Charles De Smet

Characterization of an Antigen That Is Recognized on a Melanoma Showing Partial HLA Loss by CTL Expressing an NK Inhibitory Receptor

Melanoma lines MEL.A and MEL.B were derived from metastases removed from patient LB33 in 1988 and 1993, respectively. The MEL.A cells express several antigens recognized by autologous cytolytic T lymphocytes (CTL) on HLA class I molecules. The MEL.B cells have lost expression of all class I molecules except for HLA-A24. By stimulating autologous lymphocytes with MEL.B, we obtained an HLA-A24-restricted CTL clone that lysed these cells. A novel gene, PRAME, encodes the antigen. It is expressed in a large proportion of tumors and also in some normal tissues, albeit at a lower level. Surprisingly, the CTL failed to lyse MEL.A, even though these cells expressed the gene PRAME. The CTL expresses an NK inhibitory receptor that inhibits its lytic activity upon interaction with HLA-Cw7 molecules, which are present on MEL.A cells and not on MEL.B. Such CTL, active against tumor cells showing partial HLA loss, may constitute an intermediate line of anti-tumor defense between the CTL, which recognize highly specific tumor antigens, and the NK cells, which recognize HLA loss variants.

Structure, Chromosomal Localization, and Expression of 12 Genes of the Mage Family

We reported previously that human geneMAGE-1 directs the expression of a tumor antigen recognized on a melanoma by autologous cytolytic T lymphocytes. Probing cosmid libraries with aMAGE-1 sequence, we identified 11 closely related genes. The analysis of hamster-human somatic cell hybrids indicated that the 12MAGE genes are located in the q terminal region of chromosome X. LikeMAGE-1, the 11 additionalMAGE genes have their entire coding sequence located in the last exon, which shows 64%-85% identity with that ofMAGE-1. The coding sequences of theMAGE genes predict the same main structural features for allMAGE proteins. In contrast, the promoters and first exons of the12 MAGE genes show considerable variability, suggesting that the existence of this gene family enables the same function to be expressed under different transcriptional controls. The expression of eachMAGE gene was evaluated by reverse transcription and polymerase chain reaction amplification. Six genes of theMAGE family includingMAGE-1 were found to be expressed at a high level in a number of tumors of various histological types. None was expressed in a large panel of healthy tissues, with the exception of testis and placenta.

DNA Methylation Is the Primary Silencing Mechanism for a Set of Germ Line- and Tumor-Specific Genes with a CpG-Rich Promoter

ABSTRACT A subset of male germ line-specific genes, theMAGE-type genes, are activated in many human tumors, where they produce tumor-specific antigens recognized by cytolytic T lymphocytes. Previous studies on gene MAGE-A1 indicated that transcription factors regulating its expression are present in all tumor cell lines whether or not they express the gene. The analysis of two CpG sites located in the promoter showed a strong correlation between expression and demethylation. It was also shown thatMAGE-A1 transcription was induced in cell cultures treated with demethylating agent 5′-aza-2′-deoxycytidine. We have now analyzed all of the CpG sites within the 5′ region of MAGE-A1 and show that for all of them, demethylation correlates with the transcription of the gene. We also show that the induction ofMAGE-A1 with 5′-aza-2′-deoxycytidine is stable and that in all the cell clones it correlates with demethylation, indicating that demethylation is necessary and sufficient to produce expression. Conversely, transfection experiments with in vitro-methylatedMAGE-A1 sequences indicated that heavy methylation suffices to stably repress the gene in cells containing the transcription factors required for expression. Most MAGE-type genes were found to have promoters with a high CpG content. Remarkably, although CpG-rich promoters are classically unmethylated in all normal tissues, those of MAGE-A1 and LAGE-1 were highly methylated in somatic tissues. In contrast, they were largely unmethylated in male germ cells. We conclude that MAGE-type genes belong to a unique subset of germ line-specific genes that use DNA methylation as a primary silencing mechanism.

