Monoclonal antibody 57B stains tumor tissues that express gene MAGE-A4.

Monoclonal antibody (Mab) 57B, which was raised against a recombinant MAGE-A3 protein, was tested for its ability to stain cells expressing various members of the MAGE-A gene family. COS-7 cells transfected with cDNAs encoding MAGE-A1, A2, A3, A4, A6, or A12 were stained, whereas those transfected with MAGE-A8, A9, A10, or A11 cDNAs were not. However, in tissue sections, we observed a different pattern of staining: the antibody effectively stained the tumors that expressed MAGE-A4 and only these tumors, regardless of the expression of the other MAGE-A genes. It seems, therefore, that at the level of MAGE gene expression found in tumors, a level clearly lower than that observed in transfected COS cells, only the MAGE-A4 protein can be reliably detected. We conclude that the 57B Mab should be useful for tumor diagnosis related to therapeutic vaccination involving MAGE-A4.

Expression of gene MAGE-A4 in Reed-Sternberg cells.

Genes of the MAGE-A family are expressed in several types of solid tumors but are silent in normal tissues with the exception of male germline cells, which do not carry HLA molecules.Therefore, peptides encoded by MAGE-A genes are strictly tumor-specific antigens that can be recognized by CTL and constitute promising targets for immunotherapy. The expression of 6 genes of the MAGE-A family was tested with reverse transcriptase-polymerase chain reaction in lymphoma samples. Among 38 samples of non-Hodgkin lymphoma, 1 anaplastic large cell lymphoma expressed genes MAGE-A1, -A2, -A3, -A4, and -A12, and 1 lymphoepithelioid T-cell lymphoma expressed gene MAGE-A4. Five of 18 samples (28%) from patients with Hodgkin disease expressed gene MAGE-A4. In tissue sections, staining by a monoclonal antibody that recognizes the MAGE-A4 protein was observed in 11 of 53 samples (21%) from patients with Hodgkin disease. In the positive samples, the Reed-Sternberg cells were strongly stained whereas the surrounding cells were not. These results indicate that Hodgkin disease may be a target for specific immunotherapy involving MAGE-A4 antigens.

Genes encoding tumor-specific antigens are expressed in human myeloma cells.

Genes of the MAGE, BAGE, GAGE, and LAGE-1/NY-ESO-1 families encode antigenic peptides that are presented by HLA class I molecules and that are recognized on human tumors by autologous cytolytic T lymphocytes. These genes are expressed in many solid tumor types but not in normal tissues, except male germline cells. Because the latter cells are devoid of HLA molecules, the derived antigens are strictly tumor-specific and should constitute safe immunogens for cancer immunotherapy. We detected a significant expression of these genes in a high proportion of bone marrow samples from patients with advanced multiple myeloma. This observation provides a basis for clinical trials aimed at inducing a cellular immune response directed at malignant plasma cells in advanced myeloma patients.

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-I. Using a LAGE-I 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-I, a gene recently reported to code for an antigen recognized by autologous antibodies in an esophageal squamous cell carcinoma. Gene LAGE-I maps to Xq28. It comprises 3 exons. Alternative splicing produces 2 major transcripts encoding polypeptides of 210 and 180 residues, respectively. Expression of LAGE-I 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-AI, expression of LAGE-I is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation. The expression of LAGE-I is strongly correlated with that of NY-ESO-I. It is also clearly correlated with the expression of MAGE genes.

Mutations of the beta2-microglobulin gene result in a lack of HLA class I molecules on melanoma cells of two patients immunized with MAGE peptides.

Mutations have been identified in the beta2-microglobulin gene of tumor cells of two metastatic melanoma patients who received immunizations with MAGE peptides. One mutation abolishes the start codon whereas the other introduces a premature stop codon. The second beta2-microglobulin allele of both tumors appears to be lost on the basis of sequence data and loss of microsatellite heterozygosity. The lack of beta2-microglobulin gene product results in the absence of HLA class I antigens on the surface of the tumor cells. This may explain why the tumors of both patients progressed despite the immunization treatment and shows the necessity of analyzing in depth the antigen presentation capability of the tumor cells for the interpretation of clinical trials involving anti-tumor vaccination.

