Molecular cloning and expression analysis of feline melanoma antigen (MAGE) obtained from a lymphoma cell line

Molecular cloning and expression analysis of feline melanoma antigen (MAGE) obtained from a lymphoma cell line

Veterinary Immunology and Immunopathology 83 (2001) 241±252 Molecular cloning and expression analysis of feline melanoma antigen (MAGE) obtained from...

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Veterinary Immunology and Immunopathology 83 (2001) 241±252

Molecular cloning and expression analysis of feline melanoma antigen (MAGE) obtained from a lymphoma cell line Zhiyong Ma, Tanvir S. Khatlani, Li Li, Kimikazu Sasaki, Masaru Okuda, Hisashi Inokuma, Takafumi Onishi* Laboratory of Veterinary Internal Medicine, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan Received 14 May 2001; received in revised form 4 September 2001; accepted 4 September 2001

Abstract Melanoma antigens (MAGE) are regarded as inducing tumor-speci®c immune response and thought to be potential therapeutical agents for cancer immunotherapy. We hereby report the cloning of feline MAGE cDNA obtained from a lymphoma cell line derived from cat malignant lymphoma, and its expression pattern in tumor and normal tissues. The cDNA encoding the MAGE is 1668 base pairs (bp) in length, and contains an open reading frame (ORF) of 936 bp encoding a protein of 311 amino acids. The predicted amino acid sequence has 29±46% of homology with other MAGE proteins from human and mouse. mRNA transcripts for the feline MAGE were detected in certain tumors, but not in adult cat normal tissues except in testis, by reverse transcription polymerase chain reaction (RT±PCR) analysis. This indicates that the expression pattern of feline MAGE mRNA is similar to those of other MAGE family genes in tumors and normal tissues. # 2001 Elsevier Science B.V. All rights reserved. Keywords: MAGE; Tumor antigen; Lymphoma; Cat; RT±PCR

1. Introduction For treatment of cancer, a potential new therapy is designed to activate tumor-speci®c immunoresponse by using melanoma antigen (MAGE) gene products (Gillespie and Abbreviations: bp, base pairs; FeLV, feline leukemia virus; FIV, feline immunodeficiency virus; MAGE, melanoma antigen; ORF, open reading frame; pI, isoelectric point; RT±PCR, reverse transcription polymerase chain reaction; NRAGE, neurotrophin receptor-interacting MAGE homolog * Corresponding author. Tel./fax: ‡81-83-933-5893. E-mail address: [email protected] (T. Onishi). 0165-2427/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 1 ) 0 0 3 8 4 - 1

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Coleman, 1999). The MAGE gene encoding an antigen recognized by cytolytic T lymphocytes has originally been identi®ed from a melanoma cell line (Van der Bruggen et al., 1991). Since then, a series of MAGE genes were identi®ed from human, mouse and dog. These MAGE genes have been classi®ed into three groups (MAGE-A, MAGE-B and MAGE-C) in human (De Plaen et al., 1994; Lurquin et al., 1997; Lucas et al., 1998, 2000) and two groups (Mage-a and Mage-b) in mouse (De Backer et al., 1995; De Plaen et al., 1999). In dogs, a MAGE cDNA was recently cloned and sequenced (Ma et al., 2000), which is yet to be classi®ed. Several MAGE genes are expressed in tumors of different histological types, but not expressed in normal tissues except testis and placenta, indicating that the MAGE proteins are tumor-associated testis-speci®c antigens (De Plaen et al., 1994, 1999). Although the actual function of MAGE protein is still unknown, but the short peptides of certain MAGE proteins have been found to be bound by HLA class I molecules of tumor cells and presented to cytolytic T lymphocytes (Traversari et al., 1992; Chaux et al., 1999). These MAGE proteins were therefore thought to be a potential therapeutical agents for cancer immunotherapy, and some of them, under different forms, are assayed in vivo and in vitro (Park et al., 1999; Fujie et al., 1999) and signi®cant anti-tumor activity has also been observed (Marchand et al., 1999). Recently, a new, unorthodox member of the MAGE gene family, MAGE-D, has been identi®ed in human (Pold et al., 1999). MAGE-D is expressing both in normal and tumor tissues, and differs from other MAGE genes in the expression pattern, suggesting that the biology of the MAGE gene family is more complex than previously thought (Lucas et al., 1999; Pold et al., 1999). Malignant lymphomas, often associated with feline leukemia virus (FeLV) or feline immunode®ciency virus (FIV) (Neil et al., 1991; Endo et al., 1997) are the most common neoplastic disorders in cat (Hardy et al., 1976; MacVean et al., 1978) and often result in death. Expression of certain MAGE genes have already been reported in human lymphomas (Lurquin et al., 1997; Chambost et al., 2000). However, there is no report of cloning and expression analysis of feline MAGE gene in cat tumors so far, especially in cat lymphomas. Although MAGE gene was originally discovered in human melanoma cell line, but it was also found to be expressed in non-melanocytic tumors such as lung cancers, sarcomas, mammary tumors, and colon carcinomas in human (Gillespie and Coleman, 1999). To understand the expression of feline MAGE genes and to study its role in cancer immunotherapy in cats, we undertook the present work to clone and sequence the feline cDNA encoding MAGE from a lymphoma cell line and to analyze its expression pattern in tumor and normal tissues of cats. 2. Material and methods 2.1. Cell line and tissue samples A feline T-cell lymphoma cell line, KO-1, derived from a malignant lymphoma patient naturally infected with FeLV (unpublished data) was used as basic material for cloning of feline MAGE. This lymphoma cell line and other four feline lymphoma cell lines established in our laboratory for expressional analysis were maintained in RPMI1640