Myc represses transcription through recruitment of DNA methyltransferase corepressor

The Myc transcription factor is an essential mediator of cell growth and proliferation through its ability to both positively and negatively regulate transcription. The mechanisms by which Myc silences gene expression are not well understood. The current model is that Myc represses transcription through functional interference with transcriptional activators. Here we show that Myc binds the corepressor Dnmt3a and associates with DNA methyltransferase activity in vivo. In cells with reduced Dnmt3a levels, we observe specific reactivation of the Myc‐repressed p21Cip1 gene, whereas the expression of Myc‐activated E‐boxes genes is unchanged. In addition, we find that Myc can target Dnmt3a selectively to the promoter of p21Cip1. Myc is known to be recruited to the p21Cip1 promoter by the DNA‐binding factor Miz‐1. Consistent with this, we observe that Myc and Dnmt3a form a ternary complex with Miz‐1 and that this complex can corepress the p21Cip1 promoter. Finally, we show that DNA methylation is required for Myc‐mediated repression of p21Cip1. Our data identify a new mechanism by which Myc can silence gene expression not only by passive functional interference but also by active recruitment of corepressor proteins. Furthermore, these findings suggest that targeting of DNA methyltransferases by transcription factors is a wide and general mechanism for the generation of specific DNA methylation patterns within a cell.

LAGE-1, a new gene with tumor specificity.

Representational difference analysis was used to identify genes that are expressed in a human melanoma cell line and not in normal skin. A cDNA clone that appeared to be specific for tumors was obtained and the corresponding gene was sequenced. This new gene was named LAGE‐1. Using a LAGE‐1 probe to screen a cDNA library from the same melanoma cell line, we identified a closely related gene, which proved to be identical to NY‐ESO‐1, a gene recently reported to code for an antigen recognized by autologous antibodies in an esophageal squamous cell carcinoma. Gene LAGE‐1 maps to Xq28. It comprises 3 exons. Alternative splicing produces 2 major transcripts encoding polypeptides of 210 and 180 residues, respectively. Expression of LAGE‐1 was observed in 25–50% of tumor samples of melanomas, non‐small‐cell lung carcinomas, bladder, prostate and head and neck cancers. The only normal tissue that expressed the gene was testis. As for MAGE‐A1, expression of LAGE‐1 is induced by deoxy‐azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation. The expression of LAGE‐1 is strongly correlated with that of NY‐ESO‐1. It is also clearly correlated with the expression of MAGE genes. Int. J. Cancer 76:903–908, 1998.© 1998 Wiley‐Liss, Inc.

Promoter-dependent mechanism leading to selective hypomethylation within the 5 ' region of gene MAGE-A1 in tumor cells

ABSTRACT Several male germ line-specific genes, including MAGE-A1, rely on DNA methylation for their repression in normal somatic tissues. These genes become activated in many types of tumors in the course of the genome-wide demethylation process which often accompanies tumorigenesis. We show that in tumor cells expressing MAGE-A1, the 5′ region is significantly less methylated than the other parts of the gene. The process leading to this site-specific hypomethylation does not appear to be permanent in these tumor cells, since in vitro-methylated MAGE-A1 sequences do not undergo demethylation after being stably transfected. However, in these cells there is a process that inhibits de novo methylation within the 5′ region of MAGE-A1, since unmethylated MAGE-A1 transgenes undergo remethylation at all CpGs except those located within the 5′ region. This local inhibition of methylation appears to depend on promoter activity. We conclude that the site-specific hypomethylation of MAGE-A1 in tumor cells relies on a transient process of demethylation followed by a persistent local inhibition of remethylation due to the presence of transcription factors.

