乳腺癌中MAGE-A9、MAGE-A11、MAGE-C1和MAGE-C2基因的表达及其表达机制的研究
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摘要
乳腺癌是当代影响女性健康最严重的疾病之一，近年来在我国的发病率呈上升趋势。乳腺癌目前的治疗仍以手术治疗为主，放、化疗和内分泌治疗为辅。然而这些治疗方法对于肿瘤的复发、转移以及晚期肿瘤，却难以获得理想疗效，因此建立特异性的肿瘤免疫疗法，使其成为重要的辅助治疗甚至主要治疗手段，是目前研究的热点。
癌/睾丸抗原（CTA）是肿瘤相关抗原的一种，其在除睾丸和胎盘以外的正常人体组织中均不表达，但在多种不同组织类型的肿瘤中有较高的表达，因此，CTA可作为肿瘤特异性免疫治疗的理想靶点。MAGE是CTA的一个亚家族成员，和CTA有着相同的表达模式，其编码的抗原在细胞内经加工产生抗原肽，与HLA-Ⅰ类分子结合成复合物，可被自体细胞毒性T淋巴细胞（CTL）特异性识别，诱导出对相应肿瘤细胞的特异性杀伤，从而达到治疗肿瘤的目的。
导师所领导课题组近年来一直致力于对MAGE家族的研究。本研究首次采用RT-PCR和免疫组织化学方法对60例乳腺正常组织、60例乳腺良性病变（纤维瘤及腺病）以及60例原发性乳腺癌组织中MAGE-A9、MAGE-A11、MAGE-C1和MAGE-C2的mRNA及蛋白表达情况进行了研究，并分析其表达与乳腺癌临床病理学指标以及预后之间的关系；应用不同浓度的DNA甲基化抑制剂和组蛋白去乙酰化酶抑制剂处理两种乳腺癌细胞株，观察药物处理前后MAGE-A9、MAGE-A11、MAGE-C1、MAGE-C2基因表达水平的变化，初步探索MAGE-A9、MAGE-A11、MAGE-C1、MAGE-C2表达的分子生物学机制。
主要研究内容和结果如下：
第一部分肿瘤抗原MAGE-A9和MAGE-A11在乳腺癌组织中的表达及与乳腺癌临床病理学指标和预后的关系
目的：检测MAGE-A9和MAGE-A11在乳腺正常组织、乳腺良性病变以及乳腺癌组织中的表达，并分析其表达与乳腺癌临床病理学指标以及预后之间的关系。
方法：采用RT-PCR和免疫组织化学的实验方法分别检测了MAGE-A9和MAGE-A11mRNA及蛋白在60例乳腺正常组织、60例乳腺良性病变和60例乳腺癌组织中的表达情况，并分析两者的表达与乳腺癌临床病理学指标（包括患者年龄、肿瘤大小、病理类型、组织学分级、临床分期、腋淋巴结转移、有无脉管瘤栓、ER、PR和HER-2）之间的关系。通过病例随访，分析MAGE-A9和MAGE-A11蛋白表达与乳腺癌患者预后之间的关系。
结果：
1MAGE-A9和MAGE-A11mRNA在乳腺正常组织、乳腺良性病变和乳腺癌组织中的表达及与乳腺癌临床病理学指标之间的关系
60例乳腺正常组织及60例乳腺良性病变中未见MAGE-A9和MAGE-A11mRNA的表达，而60例乳腺癌组织标本中MAGE-A9和MAGE-A11mRNA阳性表达率分别为45%和66.7%。
MAGE-A9和MAGE-A11mRNA的表达与ER及HER-2的表达呈正相关。ER表达阳性的乳腺癌患者，其MAGE-A9和MAGE-A11mRNA的阳性表达率分别高于ER表达阴性的乳腺癌患者（A9: χ2=4.865，P=0.027;A11: χ2=5.742，P=0.017）。HER-2表达阳性的乳腺癌患者，其MAGE-A9和MAGE-A11mRNA的表达阳性率分别高于HER-2表达阴性的乳腺癌患者（A9: χ2=6.16，P=0.013; χ2=4.266，P=0.039）。
MAGE-A9mRNA的表达与乳腺癌患者的年龄（χ2=2.372，P=0.366）、肿瘤大小（χ2=3.125，P=0.246）、病理类型（χ2=5.104，P=0.08）、临床分期（χ2=3.326，P=0.190）、组织学分级（χ2=1.304，P=0.562）、淋巴结转移（χ2=0.180，P=0.672）、瘤栓（χ2=2.095，P=0.148）及PR表达（χ2=1.358，P=0.244）均无相关性。
MAGE-A11mRNA的表达与乳腺癌患者的年龄（χ2=0.399，P=0.895）、肿瘤大小（χ2=2.687，P=0.263）、病理类型（χ2=5.312，P=0.064）、临床分期（χ2=2.461，P=0.305）、组织学分级（χ2=0.525，P=0.893）、淋巴结转移（χ2=1.250，P=0.264）、瘤栓（χ2=0.549，P=0.459）及PR表达（χ2=0.601，P=0.438）均无相关性。
2MAGE-A9和MAGE-A11蛋白在正常睾丸组织中的表达
在正常睾丸组织中，MAGE-A9和MAGE-A11主要表达于初级精母细胞和精原细胞中，且主要定位于细胞质，部分定位于细胞核。
3MAGE-A9和MAGE-A11蛋白在乳腺正常组织、乳腺良性病变、乳腺癌组织中的表达及与乳腺癌临床病理学指标之间的关系
MAGE-A9和MAGE-A11蛋白主要表达于乳腺癌细胞的胞质中，部分表达于细胞核，乳腺癌组织中MAGE-A9和MAGE-A11蛋白阳性表达率分别为43.3%和63.3%，而60例乳腺正常组织及60例乳腺良性病变中均未见MAGE-A9和MAGE-A11蛋白表达。
MAGE-A9（r=0.967，κ=0.966，P=0.000）和MAGE-A11（r=0.929，κ=0.927，P=0.000）mRNA与蛋白表达具有一致性，呈正相关。
MAGE-A9和MAGE-A11蛋白的表达与ER及HER-2的表达呈正相关，ER表达阳性的乳腺癌患者，其MAGE-A9和MAGE-A11蛋白的阳性表达率分别高于ER表达阴性的乳腺癌患者（A9: χ2=4.344，P=0.037; A11:χ2=4.311, P=0.038）。HER-2表达阳性的乳腺癌患者，其MAGE-A9和MAGE-A11蛋白的阳性表达率分别高于HER-2表达阴性的乳腺癌患者（A9: χ2=4.671, P=0.031; A11: χ2=5.967, P=0.015）。
MAGE-A9蛋白的表达与乳腺癌患者的年龄（χ2=3.621，P=0.209）、肿瘤大小（χ2=4.188，P=0.116）、病理类型（χ2=4.186，P=0.122）、临床分期（χ2=2.658，P=0.273）、组织学分级（χ2=1.006，P=0.618）、淋巴结转移（χ2=0.045，P=0.832）、瘤栓（χ2=2.807，P=0.094）及PR表达（χ2=1.067，P=0.302）均无相关性。
MAGE-A11蛋白的表达与乳腺癌患者的年龄（χ2=0.548，P=0.808）、肿瘤大小（χ2=2.943，P=0.231）、病理类型（χ2=3.257，P=0.203）、临床分期（χ2=4.232，P=0.117）、组织学分级（χ2=0.964，P=0.732）、淋巴结转移（χ2=3.062，P=0.08）、瘤栓（χ2=1.386，P=0.239）及PR表达（χ2=1.270，P=0.260）均无相关性。4MAGE-A9和MAGE-A11蛋白表达与乳腺癌患者生存率的关系
MAGE-A9和MAGE-A11蛋白表达阳性的乳腺癌患者的生存率分别低于MAGE-A9和MAGE-A11蛋白表达阴性的乳腺癌患者（A9: χ2=5.348,P=0.021; A11: χ2=4.192, P=0.041）。
结论：
1乳腺正常组织和乳腺良性病变中均未发现MAGE-A9和MAGE-A11mRNA及蛋白的表达。乳腺癌组织中MAGE-A9和MAGE-A11mRNA表达阳性率分别为45%和66.7%，蛋白阳性表达率分别为43.3%和63.3%，提示MAGE-A9和MAGE-A11是肿瘤特异性抗原，可作为乳腺癌免疫治疗的靶点。
2MAGE-A9和MAGE-A11mRNA和蛋白表达与乳腺癌患者的年龄、病理类型、组织学分级、临床分期、肿瘤大小、淋巴结转移、瘤栓、PR表达均无相关性，但与ER和HER-2的表达呈正相关。ER和HER-2表达阳性的乳腺癌患者，其MAGE-A9和MAGE-A11mRNA和蛋白的表达率明显高于ER和HER-2表达阴性的乳腺癌患者，提示MAGE-A9和MAGE-A11有望成为指导乳腺癌治疗及监测预后的重要指标。
3MAGE-A9和MAGE-A11蛋白表达阳性的乳腺癌患者的生存率分别低于MAGE-A9和MAGE-A11蛋白表达阴性的乳腺癌患者，说明MAGE-A9和MAGE-A11高表达可能是乳腺癌患者不良预后的一个重要标志。
第二部分肿瘤抗原MAGE-C1和MAGE-C2在乳腺癌组织中的表达及与乳腺癌临床病理学指标和预后的关系
目的：检测MAGE-C1和MAGE-C2在乳腺正常组织、乳腺良性病变以及乳腺癌组织中的表达，并分析其表达与乳腺癌临床病理学指标以及预后之间的关系。
方法：采用RT-PCR和免疫组织化学的实验方法分别检测了MAGE-C1和MAGE-C2mRNA及蛋白在60例乳腺正常组织、60例乳腺良性病变和60例乳腺癌组织中的表达情况，并分析两者的表达与乳腺癌临床病理学指标之间的关系。通过病例随访，分析MAGE-C1和MAGE-C2蛋白表达与乳腺癌患者预后之间的关系。
结果：
1MAGE-C1和MAGE-C2mRNA在乳腺正常组织、乳腺良性病变和乳腺癌组织中的表达及其与乳腺癌临床病理学指标之间的关系
60例乳腺正常组织及60例乳腺良性病变中均未见MAGE-C1和MAGE-C2mRNA的表达，而60例乳腺癌组织标本中MAGE-C1和MAGE-C2mRNA阳性表达率分别为43.3%和61.7%。
MAGE-C1和MAGE-C2mRNA的表达与乳腺癌的组织学分级呈正相关，组织学分级为Ⅲ级的乳腺癌患者，其MAGE-C1和MAGE-C2mRNA阳性表达率分别高于组织学分级Ⅰ级的乳腺癌患者（C1: χ2=8.832, P=0.01;C2: χ2=8.575, P=0.01）。
MAGE-C2mRNA表达还与乳腺癌的病理类型及瘤栓表达呈正相关。瘤栓表达阳性的乳腺癌患者，其MAGE-C2mRNA的阳性表达率高于瘤栓表达阴性的乳腺癌患者（χ2=6.094，P=0.014）。病理类型为浸润性导管癌的患者，其MAGE-C2mRNA的阳性表达率分别高于浸润性小叶癌和髓样癌的患者（χ2=11.707，P=0.002）。
MAGE-C1mRNA的表达与乳腺癌患者的年龄（χ2=0.403，P=0.907）、肿瘤大小（χ2=0.976，P=0.671）、病理类型（χ2=2.256，P=0.352）、临床分期（χ2=0.684，P=0.765）、淋巴转移（χ2=0.102，P=0.75）、瘤栓（χ2=0.008，P=0.93）、ER（χ2=0.611，P=0.434）、PR（χ2=0.16，P=0.689）及HER-2表达（χ2=0.307，P=0.58）均无相关性。
MAGE-C2mRNA的表达与乳腺癌患者的年龄（χ2=0.228，P=1.0）、肿瘤大小（χ2=0.579，P=0.815）、临床分期（χ2=4.059，P=0.115）、淋巴转移（χ2=2.303，P=0.129）、ER（χ2=0.004，P=0.947）、PR（χ2=0.000，P=1.0）及HER-2表达（χ2=2.366，P=0.124）均无相关性。
2MAGE-C1和MAGE-C2蛋白在正常睾丸组织中的表达
在正常睾丸组织中，MAGE-C1和MAGE-C2主要表达于初级精母细胞和精原细胞中，且主要定位于细胞质，部分定位于细胞核。
3MAGE-C1和MAGE-C2蛋白在乳腺正常组织、乳腺良性病变、乳腺癌组织中的表达及与乳腺癌临床病理学指标之间的关系
MAGE-C1和MAGE-C2蛋白主要表达于乳腺癌细胞的胞质中，部分表达于细胞核，60例乳腺癌组织中MAGE-C1和MAGE-C2蛋白阳性表达率分别为38.3%和58.3%，而60例乳腺正常组织及60例乳腺良性病变中均未见MAGE-C1和MAGE-C2蛋白表达。
MAGE-C1（r=0.902，κ=0.897，P=0.000）和MAGE-C2（r=0.933，κ=0.931，P=0.000）mRNA与蛋白表达具有一致性，呈正相关。
MAGE-C1和MAGE-C2蛋白表达与乳腺癌的组织学分级呈正相关，组织学分级为Ⅲ级的乳腺癌患者，其MAGE-C1和MAGE-C2蛋白的阳性表达率分别高于组织学分级Ⅰ级的乳腺癌患者（C1: χ2=6.233, P=0.038;C2: χ2=7.471, P=0.017）。
MAGE-C2蛋白表达还与乳腺癌的病理类型、瘤栓表达呈正相关。瘤栓表达阳性的乳腺癌患者，其MAGE-C2蛋白的阳性表达率高于瘤栓表达阴性的乳腺癌患者（χ2=5.503，P=0.019）。病理类型为浸润性导管癌的患者，其MAGE-C2蛋白的阳性表达率分别高于浸润性小叶癌和髓样癌的患者（χ2=10.889，P=0.004）。
MAGE-C1蛋白的表达与乳腺癌患者的年龄（χ2=1.115，P=0.729）、肿瘤大小（χ2=1.659，P=0.435）、病理类型（χ2=3.173，P=0.206）、临床分期（χ2=0.383，P=0.87）、淋巴转移（χ2=0.012，P=0.914）、瘤栓（χ2=0.099，P=0.753）、ER（χ2=0.533，P=0.465）、PR（χ2=0.097，P=0.755）及HER-2表达（χ2=1.422，P=0.233）均无相关性。
MAGE-C2蛋白的表达与乳腺癌患者的年龄（χ2=0.525，P=0.903）、肿瘤大小（χ2=0.359，P=0.936）、临床分期（χ2=4.022，P=0.15）、淋巴转移（χ2=1.143，P=0.285）、ER（χ2=0.429，P=0.513）、PR（χ2=0.07，P=0.791）及HER-2表达（χ2=1.936，P=0.164）均无相关性。4MAGE-C1和MAGE-C2蛋白表达与乳腺癌患者生存率的关系
MAGE-C1和MAGE-C2蛋白表达阳性的乳腺癌患者，其生存率分别低于MAGE-C1和MAGE-C2蛋白表达阴性的乳腺癌患者（C1: χ2=4.213,P=0.04; C2: χ2=4.467, P=0.035）。
结论：
1乳腺正常组织和乳腺良性病变中均未发现MAGE-C1和MAGE-C2mRNA及蛋白的表达。乳腺癌组织中MAGE-C1和MAGE-C2mRNA阳性表达率分别为43.3%和61.7%，MAGE-C1和MAGE-C2蛋白阳性表达率分别为38.3%和58.3%，提示MAGE-C1和MAGE-C2可作为肿瘤标志物检测乳腺癌的发生，也可成为乳腺癌免疫治疗的理想靶点。
2MAGE-C1mRNA和蛋白表达与乳腺癌患者的年龄、病理类型、临床分期、肿瘤大小、淋巴转移、瘤栓、ER、PR、HER-2表达均无相关性，MAGE-C2mRNA和蛋白表达与乳腺癌患者的年龄、临床分期、肿瘤大小、淋巴转移、ER、PR、HER-2表达均无相关性。MAGE-C1和MAGE-C2mRNA和蛋白表达与乳腺癌的组织学分级呈正相关。MAGE-C2mRNA和蛋白表达还与乳腺癌的病理类型、瘤栓表达呈正相关，提示MAGE-C1和MAGE-C2的表达和乳腺癌的恶性程度密切相关，且不同病理类型的肿瘤表达MAGE-C2的频率不同，以癌睾丸抗原为基础进行的肿瘤免疫治疗可能更适合在某些病理类型的肿瘤中进行。
3MAGE-C1和MAGE-C2蛋白的高表达与乳腺癌患者的低生存率正相关，说明MAGE-C1和MAGE-C2高表达可能是乳腺癌患者不良预后的一个重要标志。
第三部分乳腺癌中肿瘤抗原MAGE-A9、MAGE-A11、MAGE-C1和MAGE-C2表达机制的研究
目的：应用DNA甲基化抑制剂5-aza-CdR和组蛋白去乙酰化酶抑制剂TSA单独或联合处理MCF-7、MDA-MB-231两种乳腺癌细胞，分析药物处理前后MAGE-A9、MAGE-A11、MAGE-C1、MAGE-C2基因表达的变化，初步探索它们表达的分子生物学机制。方法：将实验细胞分为4组：（1）对照组:同期培养的不加任何药物处理的细胞；（2）5-aza-CdR组：分别用含2.5μmol/L、5μmol/L5-aza-CdR的培养基培养细胞72h；（3）TSA组：分别用含0.3μmol/L、0.5μmol/L TSA的培养基培养细胞24h；（4）5-aza-CdR及TSA组：用含5μmol/L5-aza-CdR的培养基培养细胞48h后再加入含0.5μmol/LTSA的培养基继续培养24h。药物刺激72小时后，收集细胞，提取RNA，每组实验重复三次。
结果：
1药物处理前后MCF-7、MDA-MB-231细胞中MAGE-A9和MAGE-A11mRNA表达水平的变化RT-PCR结果显示，未经药物处理的MCF-7、MDA-MB-231细胞未见MAGE-A9和MAGE-A11mRNA表达。应用2.5μmol/L、5μmol/L5-aza-CdR分别作用72小时后可见MAGE-A9和MAGE-A11基因重新表达，且随着药物浓度增高，MAGE-A9和MAGE-A11基因表达逐渐增强。与单独应用5-aza-CdR相比较，联合应用5μmol/L5-aza-CdR和0.5μmol/L TSA可使MAGE-A9和MAGE-A11基因表达进一步增加。而0.3μmol/L、0.5μmol/L TSA单独作用均不能诱导出MAGE-A9和MAGE-A11基因的重新表达。
2药物处理前后MCF-7、MDA-MB-231细胞中MAGE-C1和MAGE-C2mRNA表达水平的变化
RT-PCR结果显示，未经药物处理的MCF-7、MDA-MB-231细胞未见MAGE-C1mRNA表达。单独应用2.5μmol/L、5μmol/L5-aza-CdR及0.3μmol/L、0.5μmol/L TSA均不能诱导出MAGE-C1基因的表达。而联合应用5μmol/L5-aza-CdR和0.5μmol/L TSA也未能诱导出MAGE-C1基因的表达。
RT-PCR结果显示，未经药物处理的MCF-7、MDA-MB-231细胞未见MAGE-C2mRNA表达。应用2.5μmol/L、5μmol/L5-aza-CdR分别作用72小时后可见MAGE-C2基因重新表达，且随着药物浓度增高，基因表达逐渐增强。与单独应用5-aza-CdR相比较，联合应用5μmol/L5-aza-CdR和0.5μmol/L TSA可使MAGE-C2基因表达进一步增加。而0.3μmol/L、0.5μmol/L TSA单独作用均不能诱导出MAGE-C2基因的重新表达。
结论：
1单独应用5-aza-CdR处理MCF-7及MDA-MB-231细胞可诱导MAGE-A9、MAGE-A11、MAGE-C2基因的表达，提示DNA去甲基化是MAGE-A9、MAGE-A11和MAGE-C2基因表达重要机制。
2联合应用5-aza-CdR和TSA可使MCF-7及MDA-MB-231细胞中MAGE-A9、MAGE-A11和MAGE-C2基因的表达进一步增加，而TSA单独作用对基因表达没有影响，说明DNA去甲基化和组蛋白乙酰化在MAGE基因的表达中具有协同作用，DNA去甲基化扮演了主导作用。
3单独或联合应用不同浓度的5-aza-CdR和TSA均不能诱导出MCF-7及MDA-MB-231细胞中MAGE-C1的表达，说明DNA甲基化和组蛋白乙酰化并不是调节MAGE基因表达的唯一机制。
Breast cancer is the most common malignancy in women. However,therapeutic options for the treatment of patients with this tumor are limited to3fundamental modalities: surgical resection, radiation therapy andchemotherapy. Against advanced carcinomas, therapeutic options are limitedto radiation therapy and chemotherapy; however, these modalities do not yieldresults. Therefore, cancer-specific immunotherapy may be expected to becomea novel treatment modality for breast carcinomas.
Melanoma-associated antigens (MAGE) are a group of well-characterizedmembers of the cancer-testis antigen (CTA) family that are expressed invarious tumor cells, but not in healthy tissues except for the testis and placenta.The MAGE gene family encodes tumor-associated antigens, which arerecognized by CTLs in conjunction with MHC class I molecules of varioushaplotypes on the tumor cell surface. Thus, MAGE are appealing targets forcancer immunotherapy.
