Expression of MAGE genes on human colon cancers and its biological significance.

MAGE-1基因是从MZ2-MEL黑色素瘤亚克隆的基因组DNA中分离出来的，方法是将其cosmid文库转染到e抗原缺失变体MZ-2MEL.2.2中，然后对CD8^+ CTL克隆进行易感性筛选（Van der Bruggen et al., 1991）。MAGE多基因家族包括MAGE-1和MAGE -3，它们编码被细胞毒性t淋巴细胞（CTL）识别的HLA-A1上的肿瘤排斥抗原。MAGE基因似乎是一个多基因家族，其成员具有高度的同源性。然而，人们对MAGE-4、-41和-6基因知之甚少。因此，我们首先从癌细胞中扩增了1040-bp (MAGE-1), 1061-bp （MAGE-3和-6）和1064-bp (MAGE-4和-41)cDNA片段，包括整个编码序列（927 ~ 951 bp），然后使用逆转录聚合酶链反应（RT-PCR）方法进行核苷酸（nt）测序。其中一个成员在nt或推导出的氨基酸（aa）水平上与其他成员的同源性分别大于80%或66%。MAGE-3和-6的同源性较高（98%），MAGE-4和-41的同源性也较高（98%）。本研究结果表明MAGE家族成员在编码序列水平上具有较高的同源性，但也存在明显的差异。MAGE-1、-2、-3、-4a、-6和-12基因在mRNA水平上被研究在许多不同的癌症中，包括结肠癌、食管癌和肺癌。这些基因中至少有一种表达了大约35%到50%的癌症。例如，53例肺癌（非小细胞肺癌50例，小细胞肺癌3例）中，MAGE-1、-2、-31-6、-4基因分别在6、7、20、7例中mRNA水平表达。相比之下，除了睾丸和胎盘外，正常细胞和正常组织都没有mRNA水平表达MAGE基因。这些结果表明，MAGE基因产物是人类癌症特异性免疫治疗的合适靶分子。然而，没有可用的定量方法来测量细胞MAGE蛋白在人类癌症中的表达。因此，我们建立了酶联免疫吸附法（ELISA），利用重组MAGE-4b蛋白的单克隆抗体（mAb）和多克隆抗体（Ab）检测人肿瘤细胞上表达的MAGE-4蛋白（MAGE-4a和/或-4b）。该ELISA与其他MAGE基因产物（MAGE-1、-2、-3、-6和-12）无明显的交叉反应性。测定MAGE-4蛋白的最低检测水平为10 pg/孔（100pg/ml）。结果表明，该酶联免疫吸附试验是一种可靠的定量测定细胞MAGE-4蛋白的方法，MAGE-4蛋白是人类癌症特异性免疫治疗的潜在靶分子。少

MAGE-1 gene was isolated from the genomic DNA of MZ2-MEL melanoma subclone by transfection of its cosmid library into E-antigen-loss variant MZ-2MEL.2.2 followed by screening with the susceptibility to CD8^+ CTL clones (Van der Bruggen et al., 1991). The MAGE multigene family includes the MAGE-1 and -3 henes that encode tumor-rejection antigens on HLA-A1 recognized by cytotoxic T-lymphocytes (CTL). MAGE genes seem to be a multigene family, the members of which share a high degree of homology. However, little is known about the MAGE-4, -41 and -6 genes. Therefore, we initialy amplified 1040-bp (MAGE-1), 1061-bp (MAGE-3 and -6) and 1064-bp (MAGE-4 and -41) cDNA fragments from cancer cells, including the entire coding sequences (927 to 951 bp), using the reverse transcription-polymerase chain reaction (RT-PCR) method followed by nucleotide (nt) sequencing. One member had greater than 80 or 66% homology with the other members at the nt or deduced amino acid (aa) levels, respectively. Highe … More r homology was found between MAGE-3 and-6 (98% at the nt level) and also between MAGE-4 and -41 (98%). The results of this investigation demonstrated the high homology, as well as the clear differences between the members of the MAGE family at the coding sequence level.MAGE-1, -2, -3, -4a, -6, and -12 genes are investigated at the mRNA level in many different cancers including, colon cancers, esophageal cancers, and lung cancers. At least one of these genes were expressed approximately 35 to 50% of these cancers. For example, MAGE-1, -2, -31-6, and -4 genes were respectively expressed at the mRNA level on 6,7,20, and 7 of 53 lung cancers (50 non-small-cell-lung cancers and 3 small-cell-lung cancers). In contrast, neither normal cells nor normal tissues other than testis and placenta express the MAGE gene at the mRNA level. These results suggest that the MAGE gene products are appropriate target molecules for spcific immunotherapy of human cancers.However, there was no available quantitative method to measure cellular MAGE protein expressed on human cancers. Therefore, an enzyme-linked immunosorbent assay (ELISA) was established for measuring cellular MAGE-4 protein (MAGE-4a and/or -4b) expressed on human tumor cells using the monoclonal antibody (mAb) and the polyclonal Ab to recombinant MAGE-4b protein. This ELISA also showed no apparent cross-reactivity with the other MAGE gene products (MAGE-1, -2, -3, -6, and -12). The minimum detectable level of MAGE-4 protein was determined to be 10 pg/well (100pg/ml).The results suggest that this ELISA is a reliable and quantitative method to measure cellular MAGE-4 protein that is a potential target molecule for specific immunotherapy of human cancers. Less