The IL-9 receptor gene (IL9R): genomic structure, chromosomal localization in the pseudoautosomal region of the long arm of the sex chromosomes, and identification of IL9R pseudogenes at 9qter, 10pter, 16pter, and 18pter.

Cosmids containing the human IL-9 receptor (R) gene (IL9R) have been isolated from a genomic library using the IL9R cDNA as a probe. We have shown that the human IL9R cDNA as a probe. We have shown that hte human IL9R gene is composed of 11 exons and 10 introns, stretching over approximately 17 kb, and is located within the pseudoautosomal region of the Xq and Yq chromosome, in the vicinity of the telomere. Analysis f the 5' flanking region revealed multiple transcription initiation sites as well as potential binding motifs for AP1, AP2, AP3, Sp1, and NF-kB, although this region lacks a TATA box. Using the human IL9R cosmid as a probe to perform fluorescence in situ hybridization, additional signals were identified in the subtelomeric regions of chromosomes 9q, 10p, 16p, and 18p. IL9R homologs located on chromosomes 16 and 10 were completely sequenced. Although they are similar to the IL9R gene (approximately 90% identity), none of these copies encodes a functional receptor: none of them contains sequences homologous to the 5' flanking region or exon 1 of the IL9R gene, and the remaining ORFs have been inactivated by various point mutations and deletions. Taken together, our results indicate that the IL9R gene is located at Xq28 and Yq12, in the long arm pseudoautosomal region, and that four IL9R pseudogenes are located on 9q34, 10p15, 16p13.3, and 18p11.3, probably dispersed as the result of translocations during evolution.

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.

The tumor protein MAGE-1 is located in the cytosol of human melanoma cells.

The MAGE-1 protein has been localized to the cytosol of human melanoma culture cells (MZ2-MEL.43) by subcellular fractionation. Its distribution between nuclear, mitochondrial, microsomal and cytosolic fractions was established by SDS-PAGE and immunoblotting with a rabbit antiserum and compared to that of markers for the main cell compartments. The immunoreactive material was recovered in the cytosolic fraction as a polypeptide migrating at 42 kDa, which has been further identified as the MAGE-1 protein because it comigrates with the product of cell-free transcription-translation of the MAGE-1 cDNA. Location to the cytosol was confirmed by immunofluorescence microscopy.

Identification and characterization of the tumor-specific P1A gene product.

In murine mastocytoma P815, gene P1A directs the expression of antigens P815A and B which are the target of a T cell-mediated rejection response in syngeneic animals. This gene is expressed at a high level in various tumors, but is silent in normal tissues except testis and placenta; its activation is thus possibly related to malignant transformation. An anti-synthetic peptide rabbit antiserum reacted by immunoblotting with a cellular protein migrating near 40 kDa on SDS-PAGE. The immunoreactive protein was detected only in lysates from cells which express antigen P815A: P1.HTR mastocytoma cells and, after transfection with cosmids carrying the P1A gene, the antigen-loss variant P0.HTR cells and DAP-3 H-2Ld fibroblasts. The identity of this protein as the P1A gene product was confirmed by cell-free transcription-translation of the P1A cDNA, the product of which also migrated near 40 kDa in SDS-PAGE and was captured by protein A-Sepharose in the presence of the antiserum. Subcellular fractionation by differential and isopycnic centrifugation indicated that the P1A protein is associated with cytoplasmic membranes demonstrating a broad distribution with respect to size and density. Immunofluorescence microscopy also revealed a cytoplasmic signal, particularly intense in small vesicles, which coincides with that produced by an anti-mouse type I collagen guinea pig antiserum except near the cell periphery where the P1A signal is weaker. We conclude that the P1A protein is bound to membranes of the secretory pathway, at a concentration which goes increasing from the endoplasmic reticulum to secretion vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

The tum- antigens P91A and P198 derive from proteins located in the cytosolic compartment of cells.

