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Cancer Cell IntCancer Cell International1475-2867BioMed Central London 1475-2867-5-41571590910.1186/1475-2867-5-4ReviewCancer/testis antigens and gametogenesis: a review and "brain-storming" session Kalejs Martins 1m.kalejs@no.lvErenpreisa Jekaterina 1katrina@biomed.lu.lv1 Biomedical Research and Study Centre of the Latvian University, Riga, Latvia2005 16 2 2005 5 4 4 9 12 2004 16 2 2005 Copyright © 2005 Kalejs and Erenpreisa; licensee BioMed Central Ltd.2005Kalejs and Erenpreisa; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Genes expressed both in normal testis and in malignancies (Cancer/ Testis associated genes – CTA) have become the most extensively studied antigen group in the field of tumour immunology. Despite this, many fundamentally important questions remain unanswered: what is the connection between germ-cell specific genes and tumours? Is the expression of these genes yet another proof for the importance of genome destabilisation in the process of tumorigenesis?, or maybe activation of these genes is not quite random but instead related to some programme giving tumours a survival advantage?

This review collates most of the recent information available about CTAs expression, function, and regulation. The data suggests a programme related to ontogenesis, mostly to gametogenesis. In the "brain-storming" part, facts in conflict with the hypothesis of random CTA gene activation are discussed. We propose a programme borrowed from organisms phylogenetically much older than humans, which existed before the differentiation of sexes. It is a programme that has served as a life cycle with prominent ploidy changes, and from which, as we know, the germ-cell ploidy cycle – meiosis – has evolved. Further work may show whether this hypothesis can lead to a novel anti-tumour strategy.

CTA genesgametogenesispolyploidy
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Introduction
Cancer/Testis (CT) antigens are a group of tumour antigens with gene expression restricted to male germ cells in the testis and to various malignancies. Their function in tumours is enigmatic and a common between testis genes (gametogenesis) and cancer remains elusive. When the causal link is not evident, it is tempting to believe the association is random, and assign it to general aspects of "genome instability in cancer". However, we believe a more direct link may exist. As suggested in this review, possible clues may be found in the common evolutionary pathway between ploidy cycles in meiosis and polyploidy in tumour cells. The latter, along with CT antigen expression, is a characteristic feature of well-progressed tumours. However, before discussing such possibilities, it is necessary to review the established literature.

The search for tumour antigens began in the 1960's with two groups identifying first alpha-fetoprotein (AFP), a serum marker for hepatoma and germ-cell tumours [1], and then carcinoembryonic antigen (CEA), a serum marker for colon and other epithelial cancers [2]. These antigens were discovered using heterogenous sera acquired by immunizing laboratory animals with human tumour material. However, only during the 90's did both cellular [3,4] and humoral [5] immune responses to human tumours get proper molecular definitions.

The first CTA, MAGEA-1, was identified in 1991 by Boon and colleagues using T-cell epitope cloning, a very complicated and time-consuming method [3]. In 1995 the SEREX (serological expression cloning) technique to identify tumour antigens was developed by Pfreundschuh and colleagues [5], which remains the leading approach to identifying new antigens that elicit humoral immune responses. Besides MAGEA1, BAGE, and GAGE1 discovered by T-cell epitope cloning, SEREX very soon displayed more tumour antigens with a cancer/testis restricted expression profile (SSX2, NY-ESO-1, and SYCP-1). The term "cancer-testis (CT) antigen" was introduced by Chen et al. [6], who recognized this group of genes had little in common except their expression profile.

By initial definition, expression of genes coding for CT antigens should be restricted in normal tissues to male germ cells in the testis and to malignancies of various histological types. However, the criteria proposed in the 90's are not true for all antigens of this group as seen today. Furthermore, for many of the recently discovered gene products with the described expression profile, no T-cell recognized epitopes have hitherto been identified. This is why CTA – "Cancer/Testis Associated" is a more appropriate name for this family of genes (and will be used in this context further in this review), because a lot of its members still need to be proven as possessing antigenic properties in cancer patients.

Attributing genes to the CTA gene family is based on several characteristic features [7,8]:

1. Predominant expression in germ cells of the testis and generally not in other normal tissues.

2. Expression in a number of malignant tumours of various histological types.

3. Mapping of the gene to the X-chromosome

4. Membership of a multigene family.

5. Antigenic properties in tumour-bearing patients.

Some exceptions to these criteria for certain CTAs will be described and discussed later.

Expression of CTA genes
To date, 89 individual CTA genes or isoforms have been described, which are organised in to 44 families (see additional file 1). From these, 19 families are testis-restricted, and 11 show additional expression in one or two somatic tissues. Nine are expressed in 3–6 tissue types besides testis, and 5 are ubiquitously somatically expressed. With the exception of the testis-restricted CTAs, the others also show expression in the pancreas but at levels as much as 10 × lower than in testis (based on mRNA expression levels from [9]). Expression of CTA was first shown in melanoma and all the classic CTA are expressed in this type of tumour, but since the 1980's, expression in various other tumours has been recognised (additional file 2).

The expression pattern of CTAs during spermatogenesis is of special interest. Functional analysis of these genes during gametogenesis might well give some clues about their possible role in tumours. Their expression is restricted exclusively to spermatogenic germ cells with other tubular cells (e.g. Leydig and Sertoli) being negative. This fits well with the findings of Yuasa et al. [10] who demonstrated that CTAs have much higher expression frequencies in the germ cell cancers (seminomas) than non-seminomas..

Different CTAs are expressed during different stages of spermatogenesis (Fig. 1.), so one may imagine that their functions are versatile, starting from regulation of mitotic cycling in spermatogonia, association with the meiotic cycle in spermatocytes, and finalizing with acrosome maturation in sperm.

Figure 1 Expression of CTAs during male germ cell development. References for Fig. 1: [25] [27] [28] [37] [40] [42] [53] [92] [93] [94] [95] [96]

In normal tissues, expression of NY-ESO1, MAGE-A3, -A4, and -A8 through -A11 as well as of several members of the XAGE gene family is found in the placenta. NY-ESO1 and several XAGEs are also expressed in the fetal ovary [11-13].

