Genomic Organization, Incidence, and Localization of the SPAN-X Family of Cancer-Testis Antigens in Melanoma Tumors and Cell Lines

Vol. 10, 101–112, January 1, 2004 Clinical Cancer Research 101

1 1 V. Anne Westbrook, Pamela D. Schoppee, immunoreactive proteins migrated between Mr 15,000 and 1 1 Alan B. Diekman, Kenneth L. Klotz, Mr 20,000 similar to those observed in spermatozoa. Immu- Margaretta Allietta,1 Kevin T. Hogan,2 noperoxidase labeling of melanoma cells and tissue sections 2 3 demonstrated SPAN-X protein localization in the nucleus, Craig L. Slingluff, James W. Patterson, cytoplasm, or both. Ultrastructurally, in melanoma cells 3 4 Henry F. Frierson, William P. Irvin, Jr., with nuclear SPAN-X, the protein was associated with the Charles J. Flickinger,1 Michael A. Coppola,1 and nuclear envelope, a localization similar to that observed in John C. Herr1 human spermatids and spermatozoa. Significantly, the inci- 1Departments of Cell Biology, 2Surgery, 3Pathology, and 4Obstetrics dence of SPAN-X-positive immunostaining was greatest in and Gynecology, Center for Research in Contraceptive and the more aggressive skin tumors, particularly in distant, Reproductive Health, University of Virginia, Charlottesville, Virginia nonlymphatic metastatic melanomas. Conclusions: The data herein suggest that the SPAN-X protein may be a useful target in cancer immunotherapy. ABSTRACT Purpose: Members of the SPAN-X (sperm protein as- INTRODUCTION sociated with the nucleus mapped to the X chromosome) Molecular characterization of human tumor antigens rec- family of cancer-testis antigens are promising targets for ognized by the cellular or humoral immune systems of cancer tumor immunotherapy because they are normally expressed patients has generated new prospects for the development of exclusively during spermiogenesis on the adluminal side of cancer vaccines (1–3). The list of human cancer antigens de- the blood-testis barrier, an immune privileged compart- fined to date falls into three major categories: (a) differentiation ment. antigens; (b) mutational antigens; and (c) viral antigens. Cancer- Experimental Design and Results: This study analyzed testis (CT) antigens are a distinct class of differentiation anti- the human SPANX genomic organization, as well as SPAN-X gens that are viewed as attractive candidates for cancer vaccines, mRNA and protein expression in somatic and cancer cells. because the expression of CT antigens in normal tissues seems The SPANX family consists of five genes, one of which is to be restricted to the testis. Malignant transformation is asso- duplicated, all located in a gene cluster at Xq27.1. From the ciated with activation or derepression of these normally silent centromere, the arrangement of the five SPANX genes genes resulting in CT antigen gene expression in a variable mapped on one contiguous sequence is SPANXB, -C, -A1, portion of many human tumors. No tumor specimen or cell line -A2, and –D. Reverse transcription-PCR analyses demon- screened to date has been found to express all of the defined CT strated expression of SPAN-X mRNA in melanoma and antigens (1–3). ovarian cell lines, and virtual Northern analysis established CT antigens represent good targets for immunotherapy, SPANX gene expression in numerous cancer cell lines. Im- because they are widely distributed in a number of tumors and munoblot analysis using polyclonal antisera raised against are present primarily in normal testes. The testis is considered an recombinant SPAN-X confirmed the translation of SPAN-X immune privileged site due to the presence of the blood-testis proteins in melanoma and ovarian tumor cell lines. The barrier, which inhibits contact between differentiating germ-line and immune cells, and to the apparent lack of HLA class I expression on the surface of germ cells (4). These considerations suggest that only cancerous cells will be targeted by CTLs Received 4/21/03; revised 8/11/03; accepted 8/14/03. during CT antigen immunotherapy. Unfortunately, many of the Grant support: U54HD29099 and D43TW/HD00654 (J.C.H.) from the Fogarty International Center, a pilot project grant funded through the CT antigens studied to date are first expressed in spermatogonia National Cancer Institute Cancer Center, Support Grant to the Univer- or spermatocytes located in the basal compartment of the sem- sity of Virginia Cancer Center (M. A. C.), and a grant from the Cancer iniferous epithelium, the nonprotected side of the blood-testis Antigen Discovery Collaborative of the Cancer Research Institute and barrier. Thus, targeting of these antigens may reduce or elimi- the Ludwig Institute for Cancer Research (M. A. C.). nate spermatogenesis and compromise the subsequent fertility The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked of the patient. In contrast, the identification and validation of CT advertisement in accordance with 18 U.S.C. Section 1734 solely to antigens, which lie on the adluminal (protected) side of the indicate this fact. blood-testis barrier, may reduce the chances of inducing auto- Requests for reprints: John C. Herr, Department of Cell Biology, immune orchitis (5, 6) and permanent injury to the testes as a Center for Research in Contraceptive and Reproductive Health, Univer- sity of Virginia, P. O. Box 800732, 1300 Jefferson Park Avenue, result of cancer immunotherapy. Furthermore, polyvalent CT Charlottesville, Virginia 22908. Phone: (434) 924-2007; Fax: (804) 982- antigen vaccines comprised of a mixture of immunogens repre- 3912; E-mail: [email protected]. sent an important strategy for obtaining adequate therapeutic

