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Molecular Biology and Genetics |
Department of Pathology, University Hospital, 6500 HB Nijmegen, the Netherlands [A. J. W. Z., I. M. H. A. C., D. J. R., G. N. P. v. M.], and Roche Diagnostics GmbH, Pharma Research, D-82377 Penzberg, Germany [U. H. W.]
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ABSTRACT |
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A computer search showed strong homology with human genomic clones all localized on chromosome X (Xq26.3-Xq27.1) and with several expressed sequence tags, all from testis. Localization of the gene on chromosome X was confirmed by genomic PCR on a panel of human chromosome-specific rodent/human hybrid cell lines.
Northern blotting and reverse transcription-PCR on 17 different normal human tissue samples showed that the gene was only expressed in normal testis. Reverse transcription-PCR on a great number of different human tumor cell lines showed expression in 25–30% of the melanoma and bladder carcinoma cell lines. Only 2 of 29 other tumor cell lines were positive. Nested PCR analysis of a series of fresh human melanocytic tumors demonstrated expression in 7 of 10 melanomas tested. No expression was seen in benign melanocytic tumors. In addition to melanoma, some malignant tumors from other histological types were also found to be positive.
Based on these data, we conclude that the described gene, CTp11 (cancer/testis-associated protein of 11 kDa), is a novel member of the family of cancer/testis antigens.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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From many studies, it is known that metastatic spread requires multiple changes in the biological behavior and as a consequence, in the phenotype of the tumor cells. Many genes are differentially expressed during subsequent processes involved in metastasis. To identify genes that may contribute to the metastatic pathway of neoplastic cells, we compared mRNA expression of two closely related human melanoma cell lines, 1F6 and its metastatic variant, 1F6m, using mRNA differential display (2) .
Here we describe the isolation and first characterization of a cDNA clone isolated with this model system. Expression studies on a panel of human melanoma cell lines with different metastatic behavior after s.c. inoculation into nude mice correlated with the metastatic behavior.
Based on the expression profile in normal tissues and a series of different malignant tumor tissues tested and the chromosomal localization of the gene, we conclude that it belongs to the growing family of CTAs3 .
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human Tissues.
Lesions from all stages of melanocytic tumor progression (common nevi, atypical nevi, primary melanoma, and melanoma metastases) and other tumor specimens were excised from patients at the University Hospital Nijmegen, the Netherlands. As normal human tissues, we used disease-free samples from surgically removed tissues or from autopsies with a postmortem delay <4 h. Tissue samples were snap-frozen in liquid nitrogen and stored at -80°C until use.
RNA Isolation.
From cultured cells, total RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. From tissue samples, total RNA was isolated (following manufacturer’s protocol) by disrupting about 25 frozen sections of 20 µm thickness in 1 ml of RNAzolB (Campro, Veenendaal, the Netherlands) using a pestle. The RNAzolB method was followed by an additional RNeasy cleaning step.
mRNA Differential Display.
Before mRNA differential display PCR, DNaseI treatment was performed on the RNA samples using the Message-Clean kit (GenHunter Corporation, Nashville, TN). For differential display, the RNAmap protocol (GenHunter) was used with some minor modifications. Differing from the original protocol, we used [32P]-dATP instead of[35S]-dATP. For the PCR, combinations of the four T12MN primers together with six arbitrary primers, AP1, 2, 6, 7, 11, 12 (7)
, were used.
Northern Blotting.
Ten micrograms of total RNA were treated with glyoxal/DMSO (8)
, separated on a 1.2% agarose gel, and blotted onto a Hybond N+ membrane (Amersham, Aylesbury, United Kingdom). cDNA probes were radiolabeled by [32P]-dATP incorporation using a random-primed DNA labeling kit (Roche Diagnostics GmbH, Penzberg, Germany). Membranes were hybridized overnight with the radiolabeled probes at 65°C in a hybridization mix[0.25 M sodium phosphate buffer (pH 7.2), 7% SDS, 1% BSA, 1 mM EDTA, 0.1 mg/ml single-stranded Salmon sperm DNA]. Afterward, membranes were washed at 65°C with buffers containing decreasing amounts of salt[1% SDS, 1 mM EDTA, and 0.25–0.05 M sodium phosphate (pH 7.2)] and autoradiographed using Kodak Xomat-S films.
cDNA Library Screening, Sequencing, and Homology Searching.
cDNA probes were labeled as described before (8)
and hybridized to a 
ZAP cDNA library of the human melanoma cell line MV3, which was kindly provided by Dr. G. Swart (Department of Biochemistry, University of Nijmegen, the Netherlands). After isolation of a full-length cDNA, both strands were sequenced using the Dye Terminator Reaction Mix (Perkin-Elmer, Norwalk, CT). Homology searches were performed with Basic Local Alignment Search Tool (9)
and other software on all kinds of public servers of DNA and protein databases as described earlier (4)
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RT-PCR.