In recent years, our professor’s research group has been committed to theresearch of MAGE family. In the present study, we investigated the expressionstatus of MAGE-A9, MAGE-A11, MAGE-C1and MAGE-C2by reversetranscriptional-polymerase chain reaction (RT-PCR) andimmunohistochemistry in60breast benign diseases specimens (includingfibroadenoma and adenosis),60primary breast cancer specimens and60tumor free breast specimens, analyzed their correlation with theclinicopathological parameters and the overall survival of breast cancerpatients. Furthermore, we added DNA methyltransferase inhibitor5-aza-CdRand/or histone deacetylase inhibitor TSA to two breast cancer cell lines andanalyzed the expression of MAGE-A9, MAGE-A11, MAGE-C1andMAGE-C2genes before and after treating with the two inhibitors by RT-PCR in order to elucidate epigenetic mechanism of the four genes expression.
The main research contents and results were shown as follows:Part ⅠThe expression of MAGE-A9and MAGE-A11in breast cancertissues and their correlation with the clinicopathological parameters andthe prognosis
Objective: To investigate the expression of MAGE-A9and MAGE-A11inbreast benign diseases tissues, tumor-free breast tissues and breast cancertissues, explore their correlation with the clinicopathological parameters andthe prognosis of the breast cancer patients.
Methods: The expression of MAGE-A9and MAGE-A11was investigated in60breast benign diseases specimens,60tumor-free breast specimens and60breast cancer specimens by RT-PCR and immunohistochemistry, then thecorrelation between MAGE-A9, MAGE-A11expression and theclinicopathological parameters of breast cancer patients, including age of thepatients, tumor size, pathological types, histology grades, clinical stages,metastasis of axillary lymph nodes, tumor embolus, estrogen receptor (ER),progestrogen receptor (PR) and HER-2status was analyzed. The associationbetween MAGE-A9and MAGE-A11protein expression and the prognosis ofbreast cancer patients was also analyzed.
Results:
1Expression of MAGE-A9and MAGE-A11mRNA in breast benign diseasesspecimens, tumor-free breast specimens, breast cancer specimens and thecorrelation between MAGE-A9, MAGE-A11expressions andclinicopathological parameters of breast cancer patients
Overall, no MAGE-A9and MAGE-A11expression was found in60tumor-free breast specimens and60breast benign diseases specimens.Expression of MAGE-A9and MAGE-A11mRNA was detected in45%and66.7%of the breast cancer specimens, respectively.
MAGE-A9and MAGE-A11mRNA expression was positively associatedwith ER and HER-2expression. MAGE-A9and MAGE-A11mRNAexpression was more frequent in ER-positive breast carcinomas (A9:25/45, 55.6%; A11:34/45,75.6%) compared with ER-negative breast carcinomas(A9:2/15,13.3%; A11:6/15,40%)(A9: χ2=4.865，P=0.027; A11: χ2=5.742，P=0.017). MAGE-A9and MAGE-A11mRNA expression was also morefrequent in HER-2-positive breast carcinomas (A9:25/48,52.1%; A11:36/48,75%) compared with HER-2-negative breast carcinomas (A9:2/12,16.7%;A11:4/12,33.3%)(A9: χ2=6.16，P=0.013; χ2=4.266，P=0.039).
No correlation was found between MAGE-A9mRNA expression and theage of the patients (χ2=2.372，P=0.366), tumor size (χ2=3.125，P=0.246),pathological type (χ2=5.104，P=0.08), clinical stage (χ2=3.326，P=0.190)，histological grade (χ2=1.304，P=0.562), lymphatic metastasis (χ2=0.180，P=0.672), tumor embolus (χ2=2.095，P=0.148)and PR (χ2=1.358，P=0.244)status.
No correlation was found between MAGE-A11mRNA expression and theage of the patients (χ2=0.399，P=0.895), tumor size (χ2=2.687，P=0.263),pathological type (χ2=5.312，P=0.064), clinical stage (χ2=2.461，P=0.305)，histological grade (χ2=0.525，P=0.893), lymphatic metastasis (χ2=1.250，P=0.264), tumor embolus (χ2=0.549，P=0.459)and PR (χ2=0.601，P=0.438)status.2Expression of MAGE-A9and MAGE-A11proteins in normal human testistissues
As the positive control, MAGE-A9and MAGE-A11antibodies were firstlyused to stain human normal testicular tissue sections. Both MAGE-A9andMAGE-A11expressions were mainly observed on primary spermatocytes andspermatogonia, and both the nucleus and the cytoplasm were stained.3Expression of MAGE-A9and MAGE-A11protein in breast benign diseasesspecimens, tumor-free breast specimens, breast cancer specimens and thecorrelation between MAGE-A9, MAGE-A11expression andclinicopathological parameters of breast cancer patients
In most breast cancer cells, MAGE-A9and MAGE-A11staining werefound in cytoplasm but occasionally also observed in nucleus. Overall, noMAGE-A9and MAGE-A11immunoreactivity was observed in60tumor-free breast specimens and60breast benign diseases specimens, while43.3%(26out of60) and63.3%(38out of60) breast cancer specimens were foundpositive with MAGE-A9and MAGE-A11antibodies, respectively.
There was generally concordance between MAGE-A9(r=0.967，κ=0.966，P=0.000) and MAGE-A11(r=0.929，κ=0.927，P=0.000) mRNA and proteinexpression.
MAGE-A9and MAGE-A11protein expression was positively associatedwith ER and HER-2expression. MAGE-A9and MAGE-A11proteinexpression was more frequent in ER-positive breast carcinomas comparedwith ER-negative breast carcinomas (A9: χ2=4.344，P=0.037; A11: χ2=4.311,P=0.038). MAGE-A9and MAGE-A11protein expression was also morefrequent in HER-2-positive breast carcinomas compared with HER-2-negativebreast carcinomas (A9: χ2=4.671, P=0.031; A11: χ2=5.967, P=0.015).
No correlation was found between MAGE-A9protein expression and theage of the patients (χ2=3.621，P=0.209), tumor size (χ2=4.188，P=0.116),pathological type (χ2=4.186，P=0.122), clinical stage (χ2=2.658，P=0.273)，histological grade (χ2=1.006，P=0.618), lymphatic metastasis (χ2=0.045，P=0.832), tumor embolus (χ2=2.807，P=0.094)and PR (χ2=1.067，P=0.302)status.
No correlation was found between MAGE-A11protein expression and theage of the patients (χ2=0.548，P=0.808), tumor size (χ2=2.943，P=0.231),pathological type (χ2=3.257，P=0.203), clinical stage (χ2=4.232，P=0.117)，histological grade (χ2=0.964，P=0.732), lymphatic metastasis (χ2=3.062，P=0.08), tumor embolus (χ2=1.386，P=0.239)and PR (χ2=1.270，P=0.260)status.
4Correlation between MAGE-A9and MAGE-A11expression and theprognosis of breast cancer patients
Patients with MAGE-A9or MAGE-A11expression had a worse prognosisthan the patients without MAGE-A9(χ2=5.348, P=0.021) or MAGE-A11(χ2=4.192, P=0.041) expression.
Conclusions: 1Expression of MAGE-A9and MAGE-A11mRNA was detected in45%and66.7%of the breast cancer specimens, respectively. The expression rate ofMAGE-A9and MAGE-A11protein in breast cancer was43.3%and63.3%,respectively, while no MAGE-A9and MAGE-A11was observed intumor-free breast specimens and breast benign diseases specimens, suggestingthat MAGE-A9and MAGE-A11were tumor specific antigens.2MAGE-A9, MAGE-A11mRNA and protein expression were more frequentin ER or HER-2positive breast carcinomas compared with ER or HER-2negative breast carcinomas.
3Both MAGE-A9and MAGE-A11expression were significantly associatedwith reduced overall survival, suggesting that MAGE-A9and MAGE-A11may be potential markers of a poor prognosis for breast cancer patients.
Part ⅡThe expression of MAGE-C1and MAGE-C2in breast cancertissues and their correlation with the clinicopathological parameters andthe prognosis
Objective: To investigate the expression of MAGE-C1and MAGE-C2inbreast benign diseases tissues, tumor-free breast tissues and breast cancertissues, explore their correlation with the clinicopathological parameters andthe prognosis of the breast cancer patients.
Methods: The expression of MAGE-C1and MAGE-C2was investigated in60breast benign diseases specimens,60tumor-free breast specimens and60breast cancer specimens by RT-PCR and immunohistochemistry, then thecorrelation between MAGE-C1and MAGE-C2expression and theclinicopathological parameters of breast cancer patients was analyzed. Theassociation between MAGE-C1and MAGE-C2protein expression and theprognosis of breast cancer patients was also analyzed.
Results:
1Expression of MAGE-C1and MAGE-C2mRNA in breast benign diseasesspecimens, tumor-free breast specimens, breast cancer specimens and thecorrelation between MAGE-C1and MAGE-C2expression andclinicopathological parameters of breast cancer patients Overall, no MAGE-C1and MAGE-C2expression was found in60tumor-free breast specimens and60breast benign diseases specimens.Expression of MAGE-C1and MAGE-C2mRNA was detected in43.3%and61.7%of the breast cancer specimens, respectively.
MAGE-C1and MAGE-C2mRNA expression was positively correlatedwith tumor grade. MAGE-C1and MAGE-C2mRNA expression was morefrequent in high-grade tumors (grade III) compared with low-grade tumors(grade I)(C1: χ2=8.832, P=0.01; C2: χ2=8.575, P=0.01).
Furthermore, MAGE-C2mRNA expression was also significantlyassociated with tumor embolus and histological type. Higher frequencies ofMAGE-C2mRNA expression was seen in invasive ductal breast cancercompared to invasive lobular breast cancer and medullary breast cancer(χ2=11.707，P=0.002). MAGE-C2expression was also more frequent in breastcarcinomas with positive tumor embolus compared to breast carcinomas withnegative tumor embolus (χ2=6.094，P=0.014).