responses given the variable incidence of CT antigen expression reverse transcriptase kit (Qiagen, Valencia, CA) according to in tumors. For these reasons, it is important to identify and the protocol provided therein. An RNase inhibitor (10 units) was ␮ characterize new CT antigens, particularly those that are ex- added to the reaction and oligo(dT)18 (1 M) was used as the pressed postmeiotically and are immunogenic in humans. primer. The reactions were incubated at 37°C for 60 min and We have characterized a testis-specific protein designated then at 95°C for 5 min to inactivate the enzyme. Subsequently, SPAN-X (sperm protein associated with the nucleus on the X PCR was performed on 2 ␮l of cDNA using the HotStarTaq chromosome; Refs.7–9). Ultrastructurally, the proteins are as- DNA polymerase and master mix kit (Qiagen). The reaction sociated with the nuclear envelope of ejaculated spermatozoa. In contained specific primers published previously (10) with mod- transfected mammalian CV1 cells the SPAN-Xa protein local- ifications. The forward primers were 5Ј-CTGCCRCWGACAT- ized to the nucleus, whereas SPAN-Xb localized to the cyto- TGAAGAA-3Ј (SPAN-XA, C and D) and 5Ј-CTACTGTA- plasm. SPAN-X mRNAs were expressed exclusively after mei- GATCGAAGAA-3Ј (SPAN-XB) mixed at a 3:1 molar ratio; the osis in the testis in round and elongating spermatids (7). The reverse primer was 5Ј-TCYATGAATTCCTCCTCCTC-3Ј. Am- human SPANX gene was mapped to chromosome Xq27.1. The plification was performed over 30 cycles at 94°C for 1 min, expression of SPAN-X, an X-linked gene product, exclusively 58°C for 30 s, and 72°C for 30 s. in haploid spermatids leads to interesting questions regarding Western Blot Analysis. For SDS-PAGE, snap-frozen the transcription of sex-linked genes in a precise temporal and cell pellets were resuspended in 2ϫ Laemmli sample buffer (7), spatial sequence during spermiogenesis and transcriptional reg- vortexed, boiled for 10 min, and centrifuged at 16,000 ϫ g for ulation of this highly regulated gene during tumorigenesis. 15 min. The supernatants were transferred to clean tubes and SPAN-Xc, also designated CTp11, was identified previ- diluted with an equal volume of distilled water. One dimen- ously as a CT gene by comparing mRNA expression between a sional SDS-PAGE was performed on 15% acrylamide gels 6 human parental melanoma cell line and its metastatic variant using 2 ϫ 10 cell equivalents of protein extract per lane and the using mRNA differential display (10). A recent study demon- polypeptides transferred onto nitrocellulose (14). A 1% SDS strated the expression of SPAN-Xb in myeloma and other he- extract of sperm (40 ␮g per lane) was included as a positive matological cancers (11). Significantly, high titer antibodies control for the SPAN-X protein. All of the samples were ob- against SPAN-X were present in sera of these patients, but tained with informed consent using forms approved by the absent in healthy individuals, confirming the immunogenicity of University of Virginia Human Investigation Committee. After the SPAN-X differentiation antigen. In the present study, we transfer, the blots were washed with PBS [150 mM NaCl and 10 examined the incidence of SPAN-X expression in several tumor mM sodium phosphate (pH 7.4)], incubated with blocking buffer types and placed particular emphasis on SPAN-X protein ex- (PBS, 0.05% Tween 20, and 5.0% nonfat dry milk), and then pression in melanomas. We determined that SPAN-X protein, a with guinea pig anti-SPAN-X antiserum diluted 1:1000 in postmeiotic, germ cell marker, was present in more than half the blocking buffer. SPAN-X antiserum was produced by immuniz- melanoma tumors examined and that its presence correlated ing guinea pigs with recombinant SPAN-X protein as described with tumor progression. These findings prompted a detailed previously (8). Pre-immune serum was used as a negative con- study of SPANX gene organization that revealed a multigene trol for immunostaining. After washing three times in PBS- family of five members closely linked on Xq27.1. In coopera- Tween, blots were incubated with horseradish peroxidase- ϩ Ј tion with the Human Genome Nomenclature Committee, the conjugated donkey antiguinea pig IgG (H L) F(ab )2 fragments nomenclature for the SPANX gene family was established, and (Jackson ImmunoResearch, West Grove, PA) diluted 1:2000 in the ordering of the SPANX gene cluster was determined. blocking buffer and washed again. Immunoreactive proteins were visualized with TMB membrane peroxide substrate (KPL, Gaithersburg, MD). MATERIALS AND METHODS Immunoperoxidase Labeling of Tissue Sections. Cell Culture. Melanoma cell lines were derived from SPAN-X protein localization in tumor cells was examined by tumor digests obtained from patients at the University of Vir- immunoperoxidase labeling of paraffin-embedded tumor sec- ginia as described (12, 13). The ovarian cancer cell lines tions. For fresh specimens, washed cells or tissues were fixed CaOV3, ES-2, MDAH2774, OvCar3, SKOV3, and SW626 for several hours with 10% neutral-buffered formalin (Sigma). were obtained from the American Type Culture Collection The samples were dehydrated through an ethanol series, embed- (Manassas, VA). All of the cell lines were cultured in RPMI ded in paraffin, sectioned, and mounted onto slides. In addition, 1640 supplemented with 10% fetal bovine serum, penicillin, and multiple archival samples of paraffin-embedded tumor speci- streptomycin. Cells were washed once with Dulbecco’s PBS, mens and tissue microarrays were obtained from the Department and the pellets were snap-frozen in liquid nitrogen and stored at of Pathology, University of Virginia. Normal testis sections Ϫ80°C for subsequent protein or RNA extraction. served as positive controls for SPAN-X protein localization. Reverse Transcription-PCR (RT-PCR) Analysis of Before use, sections were dewaxed, rehydrated, and treated with Tumor Cell Lines and Clinical Tumor Specimens. mRNA 0.25% hydrogen peroxide to block endogenous peroxidase ac- was isolated with Tri Reagent (Molecular Research Center, Inc., tivity. Nonspecific protein binding sites were blocked by incu- Cincinnati, OH). To detect low abundance SPAN-X transcripts, bating the slides in PBS with 5% normal goat serum. Slides reverse transcription of total RNA followed by PCR amplifica- were incubated with immune guinea pig anti-SPAN-X antisera tion of the SPAN-X cDNA (or control ␤-actin cDNA) was diluted in PBS with 1% normal goat serum. SPAN-X antiserum performed. Briefly, 2 ␮g of total RNA from each tissue were was produced by immunizing guinea pigs with recombinant reverse transcribed in a 20-␮l reaction using the Omniscript SPAN-X protein as described previously (8). The slides were

washed and incubated with horseradish peroxidase-conjugated SPANXD gene expression in the testis has been confirmed Ј 10 F(ab )2 fragments of goat antiguinea pig IgG (Jackson Immu- recently in our laboratory. noResearch) in PBS-normal goat serum. Preimmune serum was Each of the five genes, including SPANXB, is represented used as a negative control for immunostaining. Slides were by two exons with a small intron of 648 bp. The order of the washed with PBS, immunoreactive proteins visualized by stain- SPANX genes from the centromere is SPANXB, SPANXC, ing with the 3,3Ј-diaminobenzidine peroxidase substrate, and SPANXA1, SPANXA2, and SPANXD. These results demon- counterstained with H&E. Slides were visualized using an strated that SPANX comprises a multigene family consisting of Olympus BH-2 microscope, and digital images were obtained at least four unique members, one of which is duplicated in the using Image-Pro Plus software (Media Cybernetics, Des human genome, all of which are tightly clustered on Xq27.1. Moines, IA). Other SPAN-X cDNA sequences, designated SPANXE Immunoelectron Microscopy. For postembedding im- (AJ457795), SPANXF1 (AJ457796), and SPANXF2, have been munolabeling, washed VMM150 cells were fixed on ice with annotated in the NCBI database. It seems unlikely that these 4% formaldehyde, 0.5% glutaraldehyde, and 1% tannic acid in sequences represent additional SPANX loci but rather represent 0.1 M sodium phosphate buffer (pH 7.4), rinsed in buffer, polymorphisms of the loci described above. For example, the dehydrated through an ethanol series, and embedded in Lowic- SPAN-Xe (AJ457795) sequence maps to the SPANXD locus and ryl K4M resin (Electron Microscopy Sciences, Ft. Washington, exhibits only three nucleotide substitutions compared with the PA). Thin sections were mounted on nickel grids and immuno- SPAN-Xd mRNA (AF312765). SPAN-Xf1 and -f2 map to the stained. Primary guinea pig antibodies were used at a dilution of SPANXB locus and exhibit only one nucleotide difference com- 1:200, and 5-nm gold-conjugated secondary goat antiguinea pig pared with the SPAN-Xb mRNA (AF098307), and this substi- antibodies (Goldmark, Phillipsburg, NJ) were used at a dilution tution does not change the amino acid sequence. Each of these of 1:50. Grids were rinsed with PBS, rinsed with water, stained nucleotide differences has been described in the single nucleo- with uranyl acetate, and carbon coated. Specimens were viewed tide polymorphism database. Thus, the SPANX gene family and photographed with a JEOL 100CX electron microscope. consists of five closely linked genes with multiple polymor- Darkfield images were obtained from the photographs using phisms. Image-Pro Plus software (Media Cybernetics). The proteins encoded by the SPANX genes are presented in Bioinformatics. Analyses of the SPAN-X genes were Fig. 2. The SPANXA1, SPANXA2, SPANXC, and SPANXD performed using Basic Local Alignment Search Tool (BLAST) genes encode highly similar proteins of 97 amino acids. the Human Genome5 and National Center for Biotechnology SPANXB encodes the SPAN-Xb protein that is 103 amino acids Information (NCBI) Map Viewer.6 Multiple sequence alignment in length. The SPAN-Xb protein sequence differs from SPAN- was performed using the ClustalW program at the European Xa, SPAN-Xc, and SPAN-Xd at the NH2 and COOH termini, Molecular Biology Laboratory-European Bioinformatics Insti- and contains a six amino acid insertion. No homologous se- tute.7 Virtual Northern analysis was performed against SAGE quences or motifs, with the exception of consensus nuclear (serial analysis of gene expression) libraries using identified localization signals described previously (8), have been identi- SAGE tags.8 Analysis of the expressed sequence tags databases fied to date for the SPAN-X proteins. was performed using nucleotide BLAST and MEGABLAST at SPAN-X mRNA Is Expressed in a Variety of Tumor NCBI. Single nucleotide polymorphisms were identified from Cell Lines. To examine the expression of the SPANX gene in the dbSNP database at NCBI.9 tumors, virtual Northern analysis was performed by searching the SAGE and expressed sequence tag databases with SPAN-X RESULTS cDNA sequences. The SAGE database uses serial analysis of SPANX Is a MultiGene Family. Genomic analysis dem- gene expression to quantify transcript levels in both malignant onstrated that the SPANX genes comprise a multigene family on and normal human tissues. Using the SAGE tag sequence, Ј Ј Xq27.1. SPANX genomic sequences include nonredundant hu- 5 -GAAATAATGG-3 , present in SPAN-Xa, SPAN-Xc, and man DNA sequences on one contiguous genomic sequence SPAN-Xd, a variety of cancer cell lines expressing SPANX, (NT_011786.13) at 138 M to 138.7 M on the X chromosome were identified (Table 1). These cell lines include: (a) GBM from human genome build 33 (Fig. 1). Within this contiguous H1110, a glioblastoma multiforme primary tumor derived from sequence, two identical copies of SPANXA, designated a 51-year-old male; (b) three treatment groups of MCF7, derived SPANXA1 and SPANXA2, were identified in opposing orienta- from a mammary gland carcinoma; (c) DCIS-3 and DCIS-5, tions. The SPANXA1 and SPANXA2 genes encode the SPAN-Xa derived from two patients with mammary gland ductal in situ mRNA and protein. One copy each of SPANXB, SPANXC, and high grade carcinomas; (d) DCIS-6 from a vascular endothelium SPANXD have also been identified on this contiguous sequence. breast carcinoma; (e) a mammary gland carcinoma metastasis, designated B2; (f) ES2–1, an ovarian clear cell carcinoma cell

