but spanned a larger part of chromosome 18p11.2 especially in the 3′ direction. The open reading frame of cTAGE-2 and the predicted terminal exon largely overlap, but the predicted structure at the 5′ end differed from all experimentally derived mRNA sequences (cTAGE-1, cTAGE-2, or expressed sequence tags). Thus this gene may indeed be spliced into several variants. RT-PCR analysis revealed that cTAGE-1 was present in 35% of the CTCL tumor specimens and all four CTCL cell lines, but a second primer pair II, which should detect cTAGE-2, could not be found in any of the tumor specimens (Table I). This indicates the expression of a shorter splicing variant of cTAGE-2 instead of the full-length cTAGE-2 within the normal tissues, as cTAGE-1-specific primer set I would also detect the latter. In fact, an additional 3′RACE starting within cTAGE-2 and using testis as template led to several products, among them the already known 3′ end given by cTAGE-1 (Figure 1) and another, shorter product, which can explain the above-mentioned RT-PCR results and was named cTAGE-2s (Figure 1). Thus, cTAGE-1 and cTAGE-2s were never coexpressed with the exception of testis tissue.Table 1The cTAGE gene family: chromosomal localizations and expression analyzed by RT-PCRCTCL tissues (n)cMF, mycosis fungoides; SS, Sézary syndrome; TZL, T-zone lymphoma; CD30+, CD30-positive CTCL.mRNA (bp)Putative chromosomal localizationacTAGE-1/2 belong to the same gene on chromosome 18p11.2 (cf. Figure 1), whereas cTAGE-5A through cTAGE-5D belong to the same gene on chromosome 14 (cf. Figure 4). All similarities between mRNA sequences and chromosomal sequences were larger than 99.1%.Controls (various organs)bPositive tested normal tissues: cTAGE-1, testis; cTAGE-1B, testis; cTAGE-1C, fetal brain, lung, skeletal muscle and testis; cTAGE-2s, ovary, skin, spleen, stomach, testis, and trachea; cTAGE-2, testis only; cTAGE-3, fetal brain, fetal heart, fetal spleen, colon, mammary gland, ovary, placenta, stomach, and testis; cTAGE-4, placenta, skin, and testis; cTAGE-5A, testis only; cTAGE-5B, placenta, small intestine, stomach, and testis; cTAGE-5C/MGEA-6, bone marrow, brain, colon, placenta, prostate, skeletal muscle, small intestine, testis, and trachea; cTAGE-5C/MGEA-11, colon, prostate, skeletal muscle, and testis; all other normal tissues were tested negative.MFSSTZLCD30+TotalCTCL cell lines (n=4)cTAGE-1128418p11.2285/141/3+–37%100%fPrimer set I and II positive; thus uncertain expression profile in this case.cTAGE-1B229618p11.21/280/60/3––0%100%fPrimer set I and II positive; thus uncertain expression profile in this case.cTAGE-1C134718p11.24/280/60/3––0%100%fPrimer set I and II positive; thus uncertain expression profile in this case.cTAGE-2s360718p11.26/280/60/3––0%0%cTAGE-2434518p11.21/280/60/3––0%0%cTAGE-3227713dChromosomal localization uncertain; for details see text.9/284/110/3+–31%0%cTAGE-419677eSequences from chromosome 7q33 and 6 showed a slightly lower similarity (see text).3/282/110/3––13%0%cTAGE-5A2778141/284/110/3––25%100%cTAGE-5B4110144/282/110/3+–19%0%cTAGE-5C/MGEA-63676149/281/60/3––9%25%cTAGE-5D/MGEA-112814144/280/60/3––0%25%a cTAGE-1/2 belong to the same gene on chromosome 18p11.2 (cf. Figure 1), whereas cTAGE-5A through cTAGE-5D belong to the same gene on chromosome 14 (cf. Figure 4). All similarities between mRNA sequences and chromosomal sequences were larger than 99.1%.b Positive tested normal tissues: cTAGE-1, testis; cTAGE-1B, testis; cTAGE-1C, fetal brain, lung, skeletal muscle and testis; cTAGE-2s, ovary, skin, spleen, stomach, testis, and trachea; cTAGE-2, testis only; cTAGE-3, fetal brain, fetal heart, fetal spleen, colon, mammary gland, ovary, placenta, stomach, and testis; cTAGE-4, placenta, skin, and testis; cTAGE-5A, testis only; cTAGE-5B, placenta, small intestine, stomach, and testis; cTAGE-5C/MGEA-6, bone marrow, brain, colon, placenta, prostate, skeletal muscle, small intestine, testis, and trachea; cTAGE-5C/MGEA-11, colon, prostate, skeletal muscle, and testis; all other normal tissues were tested negative.c MF, mycosis fungoides; SS, Sézary syndrome; TZL, T-zone lymphoma; CD30+, CD30-positive CTCL.d Chromosomal localization uncertain; for details see text.e Sequences from chromosome 7q33 and 6 showed a slightly lower similarity (see text).f Primer set I and II positive; thus uncertain expression profile in this case. Open table in a new tab The expression of the different splicing forms could be deduced from the combination of three different primer sets (cTAGE-1 mRNA will be amplified by primer set I, cTAGE-1B by primer sets I and II, cTAGE-1C by primer set II, cTAGE-2 by all three primer sets, and cTAGE-2s by primer sets II and III; see Figure 1). All cTAGE-1/2 splicing forms were isolated from testis libraries; thus all of them are expressed in testis tissues. Among 28 analyzed normal tissues cTAGE-1, cTAGE-1B, and cTAGE-2 could be detected only in testis, whereas cTAGE-2s and cTAGE-1C expression analysis gave positive results in 21% and 14% of the normal tissues, respectively" @default.
• W1988630685 created "2016-06-24" @default.
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• W1988630685 date "2003-07-01" @default.
• W1988630685 modified "2023-09-28" @default.
• W1988630685 title "cTAGE: A Cutaneous T Cell Lymphoma Associated Antigen Family with Tumor-Specific Splicing" @default.
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• W1988630685 doi "https://doi.org/10.1046/j.1523-1747.2003.12318.x" @default.
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