No correlation was found between MAGE-C1mRNA expression and theage of the patients (χ2=0.403，P=0.907), tumor size (χ2=0.976，P=0.671),pathological type (χ2=2.256，P=0.352), clinical stage (χ2=0.684，P=0.765)，lymphatic metastasis (χ2=0.102，P=0.75), tumor embolus (χ2=0.008，P=0.93),ER (χ2=0.611，P=0.434), PR (χ2=0.16，P=0.689) and HER-2(χ2=0.307，P=0.58) status.
No correlation was found between MAGE-C2mRNA expression and theage of the patients (χ2=0.228，P=1.0), tumor size (χ2=0.579，P=0.815), clinicalstage (χ2=4.059，P=0.115)，lymphatic metastasis (χ2=2.303，P=0.129), ER(χ2=0.004，P=0.947), PR (χ2=0.000，P=1.0) and HER-2(χ2=2.366，P=0.124)status.
2Expression of MAGE-C1and MAGE-C2proteins in normal human testistissues
As the positive control, MAGE-C1and MAGE-C2antibodies were firstlyused to stain human normal testicular tissue sections. Both MAGE-C1andMAGE-C2expressions were mainly observed on primary spermatocytes and spermatogonia, and both the nucleus and the cytoplasm were stained.
3Expression of MAGE-C1and MAGE-C2proteins in breast benign diseasesspecimens, tumor-free breast specimens, breast cancer specimens and thecorrelation between MAGE-C1and MAGE-C2expression andclinicopathological parameters of breast cancer patients
In most breast cancer cells, MAGE-C1and MAGE-C2staining were foundin cytoplasm but occasionally also observed in nucleus. Overall, noMAGE-C1and MAGE-C2immunoreactivity was observed in60tumor-freebreast specimens and60breast benign diseases specimens, while38.3%and58.3%breast cancer specimens were found positive with MAGE-C1andMAGE-C2antibodies, respectively.
There was generally concordance between MAGE-C1(r=0.902，κ=0.897，P=0.000) and MAGE-C2(r=0.933，κ=0.931，P=0.000) mRNA and proteinexpression.
MAGE-C1and MAGE-C2protein expressions were positively correlatedwith tumor grade. MAGE-C1and MAGE-C2protein expressions were morefrequent in high-grade tumors (grade III) compared with low-grade tumors(grade I)(C1: χ2=6.233, P=0.038; C2: χ2=7.471, P=0.017).
Furthermore, MAGE-C2mRNA protein was also significantly associatedwith tumor embolus and histological type. Higher frequencies of MAGE-C2protein expression was seen in invasive ductal breast cancer compared toinvasive lobular breast cancer and medullary breast cancer (χ2=10.889，P=0.004). MAGE-C2protein expression was also more frequent in breastcarcinomas with positive tumor embolus compared to breast carcinomas withnegative tumor embolus (χ2=5.503，P=0.019).
No correlation was found between MAGE-C1protein expression and theage of the patients (χ2=1.115，P=0.729), tumor size (χ2=1.659，P=0.435),pathological type (χ2=3.173，P=0.206), clinical stage (χ2=0.383，P=0.87)，lymphatic metastasis (χ2=0.012， P=0.914), tumor embolus (χ2=0.099，P=0.753), ER (χ2=0.533，P=0.465), PR (χ2=0.097，P=0.755) and HER-2(χ2=1.422，P=0.233) status.
No correlation was found between MAGE-C2protein expression and theage of the patients (χ2=0.525，P=0.903), tumor size (χ2=0.359，P=0.936),clinical stage (χ2=4.022，P=0.15)，lymphatic metastasis (χ2=1.143，P=0.285),ER (χ2=0.429，P=0.513), PR (χ2=0.07，P=0.791) and HER-2(χ2=1.936，P=0.164) status.
4Correlation between MAGE-C1and MAGE-C2expressions and theprognosis of breast cancer patientsPatients with MAGE-C1or MAGE-C2expression had a worse prognosisthan the patients without MAGE-C1(χ2=4.213, P=0.04) or MAGE-C2(χ2=4.467, P=0.035) expression.
Conclusions:
1Expression of MAGE-C1and MAGE-C2mRNA was detected in43.3%and61.7%of the breast cancer specimens, respectively. The expression rate ofMAGE-C1and MAGE-C2protein in breast cancer was38.3%and58.3%,respectively, while no MAGE-C1and MAGE-C2was observed in tumor-freebreast specimens and breast benign diseases specimens, suggesting thatMAGE-C1and MAGE-C2can be used not only as appropriate candidates fortargets of cancer-specific immunotherapy but also as tumor biomarkers forbreast cancer.
2MAGE-C1and MAGE-C2expression correlated significantly with tumorgrade, MAGE-C2-positive expression was also significantly associated withpresence of tumor embolus and histological type, suggesting that there was anassociation between MAGE-C1, MAGE-C2expression and features of a moreaggressive clinical behavior in breast cancer, and some carcinoma might bemore suitable for CTA based immunotherapy than others, depending on cellmorphology.
3Both MAGE-C1and MAGE-C2expression were significantly associatedwith reduced overall survival, suggesting that MAGE-C1and MAGE-C2maybe potential markers of a poor prognosis for breast cancer patients.
Part Ⅲ The expression mechanism of MAGE-A9, MAGE-A11,MAGE-C1and MAGE-C2in breast cancers
Objective: We examined the influence of the DNA methylase inhibitor5-aza-2'-deoxycytidine (5-aza-CdR) together with the histone deacetylaseinhibitor trichostatin A (TSA) on the expression of MAGE-A9, MAGE-A11,MAGE-C1and MAGE-C2genes in two breast cancer cell lines.
Methods: Cell lines were divided into four groups:(1)5-aza-CdR group:Cells were stimulated by2.5μmol/L and5μmol/L5-aza-CdR for72hours,respectively;(2) TSA group: Cells were stimulated by0.3μmol/L and0.5μmol/L TSAfor24hours, respectively;(3)5-aza-CdR+TSA group: Cells werestimulated by0.5μmol/L TSA for24hours after the48-hour5μmol/L5-aza-CdR incubation;(4) Control group: Cells were mock-treated with thesame volume of culture medium. Then the cells were collected to extract totalRNA for evaluate MAGE-A9, MAGE-A11, MAGE-C1and MAGE-C2geneexpressions.
Results:
1Expression patterns of MAGE-A9and MAGE-A11genes in MCF-7andMDA-MB-231cells before and after treatment with5-aza-CdR and/or TSA
Treatment of the two breast cancer cells with2.5μmol/L and5μmol/L5-aza-CdR alone could induce the expression of MAGE-A9and MAGE-A11,and the expression was enhanced with5-aza-CdR concentration increased. Weobserved robust expression of both genes after addition of0.5μmol/L TSA for24hours following48hours of5μmol/L5-aza-CdR treatment. However,0.3μmol/L and0.5μmol/L TSA treatment alone had no influence on MAGE-Agene expression in the two cell lines.
2Expression patterns of MAGE-C1and MAGE-C2genes in MCF-7andMDA-MB-231cells before and after treatment with5-aza-CdR and/or TSA
Treatment of the two breast cancer cells with2.5μmol/L and5μmol/L5-aza-CdR alone could only induce the expression of MAGE-C2, and theexpression was increased with5-aza-CdR concentration evaluated. Weobserved robust expression of MAGE-C2genes after addition of0.5μmol/LTSA for24hours following48hours of5μmol/L5-aza-CdR treatment.However,0.3μmol/L and0.5μmol/L TSA treatment alone had no influence on MAGE-C2gene expression in the two cell lines.
However, addition of2.5μmol/L and5μmol/L5-aza-CdR alone or0.3μmol/L and0.5μmol/L TSA alone could not activate MAGE-C1geneexpression in the two breast cancer cells.5μmol/L5-aza-CdR in combinationwith0.5μmol/L TSA also failed to activate MAGE-C1gene expression in thetwo breast cancer cells.
Conclusions:
1Treatment of MCF-7, MDA-MB-231cells with5-aza-CdR could induce theexpression of MAGE-A9, MAGE-A11and MAGE-C2, suggesting that DNAmethylation is an important mechanism in the regulation of MAGE-A9,MAGE-A11and MAGE-C2.
2DNA methylation and histone deacetylation appear to act as synergisticlayers for the silencing of MAGE-A9, MAGE-A11and MAGE-C2genes, andDNA methylation is dominant for the stable maintenance of a silent state.
3MAGE-C1expressions could not be reactivating in the two breast cancercells despite treatment with5-aza-CdR and/or TSA, suggesting that DNAmethylation and histone acetylation are not the only mechanism in theregulation of MAGE gene expression.
癌/睾丸抗原（CTA）是肿瘤相关抗原的一种，其在除睾丸和胎盘以外的正常人体组织中均不表达，但在多种不同组织类型的肿瘤中有较高的表达，因此，CTA可作为肿瘤特异性免疫治疗的理想靶点。MAGE是CTA的一个亚家族成员，和CTA有着相同的表达模式，其编码的抗原在细胞内经加工产生抗原肽，与HLA-Ⅰ类分子结合成复合物，可被自体细胞毒性T淋巴细胞（CTL）特异性识别，诱导出对相应肿瘤细胞的特异性杀伤，从而达到治疗肿瘤的目的。
导师所领导课题组近年来一直致力于对MAGE家族的研究。本研究首次采用RT-PCR和免疫组织化学方法对60例乳腺正常组织、60例乳腺良性病变（纤维瘤及腺病）以及60例原发性乳腺癌组织中MAGE-A9、MAGE-A11、MAGE-C1和MAGE-C2的mRNA及蛋白表达情况进行了研究，并分析其表达与乳腺癌临床病理学指标以及预后之间的关系；应用不同浓度的DNA甲基化抑制剂和组蛋白去乙酰化酶抑制剂处理两种乳腺癌细胞株，观察药物处理前后MAGE-A9、MAGE-A11、MAGE-C1、MAGE-C2基因表达水平的变化，初步探索MAGE-A9、MAGE-A11、MAGE-C1、MAGE-C2表达的分子生物学机制。
主要研究内容和结果如下：
第一部分肿瘤抗原MAGE-A9和MAGE-A11在乳腺癌组织中的表达及与乳腺癌临床病理学指标和预后的关系
目的：检测MAGE-A9和MAGE-A11在乳腺正常组织、乳腺良性病变以及乳腺癌组织中的表达，并分析其表达与乳腺癌临床病理学指标以及预后之间的关系。
方法：采用RT-PCR和免疫组织化学的实验方法分别检测了MAGE-A9和MAGE-A11mRNA及蛋白在60例乳腺正常组织、60例乳腺良性病变和60例乳腺癌组织中的表达情况，并分析两者的表达与乳腺癌临床病理学指标（包括患者年龄、肿瘤大小、病理类型、组织学分级、临床分期、腋淋巴结转移、有无脉管瘤栓、ER、PR和HER-2）之间的关系。通过病例随访，分析MAGE-A9和MAGE-A11蛋白表达与乳腺癌患者预后之间的关系。
结果：
1MAGE-A9和MAGE-A11mRNA在乳腺正常组织、乳腺良性病变和乳腺癌组织中的表达及与乳腺癌临床病理学指标之间的关系
60例乳腺正常组织及60例乳腺良性病变中未见MAGE-A9和MAGE-A11mRNA的表达，而60例乳腺癌组织标本中MAGE-A9和MAGE-A11mRNA阳性表达率分别为45%和66.7%。
MAGE-A9和MAGE-A11mRNA的表达与ER及HER-2的表达呈正相关。ER表达阳性的乳腺癌患者，其MAGE-A9和MAGE-A11mRNA的阳性表达率分别高于ER表达阴性的乳腺癌患者（A9: χ2=4.865，P=0.027;A11: χ2=5.742，P=0.017）。HER-2表达阳性的乳腺癌患者，其MAGE-A9和MAGE-A11mRNA的表达阳性率分别高于HER-2表达阴性的乳腺癌患者（A9: χ2=6.16，P=0.013; χ2=4.266，P=0.039）。
MAGE-A9mRNA的表达与乳腺癌患者的年龄（χ2=2.372，P=0.366）、肿瘤大小（χ2=3.125，P=0.246）、病理类型（χ2=5.104，P=0.08）、临床分期（χ2=3.326，P=0.190）、组织学分级（χ2=1.304，P=0.562）、淋巴结转移（χ2=0.180，P=0.672）、瘤栓（χ2=2.095，P=0.148）及PR表达（χ2=1.358，P=0.244）均无相关性。
MAGE-A11mRNA的表达与乳腺癌患者的年龄（χ2=0.399，P=0.895）、肿瘤大小（χ2=2.687，P=0.263）、病理类型（χ2=5.312，P=0.064）、临床分期（χ2=2.461，P=0.305）、组织学分级（χ2=0.525，P=0.893）、淋巴结转移（χ2=1.250，P=0.264）、瘤栓（χ2=0.549，P=0.459）及PR表达（χ2=0.601，P=0.438）均无相关性。
2MAGE-A9和MAGE-A11蛋白在正常睾丸组织中的表达
在正常睾丸组织中，MAGE-A9和MAGE-A11主要表达于初级精母细胞和精原细胞中，且主要定位于细胞质，部分定位于细胞核。
3MAGE-A9和MAGE-A11蛋白在乳腺正常组织、乳腺良性病变、乳腺癌组织中的表达及与乳腺癌临床病理学指标之间的关系