5 Internet address: http://www.ncbi.nlm.nih.gov/genome/seq/page.c- gi?F ϭ HsBlast.html&&ORG ϭ Hs. 10 V. A. Westbrook, P. D. Schoppee, A. B. Diekman, C. J. Flickinger, 6 Internet address: http://www.ncbi.nlm.nih.gov/mapview/. M. A. Coppola, J. C. Herr. The SPAN-X family of genes are differen- 7 Internet address: http://www.ebi.ac.uk/clustalw/. tially expressed during spermiogenesis and SPAN-X segregates in hu- 8 Internet address: http://www.ncbi.nlm.nih.gov/SAGE/. man and chimpanzee spermatids to non-acrosomal domains during 9 Internet address: http://www.ncbi.nlm.nih.gov/SNP/. nuclear envelope morphogenesis, manuscript in preparation.

Fig. 1 Mapping of the SPANX gene family to the genomic contig NT_011786.13 between 138 M and 138.7 M on human chromosome X. Five SPANX genes were identified, in differing orientation (arrows) on the DNA strands, including identical duplications of SPANXA. Other genes or putative genes that have been mapped to this region are indicated, including six cancer-testis antigens of the MAGE gene family. The SPANX family is tightly clustered. Only two genes intervene between the SPANXB and SPANXC genes, and two also intervene between SPANXC and SPANXA, whereas SPANXA and SPANXD are adjacent in build 33. Two of the intervening genes are pseudogenes.

line; (g) a prostate carcinoma cell line PC3 AS2; (h) two brain were also identified in prostate tumors (15), chronic myeloge- ependymomas R582 and R1023; and (i) a normal first trimester nous leukemia (accession no. BF212177), and amelanocytic placental cell line. No SAGE tags were identified in the melanoma (accession no. BM554791). These data indicate that, SPAN-Xb sequence by BLAST analysis of expressed sequence whereas in normal tissue SPANX is expressed exclusively in the tag databases; SPANX gene expression, including sequences testis, SPANX is expressed in a number of histologically distinct corresponding to all four of the SPANX gene transcripts, was tumors, a characteristic shared with other CT antigens. identified in several libraries from normal testes and germ cells SPAN-X mRNA expression in melanoma and ovarian can- but not from any other normal somatic tissues. These data are cer cell lines was also examined using RT-PCR (Fig. 3). A consistent with the testis-specific expression of SPAN-X ob- 297-bp band at the expected size for SPAN-X was observed in served previously for somatic tissues by Northern and dot blot normal human testis, the melanoma cell lines VMM150 and analyses (9). Expressed sequence tags homologous to SPAN-X VMM12, and the ovarian cancer cell lines MDAH2774,

Fig. 2 Comparison of the deduced amino acid sequences of SPAN-Xa, SPAN-Xb, SPAN-Xc, and SPAN-Xd. In all of the peptide sequences, three overlapping consensus nuclear localization signals were observed (boxed). The deduced amino acid sequences contained an unusually large percentage of charged amino acid residues. One potential site of N-linked glycosylation was observed in the SPAN-Xb deduced peptide sequence within a six amino acid insertion (underline).

Table 1 The table summarizes the results of SAGEa analysis of the query sequence GAAATAATGG specific to SPAN-Xa, c, and d Library search query: GAAATAATGG Total tags in library Tags per 200,000 SAGE breast carcinoma CL MCF7, control 0h 59877 46 SAGE breast carcinoma CL MCF7, estradiol 10H 59583 16 SAGE breast carcinoma CL MCF7, estradiol 3h 59683 16 SAGE breast carcinoma B DCIS-5 43098 9 SAGE ovary carcinoma CL ES2-1 31159 6 SAGE breast carcinoma metastasis B 2 49794 4 SAGE prostate carcinoma CL PC3 AS2 40768 4 SAGE brain ependymoma B R582 52189 3 SAGE breast carcinoma AP DCIS-3 57402 3 SAGE universal reference human RNA CL 51729 3 SAGE vascular endothelium breast carcinoma AP DCIS6 65314 3 SAGE brain glioblastoma B H1110 68986 2 SAGE placenta first trimester normal B 1 89265 2 SAGE brain ependymoma B R1023 122690 1 a SAGE, serial analysis of gene expression.

ϳ high molecular weight band of Mr 90,000 was present in all of the cell lines examined but not in spermatozoa. Interestingly, this band was consistently detected in cultured human cell lines, but not in extracts of any normal or cancerous primary human tissue samples examined to date (data not shown). This may represent a cross-reactive protein that shares a SPAN-X epitope or epitopes or a nonspecific reactivity induced in guinea pigs after administration of Complete Freund’s Adjuvant. No immunoreactive bands were observed in preimmune serum. These data are consistent with the expression of SPAN-X mRNA in

Fig. 3 Reverse transcription-PCR analysis of SPAN-X mRNA expression in melanoma and ovarian cancer cell lines. A 297-bp band at the expected size for SPAN-X was observed in normal human testis, the melanoma cell lines VMM150 and VMM12, and the ovarian cancer cell lines MDAH2774, SKOV3, SW626, OVCar3, and ES-2. No SPAN-X transcripts were observed in normal ovary, the ovarian cancer cell line CaOV3, or the melanoma cell lines VMM5 and VMM18.