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medium containing 10% fetal bovine serum, and supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin. Normal tissue from 10 healthy cats and tumor tissue specimens from 15 individual cats suffering from cancer were obtained by surgical or biopsy. These specimens were immediately frozen in liquid nitrogen and maintained at 80 8C until RNA extraction. 2.2. RNA extraction and cDNA synthesis Total RNAs were isolated from the frozen tissue samples and cell lines using a RNesy Mini Kit (QIAGEN, Hilden, Germany). cDNAs were synthesized by using reverse transcriptase and an oligo-dT adapter primer (Table 1) provided with the 30 RACE System Kit (Gibco/BRL, Rockville, MD). 2.3. Primers Primers used in this study are shown in Table 1 and the positions of the respective primers in feline MAGE cDNA sequence are shown in Fig. 1. The primer pair, SP1 and AP1 were designed on the basis of the conserved regions of the human, mouse, and dog MAGE gene sequences, and other primers were designed from the cloned feline MAGE transcript sequence. The primer pair, FBAIS and FBAIR were used to amplify 389 bp feline beta-actin (GenBank accession number AB051104) as control. 2.4. Cloning a part of feline MAGE cDNA cDNA synthesized from mRNA, obtained from the cell line KO-1 was used as template in polymerase chain reaction (PCR). AmpliTaq Gold polymerase (Perkin Elmer, Foster City, CA) was used in PCR with primer pair SP1±AP1 for ampli®cation of a part of feline Table 1 Sequences of oligonucleotide primers used for RT±PCR and PCR amplification Primers

Direction

AP SP1 AP1 P0 AUAP SP2 AP2 SP3 AP3 FBAIS FBAIR a

Sense Anti-sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense

50 ±30 Sequence

Purpose

GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTTa GGYGTGATCTTCWYRAAKGb TCSAGGACTTTCATCTTGSb CATAGGCTCTTGGc GGCCACGCGTCGACTAGTACa TCCATGCCGTGAGTTCCTGT CCCATCACATTAAGCACTTCCCAC CCCTCATTGATGCCTACTGTC TGAGGAAAGTGCTTCAGGTC GGGGTCACCCACACTGTGCCCATC ACGTCACACTTCATGATGGAGTTG

For synthesis of first strand cDNA For amplifying a part of feline MAGE cDNA For amplifying cDNA ends For amplifying full coding region and expressional detection For amplifying 389 bp of feline beta-actin cDNA

Provided with the 30 RACE System Kit (Gibco/BRL, Rockville, MD). K ˆ G=T; R ˆ A=G; S ˆ C=G; W ˆ A=T; Y ˆ C=T. c 5-End of the primer was phosphorylated. b

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Fig. 1. Nucleotide and deduced amino acid sequence of feline MAGE (GenBank accession number AF325359). Asterisk is positioned to show the stop codon. The polyadenylation signal (AATAAA) is boxed. The primers (arrows), SP1 and AP1 were designed for amplification of 207 bp of feline MAGE cDNA fragment, P0, SP2 and AP2 for cDNA ends, and the pair of primer SP3±AP3 for cloning the full coding region and detecting the expression of feline MAGE mRNA by RT±PCR in tissue and cell line samples. The 738 bp of fragment used for preparing the probe generated by double digestion with HindIII and EcoRI is shadowed.