MAGE-A9和MAGE-A11蛋白主要表达于乳腺癌细胞的胞质中，部分表达于细胞核，乳腺癌组织中MAGE-A9和MAGE-A11蛋白阳性表达率分别为43.3%和63.3%，而60例乳腺正常组织及60例乳腺良性病变中均未见MAGE-A9和MAGE-A11蛋白表达。
MAGE-A9（r=0.967，κ=0.966，P=0.000）和MAGE-A11（r=0.929，κ=0.927，P=0.000）mRNA与蛋白表达具有一致性，呈正相关。
MAGE-A9和MAGE-A11蛋白的表达与ER及HER-2的表达呈正相关，ER表达阳性的乳腺癌患者，其MAGE-A9和MAGE-A11蛋白的阳性表达率分别高于ER表达阴性的乳腺癌患者（A9: χ2=4.344，P=0.037; A11:χ2=4.311, P=0.038）。HER-2表达阳性的乳腺癌患者，其MAGE-A9和MAGE-A11蛋白的阳性表达率分别高于HER-2表达阴性的乳腺癌患者（A9: χ2=4.671, P=0.031; A11: χ2=5.967, P=0.015）。
MAGE-A9蛋白的表达与乳腺癌患者的年龄（χ2=3.621，P=0.209）、肿瘤大小（χ2=4.188，P=0.116）、病理类型（χ2=4.186，P=0.122）、临床分期（χ2=2.658，P=0.273）、组织学分级（χ2=1.006，P=0.618）、淋巴结转移（χ2=0.045，P=0.832）、瘤栓（χ2=2.807，P=0.094）及PR表达（χ2=1.067，P=0.302）均无相关性。
MAGE-A11蛋白的表达与乳腺癌患者的年龄（χ2=0.548，P=0.808）、肿瘤大小（χ2=2.943，P=0.231）、病理类型（χ2=3.257，P=0.203）、临床分期（χ2=4.232，P=0.117）、组织学分级（χ2=0.964，P=0.732）、淋巴结转移（χ2=3.062，P=0.08）、瘤栓（χ2=1.386，P=0.239）及PR表达（χ2=1.270，P=0.260）均无相关性。4MAGE-A9和MAGE-A11蛋白表达与乳腺癌患者生存率的关系
MAGE-A9和MAGE-A11蛋白表达阳性的乳腺癌患者的生存率分别低于MAGE-A9和MAGE-A11蛋白表达阴性的乳腺癌患者（A9: χ2=5.348,P=0.021; A11: χ2=4.192, P=0.041）。
结论：
1乳腺正常组织和乳腺良性病变中均未发现MAGE-A9和MAGE-A11mRNA及蛋白的表达。乳腺癌组织中MAGE-A9和MAGE-A11mRNA表达阳性率分别为45%和66.7%，蛋白阳性表达率分别为43.3%和63.3%，提示MAGE-A9和MAGE-A11是肿瘤特异性抗原，可作为乳腺癌免疫治疗的靶点。
2MAGE-A9和MAGE-A11mRNA和蛋白表达与乳腺癌患者的年龄、病理类型、组织学分级、临床分期、肿瘤大小、淋巴结转移、瘤栓、PR表达均无相关性，但与ER和HER-2的表达呈正相关。ER和HER-2表达阳性的乳腺癌患者，其MAGE-A9和MAGE-A11mRNA和蛋白的表达率明显高于ER和HER-2表达阴性的乳腺癌患者，提示MAGE-A9和MAGE-A11有望成为指导乳腺癌治疗及监测预后的重要指标。
3MAGE-A9和MAGE-A11蛋白表达阳性的乳腺癌患者的生存率分别低于MAGE-A9和MAGE-A11蛋白表达阴性的乳腺癌患者，说明MAGE-A9和MAGE-A11高表达可能是乳腺癌患者不良预后的一个重要标志。
第二部分肿瘤抗原MAGE-C1和MAGE-C2在乳腺癌组织中的表达及与乳腺癌临床病理学指标和预后的关系
目的：检测MAGE-C1和MAGE-C2在乳腺正常组织、乳腺良性病变以及乳腺癌组织中的表达，并分析其表达与乳腺癌临床病理学指标以及预后之间的关系。
方法：采用RT-PCR和免疫组织化学的实验方法分别检测了MAGE-C1和MAGE-C2mRNA及蛋白在60例乳腺正常组织、60例乳腺良性病变和60例乳腺癌组织中的表达情况，并分析两者的表达与乳腺癌临床病理学指标之间的关系。通过病例随访，分析MAGE-C1和MAGE-C2蛋白表达与乳腺癌患者预后之间的关系。
结果：
1MAGE-C1和MAGE-C2mRNA在乳腺正常组织、乳腺良性病变和乳腺癌组织中的表达及其与乳腺癌临床病理学指标之间的关系
60例乳腺正常组织及60例乳腺良性病变中均未见MAGE-C1和MAGE-C2mRNA的表达，而60例乳腺癌组织标本中MAGE-C1和MAGE-C2mRNA阳性表达率分别为43.3%和61.7%。
MAGE-C1和MAGE-C2mRNA的表达与乳腺癌的组织学分级呈正相关，组织学分级为Ⅲ级的乳腺癌患者，其MAGE-C1和MAGE-C2mRNA阳性表达率分别高于组织学分级Ⅰ级的乳腺癌患者（C1: χ2=8.832, P=0.01;C2: χ2=8.575, P=0.01）。
MAGE-C2mRNA表达还与乳腺癌的病理类型及瘤栓表达呈正相关。瘤栓表达阳性的乳腺癌患者，其MAGE-C2mRNA的阳性表达率高于瘤栓表达阴性的乳腺癌患者（χ2=6.094，P=0.014）。病理类型为浸润性导管癌的患者，其MAGE-C2mRNA的阳性表达率分别高于浸润性小叶癌和髓样癌的患者（χ2=11.707，P=0.002）。
MAGE-C1mRNA的表达与乳腺癌患者的年龄（χ2=0.403，P=0.907）、肿瘤大小（χ2=0.976，P=0.671）、病理类型（χ2=2.256，P=0.352）、临床分期（χ2=0.684，P=0.765）、淋巴转移（χ2=0.102，P=0.75）、瘤栓（χ2=0.008，P=0.93）、ER（χ2=0.611，P=0.434）、PR（χ2=0.16，P=0.689）及HER-2表达（χ2=0.307，P=0.58）均无相关性。
MAGE-C2mRNA的表达与乳腺癌患者的年龄（χ2=0.228，P=1.0）、肿瘤大小（χ2=0.579，P=0.815）、临床分期（χ2=4.059，P=0.115）、淋巴转移（χ2=2.303，P=0.129）、ER（χ2=0.004，P=0.947）、PR（χ2=0.000，P=1.0）及HER-2表达（χ2=2.366，P=0.124）均无相关性。
2MAGE-C1和MAGE-C2蛋白在正常睾丸组织中的表达
在正常睾丸组织中，MAGE-C1和MAGE-C2主要表达于初级精母细胞和精原细胞中，且主要定位于细胞质，部分定位于细胞核。
3MAGE-C1和MAGE-C2蛋白在乳腺正常组织、乳腺良性病变、乳腺癌组织中的表达及与乳腺癌临床病理学指标之间的关系
MAGE-C1和MAGE-C2蛋白主要表达于乳腺癌细胞的胞质中，部分表达于细胞核，60例乳腺癌组织中MAGE-C1和MAGE-C2蛋白阳性表达率分别为38.3%和58.3%，而60例乳腺正常组织及60例乳腺良性病变中均未见MAGE-C1和MAGE-C2蛋白表达。
MAGE-C1（r=0.902，κ=0.897，P=0.000）和MAGE-C2（r=0.933，κ=0.931，P=0.000）mRNA与蛋白表达具有一致性，呈正相关。
MAGE-C1和MAGE-C2蛋白表达与乳腺癌的组织学分级呈正相关，组织学分级为Ⅲ级的乳腺癌患者，其MAGE-C1和MAGE-C2蛋白的阳性表达率分别高于组织学分级Ⅰ级的乳腺癌患者（C1: χ2=6.233, P=0.038;C2: χ2=7.471, P=0.017）。
MAGE-C2蛋白表达还与乳腺癌的病理类型、瘤栓表达呈正相关。瘤栓表达阳性的乳腺癌患者，其MAGE-C2蛋白的阳性表达率高于瘤栓表达阴性的乳腺癌患者（χ2=5.503，P=0.019）。病理类型为浸润性导管癌的患者，其MAGE-C2蛋白的阳性表达率分别高于浸润性小叶癌和髓样癌的患者（χ2=10.889，P=0.004）。
MAGE-C1蛋白的表达与乳腺癌患者的年龄（χ2=1.115，P=0.729）、肿瘤大小（χ2=1.659，P=0.435）、病理类型（χ2=3.173，P=0.206）、临床分期（χ2=0.383，P=0.87）、淋巴转移（χ2=0.012，P=0.914）、瘤栓（χ2=0.099，P=0.753）、ER（χ2=0.533，P=0.465）、PR（χ2=0.097，P=0.755）及HER-2表达（χ2=1.422，P=0.233）均无相关性。
MAGE-C2蛋白的表达与乳腺癌患者的年龄（χ2=0.525，P=0.903）、肿瘤大小（χ2=0.359，P=0.936）、临床分期（χ2=4.022，P=0.15）、淋巴转移（χ2=1.143，P=0.285）、ER（χ2=0.429，P=0.513）、PR（χ2=0.07，P=0.791）及HER-2表达（χ2=1.936，P=0.164）均无相关性。4MAGE-C1和MAGE-C2蛋白表达与乳腺癌患者生存率的关系
MAGE-C1和MAGE-C2蛋白表达阳性的乳腺癌患者，其生存率分别低于MAGE-C1和MAGE-C2蛋白表达阴性的乳腺癌患者（C1: χ2=4.213,P=0.04; C2: χ2=4.467, P=0.035）。
结论：
1乳腺正常组织和乳腺良性病变中均未发现MAGE-C1和MAGE-C2mRNA及蛋白的表达。乳腺癌组织中MAGE-C1和MAGE-C2mRNA阳性表达率分别为43.3%和61.7%，MAGE-C1和MAGE-C2蛋白阳性表达率分别为38.3%和58.3%，提示MAGE-C1和MAGE-C2可作为肿瘤标志物检测乳腺癌的发生，也可成为乳腺癌免疫治疗的理想靶点。
2MAGE-C1mRNA和蛋白表达与乳腺癌患者的年龄、病理类型、临床分期、肿瘤大小、淋巴转移、瘤栓、ER、PR、HER-2表达均无相关性，MAGE-C2mRNA和蛋白表达与乳腺癌患者的年龄、临床分期、肿瘤大小、淋巴转移、ER、PR、HER-2表达均无相关性。MAGE-C1和MAGE-C2mRNA和蛋白表达与乳腺癌的组织学分级呈正相关。MAGE-C2mRNA和蛋白表达还与乳腺癌的病理类型、瘤栓表达呈正相关，提示MAGE-C1和MAGE-C2的表达和乳腺癌的恶性程度密切相关，且不同病理类型的肿瘤表达MAGE-C2的频率不同，以癌睾丸抗原为基础进行的肿瘤免疫治疗可能更适合在某些病理类型的肿瘤中进行。
3MAGE-C1和MAGE-C2蛋白的高表达与乳腺癌患者的低生存率正相关，说明MAGE-C1和MAGE-C2高表达可能是乳腺癌患者不良预后的一个重要标志。
第三部分乳腺癌中肿瘤抗原MAGE-A9、MAGE-A11、MAGE-C1和MAGE-C2表达机制的研究
目的：应用DNA甲基化抑制剂5-aza-CdR和组蛋白去乙酰化酶抑制剂TSA单独或联合处理MCF-7、MDA-MB-231两种乳腺癌细胞，分析药物处理前后MAGE-A9、MAGE-A11、MAGE-C1、MAGE-C2基因表达的变化，初步探索它们表达的分子生物学机制。方法：将实验细胞分为4组：（1）对照组:同期培养的不加任何药物处理的细胞；（2）5-aza-CdR组：分别用含2.5μmol/L、5μmol/L5-aza-CdR的培养基培养细胞72h；（3）TSA组：分别用含0.3μmol/L、0.5μmol/L TSA的培养基培养细胞24h；（4）5-aza-CdR及TSA组：用含5μmol/L5-aza-CdR的培养基培养细胞48h后再加入含0.5μmol/LTSA的培养基继续培养24h。药物刺激72小时后，收集细胞，提取RNA，每组实验重复三次。
结果：
1药物处理前后MCF-7、MDA-MB-231细胞中MAGE-A9和MAGE-A11mRNA表达水平的变化RT-PCR结果显示，未经药物处理的MCF-7、MDA-MB-231细胞未见MAGE-A9和MAGE-A11mRNA表达。应用2.5μmol/L、5μmol/L5-aza-CdR分别作用72小时后可见MAGE-A9和MAGE-A11基因重新表达，且随着药物浓度增高，MAGE-A9和MAGE-A11基因表达逐渐增强。与单独应用5-aza-CdR相比较，联合应用5μmol/L5-aza-CdR和0.5μmol/L TSA可使MAGE-A9和MAGE-A11基因表达进一步增加。而0.3μmol/L、0.5μmol/L TSA单独作用均不能诱导出MAGE-A9和MAGE-A11基因的重新表达。
2药物处理前后MCF-7、MDA-MB-231细胞中MAGE-C1和MAGE-C2mRNA表达水平的变化
RT-PCR结果显示，未经药物处理的MCF-7、MDA-MB-231细胞未见MAGE-C1mRNA表达。单独应用2.5μmol/L、5μmol/L5-aza-CdR及0.3μmol/L、0.5μmol/L TSA均不能诱导出MAGE-C1基因的表达。而联合应用5μmol/L5-aza-CdR和0.5μmol/L TSA也未能诱导出MAGE-C1基因的表达。
RT-PCR结果显示，未经药物处理的MCF-7、MDA-MB-231细胞未见MAGE-C2mRNA表达。应用2.5μmol/L、5μmol/L5-aza-CdR分别作用72小时后可见MAGE-C2基因重新表达，且随着药物浓度增高，基因表达逐渐增强。与单独应用5-aza-CdR相比较，联合应用5μmol/L5-aza-CdR和0.5μmol/L TSA可使MAGE-C2基因表达进一步增加。而0.3μmol/L、0.5μmol/L TSA单独作用均不能诱导出MAGE-C2基因的重新表达。
结论：
1单独应用5-aza-CdR处理MCF-7及MDA-MB-231细胞可诱导MAGE-A9、MAGE-A11、MAGE-C2基因的表达，提示DNA去甲基化是MAGE-A9、MAGE-A11和MAGE-C2基因表达重要机制。
2联合应用5-aza-CdR和TSA可使MCF-7及MDA-MB-231细胞中MAGE-A9、MAGE-A11和MAGE-C2基因的表达进一步增加，而TSA单独作用对基因表达没有影响，说明DNA去甲基化和组蛋白乙酰化在MAGE基因的表达中具有协同作用，DNA去甲基化扮演了主导作用。
3单独或联合应用不同浓度的5-aza-CdR和TSA均不能诱导出MCF-7及MDA-MB-231细胞中MAGE-C1的表达，说明DNA甲基化和组蛋白乙酰化并不是调节MAGE基因表达的唯一机制。
Breast cancer is the most common malignancy in women. However,therapeutic options for the treatment of patients with this tumor are limited to3fundamental modalities: surgical resection, radiation therapy andchemotherapy. Against advanced carcinomas, therapeutic options are limitedto radiation therapy and chemotherapy; however, these modalities do not yieldresults. Therefore, cancer-specific immunotherapy may be expected to becomea novel treatment modality for breast carcinomas.
Melanoma-associated antigens (MAGE) are a group of well-characterizedmembers of the cancer-testis antigen (CTA) family that are expressed invarious tumor cells, but not in healthy tissues except for the testis and placenta.The MAGE gene family encodes tumor-associated antigens, which arerecognized by CTLs in conjunction with MHC class I molecules of varioushaplotypes on the tumor cell surface. Thus, MAGE are appealing targets forcancer immunotherapy.
In recent years, our professor’s research group has been committed to theresearch of MAGE family. In the present study, we investigated the expressionstatus of MAGE-A9, MAGE-A11, MAGE-C1and MAGE-C2by reversetranscriptional-polymerase chain reaction (RT-PCR) andimmunohistochemistry in60breast benign diseases specimens (includingfibroadenoma and adenosis),60primary breast cancer specimens and60tumor free breast specimens, analyzed their correlation with theclinicopathological parameters and the overall survival of breast cancerpatients. Furthermore, we added DNA methyltransferase inhibitor5-aza-CdRand/or histone deacetylase inhibitor TSA to two breast cancer cell lines andanalyzed the expression of MAGE-A9, MAGE-A11, MAGE-C1andMAGE-C2genes before and after treating with the two inhibitors by RT-PCR in order to elucidate epigenetic mechanism of the four genes expression.
The main research contents and results were shown as follows:Part ⅠThe expression of MAGE-A9and MAGE-A11in breast cancertissues and their correlation with the clinicopathological parameters andthe prognosis
Objective: To investigate the expression of MAGE-A9and MAGE-A11inbreast benign diseases tissues, tumor-free breast tissues and breast cancertissues, explore their correlation with the clinicopathological parameters andthe prognosis of the breast cancer patients.
Methods: The expression of MAGE-A9and MAGE-A11was investigated in60breast benign diseases specimens,60tumor-free breast specimens and60breast cancer specimens by RT-PCR and immunohistochemistry, then thecorrelation between MAGE-A9, MAGE-A11expression and theclinicopathological parameters of breast cancer patients, including age of thepatients, tumor size, pathological types, histology grades, clinical stages,metastasis of axillary lymph nodes, tumor embolus, estrogen receptor (ER),progestrogen receptor (PR) and HER-2status was analyzed. The associationbetween MAGE-A9and MAGE-A11protein expression and the prognosis ofbreast cancer patients was also analyzed.
Results:
1Expression of MAGE-A9and MAGE-A11mRNA in breast benign diseasesspecimens, tumor-free breast specimens, breast cancer specimens and thecorrelation between MAGE-A9, MAGE-A11expressions andclinicopathological parameters of breast cancer patients
Overall, no MAGE-A9and MAGE-A11expression was found in60tumor-free breast specimens and60breast benign diseases specimens.Expression of MAGE-A9and MAGE-A11mRNA was detected in45%and66.7%of the breast cancer specimens, respectively.