Regulation of CTA expression
The mechanisms involved in regulation of CTA expression have recently been comprehensively reviewed by Albert Zendman et al [14]. Thus, only a short recitation of some of the main points is provided here.

Mostly, methylation processes are responsible for the ectopic derepression of CTA genes. Using the demethylating agent, 5-aza-2-deoxycytidine (5DC), expression of several CTAs in cultured tumour cell lines can be induced/upregulated [15]. 5DC entraps DNA methyltransferases in a complex with DNA, which leads to progressive loss of DNA methylation, thereby releasing transcriptional blockage. Such upregulated expression has been reported for several MAGE members – LAGE-1, SSX-2, CAGE, NY-ESO-1. For MAGE-A1, demethylation is necessary and sufficient for gene expression, suggesting demethylation is the primary mechanism of transcription control [16]. In this context, a recent discovery of a CTA, viz. 'Boris', is interesting. Boris is reported as being expressed in several types of malignancies, and normally plays a major role in regulating methylation processes during spermatogenesis – it removes imprinting from genes during the last mitotic division of spermatocytes (reviewed in [17]). Several lines of evidence indicate that expression of some CTAs is dependent not only on demethylation, but on other transcriptional mechanisms.

Histone deacetylase (HDAC) inhibitors, on their own or in combination with 5DC, can also induce CTA expression, including MAGE, SSX, and NY-ESO-1 family members [15]. The CTA-rich region in Xp11.21-22 (e.g. SSX, MAGE-B) may escape X-chromosomal inactivation, but these genes are not normally expressed in females [18]. While global hypomethylation is common and prominent in colorectal cancer, few CTAs have ever been reported as expressed in this type of cancer [19]. Non-demethylation dependent induction of MAGE expression has been demonstrated by Park et al. [20], demonstrating that 40 mM NaCl induces the transcriptional and translational activation of MAGE-B1 and -B2 in specific tissues at hypertonic conditions.

There exist definite expression patterns (sets) of different CTAs in certain tumours. Marked heterogeneity of CTA expression is found in cells of some tumours, which cannot easily be explained by a global demethylation process [21-24]. The mechanisms of ectopic transcriptional activation of CTA genes clearly needs more investigation.

Function
Information regarding the function and cellular localization of CTAs is far less comprehensive. Often the proposed function is based purely on sequence homology with another protein of a known function. The only CTA proteins functionally established in gametogenesis are SCP-1, involved in chromosome pairing during meiosis [7], OY-TES-1 which functions in acrosin packaging in the acrosome of sperm heads [25], SPO11 acting as a meiosis-specific endonuclease [26], and BORIS, which is involved in cancellation of imprinting by epigenetic reprogramming during the final round of mitosis in spermatogenesis [27]. However, contrary to the situation in meiosis where it is rapidly degraded after the meiotic prophase in spermatocytes, SCP-1 expression in tumours is not cell cycle restricted [7]. BORIS is a paralog of CTCF. CTCF is a highly versatile 11 zinc-finger factor involved in various aspects of gene regulation – X chromosome inactivation, reading of imprinting sites, etc. [27]. During spermatogenesis, Boris is expressed later than many other CTA genes ([27]; Fig. 1.).

Suggestions for the functions of other CTAs mostly arose from studies of their homology with some well-known proteins and their domains, TSP50 being protease-like, CT17 phospholipase-like, and CT15 metalloproteinase-like [28]. The CT15 gene encodes a disintegrin and metalloproteinase (ADAM) domain 2, which is a member of the ADAM protein family [29]. Members of this family are membrane-anchored proteins structurally related to snake venom disintegrins, which have been implicated in a variety of biologic processes involving cell-cell and cell-matrix interactions, including fertilization, muscle development, and neurogenesis. This member is a subunit of an integral sperm membrane glycoprotein called fertilin, which plays an important role in sperm-egg interactions [30]. It is a membrane metalloproteinase with a possible role in tumour evasion and metastasis.

LDHC, the germ cell-specific member of the lactate dehydrogenase family, escapes from transcriptional repression, resulting in significant expression levels in virtually all tumour types tested. It might contribute to the constitutive activation of an anaerobic pathway in tumours, because its expression in tumours is not dependent on hypoxia [31].

Suggestions as to the role of MAGEs with a CTA expression profile mainly depend on studies of their ubiquitously expressed family members (e.g. Necdin, MAGE-D1, NRAGE, Dlxin-1). In general, the data point to a role for MAGEs via transcriptional regulation in cell cycle control and apoptosis. F.ex, Necdin-related MAGE proteins differentially interact with the E2F1 transcription factor and the p75 neurotrophin receptor [32]. The high level of homology among members of the MAGE family in both mouse and human suggests an important function both in testis and cancer.

The products of the SSX genes belong to the family of highly homologous synovial sarcoma X (SSX) breakpoint proteins. These proteins may function as transcriptional repressors; SSX1, SSX2 and SSX4 genes have been involved in the t (X;18) translocation characteristically found in all synovial sarcomas [33-35]. This translocation results in the fusion of the synovial sarcoma translocation gene (SYT) on chromosome 18 to one of the SSX genes on chromosome X. The encoded hybrid proteins are probably responsible for transforming activity. In the nucleus of sarcoma cells, both diffuse and speckled localisations of SSX protein have been reported [36,37].

The HOM-TES-85 protein has structural peculiarities that are shared exclusively with the N-myc oncoprotein. However, functional studies are required for confirmation [38]. Besides, the C-myc proto-oncogene is a normal participant of spermatogenesis (Fig. 2).

Figure 2 Oncogene expression during spermatogenesis (mainly in mice). From [70].

BRDT is similar to the RING3 protein family. It possesses 2 bromodomain motifs and a PEST sequence (a proline, glutamic acid, serine, and threonine cluster) characteristic of proteins that undergo rapid intracellular degradation). The bromodomain is found in proteins that regulate transcription [39].

