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O De Backer

Publications and source records attributed to O De Backer.

18 recordsLinked to original sources
PubMed2001

MAGE-A4, a germ cell specific marker, is expressed differentially in testicular tumors.

BACKGROUND: Testicular germ cell tumors are the most common malignancy in young males, and the frequency of these tumors has risen dramatically over the last century. Because it is known that the MAGE genes are expressed in a wide variety of tumors but are expressed only in the mitotic spermatogonia (germ cells) and in the primary spermatocytes in the normal testis, the authors screened the expression of MAGE-A4 in a panel of testicular germ cell tumors. METHODS: Monoclonal antibody 57B raised against MAGE-A4 was tested immunohistochemically on 12 classical seminomas, 5 anaplastic seminomas, 10 various specimens of nonseminomatous germ cell tumors (NSGCTs), 2 combined tumors containing seminoma components, 1 Sertoli cell tumor, 2 Leydig cell tumors, and 15 carcinomas in situ (CIS). In addition, monoclonal antibody 57B was tested on embryonic gonad (age 8 weeks) and fetal gonads (ages 15 weeks, 17 weeks, and 28 weeks). RESULTS: Classical seminomas uniformly and specifically expressed MAGE-A4 compared with anaplastic seminomas and NSGCTs, which were negative for this antigen. Specific expression of MAGE-A4 also was seen in subpopulations of CIS cells, providing additional evidence for heterogeneity of the phenotype of these cells, in which it is believed that differentiation and proliferation generate seminomas and NSGCTs. Finally, MAGE-A4 was expressed in the fetal precursors of the stem germ cells from 17 weeks of gestation onward, in accordance the fact that CIS can arise from prespermatogonia in the fetus. CONCLUSIONS: MAGE-A4 can be considered a potential specific marker for normal premeiotic germ cells and germ cell tumors and can be used to characterize classical seminomas.

Antigens, Neoplasm↗
PubMed2001

An overview of the MAGE gene family with the identification of all human members of the family.

The first human members of the MAGE gene family that have been described are expressed in tumor cells but silent in normal adult tissues except in the male germ line. Hence, they encode strictly tumor-specific antigens that represent attractive targets for cancer immunotherapy. However, other members of the family were recently found to be expressed in normal cells, indicating that the family is larger and more disparate than initially expected. We therefore performed a database screening to identify all of the recorded members of both classes of human MAGE genes. This report provides an overview of the MAGE family and proposes a general nomenclature for all of the MAGE genes identified thus far. We found that the MAGE-D genes were particularly well conserved between man and mouse, suggesting that they exert important functions. In addition, the genomic structure of the MAGE-D genes indicates that one of them corresponds to the founder member of the family, and that all of the other MAGE genes are retrogenes derived from that common ancestral gene. Intriguingly, the COOH-terminal domain of MAGE-D3 was found to be identical to trophinin, a previously described protein believed to be involved in embryo implantation.

Amino Acid Sequence↗
PubMed1999

Characterization of the GAGE genes that are expressed in various human cancers and in normal testis.

The GAGE-1 gene was identified previously as a gene that codes for an antigenic peptide, YRPRPRRY, which was presented on a human melanoma by HLA-Cw6 molecules and recognized by a clone of CTLs derived from the patient bearing the tumor. By screening a cDNA library from this melanoma, we identified five additional, closely related genes named GAGE-2-6. We report here that further screening of this library led to the identification of two more genes, GAGE-7B and -8. GAGE-1, -2, and -8 code for peptide YRPRPRRY. Using another antitumor CTL clone isolated from the same melanoma patient, we identified antigenic peptide, YYWPRPRRY, which is encoded by GAGE-3, -4, -5, -6, and -7B and which is presented by HLA-A29 molecules. Genomic cloning of GAGE-7B showed that it is composed of five exons. Sequence alignment showed that an additional exon, which is present only in the mRNA of GAGE-1, has been disrupted in gene GAGE-7B by the insertion of a long interspersed repeated element retroposon. These GAGE genes are located in the p11.2-p11.4 region of chromosome X. They are not expressed in normal tissues, except in testis, but a large proportion of tumors of various histological origins express at least one of these genes. Treatment of normal and tumor cultured cells with a demethylating agent, azadeoxycytidine, resulted in the transcriptional activation of GAGE genes, suggesting that their expression in tumors results from a demethylation process.

Amino Acid Sequence↗
PubMed1999

A new family of mouse genes homologous to the human MAGE genes.