MAGE-A9and MAGE-A11mRNA expression was positively associatedwith ER and HER-2expression. MAGE-A9and MAGE-A11mRNAexpression was more frequent in ER-positive breast carcinomas (A9:25/45, 55.6%; A11:34/45,75.6%) compared with ER-negative breast carcinomas(A9:2/15,13.3%; A11:6/15,40%)(A9: χ2=4.865，P=0.027; A11: χ2=5.742，P=0.017). MAGE-A9and MAGE-A11mRNA expression was also morefrequent in HER-2-positive breast carcinomas (A9:25/48,52.1%; A11:36/48,75%) compared with HER-2-negative breast carcinomas (A9:2/12,16.7%;A11:4/12,33.3%)(A9: χ2=6.16，P=0.013; χ2=4.266，P=0.039).
No correlation was found between MAGE-A9mRNA expression and theage of the patients (χ2=2.372，P=0.366), tumor size (χ2=3.125，P=0.246),pathological type (χ2=5.104，P=0.08), clinical stage (χ2=3.326，P=0.190)，histological grade (χ2=1.304，P=0.562), lymphatic metastasis (χ2=0.180，P=0.672), tumor embolus (χ2=2.095，P=0.148)and PR (χ2=1.358，P=0.244)status.
No correlation was found between MAGE-A11mRNA expression and theage of the patients (χ2=0.399，P=0.895), tumor size (χ2=2.687，P=0.263),pathological type (χ2=5.312，P=0.064), clinical stage (χ2=2.461，P=0.305)，histological grade (χ2=0.525，P=0.893), lymphatic metastasis (χ2=1.250，P=0.264), tumor embolus (χ2=0.549，P=0.459)and PR (χ2=0.601，P=0.438)status.2Expression of MAGE-A9and MAGE-A11proteins in normal human testistissues
As the positive control, MAGE-A9and MAGE-A11antibodies were firstlyused to stain human normal testicular tissue sections. Both MAGE-A9andMAGE-A11expressions were mainly observed on primary spermatocytes andspermatogonia, and both the nucleus and the cytoplasm were stained.3Expression of MAGE-A9and MAGE-A11protein in breast benign diseasesspecimens, tumor-free breast specimens, breast cancer specimens and thecorrelation between MAGE-A9, MAGE-A11expression andclinicopathological parameters of breast cancer patients
In most breast cancer cells, MAGE-A9and MAGE-A11staining werefound in cytoplasm but occasionally also observed in nucleus. Overall, noMAGE-A9and MAGE-A11immunoreactivity was observed in60tumor-free breast specimens and60breast benign diseases specimens, while43.3%(26out of60) and63.3%(38out of60) breast cancer specimens were foundpositive with MAGE-A9and MAGE-A11antibodies, respectively.
There was generally concordance between MAGE-A9(r=0.967，κ=0.966，P=0.000) and MAGE-A11(r=0.929，κ=0.927，P=0.000) mRNA and proteinexpression.
MAGE-A9and MAGE-A11protein expression was positively associatedwith ER and HER-2expression. MAGE-A9and MAGE-A11proteinexpression was more frequent in ER-positive breast carcinomas comparedwith ER-negative breast carcinomas (A9: χ2=4.344，P=0.037; A11: χ2=4.311,P=0.038). MAGE-A9and MAGE-A11protein expression was also morefrequent in HER-2-positive breast carcinomas compared with HER-2-negativebreast carcinomas (A9: χ2=4.671, P=0.031; A11: χ2=5.967, P=0.015).
No correlation was found between MAGE-A9protein expression and theage of the patients (χ2=3.621，P=0.209), tumor size (χ2=4.188，P=0.116),pathological type (χ2=4.186，P=0.122), clinical stage (χ2=2.658，P=0.273)，histological grade (χ2=1.006，P=0.618), lymphatic metastasis (χ2=0.045，P=0.832), tumor embolus (χ2=2.807，P=0.094)and PR (χ2=1.067，P=0.302)status.
No correlation was found between MAGE-A11protein expression and theage of the patients (χ2=0.548，P=0.808), tumor size (χ2=2.943，P=0.231),pathological type (χ2=3.257，P=0.203), clinical stage (χ2=4.232，P=0.117)，histological grade (χ2=0.964，P=0.732), lymphatic metastasis (χ2=3.062，P=0.08), tumor embolus (χ2=1.386，P=0.239)and PR (χ2=1.270，P=0.260)status.
4Correlation between MAGE-A9and MAGE-A11expression and theprognosis of breast cancer patients
Patients with MAGE-A9or MAGE-A11expression had a worse prognosisthan the patients without MAGE-A9(χ2=5.348, P=0.021) or MAGE-A11(χ2=4.192, P=0.041) expression.
Conclusions: 1Expression of MAGE-A9and MAGE-A11mRNA was detected in45%and66.7%of the breast cancer specimens, respectively. The expression rate ofMAGE-A9and MAGE-A11protein in breast cancer was43.3%and63.3%,respectively, while no MAGE-A9and MAGE-A11was observed intumor-free breast specimens and breast benign diseases specimens, suggestingthat MAGE-A9and MAGE-A11were tumor specific antigens.2MAGE-A9, MAGE-A11mRNA and protein expression were more frequentin ER or HER-2positive breast carcinomas compared with ER or HER-2negative breast carcinomas.
3Both MAGE-A9and MAGE-A11expression were significantly associatedwith reduced overall survival, suggesting that MAGE-A9and MAGE-A11may be potential markers of a poor prognosis for breast cancer patients.
Part ⅡThe expression of MAGE-C1and MAGE-C2in breast cancertissues and their correlation with the clinicopathological parameters andthe prognosis
Objective: To investigate the expression of MAGE-C1and MAGE-C2inbreast benign diseases tissues, tumor-free breast tissues and breast cancertissues, explore their correlation with the clinicopathological parameters andthe prognosis of the breast cancer patients.
Methods: The expression of MAGE-C1and MAGE-C2was investigated in60breast benign diseases specimens,60tumor-free breast specimens and60breast cancer specimens by RT-PCR and immunohistochemistry, then thecorrelation between MAGE-C1and MAGE-C2expression and theclinicopathological parameters of breast cancer patients was analyzed. Theassociation between MAGE-C1and MAGE-C2protein expression and theprognosis of breast cancer patients was also analyzed.
Results:
1Expression of MAGE-C1and MAGE-C2mRNA in breast benign diseasesspecimens, tumor-free breast specimens, breast cancer specimens and thecorrelation between MAGE-C1and MAGE-C2expression andclinicopathological parameters of breast cancer patients Overall, no MAGE-C1and MAGE-C2expression was found in60tumor-free breast specimens and60breast benign diseases specimens.Expression of MAGE-C1and MAGE-C2mRNA was detected in43.3%and61.7%of the breast cancer specimens, respectively.
MAGE-C1and MAGE-C2mRNA expression was positively correlatedwith tumor grade. MAGE-C1and MAGE-C2mRNA expression was morefrequent in high-grade tumors (grade III) compared with low-grade tumors(grade I)(C1: χ2=8.832, P=0.01; C2: χ2=8.575, P=0.01).
Furthermore, MAGE-C2mRNA expression was also significantlyassociated with tumor embolus and histological type. Higher frequencies ofMAGE-C2mRNA expression was seen in invasive ductal breast cancercompared to invasive lobular breast cancer and medullary breast cancer(χ2=11.707，P=0.002). MAGE-C2expression was also more frequent in breastcarcinomas with positive tumor embolus compared to breast carcinomas withnegative tumor embolus (χ2=6.094，P=0.014).
No correlation was found between MAGE-C1mRNA expression and theage of the patients (χ2=0.403，P=0.907), tumor size (χ2=0.976，P=0.671),pathological type (χ2=2.256，P=0.352), clinical stage (χ2=0.684，P=0.765)，lymphatic metastasis (χ2=0.102，P=0.75), tumor embolus (χ2=0.008，P=0.93),ER (χ2=0.611，P=0.434), PR (χ2=0.16，P=0.689) and HER-2(χ2=0.307，P=0.58) status.
No correlation was found between MAGE-C2mRNA expression and theage of the patients (χ2=0.228，P=1.0), tumor size (χ2=0.579，P=0.815), clinicalstage (χ2=4.059，P=0.115)，lymphatic metastasis (χ2=2.303，P=0.129), ER(χ2=0.004，P=0.947), PR (χ2=0.000，P=1.0) and HER-2(χ2=2.366，P=0.124)status.
2Expression of MAGE-C1and MAGE-C2proteins in normal human testistissues
As the positive control, MAGE-C1and MAGE-C2antibodies were firstlyused to stain human normal testicular tissue sections. Both MAGE-C1andMAGE-C2expressions were mainly observed on primary spermatocytes and spermatogonia, and both the nucleus and the cytoplasm were stained.
3Expression of MAGE-C1and MAGE-C2proteins in breast benign diseasesspecimens, tumor-free breast specimens, breast cancer specimens and thecorrelation between MAGE-C1and MAGE-C2expression andclinicopathological parameters of breast cancer patients
In most breast cancer cells, MAGE-C1and MAGE-C2staining were foundin cytoplasm but occasionally also observed in nucleus. Overall, noMAGE-C1and MAGE-C2immunoreactivity was observed in60tumor-freebreast specimens and60breast benign diseases specimens, while38.3%and58.3%breast cancer specimens were found positive with MAGE-C1andMAGE-C2antibodies, respectively.
There was generally concordance between MAGE-C1(r=0.902，κ=0.897，P=0.000) and MAGE-C2(r=0.933，κ=0.931，P=0.000) mRNA and proteinexpression.
MAGE-C1and MAGE-C2protein expressions were positively correlatedwith tumor grade. MAGE-C1and MAGE-C2protein expressions were morefrequent in high-grade tumors (grade III) compared with low-grade tumors(grade I)(C1: χ2=6.233, P=0.038; C2: χ2=7.471, P=0.017).
Furthermore, MAGE-C2mRNA protein was also significantly associatedwith tumor embolus and histological type. Higher frequencies of MAGE-C2protein expression was seen in invasive ductal breast cancer compared toinvasive lobular breast cancer and medullary breast cancer (χ2=10.889，P=0.004). MAGE-C2protein expression was also more frequent in breastcarcinomas with positive tumor embolus compared to breast carcinomas withnegative tumor embolus (χ2=5.503，P=0.019).
No correlation was found between MAGE-C1protein expression and theage of the patients (χ2=1.115，P=0.729), tumor size (χ2=1.659，P=0.435),pathological type (χ2=3.173，P=0.206), clinical stage (χ2=0.383，P=0.87)，lymphatic metastasis (χ2=0.012， P=0.914), tumor embolus (χ2=0.099，P=0.753), ER (χ2=0.533，P=0.465), PR (χ2=0.097，P=0.755) and HER-2(χ2=1.422，P=0.233) status.
No correlation was found between MAGE-C2protein expression and theage of the patients (χ2=0.525，P=0.903), tumor size (χ2=0.359，P=0.936),clinical stage (χ2=4.022，P=0.15)，lymphatic metastasis (χ2=1.143，P=0.285),ER (χ2=0.429，P=0.513), PR (χ2=0.07，P=0.791) and HER-2(χ2=1.936，P=0.164) status.
4Correlation between MAGE-C1and MAGE-C2expressions and theprognosis of breast cancer patientsPatients with MAGE-C1or MAGE-C2expression had a worse prognosisthan the patients without MAGE-C1(χ2=4.213, P=0.04) or MAGE-C2(χ2=4.467, P=0.035) expression.
Conclusions:
1Expression of MAGE-C1and MAGE-C2mRNA was detected in43.3%and61.7%of the breast cancer specimens, respectively. The expression rate ofMAGE-C1and MAGE-C2protein in breast cancer was38.3%and58.3%,respectively, while no MAGE-C1and MAGE-C2was observed in tumor-freebreast specimens and breast benign diseases specimens, suggesting thatMAGE-C1and MAGE-C2can be used not only as appropriate candidates fortargets of cancer-specific immunotherapy but also as tumor biomarkers forbreast cancer.
2MAGE-C1and MAGE-C2expression correlated significantly with tumorgrade, MAGE-C2-positive expression was also significantly associated withpresence of tumor embolus and histological type, suggesting that there was anassociation between MAGE-C1, MAGE-C2expression and features of a moreaggressive clinical behavior in breast cancer, and some carcinoma might bemore suitable for CTA based immunotherapy than others, depending on cellmorphology.
3Both MAGE-C1and MAGE-C2expression were significantly associatedwith reduced overall survival, suggesting that MAGE-C1and MAGE-C2maybe potential markers of a poor prognosis for breast cancer patients.
Part Ⅲ The expression mechanism of MAGE-A9, MAGE-A11,MAGE-C1and MAGE-C2in breast cancers
Objective: We examined the influence of the DNA methylase inhibitor5-aza-2'-deoxycytidine (5-aza-CdR) together with the histone deacetylaseinhibitor trichostatin A (TSA) on the expression of MAGE-A9, MAGE-A11,MAGE-C1and MAGE-C2genes in two breast cancer cell lines.
Methods: Cell lines were divided into four groups:(1)5-aza-CdR group:Cells were stimulated by2.5μmol/L and5μmol/L5-aza-CdR for72hours,respectively;(2) TSA group: Cells were stimulated by0.3μmol/L and0.5μmol/L TSAfor24hours, respectively;(3)5-aza-CdR+TSA group: Cells werestimulated by0.5μmol/L TSA for24hours after the48-hour5μmol/L5-aza-CdR incubation;(4) Control group: Cells were mock-treated with thesame volume of culture medium. Then the cells were collected to extract totalRNA for evaluate MAGE-A9, MAGE-A11, MAGE-C1and MAGE-C2geneexpressions.
Results:
1Expression patterns of MAGE-A9and MAGE-A11genes in MCF-7andMDA-MB-231cells before and after treatment with5-aza-CdR and/or TSA
Treatment of the two breast cancer cells with2.5μmol/L and5μmol/L5-aza-CdR alone could induce the expression of MAGE-A9and MAGE-A11,and the expression was enhanced with5-aza-CdR concentration increased. Weobserved robust expression of both genes after addition of0.5μmol/L TSA for24hours following48hours of5μmol/L5-aza-CdR treatment. However,0.3μmol/L and0.5μmol/L TSA treatment alone had no influence on MAGE-Agene expression in the two cell lines.
2Expression patterns of MAGE-C1and MAGE-C2genes in MCF-7andMDA-MB-231cells before and after treatment with5-aza-CdR and/or TSA
Treatment of the two breast cancer cells with2.5μmol/L and5μmol/L5-aza-CdR alone could only induce the expression of MAGE-C2, and theexpression was increased with5-aza-CdR concentration evaluated. Weobserved robust expression of MAGE-C2genes after addition of0.5μmol/LTSA for24hours following48hours of5μmol/L5-aza-CdR treatment.However,0.3μmol/L and0.5μmol/L TSA treatment alone had no influence on MAGE-C2gene expression in the two cell lines.
However, addition of2.5μmol/L and5μmol/L5-aza-CdR alone or0.3μmol/L and0.5μmol/L TSA alone could not activate MAGE-C1geneexpression in the two breast cancer cells.5μmol/L5-aza-CdR in combinationwith0.5μmol/L TSA also failed to activate MAGE-C1gene expression in thetwo breast cancer cells.
Conclusions:
1Treatment of MCF-7, MDA-MB-231cells with5-aza-CdR could induce theexpression of MAGE-A9, MAGE-A11and MAGE-C2, suggesting that DNAmethylation is an important mechanism in the regulation of MAGE-A9,MAGE-A11and MAGE-C2.