PLU-1, a large multi-domain nuclear protein also has a strong transcriptional repression activity. It is a member of the ARID family of DNA-binding proteins. Plu-1 mRNA and PLU-1 protein are both highly expressed in the mitotic spermatogonia. The expression is reduced in the early prophase I stages (leptotene, zygotene), but reappears at pachytene, still being detectable in diplotene cells. It is located diffusely over the nucleus. PLU-1 might have a role in regulating meiotic transcription, restricted to certain meiotic stages [40].

The protein encoded by the SPANX gene targets the nucleus and associates with nuclear vacuoles and the redundant nuclear envelope in sperm cells [41]. In situ hybridization of human testis sections showed SPAN-X mRNA expression in round and elongated spermatids [42]. These redundant nuclear envelopes have a unique structure of limited chromatin sheets continued as annulate lamellae. Both these enigmatic structures have also been described in intact lymphomas [43] and irradiated lymphomas [44].

The protein encoded by IL13RA1 gene is a subunit of the interleukin-13 receptor. This subunit forms a receptor complex with IL-4 receptor alpha, a subunit shared by IL-13 and IL-4 receptors. This subunit serves as a primary IL-13-binding subunit of the IL-13 receptor, and may also be a component of IL-4 receptors. This protein binds tyrosine kinase TYK2, and thus may mediate the signalling processes that lead to the activation of JAK1, STAT3 and STAT6 induced by IL-13 and IL-4 [45].

SGY-1 (soggy-1), a secreted protein related to the Dickkopf protein family, is involved in suppressing the Wnt signal-transduction pathway controlling transcription activation of genes such as c-myc, c-jun, Fra, and cyclin D1 by preventing the accumulation of beta-catenin [46,47]. Wnt proteins are implicated in a wide variety of biologic processes including cell fate determination and patterning in early embryos, and in cell growth and/or differentiation in certain adult mammalian tissues [48]. Wnts can induce proliferation in different types of stem cells [49,50]. The importance of Wnt signalling during tumorigenesis has been recently emphasised [51,52].

NY-ESO-1 is one of the most immunogenic and therapeutically promising CTAs but functional studies on this gene are severely lagging behind its practical application. Unlike the majority of CTA genes, NY-ESO-1 stops its expression in well-progressed tumours, so it can be used as a marker to follow the early progression of testicular tumorigenesis [53].

CAGE [54] and HAGE [55] code for proteins with helicase-like features. Probably, it might be involved in recombination exchange in testis and recombination DNA repair in tumours.

TPX-1 is now seen as an integral protein of the outer dense fibres and the acrosome of spermatids in rats [56].

In summary, we see that the functions of individual CTA, when known, are very diverse, including, for example, both activators and repressors of proliferation and transcription.

Immunogenicity
Immunogenicity in cancer patients is elicited only by short peptide sequences of CTA epitopes, which are presented on the tumour cell surface by HLA Class I molecules in the case of cytotoxic T lymphocyte (CTL) mediated immune responses and by HLA Class II molecules on the surface of APC (antigen presenting cells), in the case of T-helper cell (TH) mediated immune responses. Identification of these epitopes is one of the main goals of CTA research. Knowledge of epitopes recognized by the immune system allows the creation of the tumour-specific vaccines. In the vaccination process, T-cell epitopes are often administered together with different adjuvants or cytokines, or delivered using peptide pulsed autologous dendritic cells, all of which are aimed at enhancing the immune reaction [57]. Currently efforts are being made to identify HLA class II restricted epitopes in order to promote TH responses, which are required to support the activity of CTLs, and provide a more "complete" immune response. However, vaccines designated to prime the immune system against tumours expressing various CTA have so far shown only partial clinical success [57-59].

Since the technique used to identify candidate tumour antigens has changed from T-cell epitope cloning [3,60], and SEREX (serological analysis of cDNA expression libraries) [5,61,62] to differential gene expression analysis by various techniques like RDA (representative difference analysis) [63], DD (differential display) [64] and SSH (suppression subtractive hybridization) [65] – and even further to bioinformatics – we cannot really be sure about the adequate use of the definition "antigen" in relation to CTA gene products. Whilst the former two techniques are dependent on the immunogenicity of specific epitopes in cancer patients, the latter ones are simply based on mRNA expression levels or even sequence homologies detected via search engines and provide no answer about immunoreactivity. In additional file 1, one can see that an immune response is documented against only 19 of 44 CTAs. Immune recognition of the majority of these CTAs is cancer-related, occurring spontaneously in cancer patients, not in cancer-free individuals. The exceptions to this are humoral immune responses to MAGEB1/CT3.1 in systemic lupus erythematosus patients [66] and to SPA17/CT22 in vasectomised men [67].

CTA can be considered to be tumour specific. There exists the blood-testis barrier [68], which prevents the immune system from contacting with CTA gene products. Besides this, germinative cells do not express HLA class Ia molecules [69], so they cannot present their expressed proteins to the immune system. For these reasons, the immune system never comes into contact with these proteins and recognizes them as "non-self" structures.

Brain-storming
The function of most CTAs is unknown, although some role in regulation of gene expression (both activating and repressing) seems likely [8]. Are CTAs oncogenes? By definition, oncogenes are normal cellular genes participating in proliferation cascades, which are abnormally activated in tumours [70]. Therefore, with the exception of one (HOM-TES-85, which has structural homology with the myc-oncogene) CTAs can not formally be regarded as oncogenes. Are CTAs simply activated by the imbalanced genome, due to its instability in tumours? Some, such as SSX, whose ectopic expression is caused by the SYT-SSX fusion due to translocation t (X;18) in synovial sarcomas, certainly are. However, a large number of CTA genes are only located on the X-chromosome and this chromosome is not a specific site of chromosome breaks and translocations usually associated with tumours [71]. However, the chromosome region 20q13.2 containing the BORIS gene is commonly amplified or exhibits moderate gains of material in many human cancers. This has strengthened the idea that this region contains a major oncogene [72,73]. A preliminary report using RT-PCR has found that BORIS expression is detectable in over half of ~200 cancer cell lines studied, representing most of the major forms of human tumours, discussed in [17]. However, this observation awaits further confirmation. Theoretically, the aberrant expression of the amplified (by chance) Boris, which by analogy with CTCF may demethylate CTA genes located on X-chromosome, may in turn activate a large body of CTA genes in human malignancies. However, why are other gametogenesis-related CTA genes also activated from other chromosomes? It does not look like a chance event and therefore amplification of "Boris" may still not be random.