The human MAGE genes are expressed in a wide variety of tumors but not in normal cells, with the exception of the male germ cells, placenta, and, possibly, cells of the developing embryo. These genes encode tumor-specific antigens recognized by cytolytic T lymphocytes. The MAGE genes are located on the X chromosome, in three clusters denoted MAGE-A, B, and C, mapping at q28, p21.3, and q26, respectively. The function of these genes remains unknown. Because mice offer many advantages for the study of genes that may be involved in embryonic development, we looked for the murine equivalents of the 12 human MAGE-A genes. Using a MAGE-A probe, we isolated 8 new murine genes that are homologous to the MAGE genes. On average, the open reading frames (ORFs) of these 8 closely related genes display a slightly higher degree of nucleotide identity with the MAGE-A ORFs than with the MAGE-B or MAGE-C ORFs. Furthermore, like MAGE-A genes, they encode acidic proteins, whereas the MAGE-B genes encode basic proteins. Accordingly, these 8 murine genes were named Mage-a1 to 8 (approved symbols Magea1 to 8). Mage-a genes were mapped in two different loci on the mouse X chromosome. Mage-a4 and Mage-a7 are located in a region that is syntenic to either Xp21 or Xq28. The 6 other genes are arranged in a cluster located in a region syntenic to Xp22. Like their human counterparts, Mage-a genes were found to be transcribed in adult testis, but not in other tissues. Expression of some Mage-a genes was also detected in tumor cell lines. Two Mage-a genes were found to be expressed in blastocysts.

Animals↗
PubMed1997

Genes coding for melanoma antigens recognised by cytolytic T lymphocytes.

It is now well established that human melanoma cells express antigens that are recognised by cytolytic T lymphocytes derived from the tumour-bearing patient. The molecular definition of these antigens is progressing at an accelerated pace. The currently characterised melanoma antigens can be classified into three categories: differentiation antigens, antigens encoded by genes that are specifically expressed in tumours, and antigens encoded by mutated genes. Several of these antigens are sufficiently tumour-specific to qualify them as candidate anti-cancer vaccines in melanoma patients.

Antigens↗
PubMed1996

The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation.

Human gene MAGE-1 encodes tumor-specific antigens that are recognized on melanoma cells by autologous cytolytic T lymphocytes. This gene is expressed in a significant proportion of tumors of various histological types, but not in normal tissues except male germ-line cells. We reported previously that reporter genes driven by the MAGE-1 promoter are active not only in the tumor cell lines that express MAGE-1 but also in those that do not. This suggests that the critical factor causing the activation of MAGE-1 in certain tumors is not the presence of the appropriate transcription factors. The two major MAGE-1 promoter elements have an Ets binding site, which contains a CpG dinucleotide. We report here that these CpG are demethylated in the tumor cell lines that express MAGE-1, and are methylated in those that do not express the gene. Methylation of these CpG inhibits the binding of transcription factors, as seen by mobility shift assay. Treatment with the demethylating agent 5-aza-2'-deoxycytidine activated gene MAGE-1 not only in tumor cell lines but also in primary fibroblasts. Finally, the overall level of CpG methylation was evaluated in 20 different tumor cell lines. It was inversely correlated with the expression of MAGE-1. We conclude that the activation of MAGE-1 in cancer cells is due to the demethylation of the promoter. This appears to be a consequence of a genome-wide demethylation process that occurs in many cancers and is correlated with tumor progression.

Antigens, Neoplasm↗
PubMed1995

A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma.

Human melanoma MZ2-MEL expresses several distinct antigens that are recognized by autologous cytolytic T lymphocytes (CTL). Some of these antigens are encoded by genes MAGE-1, MAGE-3, and BAGE, which are expressed in a large fraction of tumors of various histological types but are silent in normal adult tissues with the exception of testis. We report here the identification of the gene coding for MZ2-F, another antigen recognized by autologous CTL on MZ2-MEL cells. This gene, which was named GAGE-1, is not related to any presently known gene. It belongs to a family of genes that are expressed in a variety of tumors but not in normal tissues, except for the testis. Antigenic peptide YRPRPRRY, which is encoded by GAGE-1, is recognized by anti-MZ2-F CTL on class I molecule HLA-Cw6. The two genes of the GAGE family that code for this peptide, namely GAGE-1 and GAGE-2, are expressed in a significant proportion of melanomas (24%), sarcomas (25%), non-small cell lung cancers (19%), head and neck tumors (19%), and bladder tumors (12%). About 50% of melanoma patients carry on their tumor at least one of the presently defined antigens encoded by the MAGE, BAGE, and GAGE genes.

Amino Acid Sequence↗
PubMed1995

Structure, chromosomal location, and expression pattern of three mouse genes homologous to the human MAGE genes.

The human MAGE1 gene directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. MAGE1 belongs to a family of genes that are expressed in a number of tumors of various histological types but not in normal tissues except testis. The MAGE genes are arranged in two groups that are located within two different regions of the human X chromosome (Xq26-qter and Xp21.3). By hybridizing mouse genomic libraries with a MAGE1 probe, we identified three homologous genes. Two of these mouse genes, Smage1 and Smage2, are more than 99% identical to each other and encode the same protein of 330 aa. The 5' noncoding region of Smage2 provides the potential for regulating the expression of the gene through several different promoters located in front of alternative first exons. The third gene, Smage3, has the structure of a processed transcript. It codes for a protein with only 11 aa substitutions with respect to the Smage1/2 product. Somatic cell hybrids and interspecific backcross analysis showed that Smage3 is autosomal and that Smage1 and Smage2 are located between the Dmd and the Ar loci on the mouse X chromosome. Since this region is syntenic to the human Xp21.1-p22.1 region, we conclude that Smage1 and Smage2 are homologous to the MAGE-Xp rather than to the MAGE-Xq genes. Smage1/2 transcripts were detected in several tumor and embryonal cell lines but not in normal mouse tissues with the exception of testis. Expression of Smage3 was found in embryos from Day 11 to Day 15.