2DNA methylation and histone deacetylation appear to act as synergisticlayers for the silencing of MAGE-A9, MAGE-A11and MAGE-C2genes, andDNA methylation is dominant for the stable maintenance of a silent state.
3MAGE-C1expressions could not be reactivating in the two breast cancercells despite treatment with5-aza-CdR and/or TSA, suggesting that DNAmethylation and histone acetylation are not the only mechanism in theregulation of MAGE gene expression.
引文
1李霓，郑荣寿，张思维.中国城乡女性乳腺癌发病趋势分析和预测.中国卫生统计,2012,46(8):703-707
2Scanlan, MJ，AO Gure，AA Jungbluth，et al. Cancer/testis antigens: anexpanding family of targets for cancer immunotherapy. Immunol Rev，2002，188:22-32
3Zendman, AJ，DJ Ruiter，GN Van Muijen. Cancer/testis-associated genes:identification, expression profile, and putative function. J Cell Physiol，2003，194(3):272-88
4Simpson, AJ，OL Caballero，A Jungbluth，et al. Cancer/testis antigens,gametogenesis and cancer. Nat Rev Cancer，2005，5(8):615-25
5Meek DW, Marcar L. MAGE-A antigens as targets in tumour therapy.Cancer Lett,2012,324:126-132
6Rogner, U.C.，K. Wilke，E. Steck，et al. The melanoma antigen gene(MAGE) family is clustered in the chromosomal band Xq28.Genomics，1995，29(3):725-31
7De Plaen E, Arden K, Traversari C, et al. Structure, chromosomallocalization, and expression of12genes of the MAGE family.Immunogenetics,1994,40(5):360-369
8Sang M, Lian Y, Zhou X, et al. MAGE-A family: attractive targets forcancer immunotherapy. Vaccine,2011,29(47):8496-8500
9Van der Bruggen P,Travbersari C, Chomez P, etal. A gene encoding anantigen recognized by cytolytic T lymphocytes on a human melanoma[J].Science,1991,254(5038):1643-1647
10Ohman Forslund K, Nordqvist K. The melanoma antigen genes-any cluesto their functions in normal tissues? Exp Cell Res,2001,265(2):185-194
11马鸣，俞莉章.肿瘤特异性抗原MAGE基因的表达及意义.国外医学泌尿系统分册，2003,23:526-529
12Simpson AJ, Caballero OL, Jungbluth A, et al. Cancer/testis antigens,gametogenesis and cancer. Nat Rev Cancer,2005,5(8):615-625
13Chomez P, De Backer O, Bertrand M, et al. An overview of the MAGEgene family with the identification of all human members of the family.Cancer Res,2001,61(14):5544-5551
14Brasseur F, Rimoldi D, Liénard D, et al. Expression of MAGE genes inprimary and metastatic cutaneous melanoma. Int J Cancer1995,63:375-80
15Kufer P, Zippelius A, Lutterbuse R, et al. Heterogeneous expression ofMAGE-A genes in occult disseminated tumor cells: a novel multimarkerreverse transcription-polymerase chain reaction for diagnosis ofmicrometastatic disease. Cancer Res,2002,62(1):251-261
16Chen H, Cai S, Wang Y, et al. Expression of the MAGE-1gene in humanhepatocellular carcinomas. Clin Med J (Engl),2000,113(12):1112-1118
17Bergeron A, Picard V, LaRue H, et al. High frequency of MAGE-A4andMAGE-A9expression in high-risk bladder cancer. Int J Cancer,2009,125(6):1365-1371
18Peikert T, Specks U, Farver C, et al. Melanoma antigen A4is expressed innon-small cell lung cancers and promotes apoptosis. Cancer Res,2006,66(9):4693-4700
19Kim KH, Choi JS, Kim IJ, et al. Promoter hypomethylation andreactivation of MAGE-A1and MAGE-A3genes in colorectal cancer celllines and cancer tissues. World J Gastroenterol,2006,12(35):5651-5657
20Napoletano C, Bellati F, Tarquini E, et al. MAGE-A and NY-ESO-1expression in cervical cancer: prognostic factors and effects ofchemotherapy. Am J Obstat Gynecol,2008,198(1):99.e1-7
21Yakirevich E, Sabo E, Lavie O, et al. Expression of the MAGE-A4andNY-ESO-1cancer-testis antigens in serous ovarian neoplasms. Clin CancerRes,2003,9(17):6453-6460
22Yan L, Yang X, Davidson NE. Role of DNA methylation and histoneacetylation in steroid receptor expression in breast cancer. J MammaryGland Biol Neoplasia,2001,6(2):183-192
23Ross JS, Fletcher JA, Bloom KJ, et al. Targeted therapy in breast cancer:the HER-2/neu gene and protein. Mol Cell Proteomics,2004,3(4):379-398
24Yeon CH, Pegram MD. Anti-erbB-2antibody trastuzumab in the treatmentof HER2-amplified breast cancer. Invest New Drugs,2005,23(5):391-409
25Rabindran SK. Antitumor activity of HER-2inhibitors. Cancer Letters,2005,227(1):9-23
26Otte M, Zafrakas M, Riethdorf L, et al. MAGE-A gene expression patternin primary breast cancer. Cancer Res,2001,61(18):6682-6687
27Karn T, Pusztai L, Ruckhaberle E, et al. Melanoma antigen family Aidentified by the bimodality index defines a subset of triple negative breastcancers as candidates for immune response augmentation. Eur J Cancer,2012,48(1):12-23
1Early Breast Cancer Trialists’ Collaborative Group. Polychemotherapy forearly breast cancer: an overview of the randomized trials. Lancet,1998,352:930-942
2Early Breast Cancer Trialists’ Collaborative Group: Tamoxifen for earlybreast cancer: an overview of the randomised trials. Lancet,1998,351:1451-1467
3Filho PA, Lopez-Albaitero A, Xi L, Gooding W, Godfrey T, Ferris RL.Quantitative expression and immunogenicity of MAGE-3and-6in upperaerodigestive tract cancer. Int J Cancer2009;125:1912-1920
4Ma W, Germeau C, Vigneron N, Maernoudt AS, Morel S, Boon T,Coulie PG,Van den Eynde BJ. Two new tumor-specific antigenic peptides encoded bygene MAGE-C2and presented to cytolytic T lymphocytes by HLA-A2. IntJ Cancer2004;109:698-702
5Godelaine D, Carrasco J, Brasseur F, Neyns B, Thielemans K, Boon T, VanPel A. A new tumor-specific antigen encoded by MAGE-C2and presentedto cytolytic T lymphocytes by HLA-B44. Cancer Immunol Immunother2007;56:753-9
6Simpson AJ, Caballero OL, Jungbluth A, et al. Cancer/testis antigens,gametogenesis and cancer. Nat Rev Cancer,2005,5(8):615-625
7Chomez P, De Backer O, Bertrand M, et al. An overview of the MAGE genefamily with the identification of all human members of the family. CancerRes,2001,61(14):5544-5551
8De Plaen E, Arden K, Traversari C, et al. Structure, chromosomallocalization, and expression of12genes of the MAGE family.Immunogenetics,1994,40(5):360-369
9Kirkin AF, Dzhandzhugazyan KN, Zeuthen J. Cancer/testis antigens:structural and immunobiological properties. Cancer Invest,2002,20(2):222-236
10Zhuang R, Zhu Y, Fang L, et al. Generation of monoclonal antibodies tocancer/testis (CT) antigen CT10/MAGE-C2. Cancer Immun,20066:7
11Güre AO, Stockert E, Arden KC et al. CT10: a new cancer cancer-testis(CT) antigen homologous to CT7and the MAGE family, identified byrepresentational-difference analysis. Int J Cancer2000,85(5):726-732
12de Carvalho F, Alves VL, Braga WM, et al. MAGE-C1/CT7andMAGE-C2/CT10are frequently expressed in multiple myeloma and canbe explored in combined immunotherapy for this malignancy. CancerImmunol Immunother,2013,62(1):191-5
13Gure AO, Chua R, Williamson B, et al. Cancer-testis genes are coordinatelyexpressed and are markers of poor outcome in non-small cell lung cancer.Clin Cancer Res,2005,11(22):8055-62
14von Boehmer L, Keller L, Mortezavi A, et al. MAGE-C2/CT10proteinexpression is an independent predictor of recurrence in prostate cancer.PLoS One,2011,6(7):e21366
15Figueiredo DL, Mamede RC, Spagnoli GC, et al. High expression ofcancer testis antigens MAGE-A, MAGE-C1/CT7, MAGE-C2/CT10,NY-ESO-1, and gage in advanced squamous cell carcinoma of the larynx.Head Neck,2011,33(5):702-7
16Peng JR, Chen HS, Mou DC, et al. Expression of cancer/testis (CT)antigens in Chinese hepatocellular carcinoma and its correlation withclinical parameters. Cancer Lett,2005,219(2):223-32
17Chitale DA, Jungbluth AA, Marshall DS, et al. Expression of cancer-testisantigens in endometrial carcinomas using a tissue microarray. Mod Pathol,2005,18:119-126
18Zimmermann AK, Imig J, Klar A, et al. Expression of MAGE-C1/CT7andselected cancer/testis antigens in ovarian borderline tumours and primaryand recurrent ovarian carcinomas.Virchows Arch,2013,462(5):565-74
19Zendman AJ, Ruiter DJ, Van Muijen GN. Cancer/testis-associated genes:identification, expression profile, and putative function. J Cell Physiol,2003,194:272-288
20Caballero OL, Chen YT. Cancer/testis (CT) antigens: potential targets forimmunotherapy. Cancer Sci,2009,100:2014-2021
21Brasseur F, Rimoldi D, Liénard D, et al. Expression of MAGE genes inprimary and metastatic cutaneous melanoma. Int J Cancer1995,63:375-80
22Kufer P, Zippelius A, Lutterbuse R, et al. Heterogeneous expression ofMAGE-A genes in occult disseminated tumor cells: a novel multimarkerreverse transcription-polymerase chain reaction for diagnosis ofmicrometastatic disease. Cancer Res,2002,62(1):251-261
23Chen H, Cai S, Wang Y, et al. Expression of the MAGE-1gene in humanhepatocellular carcinomas. Clin Med J (Engl),2000,113(12):1112-1118
24Peikert T, Specks U, Farver C, et al. Melanoma antigen A4is expressed innon-small cell lung cancers and promotes apoptosis. Cancer Res,2006,66(9):4693-4700
25Kim KH, Choi JS, Kim IJ, et al. Promoter hypomethylation andreactivation of MAGE-A1and MAGE-A3genes in colorectal cancer celllines and cancer tissues. World J Gastroenterol,2006,12(35):5651-5657
26Napoletano C, Bellati F, Tarquini E, et al. MAGE-A and NY-ESO-1expression in cervical cancer: prognostic factors and effects ofchemotherapy. Am J Obstat Gynecol,2008,198(1):99.e1-7
27Bergeron A, Picard V, LaRue H, et al. High frequency of MAGE-A4andMAGE-A9expression in high-risk bladder cancer. Int J Cancer,2009,125(6):1365-1371
28Yakirevich E, Sabo E, Lavie O, et al. Expression of the MAGE-A4andNY-ESO-1cancer-testis antigens in serous ovarian neoplasms. Clin CancerRes,2003,9(17):6453-6460
29Moureau-Zabotto L, Ricci S, Lefranc JP, et al. Prognostic impact ofmultidrug resistance gene expression on the management of breast cancerin the context of adjuvant therapy based on a series of171patients. Br JCancer,2006,94(4):473-80
30Chitale DA, Jungbluth AA, Marshall DS, et al. Expression of cancer-testisantigens in endometrial carcinomas using a tissue microarray. Mod Pathol,2005,18:119-126
31Zimmermann AK, Imig J, Klar A, et al. Expression of MAGE-C1/CT7andselected cancer/testis antigens in ovarian borderline tumours and primaryand recurrent ovarian carcinomas.Virchows Arch,2013,462(5):565-74
32Krüger S, Ola V, Feller AC, et al. Expression of cancer-testis antigen ct7(mage-c1)in breast cancer: an immunohistochemical study with emphasison prognostic utility. Pathol Oncol Res,2007,13:91-96
33Prasad ML, Jungbluth AA, Patel SG, et al. Expression and significance ofcancer testis antigens in primary mucosal melanoma of the head and neck.Head Neck,2004,26(12):1053-7
34Atanackovic D, Luetkens T, Hildebrandt Y, et al. Longitudinal analysis andprognostic effect of cancer-testis antigen expression in multiple myeloma.Clin Cancer Res,2009,15(4):1343-52
35Perez D, Hauswirth F, J ger D, et al. Protein expression of cancer testisantigens predicts tumor recurrence and treatment response to imatinib ingastrointestinal stromal tumors. Int J Cancer,2011,128(12):2947-52
36Riener MO, Wild PJ, Soll C, et al. Frequent expression of the novel cancertestis antigen MAGE-C2/CT-10in hepatocellular carcinoma. Int J Cancer,2009,124(2):352-7
37Montoro JR, Mamede RC, Neder Serafini L, et al. Expression ofcancer-testis antigens MAGE-A4and MAGE-C1in oral squamous cellcarcinoma. Head Neck,2012,34(8):1123-8
1WolffeA P, MatzkeM A. Epigenetics: regulation through repression. Science,1999,286(5439):481-486
2Rakyan, V.，E. Whitelaw. Transgenerational epigenetic inheritance. Curr Biol,2003,13(1):R6
3Wu, C，JR Morris. Genes, genetics, and epigenetics: a correspondence.Science,2001,293(5532):1103-5
4Baylin, SB, JG Herman. DNA hypermethylation in tumorigenesis:epigenetics joins genetics. Trends Genet,2000,16(4):168-74
5Takai D, Jones PA. Comprehensive analysis of CpG islands in humanchromosomes21and22. Proc Natl Acad Sci USA,2002,19,99(6):3740-3745
6Adair SJ, Hogan KT. Treatment of ovarian cancer cell lines with5-aza-2'-deoxycytidine upregulates the expression of cancer-testis antigensand class I major histocompatibility complex-encoded molecules. CancerImmunol Immunother,2009,58:589-601
7De Smet C，O De Backer，I Faraoni，et al. The activation of human geneMAGE-1in tumor cells is correlated with genome-wide demethylation.Proc Natl Acad Sci USA,1996,93(14):7149-53
8Weiser TS, Ohnmacht GA, Guo ZS, et al. Induction of MAGE-3expressionin lung and esophageal cancer cells. Ann Thorac Surg,2001,71:295-301
9Liu, G.，H. Ying，G. Zeng，et al. HER-2, gp100, and MAGE-1are expressedin human glioblastoma and recognized by cytotoxic T cells. Cancer Res,2004,64(14):4980-6
10Cameron EE, Bachman KE, My h nen S, Herman JG, Baylin SB. Synergyof demethylation and histone deacetylase inhibition in the re-expression ofgenes silenced in cancer. Nat Genet.1999,21(1):103-7
11Suyama T, Ohashi H, Nagai H, et al. The MAGE-A1gene expression is notdetermined solely by methylation status of the promoter region inhematological malignancies.Leuk Res,2002,26:1113-8
12Bert T, Lubomierski N, Gangsauge S, et al. Expression spectrum andmethylation-dependent regulation of melanoma antigen-encoding genefamily members in pancreatic cancer cells. Pancreatology,2002,2:146-54
13Karpf AR, Lasek AW, Ririe TO, et al. Limited gene activation in tumor andnormal epithelial cells treated with the DNA methyltransferase inhibitor5-aza-2'-deoxycytidine. Mol Pharmacol,2004,65:18-27
14Pereira, CM，CC Gomes，J De Fatima Correia Silva，et al. Evaluation ofMAGE A1in oral squamous cell carcinoma. Oncol Rep,2012,27(6):1843-8
