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

MAGE基因在人结肠癌中的表达及其生物学意义。

• 批准号: 05671102
• 负责人: FUJIWARA Ryoichi
• 金额: $ 1.34万
• 依托单位: Kurume University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05671102/
• 关键词: MAGE gene; Tumor-rejection antigen; Colon cancer; Solid tumor; Gene expression; MAGE gene family; serum MAGE antigen; cytotoxic T lymphocytes; 血中抗体

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

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MAGE-1基因的粘粒文库导入E抗原缺失变异体MZ-2MEL.2.2，然后对CD8~+CTL克隆进行敏感性筛选(Van der Bruggen等，1991)。MAGE多基因家族包括编码细胞毒性T淋巴细胞(CTL)识别的HLA-A1上肿瘤排斥抗原的MAGE-1和MAGE-3基因。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出1040-bp(MAGE-1)、1061-bp(MAGE-3和-6)和1064-bp(MAGE-4和-41)的cDNA片段，包括全长编码序列(927-951 bp)，然后进行核苷酸测序。一个成员与其他成员在NT或推导的氨基酸(AA)水平上的同源性分别超过80%或66%。HIGHE…MAGE-3与MAGE-6的同源性为98%，MAGE-4与MAGE-41的同源性为98%。MAGE-1、MAGE-2、MAGE-3、MAGE-4、MAGE-6和MAGE-12基因在结肠癌、食道癌、肺癌等多种肿瘤中均有表达。在这些癌症中，至少有一个基因表达了大约35%到50%。例如，在53例肺癌(50例非小细胞肺癌和3例小细胞肺癌)中，MAGE-1、-2、-31-6和-4基因分别在6、7、20和7个肺癌组织中表达。相反，除了睾丸和胎盘外，正常细胞和正常组织都没有在mRNA水平表达MAGE基因。这些结果表明，MAGE基因产物是适合于肿瘤特异性免疫治疗的靶分子，但目前还没有一种有效的定量方法来检测肿瘤细胞上MAGE蛋白的表达。为此，利用重组MAGE-4b的单抗和多克隆抗体，建立了检测肿瘤细胞表达MAGE-4a和/或-4b的酶联免疫吸附试验(ELISA)。与MAGE基因产物(MAGE-1、MAGE-2、MAGE-3、MAGE-6、MAGE-12)无明显交叉反应。MAGE-4蛋白的最低检测浓度为10pg/孔(100pg/ml)，是一种可靠的定量检测细胞MAGE-4蛋白的方法。MAGE-4蛋白是人类肿瘤特异性免疫治疗的潜在靶分子。较少

项目成果

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Kyogo Itoh 等人：“江恩癌症研究专着 40”。

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Imai,Y.：“编码人类肿瘤排斥抗原的 MAGE 基因家族的序列分析。”

七條茂樹,伊東恭悟: "Annual Revicw免疫1995" 中外医学社, 140-146 (1995)

Shigeki Shichijo、Kyogo Ito：“年度回顾免疫学 1995”Chugai Igakusha，140-146 (1995)

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Seito, D.：“T 细胞受体 (TCR)α 和 β 基因在人类转移性黑色素瘤中细胞毒性 T 淋巴细胞的多克隆用途；TCRa 可能参与肿瘤细胞识别。” 58. 497-502。 (1994)