SKOV3, SW626, OVCar3, and ES-2. No SPAN-X transcripts were observed in normal ovary, the ovarian cancer cell line CaOV3, or the melanoma cell lines VMM5 and VMM18. These observations indicate that the expression of SPAN-X in tumors is variable, as has been observed for other CT antigens. SPAN-X Protein Is Translated in Tumor Cell Lines. To examine the distribution and molecular weight of SPAN-X protein in tumor cells, SDS extracts were prepared from four melanoma cell lines, the ES2 ovarian tumor cell line (American Type Culture Collection #CRL 1978), and ejaculated human Fig. 4 Western blot analysis of SPAN-X protein in tumor cell lines. In spermatozoa. The proteins were separated by reducing one- extracts from VMM150 and ES2 cell lines, postimmune sera reacted dimensional SDS-PAGE and immunoblotted as described in with bands migrating between Mr 15,000 and Mr 20,000 similar to that “Materials and Methods.” In extracts from VMM150 and ES2 observed in human spermatozoa. The lowest molecular weight isoforms of SPAN-X appear to be absent from the tumor extracts, and immuno- cell lines, postimmune sera reacted with bands migrating be- Ͼ reactive bands with apparent molecular weights Mr 20,000 are also tween Mr 15,000 and Mr 20,000 similar to that observed in seen in VMM150. VMM5, VMM12, and VMM18 protein in this human spermatozoa (Fig. 4). Immunoreactive bands with molecular weight range were not reactive with polyclonal antibodies slightly higher apparent molecular weights were also detected in generated against recSPAN-X. No immunoreactive bands were observed in preimmune serum. The immune serum also reacted with a high VMM150. Protein extracts from VMM5, VMM12, and ϳ molecular weight band of Mr 90,000 in all of the cell line extracts, but VMM18 did not show SPAN-X immunoreactivity in the ex- not in sperm or extracts from a variety of normal human tissues (data not pected Mr 15,000–20,000 molecular weight range. However, a shown).

Fig. 5 Immunohistochemical localization of SPAN-X was performed on paraffin-embedded tissues from human testis (A) and the melanoma cell lines VMM5 (B) and VMM150 (C and D). SPAN-X immunoreactive protein was observed as a brown reaction product within the nucleoplasm and was con- centrated at the nuclear periphery of round spermatids (red arrows). In elongated spermatids, SPAN-X protein staining was observed at the base of the sperm head, corresponding to the redundant nuclear envelope (purple arrowhead). A portion of the VMM150 cells were positive for nuclear staining whereas other cells were negative (blue arrows). SPAN-X staining of apparent nuclear envelope invaginations was observed in some VMM150 cells (green arrowhead). Only background staining was observed in the VMM5 melanoma cells (B).

VMM150 and ES2 observed by Northern blot and SAGE anal- tions of the nuclear envelope (Fig. 5D, green arrowhead). Not yses, respectively. Furthermore, these results confirm translation all of the VMM150 cells demonstrated SPAN-X immuno- of SPAN-X immunoreactive proteins in the melanoma cell line staining (Fig. 5C and D, blue arrows). VMM150 and the ovarian cancer cell line ES2. Immunohistochemical localization of SPAN-X was also SPAN-X Protein Localizes to the Nuclear Envelope in a performed on paraffin-embedded sections obtained from the Melanoma Cell Line and in Spermatids. To examine the tumor sample that gave rise to the VMM150 cell line (Fig. 6). localization of SPAN-X protein in tumor cells, two melanoma As in the VMM150 cell line (Fig. 6A), SPAN-X was localized cell lines, VMM150 and VMM5, and the metastatic melanoma to the interior and periphery of the nucleus of cells in this tumor, tumor from which the VMM150 cell line was derived were a distant metastatic melanoma (Fig. 6B). Preimmune serum fixed in paraformaldehyde and embedded in paraffin for immu- demonstrated only slight background staining in all of the tis- noperoxidase staining with anti-recSPAN-X antiserum. Human sues and cells examined (Fig. 6A and B). Similar localization testis sections served as a positive control for SPAN-X protein was seen in formaldehyde-fixed, air-dried cells immunolabeled localization (Fig. 5A). A brown reaction product from the 3,3Ј- with SPAN-X antiserum and fluorescent conjugated secondary diaminobenzidine substrate indicated a positive localization. antibodies (data not shown). This immunolocalization in the SPAN-X protein was localized to the nuclear envelope of round metastatic melanoma tumor is consistent with SPAN-X mRNA spermatids (Fig. 5A, red arrows) and to the posterior head, and protein expression in the derived melanoma cell line corresponding with the redundant nuclear envelope in elongat- VMM150 as observed by Northern and Western blot analyses, ing spermatids (Fig. 5A, purple arrowhead). This localization respectively. Furthermore, localization of the SPAN-X protein was consistent with that observed previously in ejaculated sper- to the nuclear envelope of the patient tumor specimen correlates matozoa (7, 8). The melanoma cell line VMM5 demonstrated no with the observations in the VMM150 melanoma cell line and expression of SPAN-X mRNA or protein as demonstrated by the localization observed previously in spermatids, spermato- Northern and Western blot analyses, respectively, and served as zoa, and SPAN-Xa transfected CV1 cells (7, 8). an additional negative control. The VMM5 cells demonstrated To examine the subcellular localization of SPAN-X protein only background staining of the cytoplasm possibly due to the in melanoma cells by electron microscopy, postembedding im- high molecular weight cross-reactive protein seen by Western munolabeling was performed on VMM150 and VMM5 mela- analysis (Fig. 5B). In contrast, the VMM150 cells demonstrated noma cells using anti-SPAN-X antibodies and gold-labeled sec- strong SPAN-X immunoreactivity at the nuclear periphery, ondary antibodies. Gold particles representing SPAN-X protein within the nucleoplasm close to the nuclear envelope, and slight were observed overlying the nuclear envelope of VMM150 cells reactivity of the surrounding cytoplasm (Fig. 5C and D). In (Fig. 7A and B). However, the association of gold particles with some VMM150 cells, SPAN-X reactivity was seen in invagina- any specific structural elements of the nuclear envelope such as

Fig. 6 Immunohistochemical localization of SPAN-X was performed on paraffin-embedded tissues from both the melanoma cell line VMM150 (A and AЈ) and from the VMM150 patient tumor (B and BЈ). A and B were immunostained with guinea pig anti-recSPAN-X immune serum, and AЈ and BЈ were incubated with guinea pig preimmune serum. SPAN-X immunoreactive protein was observed on the nuclear periphery of melanoma cells from the cell line (A) and the metastatic melanoma tumor (B).