Synthesis of cDNA (10' at 25°C, followed by 59' at 42°C) was performed on 0.5–1.0 µg of total RNA using the avian myeloblastosis virus RT kit (Roche Diagnostics GmbH). The reaction mixture was supplemented with 0.04 units of random hexadeoxynucleotide primers, 2 µl of 25 mM MgCl2, 1 µl of 10 mM deoxynucleotide triphosphates, 1 µl of RT buffer[100 mM Tris/HCl (pH 8.3), 500 µM KCl], 25 U RNasin, 10 U AMV RT, and water to a final volume of 10 µl. For amplification, one tenth of the cDNA was supplemented with 2.5 µl of PCR-buffer[200 mM (NH4)2SO4, 750 mM Tris/HCl (pH 9), 0.1% Tween], 5 µl of 1 M deoxynucleotide triphosphates, 10 pmol of each primer, 2.5 µl of 15 mM MgCl2, 0.15 units of Thermoperfectplus DNA polymerase (Integro, Zaandam, the Netherlands), and water to a final volume of 25 µl. PCR conditions were 45'' at 94°C, 1' at 59°C and 1'30'' at 72°C for 30 cycles. These cycles were preceded by 3 min of denaturation at 94°C and followed by a 5-min elongation step at 72°C. The primer combination used was: sense: 5'- CTGCCGCAGACATTGAAGAA-3'; and antisense: 5'-TCCATGAATTCCTCCTCCTC-3'. The PCR product length was 297 bp. When nested PCR was performed, the conditions were 30" at 94°C, 45" at 59°C, and 1' at 72°C for 30 cycles, again preceded by denaturation and followed by elongation steps as described for the first PCR. For this nested PCR, we used 2 µl of 100 times diluted product from the first PCR, again in a total volume of 25 µl. Nested primers used were: sense: 5'-TGTGAATCCAACGAGGTGAA-3'; and antisense: 5'-TTGATTCTGTTCTCTCGGGC-3'. Nested PCR product length was 188 bp. 
2-Microglobulin primers used were: sense: 5'-CTCGCGCTACTCTCTCTTTCT-3'; and antisense: 5'-TGTCGGATTGATGAAACCCAG-3'. The 2-microglobulin PCR product length was 136 bp. DNA molecular weight markers were from Roche Diagnostics GmbH.
Chromosomal Localization.
Chromosomal localization of the gene was determined by genomic PCR on a panel of hamster/human and mouse/human hybrid cell lines (10)
. For this PCR, we used the intron enclosing primers of the first PCR shown above, yielding a 1-kb PCR product.
Plasmid Construction and Transfection.
For localization studies, we cloned a fragment (bp 1–330) containing the full-length ORF minus the termination codon in the SacI-KpnI sites of pEGFP-N3 (Clontech, Palo Alto, CA). This fuses the eGFP COOH-terminal to our fragment with a linker coding for amino acids arginine-serine-isoleucine-alanine-threonine. The in-frame junction was confirmed by sequencing. Transfections were performed using FuGENE6 transfection reagent (Roche Diagnostics GmbH). In short, cells were seeded in 6-well plates and grown till subconfluency. Transfections were performed with 1 µg of plasmid construct and 3 µl of FuGENE6 in 2 ml of medium. Transient expression of the fusion protein was checked within 48 h. Stable transfectants were created under Geneticin (Roche Diagnostics GmbH) selection (500 µg/µl).
To visualize expression of the fusion protein, cells (grown on coverslips in 6-well plates) were fixed with 4% paraformaldehyde for 15 min at room temperature and subsequently placed for 2 min in acetone at -20°C. Air-dried coverslips were put on a glass slide and mounted with 10 µl of Tris-buffered glycerol[per 100 ml: 90 ml glycerol; 2 ml Tris/HCl (pH 8); 8 ml H2O] containing 1:4 Vectashield (Vector, Burlingame, CA) and 1:10,000 4',6-diamidino-2-phenylindole (Sigma, Zwijndrecht, the Netherlands). Fluorescent images were obtained using a fluorescence microscope equipped with a Charge Couple Device camera.