Monoclonal Anti-MAGE-3 CTL Responses in Melanoma Patients Displaying Tumor Regression after Vaccination with a Recombinant Canarypox Virus

We have analyzed the T cell responses of HLA-A1 metastatic melanoma patients with detectable disease, following vaccination with a recombinant ALVAC virus, which bears short MAGE-1 and MAGE-3 sequences coding for antigenic peptides presented by HLA-A1. To evaluate the anti-MAGE CTL responses, we resorted to antigenic stimulation of blood lymphocytes under limiting dilution conditions, followed by tetramer analysis and cloning of the tetramer-positive cells. The clones were tested for their specific lytic ability and their TCR sequences were obtained. Four patients who showed tumor regression were analyzed, and an anti-MAGE-3.A1 CTL response was observed in three of these patients. Postvaccination frequencies of anti-MAGE-3.A1 CTL were 3 × 10−6, 3 × 10−3, and 3 × 10−7 of the blood CD8 T cells, respectively. These three responses were monoclonal. No anti-MAGE-1.A1 CTL response was observed. These results indicate that, like peptide immunization, ALVAC immunization produces monoclonal responses. They also suggest that low-level antivaccine CTL responses can initiate a tumor regression process. Taken together, our analysis of anti-MAGE-3.A1 T cell responses following peptide or ALVAC vaccination shows a degree of correlation between CTL response and tumor regression, but firm conclusions will require larger numbers.

INVOLVEMENT OF TWO ETS BINDING SITES IN THE TRANSCRIPTIONAL ACTIVATION OF THE MAGE1 GENE

The MAGE1 gene codes for an antigen recognized on melanoma cell line MZ2-MEL by autologous cytolytic T lymphocytes. It is expressed in a number of tumors of different histological origins, but not in normal tissues except in testis. The MAGE1 promoter region was analyzed by performing transient transfections in MZ2-MEL cells with luciferase reporter plasmids. A fragment extending from nucleotide −792 to +118 exhibited high transcriptional activity. By deletional analysis of this fragment, we identified five activating regions designated C, A, B′, B, and D. The activity of region A depends on the presence of region B′ and vice versa. Two inverted Ets motifs contained in regions B′ and B were found to drive 90% of the activity of the MAGE1 promoter in MZ2-MEL cells. Electrophoretic mobility shift assays performed with a nuclear extract from MZ2-MEL cells and with competitor oligonucleotides containing an Ets consensus site showed that nuclear proteins bind to the Ets motif of regions B′ and B. Similar experiments suggested that region A binds transcription factors of the Sp1 family. The MAGE1 promoter was found to exert transcriptional activity in tumor cells where the MAGE1 gene is not expressed, suggesting that other mechanisms, such as demethylation, may contribute to the tumor-specific expression of the gene.

Five new human cancer-germline genes identified among 12 genes expressed in spermatogonia

An important class of tumor‐specific antigens is encoded by male germline‐specific genes, such as MAGE genes, that are activated in many cancers of various histological types as a result of the demethylation of their promoter region. A number of these genes were shown to be expressed exclusively during the spermatogonia stage of spermatogenesis. A recent study reported the isolation of a new set of mouse genes that are expressed in spermatogonia but not in somatic tissues. Here, we tested the tumoral expression of the human orthologs of 12 of these genes. A remarkably high proportion, i.e., 5 of 12 genes, was found to be activated in a significant fraction of tumor samples of various histological types. Expression levels of the 5 genes, namely, NXF2, TAF2Q, FTHL17, TDRD1 and TEX15, were evaluated in normal and tumoral tissues. Except for TEX15, these genes showed sufficiently high expression levels in tumors and low background transcription in normal somatic tissues to qualify them as genes that potentially code for tumor‐specific antigens. Like previously described cancer‐germline genes, the 5 genes were induced in cells treated with a demethylating agent.

DNA hypomethylation in cancer: Epigenetic scars of a neoplastic journey.

Cytosine methylation is a heritable modification of DNA in mammalian cells, and has a determinant impact on long-term gene repression and genome stability. Genomic methylation patterns, which remain generally stable in the adult, become profoundly altered in most human tumors. While discrete DNA segments become hypermethylated in cancer cells, many more sequences become hypomethylated. This review discusses our current understanding of the mechanisms that lead to DNA hypomethylation in tumors. Evidence suggests that methylation losses are not random, but rather evolve into mosaic hypomethylation patterns. It is proposed that such hypomethylation patterns result from a historical event of transient DNA demethylation, and that transcriptional regulators contribute to determining which regions escape remethylation and remain therefore unmethylated. Finally, possible stages of tumor development during which the transient DNA demethylation process may take place will be discussed.