the nuclear lamina or pore complexes could not be resolved cells and the absence of reactivity in VMM5 cells is consistent using this method of fixation and staining. Only background with the data on SPAN-X protein and mRNA expression in labeling was observed in VMM5 cells (data not shown). The these two cells lines. Cells incubated with preimmune serum localization of SPAN-X to the nuclear envelope of VMM150 showed only background labeling (data not shown). SPAN-X Localization in Skin Tumors Correlates with Tumor Aggressiveness. Immunohistochemical localization of SPAN-X protein was performed on paraffin-embedded tissues from a number of skin tumor patients. Table 2 summarizes the results of the immunohistochemical localization of SPAN-X on sections of 21 skin tumors of varying degrees of aggressiveness. Of these 21 tumors, 10 demonstrated positive immunoreactivity for SPAN-X (48%). Localization of SPAN-X was observed in the nucleus (60%), cytoplasm (20%), or both (20%). Significantly, immunostaining for SPAN-X protein correlated with increased aggressiveness of skin tumors, particularly in distant, nonlymphatic metastatic melanomas. Fig. 8A shows a metastatic melanoma with positive SPAN-X staining in the cytoplasm. Fig. 8B shows a metastatic melanoma demonstrating heavy staining of the nucleus. Fig. 8C and D are skin metastatic melanomas show- ing SPAN-X staining of the cytoplasm and nucleus, respectively. A distant metastatic melanoma of the parotid gland demonstrates nuclear localization of SPAN-X (Fig. 8E). Fig. 8F shows a metastatic melanoma with no SPAN-X staining. Slides incubated with preimmune serum showed no positive staining (data not shown). The nuclear localization of SPAN-X (Fig. 8B, D, and E) is consistent with localization of SPAN-Xa and SPAN-Xc in transfected mammalian cells (7, 10), whereas the cytoplasmic staining (Fig. 8A and C)is Fig. 7 Electron micrograph (A) and darkfield image (B) of VMM150 consistent with the transfection of SPAN-Xb (7). The differ- melanoma cells after postembedding immunolabeling with anti- ential localization of SPAN-X in tumor cells may be a result SPAN-X antibodies and 5 nm gold-conjugated secondary antibodies. of differential expression of the multiple SPANX genes in Gold particles representing SPAN-X protein were associated with the nuclear envelope of VMM150 cells. C ϭ cytoplasm, m ϭ mitochondria, individual tumors and may prove useful in diagnosing and/or n ϭ nucleus, and NE ϭ nuclear envelope. treating tumors.

Table 2 The table summarizes the results of the immunohistochemical localization of SPAN-X on skin tumor sections Twenty-one tumors of varying degrees of aggressiveness were examined. Of these 21 tumors, 10 demonstrated positive immunoreactivity for SPAN-X (48%; 10 of 21). In melanomas specifically, 53% (9 of 17) of the samples demonstrated SPAN-X protein. Localization of SPAN-X was observed in the nucleus, cytoplasm, or both. Significantly, SPAN-X immunostaining correlated with increased aggressiveness of skin tumors, particularly in distant, nonlymphatic metastatic melanomas. Nevi Melanoma Metastatic Staining Normal Congenital Dysplastic In situ Radial Vertical LN Other Nuclear 1 1 1 3 Cytoplasmic 11 Nuclear and cytoplasmic 1 1 Negative 1 2 1 1 2 4 Total 0/1 1/1 0/2 1/2 1/2 2/4 1/5 4/4

DISCUSSION genome as multigene families. For example, Ͼ30 melanoma SPANX Genes Comprise a MultiGene Family. From antigen (MAGE) gene loci have been identified including 13 the centromere, the arrangement of the five tightly clustered MAGEA genes, 4 MAGEB genes, and several MAGE-like hy- SPANX genes, on genome build 33, is SPANXB, SPANXC, pothetical genes for which evidence of transcription has not yet SPANXA1, SPANXA2, and SPANXD between 138 M and 138.7 been found. The majority of the MAGE genes map to human M on Xq27.1. The existence of multiple SPANX genes is con- chromosome X on both the long and short arms, and the cluster sistent with other CT antigens that are present in the human of SPANX genes is in close proximity to the loci of a number of

Fig. 8 Immunohistochemical localization of SPAN-X protein in skin tumors. Paraffin- embedded tissues were immunostained with anti-recSPAN-Xa immune serum. A, metastatic melanoma demonstrates positive SPAN-X staining in the cytoplasm. B, metastatic melanoma demonstrates nuclear localization of SPAN-X. C, skin metastatic melanoma shows SPAN-X staining of the cytoplasm. D, skin metastatic melanoma shows SPAN-X staining of the nucleus. E, distant metastatic melanoma of the parotid gland demonstrates nuclear localization of SPAN-X. F, metastatic melanoma with no SPAN-X staining.

MAGE genes and LDOC1 (leucine zipper, down-regulated in islands were identified Ͻ65,000 bp from any of the six SPANX cancer 1), also known as BCUR1 (breast cancer, up-regulated 1). coding sequences. Thus, it seems unlikely that methylation/ Interestingly, expression of these closely linked genes has been demethylation of the SPANX promoter is involved directly in implicated in tumorigenesis. For example, SPANX, MAGEC3 SPANX gene expression during spermatogenesis and/or tumor (HCA2), MAGEC1, MAGEA10, and MAGEE1 are members of progression. gene families that encode CT antigens and are found in a SPAN-X Protein Is Expressed in a Large Number of number of melanomas and other tumors (16–21). LDOC1 is Melanomas. Our results confirmed a high incidence of located adjacent to the SPANXC locus and was identified by SPAN-X protein expression in melanoma tumors, verified the screening for genes expressed in cancer cells using RNA dif- testis specificity of SPAN-X, and demonstrated a pattern of ferential display (22). The high number of cancer-related genes postmeiotic gene expression in the adluminal compartment of in this region of the X chromosome suggests a high suscepti- the blood-testis barrier. The presence of SPAN-X protein in bility to dysregulation during tumor progression. 48% of skin tumors and 53% of melanomas suggests that The mapping of SPANX to the q27 region of the X chro- SPAN-X may prove to be a useful marker for staging melanoma mosome additionally emphasizes the potential importance of tumor progression and that studies of additional cohorts of SPAN-X in tumorigenesis. Rapley et al. (23) mapped the locus tumor samples should be conducted. Detection of SPAN-X for familial testicular germ cell tumors, designated TGCT1,to protein in over half of the melanomas tested in the present study Xq27. Testicular germ-cell tumors (TGCTs) affect 1 in 500 men is generally comparable with the nested RT-PCR results of and are the most common cancer in males ages 15–40 in Zendman et al. (10), who identified SPAN-X mRNA in 70% of Western Europe. The known risk factors for TGCT include melanoma tumors, but not in normal skin. In a recent study undescended testis and a family history of the disorder, demon- using RT-PCR analysis of 52 melanoma samples in three tumor strating a greater risk in brothers consistent with X-linked in- stages (37), SPAN-X was identified predominantly in primary heritance. Linkage analysis of the set of families compatible melanomas: 45% in primary tumors, 27% in local metastases, with X linkage provided evidence for a TGCT predisposition and 10% in distant metastases. In the same study, the CT antigen locus at Xq27. In addition to TGCT1, a prostate susceptibility NY-ESO-1 was found in the majority of local metastasis (45%) gene, symbolized HPCX, has also been mapped to Xq27-q28 and distant metastatic (50%) melanomas but in only 10% of and is thought to account for 16% of hereditary prostate cancers primary tumors. It is not yet clear whether the difference in the (24, 25). Although other genes may be embedded within these incidence of mRNA expression in the two cited studies and the cancer susceptibility loci, SPANX is one of the few genes incidence of protein localization in the present study is indica- identified in this region to date. Furthermore, the unique distri- tive of translational regulation of SPAN-X. The possibility that bution of SPAN-X in 50% of human spermatozoa, its germ extremely sensitive and nonquantitative nested RT-PCR ap- cell-specific expression, and its identification in human tumors proaches may simply overestimate the incidence of protein also implicate the SPANX genes in the etiology of TGCT and/or expression for CT antigens such as SPAN-X must also be prostate cancer. considered, because these assays allow the efficient detection of The mechanism(s) of tumor expression of these testis- transcripts that may be present only at very low copy numbers specific genes is not well understood. The lack of coordinate in a small subset of cells within a specimen. Larger controlled expression of different CT antigen family members rules out a studies, including studies that correlate mRNA and protein general activation of silent X-linked genes in cancer and indi- detection with serological data, are indicated to additionally cates a selective gene-specific regional activation. This could be refine the incidences of SPAN-X gene expression and protein due to aberrant promoter/enhancer binding factors by cis-or localization in melanomas and other tumor types. trans- genes, or to specific modification in DNA methylation or The nuclear localization of SPAN-X in melanomas is con- acetylation patterns. During spermatogenesis, the germ cell ge- sistent with localization of SPAN-Xa (7) and SPAN-Xc (10) nome becomes more methylated, particularly at CpG dinucle- found in mammalian cells transfected with these constructs, otides (26). Demethylation of testis-specific gene promoters whereas the cytoplasmic localization is consistent with transfec- occurs before the activation of transcription during spermato- tion results for SPAN-Xb (7). These transfection experiments genesis (27, 28). For MAGE, both methylation and DNA bind- indicated that the nuclear localization signals found in the ing factors appear to be involved in aberrant expression of this SPAN-X protein sequences were effective in trafficking gene family (29–34). The expression of MAGE in tumor cells the SPAN-X a and c proteins to the nucleus. However, the seems to be a result of genome-wide demethylation that occurs SPAN-Xb protein apparently contains a variation that inhibits frequently during tumor progression (31, 35, 36). In addition, nuclear targeting in somatic cells. The open reading frame of the demethylating agent 5-aza-2Ј-deoxycytidine can induce the SPAN-Xb contains a consensus N-linked glycosylation site expression of the MAGE genes in MAGE-negative cells sug- within the six amino acid insert that is not present in the other gesting that gene activation results from the demethylation of SPAN-X protein sequences. Glycosylation of this asparagine in the promoter region (29–34). These data suggest that promoter the transfected cells expressing SPAN-Xb would account for the methylation is a shared mechanism involved in regulating the increased molecular weight of the SPAN-Xb protein observed expression of CT antigens in metastatic melanomas. Further- by immunoblotting and may relate to its localization in the more, the clinical use of 5-aza-2Ј-deoxycytidine may provide a cytoplasm (7). Thus, localization of the SPAN-X protein to the new chemoimmunotherapeutic strategy for melanoma patients. cytoplasm in some melanomas may be the result of SPAN-Xb Using the computer algorithm Grail CpG Islands and NCBI expression and glycosylation. Similarly, the differential local- Map Viewer on the genomic contig NT_011786.13, no CpG ization of SPAN-X to both the nucleus and cytoplasm in tumor