To characterize the proteins P91Ap and P198p, of which mutants generate the tum- antigens P91A and P198, respectively, rabbit antisera were raised with ovalbumin-coupled synthetic peptides that correspond to their respective C terminus. In immunoadsorption tests using immobilized protein A the antisera recognized the translation products synthesized by rabbit reticulocyte lysates programmed with the SP6 polymerase transcripts of the P91A and P198 cDNA. The presence of the two proteins was demonstrated by SDS-PAGE and immunoblotting in all the mouse cells and organs examined. P91Ap is a constituent of the cytosol; despite a remarkable homology to the Drosophila diphenol oxidase DOX-A2, it separates from murine catechol oxidase activity in rate zonal sedimentation analysis. P198p is a ribosomal constituent, or a factor firmly linked to both the free and membrane-bound ribosomes. These subcellular localizations strengthen other evidence that the antigens presented to T lymphocytes by class I products of the major histocompatibility complex derive from proteins of the cytosol, or in direct contact with it.

Presentation of mouse tum- P91A antigen from chimeric proteins with different subcellular localizations by class I molecules of the major histocompatibility complex.

Like many antigens presented by class I molecules the mouse Ld-binding tum- antigen P91A derives from a cytosolic protein. To decide how stringent this localization is for presentation to cytotoxic lymphocytes (CTL) the P91A template has been inserted in the cDNA of rat esterase ES-10, a protein located in the endoplasmic reticulum (ER), and in the cDNA of mouse interleukin-9, a secretory product of lymphocytes. The esterase construct was also engineered to replace the C-terminal leucine by arginine, which causes secretion of the protein, or to delete the N-terminal presequence, which prevents transfer of the nascent chain to the ER. After cell-free transcription-translation, or transfection in COS cells, the products of the chimeric cDNA had the expected size and localization; however, the truncated form of esterase remained undetected in COS cells. The various chimeric templates were transfected in P1.HTR cells (H-2d); upon challenge with Ld-restricted anti-P91A CTL the cells were lyzed almost as efficiently as cells transfected with the full-length P91A cDNA. We conclude that peptide fragments that bind to class I molecules of the major histocompatibility complex can be generated in the ER.

Comparative study of the effect of chrysotile, quartz and rutile on the release of lymphocyte-activating factor (interleukin 1) by murine peritoneal macrophages in vitro.

The release of lymphocyte-activating factor (LAF) into the medium of cultured mouse resident peritoneal macrophages was estimated from the effect of this medium on the proliferation of mouse thymocytes in the presence of phytohaemagglutinin. Untriggered macrophages released little LAF into their culture medium. Upon addition of UICC chrysotile A (25-50 micrograms/10(6) macrophages), LAF appeared in the medium, and release continued for at least 20 h. DQ12 quartz was a more potent inducer of LAF production, while rutile was nearly inactive. Although chrysotile and quartz caused cell damage (as estimated by the release of lactate dehydrogenase), it was established that LAF release was not attributable to leakage of preformed intracellular LAF. The lymphoproliferative activity detected in macrophage media was stable at 56 degrees C and nondialysable, which corresponds to the properties of interleukin 1. These biochemical observations are consistent with the non-specific stimulation of the immune system found in asbestotic and silicotic patients.

The membrane of the rough endoplasmic reticulum contains cytoplasmically exposed high affinity GTP-binding sites.

Binding experiments using [14C]GTP and rat liver rough microsomes gave Scatchard plots with Kd congruent to 0.1 microM and a binding capacity congruent to 40 pmol/mg microsomal protein. Removal of the ribosomes did not modify the binding parameters. GTP was competed out by GTP analogues but not by ATP. Limited proteolysis of rough microsomes decreased the GTP-binding capacity and prevented GTP from suppressing the latency of mannose-6-phosphatase and of the synthesis of dolichol-linked chitobiose. The GTP-binding protein is probably involved in these effects of GTP. Its function could be to act in the association-dissociation of membrane components at the ribosome-membrane junction.