MAGE cDNA. The condition of the PCR was set as 9 min at 95 8C for an initial hot start, followed by 30 cycles of 1 min denaturation at 94 8C, 1 min annealing at 50 8C and 1 min extension at 72 8C with a 10 min ®nal extension at 72 8C and soak at 4 8C. PCR products obtained were cloned into pCR2.1 vector (Invitrogen, Carlsbad, CA) and subjected to sequence analysis by using Thermo Sequenase Pre-mixed Cycle Sequencing Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) with sequencing T7 primer or M13 reverse primer.

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2.5. Amplification of the 30 cDNA end 30 -End of feline MAGE cDNA was generated by the 30 RACE System Kit. In PCR, the gene-speci®c primer SP2 and an abridged universal ampli®cation primer (AUAP) (Table 1) provided with the kit was used. PCR condition was 1 min at 94 8C, 1 min at 65 8C and 2 min at 72 8C for 30 cycles. The products obtained were cloned into pCR2.1 vector and subjected to sequence analysis. 2.6. Amplification of the 50 cDNA end 50 -Full RACE Core Set Kit (TaKaRa, Shiga, Japan) was used, following the instructions of the supplier. The ®rst cDNA strand was synthesized from total RNA of the KO-1 cell line using primer P0. PCR ampli®cation was performed for 35 cycles of 1 min at 94 8C, 1 min at 66 8C and 2 min at 72 8C with primers SP2 and AP2. The products were cloned into pCR2.1 vector and subjected to sequence analysis. 2.7. Reverse transcription polymerase chain reaction (RT±PCR) For ampli®cation of full coding region of feline MAGE and detection of its expression pattern in cell lines and tissue samples, SuperScript One-Step RT±PCR with PLATINUM Tap Kit (Gibco/BRL, Rockville, MD) was used with primer pair SP3±AP3. Five micrograms of total RNA isolated from normal and tumor tissues or cell lines were converted into cDNA at 50 8C, and the PCR was performed for 35 cycles of 1 min at 94 8C, 1 min at 64 8C and 1.5 min at 72 8C. A part (389 bp) of feline beta-actin cDNA (GenBank accession number AB051104) used as control was also ampli®ed with primer pair FBAIS±FBAIR by the RT±PCR kit. 2.8. Hybridization probe and southern blotting DNA fragment of 738 bp (Fig. 1) was used for preparation of probe, which was generated from double digestions by HindIII and EcoRI from the full coding region of feline MAGE cDNA. The probe labeled with Fluorescein-11-dUTP was synthesized from 25 ng of the DNA fragment using ECL random prime labeling and detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the instructions of the kit. Products of RT±PCR were transferred onto Hybond±N‡ membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK) by the capillary transfer method after agarose gel electrophoresis. Hybridization and signal generation was performed, following the instructions of the kit. The signals were detected by using Hyper®lm-ECL (Amersham Pharmacia Biotech, Buckinghamshire, UK). 2.9. BLAST search and sequence alignments Sequence analyses were performed by using BLAST search at the National Center for Biotechnology Information. The deduced amino acid sequence of feline MAGE and the multiple alignments of nucleotide and amino acid sequences with its counterparts from

