Monoclonal antibodies against recombinant-MAGE-1 protein identify a cross-reacting 72-kDa antigen which is co-expressed with MAGE-1 protein in melanoma cells

The MAGE-1 gene codes for tumor-associated peptides recognized by cytolytic T lymphocytes in association with MHC-class-I molecules such as HLA-AI and HLA-Cw16. In the course of a study aiming at the immunohistochemical detection of the MAGE-1 gene product in tumor samples, 2 mouse monoclonal antibodies (MAbs) directed against a full-length recombinant MAGE-1 fusion protein were found to react strongly not only with the 46-kDa MAGE-1 protein, but also with a 72-kDa product in immunoblots of lysates obtained from several MAGE-1-mRNA-positive melanoma cell lines. Pre-incubation of the antibodies with the recombinant MAGE-1 fusion protein abolished their reactivity both with MAGE-1 protein and with the 72-kDa product, thus confirming the occurrence of antigenic determinant(s) shared by the 2 proteins. The 72-kDa protein is not an alternative product of MAGE-1, since it was still detected in lysates of a MAGE-1 loss variant derived from a MAGE-1-positive melanoma cell line. Moreover, the 72-kDa protein does not appear to be a product of the other members of the MAGE gene family known to be expressed in tumors (such as MAGE-2, -3, -4 and -12). Interestingly, expression of the 72-kDa protein was found to be correlated with that of MAGE-I protein. Thus, in 30 tumor cell lines analyzed by immunoblotting and RT-PCR, the 72-kDa protein was never detected in MAGE-1-mRNA-negative cell lines, while it was co-expressed with MAGE-1 protein in 12 out of 15 cell lines expressing MAGE-1. Furthermore, the 72-kDa protein was detected in lysates of human testis, the only normal tissue known to express MAGE-1. Finally, treatment of MAGE-1-mRNA-negative cell lines with 5-Aza-2′-deoxycytidine, a hypomethylating agent known to induce MAGE-1 expression, resulted in the expression of the 72-kDa protein. Taken collectively, these findings suggest that expression of the gene encoding the 72-kDa protein identified in this study through antigenic determinant(s) shared with MAGE-1 protein is regulated in a way similar to that of MAGE-1. © 1996 Wiley-Liss, Inc.     

A member of the melanoma antigen-encoding gene (MAGE) family is expressed in human skin during wound healing

MAGE-1 has been identified as a human gene, which directs the expression of an antigen being recognized on melanoma cells by autologous cytolytic T cells. MAGE-1 is expressed in melanomas and some other tumors. It has been proposed that this gene may be linked to the transformation event and therefore might serve as an approach to precisely targeted immunotherapy. Prior to such an approach, extensive testing of normal human tissue is necessary to establish the tumor-specific nature of MAGE-1 expression. Similar to events that occur during neoplastic tumor growth and spreading, wound healing involves a complex interrelationship between various cell types which migrate, proliferate and differentiate. Therefore, we investigated the expression of MAGE-1 in skin during wound repair. We could detect MAGE-1 mRNA by RT-PCR followed by specific hybridization as well as by Northern blotting in human skin from the 1st to the 7th day after wounding. Comparison of the expression of MAGE mRNA with that of β-actin mRNA showed that it is expressed in amounts equal to about and at least one-fifth that of β-actin. Our data strongly suggest that MAGE mRNA expression is not necessarily linked to neoplastic transformation, but rather represents the function of a cellular gene which is activated during inflammation or early tissue repair.     

MAGE-11 protein is highly conserved in higher organisms and located predominantly in the nucleus

The MAGE-11 gene belongs to a family whose products were identified first in tumor tissue. The MAGE-11 gene product has not been characterized in detail. We have isolated MAGE-11 cDNA from HeLa cells and confirmed the presence of MAGE-11 protein and of at least 2 other MAGE proteins (MAGE-1 and MAGE-3) in this cell line. Monoclonal antibodies (MAbs), obtained by using a GST-MAGE-11 fusion protein, detect MAGE-11 protein in HeLa cells as a 48 kDa protein. In contrast to other known proteins of the MAGE family, MAGE-11 is found mainly in the nucleus. Immunoprecipitation out of whole-cell extracts from different species reveals that MAGE-11 protein is highly conserved among mammalian cells, suggesting a conserved and important function. Int. J. Cancer 75:762–766, 1998.© 1998 Wiley-Liss, Inc.     