Western Blotting.
Cultured cells were lysed in SDS-lysis buffer[1% SDS; 5 mM EDTA; 10 µg/ml leupeptin (Sigma); 200 µg/ml 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (Sigma), and 10 µg/ml chymostatin (Sigma) in PBS]. After centrifugation, equal protein amounts of supernatant were diluted 1:1 with nonreducing sample buffer and boiled for 5 min. These samples were size-separated using SDS PAGE on a 10% gel along with a protein marker and afterward blotted electroforetically on a nitrocellulose membrane in a blotting buffer[25 mM Tris/HCl (pH 8.6); 192 mM glycine; 20% methanol, and 0.02% SDS]. The marker-lane was separated and stained with amidoblack (0.1% amidoblack in methanol:acetic acid:water of 45:10:45) for size-reference. Previous to incubations, blots were washed for 15 min in PBS-Tween and incubated overnight at room temperature with blocking solution [PBS-Tween containing 5% low-fat milk powder and 0.01% antifoam A (Sigma)]. The blot was incubated for 1 h with anti-eGFP polyclonal rabbit antiserum as the first antibody and with peroxidase-labeled swine-antirabbit antiserum (Dako, Glostrup, Denmark) as the second antibody. All incubations were performed in blocking solution, and after each step, the blot was washed three times for 10 min in PBST. Detection was done with enhanced chemiluminescence (Roche Diagnostics GmbH) according to the manufacturer’s protocol. Blots were then exposed to Kodak Xomat-S films and developed.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. No expression could be detected in the intermediate and low metastatic cell lines.
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ZAP cDNA library of the MV3 melanoma cell line using the 300-bp cDNA fragment as a probe. A 408-bp cDNA was picked up (European Molecular Biology Laboratory: accession no. AJ238277). Sequencing revealed a perfect 3' match with the probe used and showed an ORF coding for a protein of 97 amino acids (Fig. 2)
. This putative protein contains a possible bipartite NLS (amino acids 40–57), although it is not completely consensus (11)
. Another remarkable feature is its high content of glutamic acid residues (14%) resulting in an acidic COOH-terminal cluster (amino acids 83–89). Overall, one-third of the residues are charged (18 negative; 14 positive), and the expected molecular weight is 11 kDa. The protein has a calculated isoelectric point of 5.0.
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655 bp, which is located near bp 112. The homology on the cDNA level was restricted to several human ESTs, which were all from testis (zt95b09; qg57b01; qe04h11; EST95628/EST95629) and which all code for the same putative protein. No substantial homology was found with clones of other species.
Expression Profile.
In addition to Northern blotting of our panel of human melanoma cell lines with known metastatic behavior, we also performed RT-PCR on RNA of these cell lines. The PCR results confirmed the expression pattern of the melanoma cell lines seen on Northern blots (Table 1)
. A specific product could only be detected in the highly metastatic cell lines 1F6m, MV3, and BLM. RT-PCR analysis on corresponding xenograft material also showed an expression profile that completely matched with the expression profile of the cultured cell lines (not shown). Analysis of a larger series of human melanoma cell lines not studied in our nude mouse model showed expression in 3 (BRO, E10, and 518A2) of 15 cell lines (Table 2)
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, we found expression in 5 of 17 bladder carcinoma cell lines, whereas 6 kidney carcinoma cell lines and 7 prostate carcinoma cell lines did not express the gene. Finally, of 16 cell lines from histological types other than the ones already mentioned, only 2 were positive (fibrosarcoma HT1080 and osteosarcoma U2OS).
Expression of the gene in normal human tissues determined by RT-PCR is shown in Fig. 3
. From 17 different tissue samples tested, only the testis was found to be positive.
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. Nested RT-PCR analysis showed that the PCR product was only detectable in advanced stages of melanocytic tumor progression. Three of four primary melanomas and four of six melanoma metastases were positive. No expression was found in normal skin, common naevus naevocellularis, and atypical nevus. Primary cultures of normal human foreskin melanocytes and cultures of naevus cells were also negative (not shown).