15Sewack G F, Elis T W, Hansen U. Binding of TATA binding Protein to anaturally positioned nucleosome is facilitated by histoneacetylation[J]·Mol Cell Biol,2001,21:1404-1415
16Lattal, K.M.，R.M. Barrett，M.A. Wood. Systemic or intrahippocampaldelivery of histone deacetylase inhibitors facilitates fear extinction. BehavNeurosci,2007,121(5):1125-31
17Mai, A，A Perrone，A Nebbioso，et al. Novel uracil-based2-aminoanilideand2-aminoanilide-like derivatives: histone deacetylase inhibition andin-cell activities. Bioorg Med Chem Lett，2008，18(8):2530-5
18Wang, G.G.，C.D. Allis，P. Chi. Chromatin remodeling and cancer, Part II:ATP-dependent chromatin remodeling. Trends Mol Med,2007,13(9):373-80
19Mahlknecht U, Hoelzer D. Histone acetylation modifiers in thepathogenesis of malignant disease[J]. Mol Med,2000,6(8):623-644
20Hou SY, Sang MX, Geng CZ, et al.Expressions of MAGE-A9andMAGE-A11in Breast Cancer and Their Mechanism of Expression. ArchMed Res,2014,45(1):44-51
21De Smet C, Lurquin C, Lethé B, et al. DNA methylation is the primarysilencing mechanism for a set of germ line-and tumor-specific genes witha CpG-rich promoter. Mol Cell Biol,1999,19(11):7327-7335
22Schrump DS, Fischette MR, Nguyen DM, et al. Phase I study ofdecitabine-mediated gene expression in patients with cancers involving thelungs, esophagus, or pleura. Clin Cancer Res,2006,12:5777-5785
23Bestor TH. Gene silencing. Methylation meets acetylation. Nature,1998,393(6683):311-312
24Billard LM, Magdinier F, Lenoir GM, et al. MeCP2and MBD2expressionduring normal and pathological growth of the human mammary gland.Oncogene,2002,21(17):2704-2712
25Picard V, Bergeron A, Larue H, et al. MAGE-A9mRNA and proteinexpression in bladder cancer. Int J Cancer,2007,120(10):2170-2177
26Wischnewski F, Pantel K, Schwarzenbach H. Promoter demethylation andhistone acetylation mediate gene expression of MAGE-A1,-A2,-A3,and-A12in human cancer cells. Mol Cancer Res,2006,4:339-349
27Donadelli M, Costanzo C, Faggioli L, et al. Trichostatin A, an inhibitor ofhistone deacetylases, strongly suppresses growth of pancreaticadenocarcinoma cells. Mol Carcinog,2003,38(2):59-69
28Drummond DC, Noble CO, Kirpotin DB, et al. Clinical development ofhistone deacetylase inhibitors as anticancer agents. Annu Rev PharmacolToxicol,2005,45:495-528
29Sang M, Wang L, Ding C, et al. Melanoma-associated antigen genes-anupdate. Cancer Lett2011,302:85-90
30Kim KH, Choi JS, Kim IJ, Ku JL and Park JG: Promoter hypomethylationand reactivation of MAGE-A1and MAGE-A3genes in colorectal cancercell lines and cancer tissues. World J Gastroenterol12(35):5651-5657,2006
1Van der Bruggen P,Travbersari C, Chomez P, etal. A gene encoding anantigen recognized by cytolytic T lymphocytes on a human melanoma[J].Science,1991,254(5038):1643-1647
2Ohman Forslund K, Nordqvist K. The melanoma antigen genes-any clues totheir functions in normal tissues? Exp Cell Res,2001,265(2):185-194
3马鸣，俞莉章.肿瘤特异性抗原MAGE基因的表达及意义.国外医学泌尿系统分册，2003,23:526-529
4Simpson AJ, Caballero OL, Jungbluth A, et al. Cancer/testis antigens,gametogenesis and cancer. Nat Rev Cancer,2005,5(8):615-625
5Chomez P, De Backer O, Bertrand M, et al. An overview of the MAGE genefamily with the identification of all human members of the family. CancerRes,2001,61(14):5544-5551
6De Plaen E, Arden K, Traversari C, et al. Structure, chromosomallocalization, and expression of12genes of the MAGE family.Immunogenetics,1994,40(5):360-369
7Kirkin AF, Dzhandzhugazyan KN, Zeuthen J. Cancer/testis antigens:structural and immunobiological properties. Cancer Invest,2002,20(2):222-236
8Salehi AH, Roux PP, Kubu CJ, et al. NRAGE, a novel MAGE protein,interacts with the p75neurotrophin receptor and facilitates nerve growthfactor-dependent apoptosis. Neuron,2000,27(2):279-288
9付伟，陈广生.黑色素瘤抗原家族.国际肿瘤学杂志，2006,33:173-175
10Brasseur F, Rimoldi D, Liénard D, et al. Expression of MAGE genes inprimary and metastatic cutaneous melanoma. Int J Cancer1995,63:375-80
11Otte M, Zafrakas M, Riethdorf L, et al. MAGE-A gene expression patternin primary breast cancer. Cancer Res,2001,61(18):6682-6687
12Chen H, Cai S, Wang Y, et al. Expression of the MAGE-1gene in humanhepatocellular carcinomas. Clin Med J (Engl),2000,113(12):1112-1118
13Bergeron A, Picard V, LaRue H, et al. High frequency of MAGE-A4andMAGE-A9expression in high-risk bladder cancer. Int J Cancer,2009,125(6):1365-1371
14Peikert T, Specks U, Farver C, et al. Melanoma antigen A4is expressed innon-small cell lung cancers and promotes apoptosis. Cancer Res,2006,66(9):4693-4700
15Kim KH, Choi JS, Kim IJ, et al. Promoter hypomethylation andreactivation of MAGE-A1and MAGE-A3genes in colorectal cancer celllines and cancer tissues. World J Gastroenterol,2006,12(35):5651-5657
16Liu J, Wang G, Okutomi T, et al. Expression of MAGE-A1and MAGE-A3genes in human salivary gland carcinomas. Clin Med J(Engl),2003,116(6):897-900
17Napoletano C, Bellati F, Tarquini E, et al. MAGE-A and NY-ESO-1expression in cervical cancer: prognostic factors and effects ofchemotherapy. Am J Obstat Gynecol,2008,198(1):99.e1-7
18Yakirevich E, Sabo E, Lavie O, et al. Expression of the MAGE-A4andNY-ESO-1cancer-testis antigens in serous ovarian neoplasms. Clin CancerRes,2003,9(17):6453-6460
19Chambost H, Van Baren N, Brasseur F, et al. Expression of geneMAGE-A4in Reed-Sternberg cells. Blood,2000,95(11):3530-3533
20Kim YD, Park HR, Song MH, et al. Pattern of cancer/testis antigenexpression in lung cancer patients. Int J Mol Med,2012,29(4):656-62
21Sang M, Wang L, Ding C, et al. Melanoma-associated antigen genes-anupdate. Cancer Lett,2011,302(2):85-90
22Atanackovic D, Blum I, Cao Y, et al. Expression of cancer-testis antigens aspossible targets for antigen-specific immunotherapy in head and necksquamous cell carcinoma. Cancer Biol Ther,2006,5(9):1218-1225
23Lucas S, De Smet C, Arden KC, et al. Identification of a new MAGE genewith tumor-specific expression by representational difference analysis.Cancer Res,1998,58(4):743-752
24Rimoldi D, Salvi S, Schultz-Thater E,et al. Anti-MAGE-3antibody57Band anti-MAGE-1antibody6C1can be used to study different proteins ofthe MAGE-A family. Int J Cancer,2000,86(5):749-751
25Sang M, Lian Y, Zhou X, et al. MAGE-A family: Attractive targets forcancer immunotherapy [J]. Vaccine,2011,29(47):8496-8500
26Lian Y, Sang M, Ding C, et al. Expressions of MAGE-A10andMAGE-A11in breast cancers and their prognostic significance: aretrospective clinical study. J Cancer Res Clin Oncol,2012,138(3):519-527
27Montoro JR, Mamede RC, Neder Serafini L, et al. Expression ofcancer-testis antigens MAGE-A4and MAGE-C1in oral squamous cellcarcinoma. Head Neck,2012,34(8):1123-1128
28Picard V, Bergeron A, Larue H, et al. MAGE-A9mRNA and proteinexpression in bladder cancer. Int J Cancer,2007,120:2170-2177
29Kariyama K, Higashi T, Kobayashi Y, et al. Expression of MAGE-1and-3genes and gene products in human hepatocellular carcinoma. Br J Cancer,1999,81(6):1080-1087
30桑梅香，单保恩，耿翠芝,等.人黑素瘤抗原MAGE-A4对p53转录活性的影响[J].肿瘤，2009，29(5)：428-432
31Atanackovic D, Luetkens T, Hildebrandt Y, et al. Longitudinal analysis andprognostic effect of cancer-testis antigen expression in multiple myeloma.Clin Cancer Res,2009,15(4):1343-1352
32Perez D, Hauswirth F, J ger D, et al. Protein expression of cancer testisantigens predicts tumor recurrence and treatment response to imatinib ingastrointestinal stromal tumors. Int J Cancer,2011,128(12):2947-2952
33Riener MO, Wild PJ, Soll C, et al. Frequent expression of the novel cancertestis antigen MAGE-C2/CT-10in hepatocellular carcinoma. Int J Cancer,2009,124(2):352-357
34Lian, Y.，M. Sang，C. Ding，et al. Expressions of MAGE-A10andMAGE-A11in breast cancers and their prognostic significance: aretrospective clinical study. J Cancer Res Clin Oncol,2012,138(3):519-527
35Yanagawa N, Tamura G, Oizumi H, et al. MAGE expressions mediated bydemethylation of MAGE promoters induce progression of non-small celllung cancer.Anticancer Res,2011,31(1):171-175
36Kufer P, Zippelius A, Lutterbüse R, et al. Heterogeneous expression ofMAGE-A genes in occult disseminated tumor cells: a novel multimarkerreverse transcription-polymerase chain reaction for diagnosis ofmicrometastatic disease. Cancer Res,2002,62(1):251-261
37Krüger S, Ola V, Feller AC, et al. Expression of cancer-testis antigen ct7(mage-c1) in breast cancer: an immunohistochemical study with emphasison prognostic utility. Pathol Oncol Res,2007;13:91-96
38Prasad ML, Jungbluth AA, Patel SG, et al. Expression and significance ofcancer testis antigens in primary mucosal melanoma of the head and neck.Head Neck,2004,26(12):1053-1057
39WolffeA P, MatzkeM A. Epigenetics: regulation through repression.Science,1999,286(5439):481-486
40Weber J, Salgaller M, Samid D, et al. Expression of the MAGE-1tumorantigen is up-regulated by the demethylating agent5-aza-2'-deoxycytidine.Cancer Res,1994,54(7):1766-1771
41Weiser TS, Ohnmacht GA, Guo ZS, et al. Induction of MAGE-3expressionin lung and esophageal cancer cells. Ann Thorac Surg,2001,71(1):295-301
42De Smet C, Martelange V, Lucas S, et al. Identification of humantestis-specific transcripts and analysis of their expression in tumor cells.Biochem Biophys Res Commun,1997,29,241(3):653-657
43De Smet C, Lurquin C, Lethé B, et al. DNA methylation is the primarysilencing mechanism for a set of germ line-and tumor-specific genes witha CpG-rich promoter. Mol Cell Biol,1999,19(11):7327-7335
44Schrump DS, Fischette MR, Nguyen DM, et al. Phase I study ofdecitabine-mediated gene expression in patients with cancers involving thelungs, esophagus, or pleura. Clin Cancer Res,2006,12:5777-5785
45Suyama T, Ohashi H, Nagai H, et al. The MAGE-A1gene expression is notdetermined solely by methylation status of the promoter region inhematological malignancies. Leuk Res,2002,26:1113-1118
46Bert T, Lubomierski N, Gangsauge S, et al. Expression spectrum andmethylation-dependent regulation of melanoma antigen-encoding genefamily members in pancreatic cancer cells. Pancreatology,2002,2:146-154
47Karpf AR, Lasek AW, Ririe TO, et al. Limited gene activation in tumor andnormal epithelial cells treated with the DNA methyltransferase inhibitor5-aza-2-deoxycytidine. Mol Pharmacol,2004,65:18-27
48Takai D, Jones PA. Comprehensive analysis of CpG islands in humanchromosomes21and22. Proc Natl Acad Sci USA,2002,19,99(6):3740-3745
49Bestor TH. Gene silencing. Methylation meets acetylation. Nature,1998,393(6683):311-312
50Billard LM, Magdinier F, Lenoir GM, et al. MeCP2and MBD2expressionduring normal and pathological growth of the human mammary gland.Oncogene,2002,21(17):2704-2712
51Wischnewski F, Pantel K, Schwarzenbach H. Promoter demethylation andhistone acetylation mediate gene expression of MAGE-A1,-A2,-A3,and-A12in human cancer cells. Mol Cancer Res,2006,4:339-349
52Sang M, Wang L, Ding C, et al. Melanoma-associated antigen genes-anupdate. Cancer Lett,2011,302(2):85-90
53Pascolo S, Schirle M, Guckel B, et al. A MAGE-A1HLA-A A*0201epitope identified by mass spectrometry. Cancer Res,2001,61(10):4072-4077
54Chinnasamy N, Wargo JA, Yu Z, et al. A TCR targeting theHLA-A*0201-restricted epitope of MAGE-A3recognizes multipleepitopes of the MAGE-A antigen superfamily in several types of cancer[J].J Immunol,2011,186(2):685-696
55Zhang Y, Stroobant V, Russo V, et al. A MAGE-A4peptide presented byHLA-B37is recognized on human tumors by cytolytic T lymphocytes.Tissue Antigens,2002,60(5):365-371
56Jia ZC, Tian Y, Huang ZM, et al. Identification of a new MAGE-A10antigenic peptide presented by HLA-A*0201on tumor cells [J]. CancerBiol Ther,2011,11(4):395-400
57Heidecker L, Brasseur F, Probst-Kepper M, et al. Cytolytic T lymphocytesraised against a human bladder carcinoma recognize an antigen encodedby gene MAGE-A12. J Immunol,2000,164(11):6041-6045
58Scanlan MJ, Gure AO, Jungbluth AA, et al. Cancer/testis antigens: anexpanding family of targets for cancer immunotherapy. Immunol Rev,2002,188:22-32
59Marchand M, Van Baren N, Weynants P, et al. Tumor regressions observedin patients with metastatic melanoma treated with an antigenic peptideencoded by gene MAGE-3and presented by HLA-A1[J]. Int J Cancer,1999,80(2):219-230
60Sadanaga N, Nagashima H, Mashino K, et al. Dendritic cell vaccinationwith MAGE peptide is a novel therapeutic approach for gastrointestinalcarcinomas. Clin Cancer Res,2001,7(8):2277-2284
61Russo V, Tanzarella S, Dalerba P, et al. Dendritic cells acquire the MAGE-3human tumor antigen from apoptotic cells and induce a class I-restricted Tcell response. Proc Natl Acad Sci USA,2000,97(5):2185-2190
62Goodyear O, Agathanggelou A, Novitzky-Basso I, et al. Induction of aCD8+T-cell response to the MAGE cancer testis antigen by combinedtreatment with azacitidine and sodium valproate in patients with acutemyeloid leukemia and myelodysplasia. Blood,2010,16;116(11):1908-1918
63江继发，魏海明.检测肿瘤转移指标的临床意义.国外医学肿瘤学分册,1998,25(2):82-84
64Park JW, Kwon TK, Kim IH, et al. A new strategy for the diagnosis ofMAGE-expressing cancers [J]. J Immunol Methods,2002,266(1-2):79-86
65Kufer P, Zippelius A, Lutterbüse R, et al. Heterogeneous expression ofMAGE-A genes in occult disseminated tumor cells: a novel multimarkerreverse transcription-polymerase chain reaction for diagnosis ofmicrometastatic disease. Cancer Res,2002,62(1):251-61