cells may be a result of differential expression of the multiple puberty, when the immune system has long been established, the SPANX genes in individual tumors and may prove useful in development of active tolerance to male germ cell autoantigens diagnosing and/or treating tumors. is of considerable importance. Immune privilege in the testis is The SPAN-X protein is associated with the nuclear enve- established as a result of both a physical barrier and an immu- lope in 80% of melanomas positive for SPAN-X staining. This nosuppressive microenvironment. The structural barrier, com- is consistent with the localization of SPAN-X to the redundant posed largely of tight junctional complexes between adjacent nuclear envelope in spermatozoa. Unlike the nuclear envelope Sertoli cells, separates the seminiferous epithelium into two of the anterior part of the sperm head, which is tightly apposed cellular compartments, basal and adluminal. From an immuno- to the nuclear chromatin and lacks nuclear pores, the redundant logical perspective, this barrier has two effects: (a) it prevents nuclear membranes, embedded within the cytoplasmic droplet, circulating antibodies from entering the seminiferous tubules have a wider apposition between their lamellae and possess beyond the Sertoli cell tight junctions; and (b) it isolates the numerous, regularly arranged nuclear pore complexes. Although meiotic and postmeiotic spermatogenic cells from the immune the SPAN-X peptide sequences do not contain FG-repeat system of a body in a compartment where differentiation anti- nucleoporin domains (38), the SPAN-X protein from spermato- gens are expressed and may be recognized as “foreign.” The zoa is highly insoluble suggesting that this protein is a structural lack of HLA gene expression in the testis may also be necessary protein or associated with structural elements of the nuclear to create a protecting environment to prevent T-cell-mediated envelope (7). The localization of SPAN-X to the redundant autoimmune reactions (4, 48). nuclear envelope and its structural characteristics suggests that Thus, it may be predicted that immunization of cancer SPAN-X may be a component of a large macromolecular struc- patients with the SPAN-X protein, which is expressed in normal tural complex such as the nuclear pore. Additional studies are testis after puberty and is protected from the immune system by under way to determine whether SPAN-X is associated with the the blood-testis barrier, should produce an immune response to nuclear pore complexes in spermatids, spermatozoa, and cancer this differentiation antigen in both male and female patients, cells expressing SPAN-X. Although the predominantly nuclear whereas being unlikely to induce autoimmune orchitis in males. localization of SPAN-X in tumors and tumor cell lines suggests The lack of SPAN-X expression in adult somatic tissues predicts that SPAN-X nuclear epitopes may be inaccessible to antibody that this protein will be immunogenic in humans, a prediction or T cells invoked by a therapeutic vaccine, the results of the verified by the recent demonstration that SPAN-Xb was aber- recent study by Wang et al. (11) demonstrating high-affinity rantly expressed and elicited immune responses in patients with antibodies to SPAN-X in cancer patients supports the hypothesis myeloma and hematological cancers (11). These considerations that SPAN-X peptides are presented at the surface of tumor cells support the possible inclusion of SPAN-X proteins in immuno- in the context of MHC or that SPAN-X protein is released therapeutic vaccines. Indeed, SPAN-X appears to be the first CT during necrosis. These hypotheses now require verification. antigen restricted to the adluminal compartment that has shown SPAN-X Protein Is on the Protected Side of the Blood- immunogenicity in humans. This finding indicates that the iden- Testis Barrier. In the testis, the MAGE CT antigens are tification of additional postmeiotic CT antigens may be impor- expressed in spermatogonia and primary spermatocytes (39). tant to the development of safe polyvalent CT antigen vaccines, MAGE-A1 and MAGE-A3 have been localized to the cyto- which will address the variability of CT antigen expression in plasm, but MAGE-A11 is present in the nucleus (40–42). tumors, whereas simultaneously mitigating pathological conse- MAGE proteins have limited homology to the tumor suppressor quences such as immune orchitis. protein p53 in a region important for the growth of p53 inhib- itory properties (2), suggesting that MAGE may function in the Note Added in Proof control of tumor proliferation, although no experimental evi- An article by Furlong et al., Journal of the National Cancer Insti- dence for such activity has yet been found. The CT antigen tute, 1999, indicates that the SW626 shown in Figure 3 is a cell line of NY-ESO-1 is also expressed in spermatogonia and localized to colon origin, rather than ovary as previously described. A recent article the cytoplasm (43). The CT antigen SCP-1 is a synaptonemal by Zendman et al., Gene, 2003, has recently described in part the complex protein that is expressed during meiotic prophase in genomic organization of the SPANX genes. spermatocytes and is thought to function in the alignment of homologous chromosomes during meiosis, the promotion of REFERENCES cross-over events, and chromosome segregation (44). Except for these examples, the expression pattern, localization, and func- 1. Gure, A. O., Tureci, O., Sahin, U., Tsang, S., Scanlan, M. J., Jager, E., Knuth, A., Pfreundschuh, M., Old, L. J., and Chen, Y. T. SSX: a tion of other CT antigens in the testis remain unknown despite multigene family with several members transcribed in normal testis and their growing number and importance in tumor immunology, human cancer. Int. J. Cancer, 72: 965–971, 1997. highlighting the value of generating anti-CT antigen antibodies 2. Kirkin, A. F., Dzhandzhugazyan, K., and Zeuthen, J. Melanoma- to characterize their localizations and function in normal testic- associated antigens recognized by cytotoxic T lymphocytes. Apmis, ular cells and tumors. 106: 665–679, 1998. Unlike the CT antigens noted above, SPAN-X is expressed 3. Chen, Y. T., and Old, L. J. Cancer-testis antigens: Targets for cancer immunotherapy. Cancer J. Sci. Am., 5: 16–17, 1999. in postmeiotic spermatids. This is interesting, as few X-linked 4. Fiszer, D., and Kurpisz, M. Major histocompatibility complex ex- genes exhibit postmeiotic expression (45). Thus, the expression pression on human, male germ cells: a review. Am. J. Reprod. Immu- of SPAN-X mRNA and protein in haploid spermatids places nol., 40: 172–176, 1998. SPAN-X on the protected, adluminal side of the blood testis 5. Tung, K. S., and Teuscher, C. Mechanisms of autoimmune disease in barrier (46, 47). Because spermatozoa appear in males only after the testis and ovary. Hum. Reprod. Update, 1: 35–50, 1995.