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. Using nested PCR in the normal tissue expression was only seen in the three testis samples, which were already positive after the first PCR (30 cycles). The other normal tissues did not reveal any PCR product. In the tumor samples, only sporadic expression was seen: lung (1 of 5), breast (1 of 4), colon (2 of 9), and bladder (1 of 11). Pancreas (n = 5) and esophagus (n = 6) tumors were negative. Regarding the testis lesions studied, 10 of 17 tumor samples were positive only after nested PCR, whereas three normal testis samples were positive already after the first round of PCR.
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it is evident that the transfected cells express the fusion protein. No specific band is seen in the lane containing lysate of nontransfected BLM cells. From the difference in size of eGFP (27 kDa) and the fusion protein (38 kDa), we deduced the size of the protein to be about 11 kDa. Based on the mRNA expression profile and the molecular weight, we named the protein CTp11: cancer/testis-associated protein of 11 kDa.
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, whereas COS-1 cells expressing the fusion protein showed specific nuclear localization of the product (Fig. 6, D–F)
; nucleoli appear negative for the fusion protein. Transfection of the human melanoma cell line BLM showed comparable results (not shown) with identical nuclear localization.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These CTAs, expressed in testis and cancers, are potential immunotherapy candidates (13) . Because the testis is an immunological privileged site, lacking human lymphocyte antigen expression necessary for antigen presentation and direct contact with cells of the immune system, only the cancerous cells will be targeted by the cytotoxic T cells (14 , 15) after immunotherapeutic treatment. Some preliminary results have been reported for immunotherapy with MAGE-derived peptides (16) .
The putative protein encoded by the ORF in the full-length cDNA has a bipartite-like NLS. Although this was not completely consensus, we did find a specific nuclear localization after fusing the ORF in front of eGFP, indicating that the bipartite-like nuclear localization sequence is indeed effective (Fig. 6)
. The bipartite NLS consensus comprises 2 basic amino acids [lysine (K) or arginine (R)] separated by a region of 10 amino acids from a basic cluster in which three of the next five residues must be basic (11)
. The spacer of 10 amino acids was shown to be optimal, although effective bipartite NLSs were also found with elongated spacers (17)
. This indicates the likeliness of the bipartite sequence in CTp11 (amino acids 40–57), with a 12-residue spacer being responsible for localization in the nucleus. Bipartite NLS sequences have also been found in several members of the SSX family, which also belong to the group of CTAs (18)
. The acidic COOH-terminal region with the high content of glutamic acid residues resembles a GAL4 domain shown to be effective in transcriptional activation after interaction or fusion with a DNA-binding protein or domain (19)
. The fact that CTp11, like the SSX proteins (18)
and melanocyte-specific gene 1 (20)
, lacks a DNA-binding domain strongly suggests that it interacts with the transcription-initiation complex to concert its putative transcriptional regulation. The high percentage of charged amino acids might contribute in such a protein-complex interaction.
The expression profile of CTp11 in normal tissues, tumor cell lines, and tumor samples places the gene in the group of CTAs, which already includes MAGE (21) , BAGE (22) , GAGE (23) , SSX (24) , NY-ESO-1 (14) , LAGE-1 (25) , PAGE-1 (26 , 27) , and SCP-1 (28) .
Criteria genes should fulfill to be considered as a member of the family of CTAs are formulated in the literature (14 , 29) : (a) predominant expression in testis and generally not in other normal tissues; (b) induction/activation of mRNA expression in a wide range of human tumors; (c) expression in malignancies in a lineage-nonspecific fashion; (d) often existence of multigene families; and (e) mapping of the gene, with some exceptions, on the X-chromosome. CTp11 clearly qualifies as a CTA family member because it shares criteria (a), (b), (c), and (e). Regarding the presence of CTp11 highly homologues family members, further investigation is required.
The CTp11 gene is localized on the X chromosome (Xq26.3-Xq27.1) and consists of two exons separated by an intron of about 655 bp as confirmed by PCR. This position is right next to the MAGE-C subfamily (Xq26; Ref. 21 ) and nearby CTAG (the gene for NY-ESO-1) and the MAGE-A cluster (both Xq28; Ref. 30 ).
CTp11 expression was found in 25–30% of the melanoma and bladder tumor cell lines tested, whereas cell lines established from other tumor types were only sporadically positive. For melanoma cell lines, which are the best studied regarding CTA expression, the percentage of positivity is comparable to the percentages of positivity for NY-ESO-1 (18%; Refs. 14 and 25 ), SSX-2 (25%; Ref. 31 ), and MAGE-B1 and -B2 (22% and 33%; Refs. 32 and 33 ). MAGE-A1 (66%; Refs. 15 and 34 ) has a markedly higher expression coverage in melanoma cell lines and is expressed in 41% of other human tumor cell lines.