Amino Acid Sequence↗
PubMed1995

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.

Antigens, Neoplasm↗
PubMed1994

Positive selection of recombinant plasmids based on the EcoK restriction activity of Escherichia coli K-12.

We have constructed a pTZ19R-derived vector which allows efficient positive selection of recombinant plasmids. The system uses the EcoK restriction activity of Escherichia coli K-12 to select against non-recombinant plasmids. The vector contains an EcoK site which, if deleted or disrupted by ligating a DNA fragment, yields recombinant plasmids that are no longer susceptible to EcoK restriction when transformed into a restriction-proficient E. coli host.

Base Sequence↗
PubMed1993

Sequence of the Salmonella typhimurium StyLT1 restriction-modification genes: homologies with EcoP1 and EcoP15 type-III R-M systems and presence of helicase domains.

The StyLT1 restriction-modification (R-M) system of Salmonella typhimurium has recently been suggested to belong to the type-III R-M systems [De Backer and Colson, Gene 97 (1991) 103-107]. The nucleotide sequences of StyLT1 mod and res have been determined. Two closely adjacent open reading frames were found 12 bp apart with coding capacities of 651 (Mod) and 982 (Res) amino acids (aa), respectively. The genes, lying in the same direction of transcription in the mod-res order, are transcribed as distinct units. The deduced aa sequences reveal homologies with known type-III enzymes from the Escherichia coli P1 prophage, E. coli P15 plasmid and Bacillus cereus chromosome. In addition, the StyLT1 restriction endonuclease (ENase), like other type-I and type-III ENases, contains sequence motifs characteristic of superfamily-II helicases, which may be involved in DNA unwinding at the cleavage site.

Amino Acid Sequence↗
PubMed1991

Identification of the recognition sequence for the M.StyLTI methyltransferase of Salmonella typhimurium LT7: an asymmetric site typical of type-III enzymes.

The StyLTI restriction-modification (R-M) system is encoded by chromosomal genes of Salmonella typhimurium LT7. We report here the identification of the nucleotide (nt) sequence methylated by the StyLTI modification methyltransferase (M.StyLTI). This enzyme was partially purified from an Escherichia coli strain expressing the cloned M.StyLTI-encoding gene, but lacking StyLTI restriction activity, and used to methylate DNAs of known sequence, using S-adenosyl-[methyl-3H]-methionine as the methyl donor. The [3H]methylated DNA was then digested with various endonucleases. Examination of labelled and unlabelled restriction fragments allowed us to map the M.StyLTI sites in perfectly defined regions of the DNA. Comparison of the nt sequences of DNA segments with or without M.StyLTI sites permitted us to identify the asymmetric and nondegenerate pentanucleotide, 5'-CAGAG-3', 3'-GTCTC-5' as the StyLTI sequence. M.StyLTI was found to methylate only the 3' A (see asterisk) in the upper strand of this sequence. Thus, M.StyLTI recognises and methylates the DNA in a manner very similar to that of the three known type-III MTases, M.EcoPI, M.EcoP15, and M.HinfIII. This strongly suggests that StyLTI constitutes a fourth type-III R-M system.

Adenine↗
PubMed1991

Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium.

The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.

Cloning, Molecular↗
PubMed1991

Transfer of the genes for the StyLTI restriction-modification system of Salmonella typhimurium to strains lacking modification ability results in death of the recipient cells and degradation of their DNA.

The genes encoding the restriction-modification system StyLTI of Salmonella typhimurium were inserted in vivo into the conjugative plasmid pULB21. This allowed us to transfer the StyLTI genes at a very high frequency and to monitor the fate of recipient cells after mating. Transfer of the StyLTI restriction and modification genes into a modificationless recipient was lethal and resulted in degradation of the cell's DNA. This indicates that, in contrast to any other known restriction-modification systems, StyLTI cannot be established after horizontal transfer into a naive host.

Conjugation, Genetic↗
PubMed1989

A procedure for the preparation of bacterial DNA that employs dimethyl sulfoxide to induce the lysis of cells.

A protocol for the preparation of DNA from Escherichia coli and Bacillus subtilis without the use of lysozyme as a permeabilizing agent is described. This preliminary step is carried out by treating the cells with dimethyl sulfoxide. A 5-min incubation of the cell pellet in the pure solvent, followed by the treatment with sodium dodecyl sulfate, is sufficient to induce cell lysis. The plasmid DNAs obtained by this method were equivalent in purity and quantity to the material prepared from lysozyme-digested cells and amenable to restriction and ligation. Transformation by plasmid and genomic DNAs prepared from dimethyl sulfoxide-treated cells was demonstrated.

Bacillus subtilis↗