Diversity of functions, including genes involved in ontogenesis suggest that CTAs are activated either as a result of the genome instability (however, why testicular genes?) or as part of a complex program. Also from this point, this looks like a program related to gametogenesis. The same idea was proposed by Old [8].

If CTAs generally do not enhance tumour growth except by stimulating proliferation, like oncogenes, another possibility is that they could do it by stimulating DNA repair or inhibiting apoptosis (combination of all three is possible). Indeed, some CTAs have relation to the DNA repair factors by homologous recombination. These are SPO11, SYCP1, helicase-like CAGE and HAGE acting in meiotic prophase of gametogenesis. In turn, homologous recombination in tumours was shown to act anti-apoptotically [74].

Are these CTAs restricted only to prophase of meiosis, where recombination takes place? It appears not. For example, some are associated with the spermatogonial stage (Plu-1 mRNA and PLU-1), and some with maturation of the acrosome (see Fig. 1). It is a program again, and why only male gametogenesis? But it also occurs in oogenesis [8]. Thus, prophase of meiosis (pairing and recombination) may be still the most important.

So, ectopic activation of CTA genes is not entirely random, being induced from the sexual X-chromosome but also from loci on other chromosomes, relating to gametogenesis. However, it is curious then that the very common proto-oncogenes of proliferatives cascades also participate in gametogenesis, e.g. myc, ras, jun, etc. This can be seen in Fig 2, (taken from the Janis Erenpreiss [70]). He and others postulated a link between gametogenesis and cancerogenesis before CTAs were revealed [75-77].

In turn, Old [8] looked at the problem from a new angle and suggested that CTAs provide a causal link between gametogenesis and cancer. This seems plausable, but does this illegitimate program in tumours embrace, even mosaically, only gametogenesis? May be only the DNA recombination repair component is common? But the CTA genes are repressing a wide Wnt family, which also functions in early embryogenesis.

The program sounds more like an ontogenetic (life-cycle) one. Let us remember the experiments by Mintz and Illmeisee [19] who cloned normal genetic mosaic mice by introducting the nuclei of malignant teratocarcinoma into enucleated eggs. Both gametic, parthenogenetic and trophoblastic theories of cancer have also been proposed in the past [8,70] and can be viewed as embryonal or ontogenetic theories of cancer.

Another question is whether CTA genes govern a life-cycle-like program with its key events similar to meiosis? In turn, to which process does meiosis provide a key – recombination and reduction division? Are tumours capable of these? We also have to consider why X-chromosomes are involved, and not Y-chromosomes, which are responsible for sex? Perhaps this is because the X-chromosome is better conserved evolutionary and appeared prior to sex discrimination.

So, this looks like an ontogenetic program, which evolutionary preceded sexual (amphimictic) life-cycle, with the key in recombination and reduction division; but what is it? The answer is evident – ancient ploidy cycles [78,79]. In general, ploidy cycles are displayed as a cyclic increase and reduction of ploidy (chromosome number), and these involve the pairing of homologous chromosomes and their segregation, omitting one round of DNA replication.

There may be reduction of ploidy 2n-n (sexual meiosis) or from nx to 2n DNA numbers, in the asexual ploidy cycles characteristic for a few protists, as in Amoebae and foraminiferans [80,81] as well as part of ontogenetic programs in the more developed taxons [82,83]. Contrary to gametic reduction in sexual meiosis, the reduction of ploidy in polyploid somatic cells is called "somatic reduction".

In this context, expression of some CTAs by the placenta is of interest because trophoblast and decidua are the only mammalian tissue capable of endoreduplication creating enormously large ploidies [84]. an ability shared by many tumour cells. The latest studies on a silver fox revealed somatic reduction in the giant cells of a trophoblast [85].

Now let us return to DNA recombination repair in tumours as a means of survival, as already mentioned. Repair by homologous recombination can protect malignant tumour cells from apoptosis. In particular, as shown in our laboratory, endopolyploid cells employ this mechanism [86]. Likewise, expression of CTAs – endopolyploidy – is a hallmark of malignant tumour progression where there is deficient TP53 function [87].

In turn, some giant tumour cells show the capability to segregate their genomes and return to mitosis ([86,88-91], see Fig. 3).

Figure 3 Reductional mitotic divisions generating low ploidy cells from one large polyploid cell. Non-treated Burkitt's lymphoma cell line, DNA staining with Toluidine-blue. × 2,500.

Conclusion
A hypothesis is put forward that activation of at least some of the CTA genes in p53-deficient human tumours could be due to the genetic program running "relic" ploidy cycles in tumour cells. This hypothesis offers new opportunities for the design of novel tumour treatment strategies. In particular, passive therapy using CTAs to prime the host immune system against the tumour could be replaced with gene therapy aimed to block the function of CTA gene products or even their expression. This approach is promising, because normally only the germ cells in the testis express CTA genes and they are well protected by the blood-testis barrier. Thus, there should not be any problem with tumour-specific priming, and respectively we would predict there to be no side effects.

Supplementary Material
Additional File 1
"Chromosomal localization, type of immune response, identification method and identification references for all known 44 CTA gene families".

Click here for file

 Additional File 2
" CTA Frequency (%) of expression in various tumour types".