Testis-specific expression regarding normal tissues seen by RT-PCR confirms the exclusiveness of CTp11 homology with only ESTs from testis. In fresh human tumor samples, we found melanoma to have the highest percentage of CTp11 positivity (70%; n = 10). Comparable positivity was seen in primary (three of four) as well as metastatic melanoma (four of six). This percentage may be one of the highest compared to the other CTAs in melanoma, being 8, 17, 22, 35, 44, 44, and 52% for SCP-1, GAGE, BAGE, MAGE-1, SSX-2, NY-ESO-1, and MAGE-3 (35) , respectively. The relatively high percentage of CTp11 in bladder cell lines (30%) was not detected in bladder tumor samples in which only 1 of 11 was found to be positive.
The absence of CTp11 in benign and preneoplastic stages of melanocytic tumor progression suggests an induction of expression in melanoma lesions rather than an up-regulation of expression. Demethylation of the X chromosome in testis and cancer might be an important factor in this induction of expression, as described previously for some CTAs (21 , 28 , 32) . However, specific modulation of this demethylation or additional factors are necessary to explain the differential expression patterns and the differences in percentages of positivity of the various CTAs in different tumor types (24) .
Interestingly, testis tumors showed a down-regulation of CTp11 expression compared to normal testis tissue because only 10 of 17 tumor lesions were positive after nested PCR. No positivity of testis tumor samples was seen after the first PCR. Positivity of these testis lesions, both seminomas and nonseminomas, could be caused by small amounts of normal testis tissue present. Another explanation may be that the tumor in the testis arose from a CTp11-negative cell type. Further studies, preferentially using antibodies, are necessary to address this question and to reveal which of the cells in normal testis and testis tumors express CTp11. Regarding the other CTAs, it is known that MAGE-1 is mainly expressed in germ cells of the testis (36) , and this finding is in line with the fact that seminoma have a higher positivity rate for MAGE expression than nonseminoma tumors (37) .
Concerning the function of CTp11, we can only speculate about a role in transcriptional regulation based on its nuclear presence in combination with its COOH-terminal negatively charged acidic domain, as seen in several transcription factors (19 , 38) . Biological functions for most CTAs are unknown thus far. Only for SCP-1, a meiotic protein, has aberrant expression been implicated in genomic instability of cancer cells (28) . SSX-2 characteristics point out toward a possible role in the negative control of proliferation (39) . MAGE-1 and MAGE-3 have been described as cytoplasmic proteins (40) .
In summary, we isolated and characterized a new gene which, based on several criteria described in literature, can be placed in the family of CTAs. Although expression was seen in several tumor types, melanoma had the highest percentage of expression. Like some other CTAs, the CTp11 protein is located in the cell nucleus.
For the near future, it would be interesting to see if CTp11 contributes to additional positivity of tumor samples (especially melanoma) negative for other known CTAs, thereby making it attractive for use in immunotherapeutic treatment. Thus far, coverage of about 75% of melanoma samples positive for at least one CTA can be achieved (35) . Immunotherapy using multiple tumor antigens might increase the efficacy of treatment and minimize tumor cell escape (41) . Usefulness of CTp11 in differential diagnosis of melanocytic lesions and perhaps correlation with patient survival will need more extensive studies with larger numbers of tumor lesions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was partly supported by Grant NUKC 98-1782 from the Dutch Cancer Society. 
2 To whom requests for reprints should be addressed, at Department of Pathology, University Hospital, P. O. Box 9101, 6500 HB Nijmegen, the Netherlands. Phone: 31-243614368; Fax: 31-243540520; E-mail: H. Zendman{at}pathol.azn.nl
3 The abbreviations used are: CTA, cancer/testis antigen; RT, reverse transcription; NLS, nuclear localization signal; MAGE, melanoma antigen.
Received 8/12/99. Accepted 10/15/99.
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(2)-macroglobulin receptor and its receptor-associated protein in late stages of cutaneous melanocytic tumor progression. Cancer Res., 56: 1432-1439, 1996.-smooth muscle actin expression in cultured human brain pericytes by transforming growth factor- 1. Am. J. Pathol., 144: 372-382, 1994.[Abstract]
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