Expression of mage genes in transitional-cell carcinomas of the urinary bladder

Human genes MAGE-1 and MAGE-3 code for distinct antigens, which are recognized on melanoma cells by autologous cytolytic T lymphocytes (CTL). These antigens may constitute useful targets for anti-cancer immunotherapy, since no expression of MAGE genes has been observed in normal tissues other than testis. Out of 57 samples of primary transitional-cell carcinomas of the bladder, 12 (21%) expressed MAGE-1 and 20 (35%) expressed MAGE-3. All but one of the tumors expressing MAGE-1 also expressed MAGE-3. Genes MAGE-2 and MAGE-4, which are closely related to MAGE-1 and MAGE-3, were expressed by 30% and 33% of the tumors respectively. MAGE expression was more frequent in advanced tumor stages: 61% of the invasive tumors (stage ± T2) were positive for expression of at least one of the four genes, whereas only 28% of the superficial tumors (stages Ta and Tl) expressed these genes. © 1995 Wiley-Liss, Inc.     

MAGE-1 and MAGE-3 or -6 expression in neuroblastoma-related pediatric solid tumors

MAGE-1 and MAGE-3 or -6 are genes encoding melanoma-rejection antigens recognized by cytotoxic T lymphocytes in an HLA-AI restriction manner. MAGE-1 and MAGE-3 or -6 were expressed in 5/14 (36%) and 6/14 (43%) neuroblastoma (NB) cell lines, and in 20/41 (49%) and 24/41 (59%) clinical NB-related tumors, respectively. Additionally, they were also expressed in pediatric tumors of other types such as rhabdomyo-sarcoma and Wilms' tumor. MAGE-1 expression at a functional level in tumor cells was confirmed by the cytotoxicity assay using MAGE-1-specific tumor-infiltrating lymphocytes (TIL). In clinical NB-related tumors, MAGE-3 or -6 expression demonstrated an inverse correlation to clinical stage. Furthermore, although the sample number was small, the incidence of MAGE-1 and/or MAGE-3 or -6 expression was significantly correlated to the absence of metastasis and a more favorable clinical outcome (p < 0.05). These results may suggest that NB cells silent for the expression of MAGE genes escape from the host anti-tumor immune response and consequently retain a growth advantage. Finally, NB-related tumors could be reliable candidates for immunotherapy targeted towards MAGE gene products. © 1996 Wiley-Liss, Inc.     

SSX: A multigene family with several members transcribed in normal testis and human cancer

Analysis of t(X;18) translocation in synovial sarcoma had previously led to the definition of the SSX2 gene, the fusion partner on chromosome X. Subsequent screening of testicular cDNA libraries identified 2 highly homologous genes, SSX1 and SSX3. Among these 3 genes, SSX2 has been found to be identical to HOM-MEL-40, which codes for an immunogenic tumor antigen expressed in various human cancers. SSX2 thus belongs to the family of cancer/testis (CT) antigens, i.e., immunogenic protein antigens with characteristic mRNA expression in normal testis and in cancer. To define additional CT antigens, we have immuno-screened a testicular cDNA expression library with an allogeneic serum from a melanoma patient, and both SSX2 and SSX3 were isolated. Further studies using testicular cDNA and SSX probes defined 2 new members of this gene family, SSX4 and SSX5, while a shorter cDNA variant of SSX4 was also identified. All 5 members of the SSX family shared strong sequence homology, with nucleotide homology ranging from 88 to 95% and amino acid homology ranging from 77 to 91%. Genomic cloning of a prototype SSX gene (SSX2) showed that its coding region is encoded by 6 exons, and the shortened form of SSX4 cDNA represents an alternatively spliced product lacking the 5th exon. Analysis of SSX mRNA expression by gene-specific RT-PCR confirmed that all 5 SSX genes are expressed in testis. In addition, analysis of a panel of 12 melanoma cell lines showed strong mRNA expression of either SSX1 (3/12), SSX2 (3/12), SSX4 (1/12), or SSX5 (1/12), indicating variable activation of the genes in malignant cells. Int. J. Cancer 72:965–971, 1997. © 1997 Wiley-Liss, Inc.     