Click here for file

 Acknowledgements
The authors gratefully thank Denys Wheatley and Jamie Honeychurch for his help in editing the article.
==== Refs
Abelev GI Perova SD Khramkova NI Postnikova ZA Irlin IS  Production of embryonal alpha-globulin by transplantable mouse hepatomas Transplantation 1963 1 174 180 14010646 
Gold P Freedman SO  Specific carcinoembryonic antigens of the human digestive system J Exp Med 1965 122 467 481 4953873 10.1084/jem.122.3.467 
van der BP Traversari C Chomez P Lurquin C De Plaen E Van den EB Knuth A Boon T  A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma Science 1991 254 1643 1647 1840703 
Traversari C van der BP Van den EB Hainaut P Lemoine C Ohta N Old L Boon T  Transfection and expression of a gene coding for a human melanoma antigen recognized by autologous cytolytic T lymphocytes Immunogenetics 1992 35 145 152 1537606 10.1007/BF00185107 
Sahin U Tureci O Schmitt H Cochlovius B Johannes T Schmits R Stenner F Luo G Schobert I Pfreundschuh M  Human neoplasms elicit multiple specific immune responses in the autologous host Proc Natl Acad Sci U S A 1995 92 11810 11813 8524854 
Chen YT Scanlan MJ Sahin U Tureci O Gure AO Tsang S Williamson B Stockert E Pfreundschuh M Old LJ  A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening Proc Natl Acad Sci U S A 1997 94 1914 1918 9050879 10.1073/pnas.94.5.1914 
Tureci O Sahin U Zwick C Koslowski M Seitz G Pfreundschuh M  Identification of a meiosis-specific protein as a member of the class of cancer/testis antigens Proc Natl Acad Sci U S A 1998 95 5211 5216 9560255 10.1073/pnas.95.9.5211 
Old LJ  Cancer/testis (CT) antigens - a new link between gametogenesis and cancer Cancer Immun 2001 1 1 12747762 
Scanlan MJ Simpson AJ Old LJ  The cancer/testis genes: review, standardization, and commentary Cancer Immun 2004 4 1 14738373 
Yuasa T Okamoto K Kawakami T Mishina M Ogawa O Okada Y  Expression patterns of cancer testis antigens in testicular germ cell tumors and adjacent testicular tissue J Urol 2001 165 1790 1794 11342977 10.1097/00005392-200105000-00102 
Jungbluth AA Chen YT Stockert E Busam KJ Kolb D Iversen K Coplan K Williamson B Altorki N Old LJ  Immunohistochemical analysis of NY-ESO-1 antigen expression in normal and malignant human tissues Int J Cancer 2001 92 856 860 11351307 10.1002/ijc.1282 
De Plaen E Arden K Traversari C Gaforio JJ Szikora JP De Smet C Brasseur F van der BP Lethe B Lurquin C .  Structure, chromosomal localization, and expression of 12 genes of the MAGE family Immunogenetics 1994 40 360 369 7927540 
Zendman AJ Van Kraats AA Weidle UH Ruiter DJ Van Muijen GN  The XAGE family of cancer/testis-associated genes: alignment and expression profile in normal tissues, melanoma lesions and Ewing's sarcoma Int J Cancer 2002 99 361 369 11992404 10.1002/ijc.10371 
Zendman AJ Ruiter DJ Van Muijen GN  Cancer/testis-associated genes: identification, expression profile, and putative function J Cell Physiol 2003 194 272 288 12548548 10.1002/jcp.10215 
Shichijo S Yamada A Sagawa K Iwamoto O Sakata M Nagai K Itoh K  Induction of MAGE genes in lymphoid cells by the demethylating agent 5-aza-2'-deoxycytidine Jpn J Cancer Res 1996 87 751 756 8698626 
De Smet C Lurquin C Lethe B Martelange V Boon T  DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter Mol Cell Biol 1999 19 7327 7335 10523621 
Klenova EM Morse HCIII Ohlsson R Lobanenkov VV  The novel BORIS + CTCF gene family is uniquely involved in the epigenetics of normal biology and cancer Semin Cancer Biol 2002 12 399 414 12191639 10.1016/S1044-579X(02)00060-3 
Miller AP Willard HF  Chromosomal basis of X chromosome inactivation: identification of a multigene domain in Xp11.21-p11.22 that escapes X inactivation Proc Natl Acad Sci U S A 1998 95 8709 8714 9671743 10.1073/pnas.95.15.8709 
Mintz B Illmensee K  Normal genetically mosaic mice produced from malignant teratocarcinoma cells Proc Natl Acad Sci U S A 1975 72 3585 3589 1059147 
Park JH Lee SW  Hypertonicity induction of melanoma antigen, a tumor-associated antigen Mol Cells 2002 13 288 295 12018852 
Coulie PG Van den Eynde BJ van der BP Van Pel A Boon T  Antigens recognized by T-lymphocytes on human tumours Biochem Soc Trans 1997 25 544 548 9191153 
Dalerba P Ricci A Russo V Rigatti D Nicotra MR Mottolese M Bordignon C Natali PG Traversari C  High homogeneity of MAGE, BAGE, GAGE, tyrosinase and Melan-A/MART-1 gene expression in clusters of multiple simultaneous metastases of human melanoma: implications for protocol design of therapeutic antigen-specific vaccination strategies Int J Cancer 1998 77 200 204 9650552 10.1002/(SICI)1097-0215(19980717)77:2<200::AID-IJC5>3.0.CO;2-U 
Sahin U Tureci O Chen YT Seitz G Villena-Heinsen C Old LJ Pfreundschuh M  Expression of multiple cancer/testis (CT) antigens in breast cancer and melanoma: basis for polyvalent CT vaccine strategies Int J Cancer 1998 78 387 389 9766577 10.1002/(SICI)1097-0215(19981029)78:3<387::AID-IJC22>3.0.CO;2-2 