6. Filippini, A., Riccioli, A., Padula, F., Lauretti, P., D’Alessio, A., De Tucker, K., Donald, J., Collins, F., Friedlander, M., Hogg, D., Goss, P., Cesaris, P., Gandini, L., Lenzi, A., and Ziparo, E. Control and impair- Heidenreich, A., Ormiston, W., Daly, P. A., Forman, D., Oliver, T. D., ment of immune privilege in the testis and in semen. Hum. Reprod. Leahy, M., Huddart, R., Cooper, C. S., Bodmer, J. G., et al. Localization Update, 7: 444–449, 2001. to Xq27 of a susceptibility gene for testicular germ-cell tumours. Nat. 7. Westbrook, V. A., Diekman, A. B., Naaby-Hansen, S., Coonrod, Genet., 24: 197–200, 2000. S. A., Klotz, K. L., Thomas, T. S., Norton, E. J., Flickinger, C. J., and 24. Peters, M. A., Jarvik, G. P., Janer, M., Chakrabarti, L., Kolb, S., Herr, J. C. Differential nuclear localization of the cancer/testis-associ- Goode, E. L., Gibbs, M., DuBois, C. C., Schuster, E. F., Hood, L., ated protein, SPAN-X/CTp11, in transfected cells and in 50% of human Ostrander, E. A., and Stanford, J. L. Genetic linkage analysis of prostate spermatozoa. Biol. Reprod., 64: 345–358, 2001. cancer families to Xq27–28. Hum. Hered., 51: 107–113, 2001. 8. Westbrook, V. A., Diekman, A. B., Klotz, K. L., Khole, V. V., von 25. Stephan, D. A., Howell, G. R., Teslovich, T. M., Coffey, A. J., Kap-Herr, C., Golden, W. L., Eddy, R. L., Shows, T. B., Stoler, M. H., Smith, L., Bailey-Wilson, J. E., Malechek, L., Gildea, D., Smith, J. R., Lee, C. Y., Flickinger, C. J., and Herr, J. C. Spermatid-specific expres- Gillanders, E. M., Schleutker, J., Hu, P., Steingruber, H. E., Dhami, P., sion of the novel X-linked gene product SPAN-X localized to the nucleus of human spermatozoa. Biol. Reprod., 63: 469–481, 2000. Robbins, C. M., Makalowska, I., Carpten, J. D., Sood, R., Mumm, S., Reinbold, R., Bonner, T. I., Baffoe-Bonnie, A., Bubendorf, L., Heis- 9. Westbrook, V., Diekman, A., Coonrod, S., Klotz, K., Buer, S., and kanen, M., Kallioneimi, O. P., Baxevanis, A. D., Joseph, S. S., Zucchi, Herr, J. Characterization of SPAN-X in the mammalian testis and I., Burk, R. D., Isaacs, W., Ross, M. T., and Trent, J. M. Physical and transfected cells. Biol. Reprod., 62: 582a, 2000. transcript map of the hereditary prostate cancer region at xq27. Genom- 10. Zendman, A. J., Cornelissen, I. M., Weidle, U. H., Ruiter, D. J., and ics, 79: 41–50, 2002. van Muijen, G. N. CTp11, a novel member of the family of human cancer/testis antigens. Cancer Res., 59: 6223–6229, 1999. 26. Trasler, J. M. Origin and roles of genomic methylation patterns in male germ cells. Semin Cell Dev. Biol., 9: 467–474, 1998. 11. Wang, Z., Zhang, Y., Liu, H., Salati, E., Chiriva-Internati, M., and Lim, S. H. Gene expression and immunologic consequence of 27. Ariel, M., McCarrey, J., and Cedar, H. Methylation patterns of SPAN-Xb in myeloma and other hematologic malignancies. Blood, 101: testis-specific genes. Proc. Natl. Acad. Sci. USA, 88: 2317–2321, 1991. 955–960, 2003. 28. Zhang, L. P., Stroud, J. C., Walter, C. A., Adrian, G. S., and 12. Brinckerhoff, L. H., Kalashnikov, V. V., Thompson, L. W., Yam- McCarrey, J. R. A gene-specific promoter in transgenic mice directs shchikov, G. V., Pierce, R. A., Galavotti, H. S., Engelhard, V. H., and testis-specific demethylation prior to transcriptional activation in vivo. Slingluff, C. L., Jr. Terminal modifications inhibit proteolytic degrada- Biol. Reprod., 59: 284–292, 1998. tion of an immunogenic MART-1(27–35) peptide: implications for 29. Weber, J., Salgaller, M., Samid, D., Johnson, B., Herlyn, M., peptide vaccines. Int. J. Cancer, 83: 326–334, 1999. Lassam, N., Treisman, J., and Rosenberg, S. A. Expression of the 13. Kittlesen, D. J., Thompson, L. W., Gulden, P. H., Skipper, J. C., MAGE-1 tumor antigen is up-regulated by the demethylating agent Colella, T. A., Shabanowitz, J., Hunt, D. F., Engelhard, V. H., Slingluff, 5-aza-2Ј-deoxycytidine. Cancer Res., 54: 1766–1771, 1994. C. L., Jr., and Shabanowitz, J. A. Human melanoma patients recognize 30. De Smet, C., Lurquin, C., Lethe, B., Martelange, V., and Boon, T. an HLA-A1-restricted CTL epitope from tyrosinase containing two DNA methylation is the primary silencing mechanism for a set of germ cysteine residues: implications for tumor vaccine development. J. Im- line- and tumor-specific genes with a CpG-rich promoter. Mol. Cell. munol., 160: 2099–2106, 1998. Biol., 19: 7327–7335, 1999. 14. Towbin, H., Staehelin, T., and Gordon, J. Electrophoretic transfer of 31. De Smet, C., De Backer, O., Faraoni, I., Lurquin, C., Brasseur, F., proteins from polyacrylamide gels to nitrocellulose sheets: procedure and Boon, T. The activation of human gene MAGE-1 in tumor cells is and some applications. 1979. Biotechnology, 24: 145–149, 1992. correlated with genome-wide demethylation. Proc. Natl. Acad. Sci. 15. Schmitt, A., Specht, T., Dahl, E., Hinzmann, B., Rosenthal, A., USA, 93: 7149–7153, 1996. Pilarsky, C., and Genomforschun, M. G. F. Human nucleic acid se- 32. Sigalotti, L., Coral, S., Altomonte, M., Natali, L., Gaudino, G., quences from prostate tumour tissue. Patent WO 9946374-A. Germany, Cacciotti, P., Libener, R., Colizzi, F., Vianale, G., Martini, F., Tognon, 1999. M., Jungbluth, A., Cebon, J., Maraskovsky, E., Mutti, L., and Maio, M. 16. Lucas, S., De Plaen, E., and Boon, T. MAGE-B5, MAGE-B6, Cancer testis antigens expression in mesothelioma: role of DNA meth- MAGE-C2, and MAGE-C3: four new members of the MAGE family ylation and bioimmunotherapeutic implications. Br. J. Cancer, 86: 979– with tumor-specific expression. Int. J. Cancer, 87: 55–60, 2000. 982, 2002. 17. Rogner, U. C., Wilke, K., Steck, E., Korn, B., and Poustka, A. The 33. Sigalotti, L., Coral, S., Nardi, G., Spessotto, A., Cortini, E., Catta- melanoma antigen gene (MAGE) family is clustered in the chromo- rossi, I., Colizzi, F., Altomonte, M., and Maio, M. Promoter methylation somal band Xq28. Genomics, 29: 725–731, 1995. controls the expression of MAGE2, 3 and 4 genes in human cutaneous 18. De Plaen, E., Arden, K., Traversari, C., Gaforio, J. J., Szikora, J. P., melanoma. J. Immunother., 25: 16–26, 2002. De Smet, C., Brasseur, F., van der Bruggen, P., Lethe, B., Lurquin, C., 34. Coral, S., Sigalotti, L., Altomonte, M., Engelsberg, A., Colizzi, F., et al. Structure, chromosomal localization, and expression of 12 genes Cattarossi, I., Maraskovsky, E., Jager, E., Seliger, B., and Maio, M. of the MAGE family. Immunogenetics, 40: 360–369, 1994. 5-aza-2Ј-deoxycytidine-induced expression of functional cancer testis 19. Jungbluth, A. A., Chen, Y. T., Busam, K. J., Coplan, K., Kolb, D., antigens in human renal cell carcinoma: immunotherapeutic implica- Iversen, K., Williamson, B., Van Landeghem, F. K., Stockert, E., and tions. Clin. Cancer Res., 8: 2690–2695, 2002. Old, L. J. CT7 (MAGE-C1) antigen expression in normal and neoplastic tissues. Int. J. Cancer, 99: 839–845, 2002. 35. Jones, P. A. The DNA methylation paradox. Trends Genet, 15: 34–37, 1999. 20. Lim, S. H., Bumm, K., Chiriva-Intemati, M., Xue, Y., and Wang, Z. MAGE-C1 (CT7) gene expression in multiple myeloma: relationship to 36. Kundu, T. K., and Rao, M. R. CpG islands in chromatin organiza- sperm protein 17. Eur. J. Haematol., 67: 332–334, 2001. tion and gene expression. J. Biochem., 125: 217–222, 1999. 21. Chen, Y. T., Gure, A. O., Tsang, S., Stockert, E., Jager, E., Knuth, 37. Goydos, J. S., Patel, M., and Shih, W. NY-ESO-1 and CTp11 A., and Old, L. J. Identification of multiple cancer/testis antigens by expression may correlate with stage of progression in melanoma. allogeneic antibody screening of a melanoma cell line library. Proc. J. Surg. Res., 98: 76–80, 2001. Natl. Acad. Sci. USA, 95: 6919–6923, 1998. 38. Adam, S. A. The nuclear pore complex. Genome Biol., 2: RE- 22. Nagasaki, K., Manabe, T., Hanzawa, H., Maass, N., Tsukada, T., VIEWS0007, 2001. and Yamaguchi, K. Identification of a novel gene, LDOC1, down- 39. Takahashi, K., Shichijo, S., Noguchi, M., Hirohata, M., and Itoh, regulated in cancer cell lines. Cancer Lett., 140: 227–234, 1999. K. Identification of MAGE-1 and MAGE-4 proteins in spermatogo- 23. Rapley, E. A., Crockford, G. P., Teare, D., Biggs, P., Seal, S., nia and primary spermatocytes of testis. Cancer Res., 55: 3478– Barfoot, R., Edwards, S., Hamoudi, R., Heimdal, K., Fossa, S. D., 3482, 1995.