Identification of HLA-A*0201-restricted CTL epitopes encoded by the tumor-specific MAGE-2 gene product

MAGE-2 is expressed in many tumors, including melanoma, laryngeal tumors, lung tumors and sarcomas, but not in healthy tissue, with the exception of testis. Thus, MAGE-2-derived peptides that bind to HLA class I molecules and elicit cytotoxic T lymphocyte (CTL) responses could be of significant therapeutic importance. In this study, we show that several MAGE-2-derived peptides bind with high affinity to HLA-A*0201. Three of them form complexes with HLA-A*0201 that are stable at 37°C and are immunogenic in HLA-A*0201K^b transgenic mice. Moreover, CTLs against 2 of them (M2 112-120, and M2 157-166) specifically recognize cells that express both the MAGE-2 protein and HLA-A*0201K^b. These 2 peptides are processed and presented in the context of HLA-A*0201. Therefore, these peptides are candidate components in peptide-based vaccines for the treatment and prevention of several types of MAGE-2-expressing cancers. Int. J. Cancer 73:125–130, 1997. © 1997 Wiley-Liss, Inc.     

nmb,a novel gene,is expressed in low-metastatic human melanoma cell lines and xenografts

From a subtractive cDNA library, we isolated several cDNA clones which showed differential expression between highly and lowly metastatic human melanoma cell lines. One clone, designated nmb, showed preferential expression in the low-meta-static cell lines and was chosen for further characterization. Sequence analysis revealed that this clone represents a novel gene, encoding a putative transmembrane glycoprotein which showed the highest homology to the precursor of pMEL17, a melanocyte-specific protein. nmb RNA expression was absent in most tumor-cell lines tested and not restricted to the melanocytic lineage. Transfection of a partial nmb cDNA into a highly metastatic melanoma cell line (BLM) resulted, in 2 of 3 transfectants, in slower subcutaneous tumor growth and, in 1 of 3 transfectants, in reduction of the potential for spontaneous metastasis in nude mice. © 1995 Wiley-Liss, Inc.     

Quantitative expression and immunogenicity of MAGE‐3 and ‐6 in upper aerodigestive tract cancer

The MAGE antigens are frequently expressed cancer vaccine targets. However, quantitative analysis of MAGE expression in upper aerodigestive tract (UADT) tumor cells and its association with T‐cell recognition has not been performed, hindering the selection of appropriate candidates for MAGE‐specific immunotherapy. Using quantitative RT‐PCR (QRT‐PCR), we evaluated the expression of MAGE‐3/6 in 65 UADT cancers, 48 normal samples from tumor matched sites and 7 HLA‐A*0201+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. Expression results were confirmed using Western blot. HLA‐A*0201:MAGE‐3‐ (271–279) specific cytotoxic T lymphocytes (MAGE‐CTL) from SCCHN patients and healthy donors showed that MAGE‐3/6 expression was highly associated with CTL recognition in vitro. On the basis of the MAGE‐3/6 expression, we could identify 31 (47%) of the 65 UADT tumors, which appeared to express MAGE‐3/6 at levels that correlated with efficient CTL recognition. To confirm that the level of MAGE‐3 expression was responsible for CTL recognition, 2 MAGE‐3/6 mRNA^high SCCHN cell lines, PCI‐13 and PCI‐30, were subjected to MAGE‐3/6‐specific knockdown. RNAi‐transfected cells showed that MAGE expression and MAGE‐CTL recognition were significantly reduced. Furthermore, treatment of cells expressing low MAGE‐3/6 mRNA with a demethylating agent, 5‐aza‐2′‐deoxycytidine (DAC), increased the expression of MAGE‐3/6 and CTL recognition. Thus, using QRT‐PCR UADT cancers frequently express MAGE‐3/6 at levels sufficient for CTL recognition, supporting the use of a QRT‐PCR‐based assay for the selection of candidates likely to respond to MAGE‐3/6 immunotherapy. Demethylating agents could increase the number of patients amenable for targeting epigenetically modified tumor antigens in vaccine trials. © 2009 UICC     

Expression of the extracellular matrix molecule thrombospondin inversely correlates with malignant progression in melanoma,lung and breast carcinoma cell lines

Thrombospondin (TSP) is a member of a family of extracellular matrix glycoproteins that may participate in multiple aspects of the metastatic cascade. We report an inverse correlation of steady-state Thbs-1 mRNA and protein expression with malignant progression among murine melanoma and human lung and breast carcinoma cell lines. Murine K-1735 melanoma cell lines of low metastatic potential, including K-1735 lines transfected with the murine nm23-1 cDNA, expressed higher TSP levels than related highly metastatic lines. In a model system of lung carcinoma malignant progression, immortalized human bronchial epithelial cells expressed higher TSP levels than v-Ki-ras, v-Ha-ras or n-ras transfectants, which in turn expressed higher TSP levels than tumor-derived, more aggressive variants. Among 3 unrelated breast carcinoma cell lines, Thbs-1 steady-state mRNA levels were greater in the 2 nonmetastatic lines than the metastatic line. Our data show that malignant progression in some cell lines is associated with reduced TSP expression. The suppressive effects of nm23-1 transfection on metastatic potential are also associated with increased TSP expression; ras transfection, which results in increased tumorigenesis, is associated with decreased TSP expression.     