Zendman AJ de Wit NJ van Kraats AA Weidle UH Ruiter DJ van Muijen GN  Expression profile of genes coding for melanoma differentiation antigens and cancer/testis antigens in metastatic lesions of human cutaneous melanoma Melanoma Res 2001 11 451 459 11595881 10.1097/00008390-200110000-00003 
Ono T Kurashige T Harada N Noguchi Y Saika T Niikawa N Aoe M Nakamura S Higashi T Hiraki A Wada H Kumon H Old LJ Nakayama E  Identification of proacrosin binding protein sp32 precursor as a human cancer/testis antigen Proc Natl Acad Sci U S A 2001 98 3282 3287 11248070 10.1073/pnas.041625098 
Romanienko PJ Camerini-Otero RD  Cloning, characterization, and localization of mouse and human SPO11 Genomics 1999 61 156 169 10534401 10.1006/geno.1999.5955 
Loukinov DI Pugacheva E Vatolin S Pack SD Moon H Chernukhin I Mannan P Larsson E Kanduri C Vostrov AA Cui H Niemitz EL Rasko JE Docquier FM Kistler M Breen JJ Zhuang Z Quitschke WW Renkawitz R Klenova EM Feinberg AP Ohlsson R Morse HCIII Lobanenkov VV  BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma Proc Natl Acad Sci U S A 2002 99 6806 6811 12011441 10.1073/pnas.092123699 
Shan J Yuan L Xiao Q Chiorazzi N Budman D Teichberg S Xu HP  TSP50, a possible protease in human testes, is activated in breast cancer epithelial cells Cancer Res 2002 62 290 294 11782390 
Scanlan MJ Gordon CM Williamson B Lee SY Chen YT Stockert E Jungbluth A Ritter G Jager D Jager E Knuth A Old LJ  Identification of cancer/testis genes by database mining and mRNA expression analysis Int J Cancer 2002 98 485 492 11920606 10.1002/ijc.10276 
Gupta SK Alves K Palladino LO Mark GE Hollis GF  Molecular cloning of the human fertilin beta subunit Biochem Biophys Res Commun 1996 224 318 326 8702389 10.1006/bbrc.1996.1027 
Koslowski M Tureci O Bell C Krause P Lehr HA Brunner J Seitz G Nestle FO Huber C Sahin U  Multiple splice variants of lactate dehydrogenase C selectively expressed in human cancer Cancer Res 2002 62 6750 6755 12438276 
Kuwako K Taniura H Yoshikawa K  Necdin-related MAGE proteins differentially interact with the E2F1 transcription factor and the p75 neurotrophin receptor J Biol Chem 2004 279 1703 1712 14593116 10.1074/jbc.M308454200 
de Bruijn DR dos Santos NR Kater-Baats E Thijssen J van den BL Stap J Balemans M Schepens M Merkx G van Kessel AG  The cancer-related protein SSX2 interacts with the human homologue of a Ras-like GTPase interactor, RAB3IP, and a novel nuclear protein, SSX2IP Genes Chromosomes Cancer 2002 34 285 298 12007189 10.1002/gcc.10073 
Yang K Lui WO Xie Y Zhang A Skytting B Mandahl N Larsson C Larsson O  Co-existence of SYT-SSX1 and SYT-SSX2 fusions in synovial sarcomas Oncogene 2002 21 4181 4190 12037676 10.1038/sj.onc.1205569 
Storlazzi CT Mertens F Mandahl N Gisselsson D Isaksson M Gustafson P Domanski HA Panagopoulos I  A novel fusion gene, SS18L1/SSX1, in synovial sarcoma Genes Chromosomes Cancer 2003 37 195 200 12696068 10.1002/gcc.10210 
dos Santos NR de Bruijn DR Balemans M Janssen B Gartner F Lopes JM de Leeuw B Geurts K  Nuclear localization of SYT, SSX and the synovial sarcoma-associated SYT-SSX fusion proteins Hum Mol Genet 1997 6 1549 1558 9285793 10.1093/hmg/6.9.1549 
dos Santos NR de Bruijn DR Kater-Baats E Otte AP van Kessel AG  Delineation of the protein domains responsible for SYT, SSX, and SYT-SSX nuclear localization Exp Cell Res 2000 256 192 202 10739666 10.1006/excr.2000.4813 
Tureci O Sahin U Koslowski M Buss B Bell C Ballweber P Zwick C Eberle T Zuber M Villena-Heinsen C Seitz G Pfreundschuh M  A novel tumour associated leucine zipper protein targeting to sites of gene transcription and splicing Oncogene 2002 21 3879 3888 12032826 10.1038/sj.onc.1205481 
Pivot-Pajot C Caron C Govin J Vion A Rousseaux S Khochbin S  Acetylation-dependent chromatin reorganization by BRDT, a testis-specific bromodomain-containing protein Mol Cell Biol 2003 23 5354 5365 12861021 10.1128/MCB.23.15.5354-5365.2003 
Madsen B Tarsounas M Burchell JM Hall D Poulsom R Taylor-Papadimitriou J  PLU-1, a transcriptional repressor and putative testis-cancer antigen, has a specific expression and localisation pattern during meiosis Chromosoma 2003 112 124 132 14579128 10.1007/s00412-003-0252-6 
Westbrook VA Schoppee PD Diekman AB Klotz KL Allietta M Hogan KT Slingluff CL Patterson JW Frierson HF Irvin WPJ Flickinger CJ Coppola MA Herr JC  Genomic organization, incidence, and localization of the SPAN-x family of cancer-testis antigens in melanoma tumors and cell lines Clin Cancer Res 2004 10 101 112 14734458 
Westbrook VA Diekman AB Klotz KL Khole VV Kap-Herr C Golden WL Eddy RL Shows TB Stoler MH Lee CY Flickinger CJ Herr JC  Spermatid-specific expression of the novel X-linked gene product SPAN-X localized to the nucleus of human spermatozoa Biol Reprod 2000 63 469 481 10906052 
Ghadially FN  Ultrastructural pathology of cell and matrix 2004 1 3 London, Butterworths 
Erenpreisa J Ivanov A Cragg M Selivanova G Illidge T  Nuclear envelope-limited chromatin sheets are part of mitotic death Histochem Cell Biol 2002 117 243 255 11914922 10.1007/s00418-002-0382-6 