40. Kocher, T., Schultz-Thater, E., Gudat, F., Schaefer, C., Casorati, G., 44. Tureci, O., Sahin, U., Zwick, C., Koslowski, M., Seitz, G., and Juretic, A., Willimann, T., Harder, F., Heberer, M., and Spagnoli, G. C. Pfreundschuh, M. Identification of a meiosis-specific protein as a mem- Identification and intracellular location of MAGE-3 gene product. Can- ber of the class of cancer/testis antigens. Proc. Natl. Acad. Sci. USA, 95: cer Res., 55: 2236–2239, 1995. 5211–5216, 1998. 41. Schultz-Thater, E., Noppen, C., Gudat, F., Durmuller, U., Zajac, P., 45. Jones, M. H., Zhang, Y., Tirosvoutis, K. N., Davey, P. M., Webster, Kocher, T., Heberer, M., and Spagnoli, G. C. NY-ESO-1 tumour asso- A. R., Walsh, D., Spurr, N. K., and Affara, N. A. Chromosomal ciated antigen is a cytoplasmic protein detectable by specific mono- assignment of 311 sequences transcribed in human adult testis. Genom- clonal antibodies in cell lines and clinical specimens. Br. J. Cancer, 83: ics, 40: 155–167, 1997. 204–208, 2000. 46. Dym, M., and Fawcett, D. W. The blood-testis barrier in the rat and 42. Jurk, M., Kremmer, E., Schwarz, U., Forster, R., and Winnacker, the physiological compartmentation of the seminiferous epithelium. E. L. MAGE-11 protein is highly conserved in higher organisms and Biol. Reprod., 3: 308–326, 1970. located predominantly in the nucleus. Int. J. Cancer, 75: 762–766, 1998. 47. Lui, W. Y., Mruk, D., Lee, W. M., and Cheng, C. Y. Sertoli cell 43. Satie, A. P., Rajpert-De Meyts, E., Spagnoli, G. C., Henno, S., tight junction dynamics: their regulation during spermatogenesis. Biol. Olivo, L., Jacobsen, G. K., Rioux-Leclercq, N., Jegou, B., and Samson, Reprod., 68: 1087–1097, 2003. M. The cancer-testis gene, NY-ESO-1, is expressed in normal fetal and 48. Fiszer, D., Ulbrecht, M., Fernandez, N., Johnson, J. P., Weiss, E. H., adult testes and in spermatocytic seminomas and testicular carcinoma in and Kurpisz, M. Analysis of HLA class Ib gene expression in male situ. Lab. Investig., 82: 775–780, 2002 gametogenic cells. Eur. J. Immunol., 27: 1691–1695, 1997.

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V. Anne Westbrook, Pamela D. Schoppee, Alan B. Diekman, et al.

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