Introduction
The first member of the human MAGE family was identified as a gene encoding a tumor-specific antigen in melanoma cells. Since then, the MAGE gene family has extensively increased in complexity with the conservation of the typical signature domain (Mage Homology Domain; MHD). The MAGE family has been divided in two big subfamilies: type-I and type-II MAGE family. Type-I MAGE family includes MAGE-A, B, and C, which are located as clusters on the X chromosome. They are completely silent in normal tissues, with the exception of male germ cells and placenta. Some of them are expressed in tumor cells of various histological types, where they code for antigens recognized by cytolytic T lymphocytes (CTL) in a major histocompatibility complex (MHC)-restricted way. Type-II MAGE family includes genes which encode different proteins containing MHD with undefined chromosome clustering. These genes differ from the type-I MAGE family by their genomic structure: they do not encode the antigenic peptide encoded by type-I MAGE and therefore will not trigger an immune response. This characteristic also gives them a different expression pattern compared to type-I MAGE: they are expressed in all normal tissues.
The functions of various MAGE proteins remain to be elucidated. Recent studies suggest some necdin-related type-II MAGE genes are involved in cell cycle arresting and neuronal apoptosis through interaction with E2F1. In addition, several MAGE-A genes, such as MAGE-A1, -A2, -A4, and –A11 are involved in transcription regulation through specific binding to transcription complexes. The broad range of expression of type-I MAGE in various tumor types (e.g., melanoma, lung, breast, bladder gastric carcinoma, and neuroblastoma makes them possible targets for anticancer immunotherapy. However, their heterogeneous expression leads to loss of recognition of tumor cells and their subsequent destruction by CTL.
The expression of type-I MAGE in tumors is known to be activated by promoter demethylation, as well as histone acetylation. Global DNA demethylation has been observed in carcinomas of the breast, liver, colon, and neuron, and is considered to occur in the early stages of tumor development. MAGE gene expression is epigenetically repressed by promoter region methylation in most cells and CD117 receptor tyrosine kinase activation may allow MAGE gene expression by suppressing the methylation of MAGE promoter regions in addition to global demethylation.
Neuroblastoma (NB) is a neuroectodermal tumor that affects children and is the most common cancer in infancy. NB cells have been classified into three major cell types: I-type (intermediate), N-type (neuroblastic), and S-type (substrate-adherent). I-type cells are the malignant NB stem cells and express features of both N cells and S cells.
Past studies showed that NB cells express MAGE-A genes, but lack constitutive expression of co-stimulatory molecules and surface HLA class I and II molecules. As a result, NB cells are likely to be ignored by CTL, since expression of HLA and costimulatory molecules on antigen presenting cells are an essential condition for efficient peptide presentation to CTL and for the subsequent activation and clonal expansion of the latter cells. The expression of MAGE-A genes in NB cells is unrelated to disease staging or histology. However, study in mouse embryonic stem cells (mES) showed mES can induce demethylation of MAGE-A1. This leads us to the assumption that the expression level of type-I MAGE genes may increased in I-type NB cells.
In this study, we focused on the expression pattern of both type-I and type-II MAGE genes in different NB cell lines. Result showed the differential expression patterns of MAGE A2, A3, and A12 (type-I MAGE) among the six different cell lines are very similar to each other, but D4 (type-II MAGE) is expressed equally in all six cell lines. This expression pattern is independent of cell types. To confirm the coexpression between MAGE-A genes is due to promoter demethylation or histone acetylation, I further investigated the expression pattern of the CSAG1 gene in these six NB cell lines. CSAG1 is located at Xq28 and is proximate to the MAGE-A genes, but is not related to the MAGE family. Result showed the expression of the CSAG1 gene in the six different cell lines is similar to that of MAGE A2, A3, and A12.