Aman MJ Tayebi N Obiri NI Puri RK Modi WS Leonard WJ  cDNA cloning and characterization of the human interleukin 13 receptor alpha chain J Biol Chem 1996 271 29265 29270 8910586 10.1074/jbc.271.46.29265 
Krupnik VE Sharp JD Jiang C Robison K Chickering TW Amaravadi L Brown DE Guyot D Mays G Leiby K Chang B Duong T Goodearl AD Gearing DP Sokol SY McCarthy SA  Functional and structural diversity of the human Dickkopf gene family Gene 1999 238 301 313 10570958 10.1016/S0378-1119(99)00365-0 
Karim R Tse G Putti T Scolyer R Lee S  The significance of the Wnt pathway in the pathology of human cancers Pathology 2004 36 120 128 15203747 10.1080/00313020410001671957 
Bergstein I Eisenberg LM Bhalerao J Jenkins NA Copeland NG Osborne MP Bowcock AM Brown AM  Isolation of two novel WNT genes, WNT14 and WNT15, one of which (WNT15) is closely linked to WNT3 on human chromosome 17q21 Genomics 1997 46 450 458 9441749 10.1006/geno.1997.5041 
Willert K Brown JD Danenberg E Duncan AW Weissman IL Reya T Yates JRIII Nusse R  Wnt proteins are lipid-modified and can act as stem cell growth factors Nature 2003 423 448 452 12717451 10.1038/nature01611 
Reya T Duncan AW Ailles L Domen J Scherer DC Willert K Hintz L Nusse R Weissman IL  A role for Wnt signalling in self-renewal of haematopoietic stem cells Nature 2003 423 409 414 12717450 10.1038/nature01593 
Polakis P  Wnt signaling and cancer Genes Dev 2000 14 1837 1851 10921899 
Taipale J Beachy PA  The Hedgehog and Wnt signalling pathways in cancer Nature 2001 411 349 354 11357142 10.1038/35077219 
Satie AP Rajpert-De Meyts E Spagnoli GC Henno S Olivo L Jacobsen GK Rioux-Leclercq N Jegou B Samson M  The cancer-testis gene, NY-ESO-1, is expressed in normal fetal and adult testes and in spermatocytic seminomas and testicular carcinoma in situ Lab Invest 2002 82 775 780 12065688 
Cho B Lim Y Lee DY Park SY Lee H Kim WH Yang H Bang YJ Jeoung DI  Identification and characterization of a novel cancer/testis antigen gene CAGE Biochem Biophys Res Commun 2002 292 715 726 11922625 10.1006/bbrc.2002.6701 
Martelange V De Smet C De Plaen E Lurquin C Boon T  Identification on a human sarcoma of two new genes with tumor-specific expression Cancer Res 2000 60 3848 3855 10919659 
O'Bryan MK Sebire K Meinhardt A Edgar K Keah HH Hearn MT De Kretser DM  Tpx-1 is a component of the outer dense fibers and acrosome of rat spermatozoa Mol Reprod Dev 2001 58 116 125 11144214 10.1002/1098-2795(200101)58:1<116::AID-MRD14>3.0.CO;2-8 
Stevanovic S  Identification of tumour-associated T-cell epitopes for vaccine development Nat Rev Cancer 2002 2 514 520 12094237 10.1038/nrc841 
Atanackovic D Altorki NK Stockert E Williamson B Jungbluth AA Ritter E Santiago D Ferrara CA Matsuo M Selvakumar A Dupont B Chen YT Hoffman EW Ritter G Old LJ Gnjatic S  Vaccine-induced CD4+ T cell responses to MAGE-3 protein in lung cancer patients J Immunol 2004 172 3289 3296 14978137 
Ehlken H Schadendorf D Eichmuller S  Humoral immune response against melanoma antigens induced by vaccination with cytokine gene-modified autologous tumor cells Int J Cancer 2004 108 307 313 14639620 10.1002/ijc.11537 
Wang RF Rosenberg SA  Human tumor antigens recognized by T lymphocytes: implications for cancer therapy J Leukoc Biol 1996 60 296 309 8830785 
Sahin U Tureci O Pfreundschuh M  Serological identification of human tumor antigens Curr Opin Immunol 1997 9 709 716 9368781 10.1016/S0952-7915(97)80053-2 
Tureci O Sahin U Pfreundschuh M  Serological analysis of human tumor antigens: molecular definition and implications Mol Med Today 1997 3 342 349 9269687 10.1016/S1357-4310(97)01081-2 
Hubank M Schatz DG  Identifying differences in mRNA expression by representational difference analysis of cDNA Nucleic Acids Res 1994 22 5640 5648 7838717 
Liang P Pardee AB  Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction Science 1992 257 967 971 1354393 
Diatchenko L Lau YF Campbell AP Chenchik A Moqadam F Huang B Lukyanov S Lukyanov K Gurskaya N Sverdlov ED Siebert PD  Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries Proc Natl Acad Sci U S A 1996 93 6025 6030 8650213 10.1073/pnas.93.12.6025 
McCurdy DK Tai LQ Nguyen J Wang Z Yang HM Udar N Naiem F Concannon P Gatti RA  MAGE Xp-2: a member of the MAGE gene family isolated from an expression library using systemic lupus erythematosus sera Mol Genet Metab 1998 63 3 13 9538511 10.1006/mgme.1997.2639 
Lea IA Adoyo P O'Rand MG  Autoimmunogenicity of the human sperm protein Sp17 in vasectomized men and identification of linear B cell epitopes Fertil Steril 1997 67 355 361 9022615 10.1016/S0015-0282(97)81923-1 
Bart J Groen HJ van der Graaf WT Hollema H Hendrikse NH Vaalburg W Sleijfer DT de Vries EG  An oncological view on the blood-testis barrier Lancet Oncol 2002 3 357 363 12107023 10.1016/S1470-2045(02)00776-3 
Fiszer D Kurpisz M  Major histocompatibility complex expression on human, male germ cells: a review Am J Reprod Immunol 1998 40 172 176 9764361 