Androgen regulation of the human pseudoautosomal gene MIC2, a potential marker for prostate cancer

Using the differential display–polymerase chain reaction technique to identify androgen-responsive genes in the human prostatic tumor cell line LNCaP, we cloned an expression tag homologous to the human pseudoautosomal gene MIC2. The role of MIC2 in the prostate had not previously been studied. We used a series of cell lines derived from LNCaP that varied in their degree of differentiation and metastatic potential to assess the relationship between MIC2 expression and androgen responsiveness in prostate cancer. The expression of MIC2 mRNA and its product E2 was upregulated by androgen in a dose- and time-dependent manner in the parental LNCaP line and correlated with the expression of prostate-specific antigen. In the LNCaP sublines and an androgen-repressed invasive human prostate cancer cell line (ARCaP), MIC2 gene expression was not regulated by androgen and was associated with poorer differentiation, decreased androgen sensitivity, and higher metastatic potential. Immunohistochemical analyses indicated that E2 was expressed in tissues from patients with primary prostate cancer (16 of 20), in fetal prostatic tissues (low levels in all 10 fetal tissues assessed), and sporadically in benign prostatic hyperplasia tissues (one of four). The normal prostate tissues did not show positive E2 staining, with the exception of one central-zone section from one of the eight normal prostate samples assessed. These findings suggest that deregulation of expression of the human pseudoautosomal gene MIC2 occurred in the prostate. Mol. Carcinog. 23:13–19, 1998. © 1998 Wiley-Liss, Inc.     

Development of a prostate cDNA microarray and statistical gene expression analysis package

A cDNA microarray comprising 5184 different cDNAs spotted onto nylon membrane filters was developed for prostate gene expression studies. The clones used for arraying were identified by cluster analysis of > 35 000 prostate cDNA library-derived expressed sequence tags (ESTs) present in the dbEST database maintained by the National Center for Biotechnology Information. Total RNA from two cell lines, prostate line 8.4 and melanoma line UACC903, was used to make radiolabeled probe for filter hybridizations. The absolute intensity of each individual cDNA spot was determined by phosphorimager scanning and evaluated by a bioinformatics package developed specifically for analysis of cDNA microarray experimentation. Results indicated 89% of the genes showed intensity levels above background in prostate cells compared with only 28% in melanoma cells. Replicate probe preparations yielded results with correlation values ranging from r = 0.90 to 0.93 and coefficient of variation ranging from 16 to 28%. Findings indicate that among others, the keratin 5 and vimentin genes were differentially expressed between these two divergent cell lines. Follow-up northern blot analysis verified these two expression changes, thereby demonstrating the reliability of this system. We report the development of a cDNA microarray system that is sensitive and reliable, demonstrates a low degree of variability, and is capable of determining verifiable gene expression differences between two distinct human cell lines. This system will prove useful for differential gene expression analysis in prostate-derived cells and tissue.     

Loss of expression of receptor tyrosine kinase family genes PTK7 and SEK in metastatic melanoma

Protein tyrosine kinases (PTKs) have been implicated in the development of many common human tumours including melanoma. Previously we isolated PTK gene sequences expressed in normal melanocytes. Here we examined expression of 9 of these genes in cell lines derived from defined stages of melanoma progression, by Northern blotting and in some cases immunoblotting. We also tested cells from 2 animal models of particular stages in progression, as well as uncultured biopsies of metastatic melanoma. The expression of 2 receptor kinase family members found in melanocytes, PTK7/CCK-4 and SEK/TYRO1, was decreased or lost in advanced melanomas. PTK7 mRNA was found in only 54% of melanoma cell lines and 20% of melanoma biopsies. Similarly, expression was lost in 2 advanced cell lines selected from an early melanoma line that did express PTK7 mRNA. SEK/TYRO1 expression was observed in 75% and 17% of cell lines from primary and metastastic melanomas, respectively. Conversely, mRNA for the non-receptor kinase PTK6/BRK was not detected in normal melanocytes or primary melanoma lines, but was found in 9% of metastatic melanoma cell lines. Int. J. Cancer 71: 1061-1065, 1997. © 1997 Wiley-Liss Inc.