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other species were generated by a genetic information processing software, GENETYXMAC ver. 9.0 (Software development, Tokyo, Japan). 3. Results 3.1. Cloning and sequencing of feline MAGE cDNA Using the SP1 and AP1 primers, we obtained a 207 bp of cDNA fragment by PCR. The sequence data of this cDNA fragment was then analyzed by BLAST, which showed a high similarity to the MAGE gene family. Subsequently, primers P0, SP2 and AP2 based on the sequence of this cDNA were designed to amplify the full nucleotide sequence by methods of rapid ampli®cation of cDNA ends. The complete feline MAGE cDNA (GenBank accession number AF325359) was assembled and con®rmed by PCR with primer pairs SP3 and AP3 which cover the full coding region. The full nucleotide sequence of feline MAGE as shown along with the predicted amino acid sequence in Fig. 1 is 1668 bp in length, and contains a single open reading frame (ORF) of 936 nucleotides that encodes a predicted protein of 311 amino acids. Three consensus polyadenylation signals, AATAAA, were observed in the 30 untranslated region. 3.2. Homology with the MAGE gene family Multiple alignments of nucleotide and amino acid sequences with its counterparts from other species were generated by the genetic information processing software. Percentage of nucleotide identity between the coding region of feline MAGE cDNA and those of human MAGE-A, human MAGE-B, murine Mage-a, and murine Mage-b genes is 36±62, 57±64, 53±57, and 57%, respectively. The predicted amino acid sequence shows higher homology with those of human MAGE-B proteins (40±46% identity) and murine Mage-b proteins (37±38% identity) than human MAGE-A proteins (29±43% identity) and murine Mage-a proteins (31±34% identity) (Fig. 2). It suggests that the feline MAGE is more similar to MAGE-B family. 3.3. Putative feline MAGE protein The predicted protein of feline MAGE is devoid of signal sequence at the N-terminus but contains a potential trans-membrane domain (residue 167±177) that has also been identi®ed in the most of the MAGE proteins (Lurquin et al., 1997). A remarkable hydrophobic domain ranging from residue 82 to 256 in feline MAGE was observed and the hydrophobicity pattern of this domain is highly similar to those of other MAGE proteins (Fig. 2). The hydrophobic domain known as the MAGE homology domain is conserved in all MAGE proteins and is predicted to constitute the core of MAGE protein (De Plaen et al., 1994, 1999; De Backer et al., 1995). Isoelectric point (pI) of the putative feline MAGE protein generated by computer analysis was 9.2, which indicates that feline MAGE is basic in nature. Comparing with human MAGE-A (pI 4±4.6), human MAGE-B (pI 9±10.7), murine Mage-a (4.3±4.4), and murine

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Fig. 2. Comparison of the amino acid sequences between feline MAGE (GenBank accession number AF325359) and human MAGE-A1 (GenBank accession number NM_004988), Human MAGE-B1 (GenBank accession number U93163), mouse Mage-a1 (GenBank accession number AJ005525), and mouse Mage-b1 (GenBank accession number U19031). Asterisks indicate residues similar to those of feline MAGE. The hydrophobic residues (VILFWMY) are shadowed. The putative hydrophobic trans-membrane domains conserved across the MAGE family are boxed.

Mage-b (10.3±10.5), the pI data suggest that the feline MAGE is more similar to the B family of MAGE. The homology data described in previous section also support this suggestion. 3.4. Expression pattern of feline MAGE Expression of feline MAGE in normal and tumor tissues, and lymphoma cell lines was tested by RT±PCR with primer pair of SP3 and AP3 which cover the full coding region. The results of RT±PCR were analyzed by agarose gel electrophoresis (Fig. 3, panel A) and further con®rmed by southern blotting (Fig. 3, panel B). Feline MAGE was found expressing in certain feline tumors, such as adenocarcinoma of mammary gland (one out of ®ve), squamous cell carcinoma of skin (one out of three), lymphoma (one out of seven) and

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Fig. 3. Detection of feline MAGE expression in normal, tumor tissues and lymphoma cell lines on an agarose gel and by southern blot hybridization. The products (993 bp) of RT±PCR were visualized by ethidium bromide staining after agarose gel electrophoresis (panel A) and detected with fluorescein-labeled probe (panel B). The KO-1 cell line was used as the positive control. Feline beta-actin cDNA (389 bp) amplified by RT±PCR was used as control (panel C). Lane numbers indicate the detection of feline MAGE in adenocarcinoma of mammary gland (1 and 2), lymphoma (3), fibrosarcoma of skin (4), hepatocellular carcinoma (5), lymphoma cell line 1 (6), lymphoma cell line 2 (7), lymphoma cell line 3 (8), and lymphoma cell line 4 (9).

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Table 2 Expression of feline MAGE in normal and tumor tissues using RT±PCR Histological type

Samples tested

Positive samples

Normal adult tissues Bladder Brain Kidney Heart Liver Lung Peripheral blood lymphocytes Ovary Skin Stomach Spleen Testis Uterus

2 2 2 2 2 2 4 3 2 2 2 4 2

0 0 0 0 0 0 0 0 0 0 0 4 0

Surgical tumor samples Adenocarcinoma of lung Adenocarcinoma of mammary gland Fibrosarcoma of skin Hepatocellular carcinoma Lymphoma Squamous cell carcinoma of skin