Erenpreiss JO  Current concepts of malignant growth Part A From a Normal Cell to Cancer  1993 Riga, Zvaigzne publishers 
Kufe D Pollock R Weicheselbaum R Bast R Gansler T Holland J Frie E  Cancer Medicine 2003 6 Hamilton (Canada), BC. Decker Inc. 
Tanner MM Grenman S Koul A Johannsson O Meltzer P Pejovic T Borg A Isola JJ  Frequent amplification of chromosomal region 20q12-q13 in ovarian cancer Clin Cancer Res 2000 6 1833 1839 10815905 
Hidaka S Yasutake T Takeshita H Kondo M Tsuji T Nanashima A Sawai T Yamaguchi H Nakagoe T Ayabe H Tagawa Y  Differences in 20q13.2 copy number between colorectal cancers with and without liver metastasis Clin Cancer Res 2000 6 2712 2717 10914715 
Raderschall E Stout K Freier S Suckow V Schweiger S Haaf T  Elevated levels of Rad51 recombination protein in tumor cells Cancer Res 2002 62 219 225 11782381 
Erenpreis YG  The function of nucleic acids in the differentiation of neoplastic processes 1964 New York, Daniel Davey a. Co 
Erenpreiss JG  Gametogenesis as a molecular model of cancerogenesis: A current view of the embryological theory of cancer Proc Latv Acad Sci Part B 1992 3 55 63 
Vinnitsky VB  Oncogerminal hypothesis of tumour growth perimental Oncology 1989 11 59 66 
Cleveland LR  The origin and evolution of meiosis Science 1947 105 287 289 17835147 
Kondrashov AS  The asexual ploidy cycle and the origin of sex Nature 1994 370 213 216 8028667 10.1038/370213a0 
Raikov IB  The Protozoan Nucleus Morphology and Evolution 1982 Wien-New York, Springer Verlag 
Raikov IB  Meiosis in protists: recent advances and persisting problems r J Protistol 1995 31 1 7 
Nagl W  Endopolyploidy and Polyteny in Differentiation and Evolution 1978 Amsterdam-New York-Oxford, North-Holland Pub.,  
Kondrashov AS  Evolutionary genetics of life cycles nu Rev Ecol Syst , 1997 391 435 10.1146/annurev.ecolsys.28.1.391 
Zybina EV Zybina TG  Polytene chromosomes in mammalian cells Int Rev Cytol 1996 165 53 119 8900957 
Zybina TG Zybina EV Kiknadze II Zhelezova AI  Polyploidization in the trophoblast and uterine glandular epithelium of the endotheliochorial placenta of silver fox (Vulpes fulvus Desm.), as revealed by the DNA content Placenta 2001 22 490 498 11373160 10.1053/plac.2001.0675 
Ivanov A Cragg MS Erenpreisa J Emzinsh D Lukman H Illidge TM  Endopolyploid cells produced after severe genotoxic damage have the potential to repair DNA double strand breaks J Cell Sci 2003 116 4095 4106 12953071 10.1242/jcs.00740 
Buglioni S D'Agnano I Vasselli S Perrone DR D'Angelo C Brenna A Benevolo M Cosimelli M Zupi G Mottolese M  p53 nuclear accumulation and multiploidy are adverse prognostic factors in surgically resected stage II colorectal cancers independent of fluorouracil-based adjuvant therapy Am J Clin Pathol 2001 116 360 368 11554164 10.1309/V7UW-UT2E-JVYH-DGWK 
Baroja A de la HC Alvarez A Ispizua A Bilbao J de Gandarias JM  Genesis and evolution of high-ploidy tumour cells evaluated by means of the proliferation markers p34(cdc2), cyclin B1, PCNA and 3[H]-thymidine Cell Prolif 1996 29 89 100 8630339 10.1046/j.1365-2184.1996.00990.x 
Baroja A de la HC Alvarez A Vielba R Sarrat R Arechaga J de Gandarias JM  Polyploidization and exit from cell cycle as mechanisms of cultured melanoma cell resistance to methotrexate Life Sci 1998 62 2275 2282 9651116 10.1016/S0024-3205(98)00208-2 
Erenpreisa JA Cragg MS Fringes B Sharakhov I Illidge TM  Release of mitotic descendants by giant cells from irradiated Burkitt's lymphoma cell line. Cell Biol Int 2000 24 635 648 10964453 10.1006/cbir.2000.0558 
Illidge TM Cragg MS Fringes B Olive P Erenpreisa JA  Polyploid giant cells provide a survival mechanism for p53 mutant cells after DNA damage Cell Biol Int 2000 24 621 633 10964452 10.1006/cbir.2000.0557 
Saburi S Nadano D Akama TO Hirama K Yamanouchi K Naito K Tojo H Tachi C Fukuda MN  The trophinin gene encodes a novel group of MAGE proteins, magphinins, and regulates cell proliferation during gametogenesis in the mouse J Biol Chem 2001 276 49378 49389 11590179 10.1074/jbc.M108584200 
Jungbluth AA Busam KJ Kolb D Iversen K Coplan K Chen YT Spagnoli GC Old LJ  Expression of MAGE-antigens in normal tissues and cancer Int J Cancer 2000 85 460 465 10699915 10.1002/(SICI)1097-0215(20000215)85:4<460::AID-IJC3>3.3.CO;2-E 
Jungbluth AA Chen YT Busam KJ Coplan K Kolb D Iversen K Williamson B Van Landeghem FK Stockert E Old LJ  CT7 (MAGE-C1) antigen expression in normal and neoplastic tissues Int J Cancer 2002 99 839 845 12115486 10.1002/ijc.10416 
Meuwissen RL Offenberg HH Dietrich AJ Riesewijk A van Iersel M Heyting C  A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes EMBO J 1992 11 5091 5100 1464329 
Tapparel C Reymond A Girardet C Guillou L Lyle R Lamon C Hutter P Antonarakis SE  The TPTE gene family: cellular expression, subcellular localization and alternative splicing Gene 2003 323 189 199 14659893 10.1016/j.gene.2003.09.038