Alteration of membrane barrier in stripped rough microsomes from rat liver on incubation with GTP: its relevance to the stimulation by this nucleotide of the dolichol pathway for protein glycosylation.

The membrane barrier of stripped rough microsomes from rat liver is markedly altered on incubation with GTP at 37 degrees C: after 30 min the structure-linked latency of mannose-6-phosphatase was considerably reduced, and esterase and nucleoside diphosphatase were partly released into the suspension medium. This phenomenon was already maximal with 30 microM GTP and was specific for this nucleotide. Similar conditions enhance the dolichol-mediated glycosylation of protein in microsomes incubated with uridine diphosphate N-acetylglucosamine and guanosine diphosphate mannose (Godelaine, D., H. Beaufay, M. Wibo, and A. Amar-Costesec, 1979, Eur. J. Biochem., 96:17-26; Godelaine, D., H. Beaufay, and M. Wibo, 1979, Eur. J. Biochem., 96:27-34). The GTP-induced permeability and glycosylation activities evolved in parallel in rough microsomes subjected to various treatments to detach the ribosomes and were maximal after removal of congruent to 60% of the RNA. In addition, GTP had no effect of this type in smooth microsome subfractions. Triton X-100, in spite of complex inhibitory effects on glycosylation reactions, mimicked the action of GTP by increasing the amount of microsomal dolichylphosphate that reacts with uridine diphosphate N-acetylglucosamine and by enhancing synthesis of dolichylpyrophosphoryl-chitobiose at concentrations greater than 2 mg/ml. Thus, GTP may activate dolichol-mediated glycosylation reactions in stripped microsomes by lowering the permeability barrier that prevents access of sugar nucleotides to the inner aspect of the membrane. The genuine role of GTP in the functioning of the endoplasmic reticulum membrane in situ remains unknown. Because GTP seems to act only on rough microsomes, we hypothesize that this role is somehow related to biosynthesis of protein by the rough endoplasmic reticulum.

The dolichol pathway of protein glycosylation in rat liver. Evidence that GTP promotes transformation of endogenous dolichyl phosphate into dolichylpyrophosphoryl-N-acetylglucosamine in stripped rough microsomes.

Tunicamycin, an antibiotic which inhibits the transfer of N-acetylglucosamine 1-phosphate to dolichyl phosphate, has been used to decide whether or not, in stripped rough microsomes incubated with UDP-N-acetylglucosamine and GDP-mannose in the absence of detergent, the earliest effect of GTP in core glycosylation of proteins is to enhance synthesis of dolichylpyrophosphoryl-N-acetylglucosamine from endogenous dolichyl phosphate, or to transform this monoglycoside derivative into dolichylpyrophosphorylchitobiose. It has been found that the presence of tunicamycin in the reaction medium annihilates incorporation of N-acetylglucosamine and mannose into all kinds of glycoside derivative of dolichyl pyrophosphate, whereas dolichylphosphorylmannose is then formed in greater amount. Incorporation of N-acetylglucosamine into protein was also abolished; that of mannose was considerably reduced. Other experiments showed that transfer of N-acetylglucosamine 1-phosphate is the only reaction of the lipid intermediates pathway that becomes limiting after addition of tunicamycin in our GTP-stimulated system. Taking these and previous results from this laboratory [Godelaine et al. (1979) Eur. J. Biochem. 96, 17-26 and 27-34] into account, we conclude that GTP enhances the transformation of endogenous dolichyl phosphate into dolichylpyrophosphoryl-N-acetylglucosamine and leads to the complete assembly of dolichol-linked oligosaccharides which become ultimately transferred to protein. Triton X-100 increased the amount of dolichol glycosylated and markedly raised the ratio of labelled dolichylpyrophosphorylchitobiose to dolichylpyrophosphoryl-N-acetylglucosamine. Such changes being induced by GTP, we suggest that this nucleotide makes it possible to overcome a structural barrier of rough microsomes.