1 5 2 1 7 3

0 1 0 0 1 1

Cell lines Lymphoma cell line

5

3

in feline lymphoma cell lines (three out of ®ve), and not detected in two ®brosarcoma of skin, one hepatocellular carcinoma and one adenocarcinoma of lung. In contrast, no expression was found in a panel of normal adult tissues of cat, with exception of testis (Table 2). This expression pattern of feline MAGE is similar to that of MAGE gene family of human in tumors and normal tissues. 4. Discussion The MAGE family is a large group of proteins that contain a well-conserved hydrophobic region of about 200 amino acids known as the MAGE homology domain (Itoh et al., 1996; Gillespie and Coleman, 1999). The MAGE proteins, which consists of this MAGE homology domain and a small amount of ¯anking sequence, were thought to be a tumorassociated testis-speci®c antigens because it expresses only in various tumors but not in normal adult tissues except testis (Gillespie and Coleman, 1999). Contrary to this, a few members of MAGE family, which consists of the MAGE homology domain and other extended domain structure, such as MAGE-D (Lucas et al., 1999; Pold et al., 1999), Necdin which is a neuron-speci®c growth suppressor that facilitates the entry of the cell into cell cycle arrest in mouse (Maruyama et al., 1991) and NRAGE (neurotrophin

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receptor-interacting MAGE homolog), interacting with the p75 neurotrophin receptor and facilitates nerve growth factor-dependent apoptosis in rat (Salehi et al., 2000), are expressed both in tumor and normal tissues. Feline MAGE protein predicted from the feline MAGE cDNA is 311 amino acids in length, similar to tumor-associated testisspeci®c MAGE proteins, which are about 300±350 amino acids in length, and contains the MAGE homology domain (Fig. 2). In addition, mRNA transcripts of feline MAGE were detected in certain tumors and lymphoma cell lines, and not in normal tissues of adult cat except in testis (Table 2), showed similar expression pattern of other tumor-associated testis-speci®c MAGE. Thus, it is believable that the present MAGE cDNA belongs to tumor-associated testis-speci®c MAGE gene family. Three different clones with high homologous cDNA sequence between themselves as well as with other MAGE genes were obtained with primer pair SP1 and AP1 by PCR ampli®cation; one of them is shown herewith (Fig. 1) whereas the remaining ones are to be cloned separately. In addition, a cDNA clone (unpublished data), which is 93.2 and 92.6% homologous in the nucleotide and amino acid sequences, respectively, to the MAGE-D (Pold et al., 1999), was also obtained by PCR ampli®cation. These results suggest the presence of MAGE gene family in cats also, like that of human and mouse. Because of the possibility of the existence of feline MAGE gene family, the RT±PCR products ampli®ed by using primer pair SP3 and AP3 from the samples of different tissue or cell lines were cloned into pCR2.1 vector and subjected to sequence analysis to test the speci®city of this primer pair. No other sequence of cDNA clones except the present feline MAGE was observed in our study, which suggests that the primer pair SP3 and AP3 is speci®c to the present feline MAGE. Thereby, it can be considered that the expression pattern of the feline MAGE identi®ed by RT±PCR is reliable. We also found that feline MAGE expressed in certain tumors and not in normal tissues of adult cat except testis (Table 2). It showed same expression patterns of the tumorassociated testis-speci®c MAGE expressing in many tumors but not in normal tissues, except in testis. Thus, it is thought to be a source of useful antigen for cancer immunotherapy. However, the information obtained regarding the expression of feline MAGE was not suf®cient for characterizing the panorama of its expression pattern in cat cancers, which will be analyzed continually in the future. The short peptides of certain MAGE proteins in human have been found to be bound by HLA class I molecules of tumor cells and presented to cytolytic T lymphocytes, to induce tumor-speci®c immune response (Traversari et al., 1992; Chaux et al., 1999; Lucas et al., 1999). The identi®cation of the peptides of feline MAGE, which can be recognized by cat cytolytic T lymphocytes is a hard work, but the results would be interesting and useful in studying its role in cancer immunotherapy in cat. References Chambost, H., Van Baren, N., Brasseur, F., Godelaine, D., Xerri, L., Landi, S.J., Theate, I., Plumas, J., Spagnoli, G.C., Michel, G., Coulie, P.G., Olive, D., 2000. Expression of gene MAGE-A4 in Reed-Sternberg cells. Blood 95, 3530±3533. Chaux, P., Luiten, R., Demotte, N., Vantomme, V., Stroobant, V., Traversari, C., Russo, V., Schultz, E., Cornelis, G.R., Boon, T., van der Bruggen, P., 1999. Identification of five MAGE-A1 epitopes recognized by cytolytic

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