Quantitative assay and subcellular distribution of enzymes acting on dolichyl phosphate in rat liver.

To establish on a quantitative basis the subcellular distribution of the enzymes that glycosylate dolichyl phosphate in rat liver, preliminary kinetic studies on the transfer of mannose, glucose, and N-acetylglucosamine-1-phosphate from the respective (14)C- labeled nucleotide sugars to exogenous dolichyl phosphate were conducted in liver microsomes. Mannosyltransferase, glucosyltransferase, and, to a lesser extent, N- acetylglucosamine-phosphotransferase were found to be very unstable at 37 degrees C in the presence of Triton X-100, which was nevertheless required to disperse the membranes and the lipid acceptor in the aqueous reaction medium. The enzymes became fairly stable in the range of 10-17 degrees C and the reactions then proceeded at a constant velocity for at least 15 min. Conditions under which the reaction products are formed in amount proportional to that of microsomes added are described. For N- acetylglucosaminephosphotransferase it was necessary to supplement the incubation medium with microsomal lipids. Subsequently, liver homogenates were fractionated by differential centrifugation, and the microsome fraction, which contained the bulk of the enzymes glycosylating dolichyl phosphate, was analyzed by isopycnic centrifugation in a sucrose gradient without any previous treatment, or after addition of digitonin. The centrifugation behavior of these enzymes was compared to that of a number of reference enzymes for the endoplasmic reticulum, the golgi complex, the plasma membranes, and mitochondria. It was very simily to that of enzymes of the endoplasmic reticulum, especially glucose-6-phosphatase. Subcellular preparations enriched in golgi complex elements, plasma membranes, outer membranes of mitochondira, or mitoplasts showed for the transferases acting on dolichyl phosphate relative activities similar to that of glucose- 6-phosphatase. It is concluded that glycosylations of dolichyl phosphate into mannose, glucose, and N-acetylglucosamine-1-phosphate derivatives is restricted to the endoplasmic reticulum in liver cells, and that the enzymes involved are similarly active in the smooth and in the rough elements.

Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes.

Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.

Coalescence of microsomal vesicles from rat liver: a phenomenon occurring in parallel with enhancement of the glycosylation activity during incubation of stripped rough microsomes with GTP.

Rough microsomes from rat liver have been subjected to various treatments and incubated afterwards with UDP-N-acetyl-[14C]glucosamine and GDP-mannose in the presence of GTP (0.5 mM), or of other nucleotides. In agreement with earlier results from this laboratory, the preparations previously treated to strip off the ribosomes and incubated in the presence of GTP assembled dolichol-linked oligosaccharides and transferred these oligosaccharides to endogenous protein acceptors much more actively than untreated preparations, or stripped preparations incubated in the absence of GTP. Thin-section and freeze-fracture electron microscopy have revealed that pyrophosphate-treated preparations incubated with GTP are aggregated and contain numerous vesicles as large as 1-4 micrometer, or more. Such large vesicles were not present before incubation and thus were considered to have been formed through coalescence of regular-sized ones. Like glycosylation, the coalescence phenomenon depends upon the removal of ribosomes, because it occurred whether ribosomes had been stripped, at least partly, with pyrophosphate, KCl, or puromycin, but not when rough microsomes had been washed with 0.25 M sucrose or with KCl and MgCl2. Like glycosylation, it also depends on the addition of GTP and was not induced by ATP, UTP, CTP, and nonhydrolysable analogues of GTP. Rough microsomes coalesced, however, when pyrophosphate-treated preparations were incubated with GTP in the absence of nucleotide sugars, or in the presence f tunicamycin, indicating that the coalescence phenomenon does not result from the glycosylation of some membrane constituents.