66Sienel W, Varwerk C, Linder A, et al. Melanoma associated antigen(MAGE)-A3expression in Stages I and II non-small cell lung cancer:results of a multi-center study. Eur J Cardiothorac Surg,2004,25(1):131-
2Scanlan, MJ，AO Gure，AA Jungbluth，et al. Cancer/testis antigens: anexpanding family of targets for cancer immunotherapy. Immunol Rev，2002，188:22-32
3Zendman, AJ，DJ Ruiter，GN Van Muijen. Cancer/testis-associated genes:identification, expression profile, and putative function. J Cell Physiol，2003，194(3):272-88
4Simpson, AJ，OL Caballero，A Jungbluth，et al. Cancer/testis antigens,gametogenesis and cancer. Nat Rev Cancer，2005，5(8):615-25
5Meek DW, Marcar L. MAGE-A antigens as targets in tumour therapy.Cancer Lett,2012,324:126-132
6Rogner, U.C.，K. Wilke，E. Steck，et al. The melanoma antigen gene(MAGE) family is clustered in the chromosomal band Xq28.Genomics，1995，29(3):725-31
7De Plaen E, Arden K, Traversari C, et al. Structure, chromosomallocalization, and expression of12genes of the MAGE family.Immunogenetics,1994,40(5):360-369
8Sang M, Lian Y, Zhou X, et al. MAGE-A family: attractive targets forcancer immunotherapy. Vaccine,2011,29(47):8496-8500
9Van der Bruggen P,Travbersari C, Chomez P, etal. A gene encoding anantigen recognized by cytolytic T lymphocytes on a human melanoma[J].Science,1991,254(5038):1643-1647
10Ohman Forslund K, Nordqvist K. The melanoma antigen genes-any cluesto their functions in normal tissues? Exp Cell Res,2001,265(2):185-194
11马鸣，俞莉章.肿瘤特异性抗原MAGE基因的表达及意义.国外医学泌尿系统分册，2003,23:526-529
12Simpson AJ, Caballero OL, Jungbluth A, et al. Cancer/testis antigens,gametogenesis and cancer. Nat Rev Cancer,2005,5(8):615-625
13Chomez P, De Backer O, Bertrand M, et al. An overview of the MAGEgene family with the identification of all human members of the family.Cancer Res,2001,61(14):5544-5551
14Brasseur F, Rimoldi D, Liénard D, et al. Expression of MAGE genes inprimary and metastatic cutaneous melanoma. Int J Cancer1995,63:375-80
15Kufer P, Zippelius A, Lutterbuse R, et al. Heterogeneous expression ofMAGE-A genes in occult disseminated tumor cells: a novel multimarkerreverse transcription-polymerase chain reaction for diagnosis ofmicrometastatic disease. Cancer Res,2002,62(1):251-261
16Chen H, Cai S, Wang Y, et al. Expression of the MAGE-1gene in humanhepatocellular carcinomas. Clin Med J (Engl),2000,113(12):1112-1118
17Bergeron A, Picard V, LaRue H, et al. High frequency of MAGE-A4andMAGE-A9expression in high-risk bladder cancer. Int J Cancer,2009,125(6):1365-1371
18Peikert T, Specks U, Farver C, et al. Melanoma antigen A4is expressed innon-small cell lung cancers and promotes apoptosis. Cancer Res,2006,66(9):4693-4700
19Kim KH, Choi JS, Kim IJ, et al. Promoter hypomethylation andreactivation of MAGE-A1and MAGE-A3genes in colorectal cancer celllines and cancer tissues. World J Gastroenterol,2006,12(35):5651-5657
20Napoletano C, Bellati F, Tarquini E, et al. MAGE-A and NY-ESO-1expression in cervical cancer: prognostic factors and effects ofchemotherapy. Am J Obstat Gynecol,2008,198(1):99.e1-7
21Yakirevich E, Sabo E, Lavie O, et al. Expression of the MAGE-A4andNY-ESO-1cancer-testis antigens in serous ovarian neoplasms. Clin CancerRes,2003,9(17):6453-6460
22Yan L, Yang X, Davidson NE. Role of DNA methylation and histoneacetylation in steroid receptor expression in breast cancer. J MammaryGland Biol Neoplasia,2001,6(2):183-192
23Ross JS, Fletcher JA, Bloom KJ, et al. Targeted therapy in breast cancer:the HER-2/neu gene and protein. Mol Cell Proteomics,2004,3(4):379-398
24Yeon CH, Pegram MD. Anti-erbB-2antibody trastuzumab in the treatmentof HER2-amplified breast cancer. Invest New Drugs,2005,23(5):391-409
25Rabindran SK. Antitumor activity of HER-2inhibitors. Cancer Letters,2005,227(1):9-23
26Otte M, Zafrakas M, Riethdorf L, et al. MAGE-A gene expression patternin primary breast cancer. Cancer Res,2001,61(18):6682-6687
27Karn T, Pusztai L, Ruckhaberle E, et al. Melanoma antigen family Aidentified by the bimodality index defines a subset of triple negative breastcancers as candidates for immune response augmentation. Eur J Cancer,2012,48(1):12-23
1Early Breast Cancer Trialists’ Collaborative Group. Polychemotherapy forearly breast cancer: an overview of the randomized trials. Lancet,1998,352:930-942
2Early Breast Cancer Trialists’ Collaborative Group: Tamoxifen for earlybreast cancer: an overview of the randomised trials. Lancet,1998,351:1451-1467
3Filho PA, Lopez-Albaitero A, Xi L, Gooding W, Godfrey T, Ferris RL.Quantitative expression and immunogenicity of MAGE-3and-6in upperaerodigestive tract cancer. Int J Cancer2009;125:1912-1920
4Ma W, Germeau C, Vigneron N, Maernoudt AS, Morel S, Boon T,Coulie PG,Van den Eynde BJ. Two new tumor-specific antigenic peptides encoded bygene MAGE-C2and presented to cytolytic T lymphocytes by HLA-A2. IntJ Cancer2004;109:698-702
5Godelaine D, Carrasco J, Brasseur F, Neyns B, Thielemans K, Boon T, VanPel A. A new tumor-specific antigen encoded by MAGE-C2and presentedto cytolytic T lymphocytes by HLA-B44. Cancer Immunol Immunother2007;56:753-9
6Simpson AJ, Caballero OL, Jungbluth A, et al. Cancer/testis antigens,gametogenesis and cancer. Nat Rev Cancer,2005,5(8):615-625
7Chomez P, De Backer O, Bertrand M, et al. An overview of the MAGE genefamily with the identification of all human members of the family. CancerRes,2001,61(14):5544-5551
8De Plaen E, Arden K, Traversari C, et al. Structure, chromosomallocalization, and expression of12genes of the MAGE family.Immunogenetics,1994,40(5):360-369
9Kirkin AF, Dzhandzhugazyan KN, Zeuthen J. Cancer/testis antigens:structural and immunobiological properties. Cancer Invest,2002,20(2):222-236
10Zhuang R, Zhu Y, Fang L, et al. Generation of monoclonal antibodies tocancer/testis (CT) antigen CT10/MAGE-C2. Cancer Immun,20066:7
11Güre AO, Stockert E, Arden KC et al. CT10: a new cancer cancer-testis(CT) antigen homologous to CT7and the MAGE family, identified byrepresentational-difference analysis. Int J Cancer2000,85(5):726-732
12de Carvalho F, Alves VL, Braga WM, et al. MAGE-C1/CT7andMAGE-C2/CT10are frequently expressed in multiple myeloma and canbe explored in combined immunotherapy for this malignancy. CancerImmunol Immunother,2013,62(1):191-5
13Gure AO, Chua R, Williamson B, et al. Cancer-testis genes are coordinatelyexpressed and are markers of poor outcome in non-small cell lung cancer.Clin Cancer Res,2005,11(22):8055-62
14von Boehmer L, Keller L, Mortezavi A, et al. MAGE-C2/CT10proteinexpression is an independent predictor of recurrence in prostate cancer.PLoS One,2011,6(7):e21366
15Figueiredo DL, Mamede RC, Spagnoli GC, et al. High expression ofcancer testis antigens MAGE-A, MAGE-C1/CT7, MAGE-C2/CT10,NY-ESO-1, and gage in advanced squamous cell carcinoma of the larynx.Head Neck,2011,33(5):702-7
16Peng JR, Chen HS, Mou DC, et al. Expression of cancer/testis (CT)antigens in Chinese hepatocellular carcinoma and its correlation withclinical parameters. Cancer Lett,2005,219(2):223-32
17Chitale DA, Jungbluth AA, Marshall DS, et al. Expression of cancer-testisantigens in endometrial carcinomas using a tissue microarray. Mod Pathol,2005,18:119-126
18Zimmermann AK, Imig J, Klar A, et al. Expression of MAGE-C1/CT7andselected cancer/testis antigens in ovarian borderline tumours and primaryand recurrent ovarian carcinomas.Virchows Arch,2013,462(5):565-74
19Zendman AJ, Ruiter DJ, Van Muijen GN. Cancer/testis-associated genes:identification, expression profile, and putative function. J Cell Physiol,2003,194:272-288
20Caballero OL, Chen YT. Cancer/testis (CT) antigens: potential targets forimmunotherapy. Cancer Sci,2009,100:2014-2021
21Brasseur F, Rimoldi D, Liénard D, et al. Expression of MAGE genes inprimary and metastatic cutaneous melanoma. Int J Cancer1995,63:375-80
22Kufer P, Zippelius A, Lutterbuse R, et al. Heterogeneous expression ofMAGE-A genes in occult disseminated tumor cells: a novel multimarkerreverse transcription-polymerase chain reaction for diagnosis ofmicrometastatic disease. Cancer Res,2002,62(1):251-261
23Chen H, Cai S, Wang Y, et al. Expression of the MAGE-1gene in humanhepatocellular carcinomas. Clin Med J (Engl),2000,113(12):1112-1118
24Peikert T, Specks U, Farver C, et al. Melanoma antigen A4is expressed innon-small cell lung cancers and promotes apoptosis. Cancer Res,2006,66(9):4693-4700
25Kim KH, Choi JS, Kim IJ, et al. Promoter hypomethylation andreactivation of MAGE-A1and MAGE-A3genes in colorectal cancer celllines and cancer tissues. World J Gastroenterol,2006,12(35):5651-5657
26Napoletano C, Bellati F, Tarquini E, et al. MAGE-A and NY-ESO-1expression in cervical cancer: prognostic factors and effects ofchemotherapy. Am J Obstat Gynecol,2008,198(1):99.e1-7
27Bergeron A, Picard V, LaRue H, et al. High frequency of MAGE-A4andMAGE-A9expression in high-risk bladder cancer. Int J Cancer,2009,125(6):1365-1371
28Yakirevich E, Sabo E, Lavie O, et al. Expression of the MAGE-A4andNY-ESO-1cancer-testis antigens in serous ovarian neoplasms. Clin CancerRes,2003,9(17):6453-6460
29Moureau-Zabotto L, Ricci S, Lefranc JP, et al. Prognostic impact ofmultidrug resistance gene expression on the management of breast cancerin the context of adjuvant therapy based on a series of171patients. Br JCancer,2006,94(4):473-80
30Chitale DA, Jungbluth AA, Marshall DS, et al. Expression of cancer-testisantigens in endometrial carcinomas using a tissue microarray. Mod Pathol,2005,18:119-126
31Zimmermann AK, Imig J, Klar A, et al. Expression of MAGE-C1/CT7andselected cancer/testis antigens in ovarian borderline tumours and primaryand recurrent ovarian carcinomas.Virchows Arch,2013,462(5):565-74
32Krüger S, Ola V, Feller AC, et al. Expression of cancer-testis antigen ct7(mage-c1)in breast cancer: an immunohistochemical study with emphasison prognostic utility. Pathol Oncol Res,2007,13:91-96
33Prasad ML, Jungbluth AA, Patel SG, et al. Expression and significance ofcancer testis antigens in primary mucosal melanoma of the head and neck.Head Neck,2004,26(12):1053-7
34Atanackovic D, Luetkens T, Hildebrandt Y, et al. Longitudinal analysis andprognostic effect of cancer-testis antigen expression in multiple myeloma.Clin Cancer Res,2009,15(4):1343-52
35Perez D, Hauswirth F, J ger D, et al. Protein expression of cancer testisantigens predicts tumor recurrence and treatment response to imatinib ingastrointestinal stromal tumors. Int J Cancer,2011,128(12):2947-52
36Riener MO, Wild PJ, Soll C, et al. Frequent expression of the novel cancertestis antigen MAGE-C2/CT-10in hepatocellular carcinoma. Int J Cancer,2009,124(2):352-7
37Montoro JR, Mamede RC, Neder Serafini L, et al. Expression ofcancer-testis antigens MAGE-A4and MAGE-C1in oral squamous cellcarcinoma. Head Neck,2012,34(8):1123-8
1WolffeA P, MatzkeM A. Epigenetics: regulation through repression. Science,1999,286(5439):481-486
2Rakyan, V.，E. Whitelaw. Transgenerational epigenetic inheritance. Curr Biol,2003,13(1):R6
3Wu, C，JR Morris. Genes, genetics, and epigenetics: a correspondence.Science,2001,293(5532):1103-5
4Baylin, SB, JG Herman. DNA hypermethylation in tumorigenesis:epigenetics joins genetics. Trends Genet,2000,16(4):168-74
5Takai D, Jones PA. Comprehensive analysis of CpG islands in humanchromosomes21and22. Proc Natl Acad Sci USA,2002,19,99(6):3740-3745
6Adair SJ, Hogan KT. Treatment of ovarian cancer cell lines with5-aza-2'-deoxycytidine upregulates the expression of cancer-testis antigensand class I major histocompatibility complex-encoded molecules. CancerImmunol Immunother,2009,58:589-601
7De Smet C，O De Backer，I Faraoni，et al. The activation of human geneMAGE-1in tumor cells is correlated with genome-wide demethylation.Proc Natl Acad Sci USA,1996,93(14):7149-53
8Weiser TS, Ohnmacht GA, Guo ZS, et al. Induction of MAGE-3expressionin lung and esophageal cancer cells. Ann Thorac Surg,2001,71:295-301
9Liu, G.，H. Ying，G. Zeng，et al. HER-2, gp100, and MAGE-1are expressedin human glioblastoma and recognized by cytotoxic T cells. Cancer Res,2004,64(14):4980-6
10Cameron EE, Bachman KE, My h nen S, Herman JG, Baylin SB. Synergyof demethylation and histone deacetylase inhibition in the re-expression ofgenes silenced in cancer. Nat Genet.1999,21(1):103-7
11Suyama T, Ohashi H, Nagai H, et al. The MAGE-A1gene expression is notdetermined solely by methylation status of the promoter region inhematological malignancies.Leuk Res,2002,26:1113-8
12Bert T, Lubomierski N, Gangsauge S, et al. Expression spectrum andmethylation-dependent regulation of melanoma antigen-encoding genefamily members in pancreatic cancer cells. Pancreatology,2002,2:146-54
13Karpf AR, Lasek AW, Ririe TO, et al. Limited gene activation in tumor andnormal epithelial cells treated with the DNA methyltransferase inhibitor5-aza-2'-deoxycytidine. Mol Pharmacol,2004,65:18-27
14Pereira, CM，CC Gomes，J De Fatima Correia Silva，et al. Evaluation ofMAGE A1in oral squamous cell carcinoma. Oncol Rep,2012,27(6):1843-8
15Sewack G F, Elis T W, Hansen U. Binding of TATA binding Protein to anaturally positioned nucleosome is facilitated by histoneacetylation[J]·Mol Cell Biol,2001,21:1404-1415
16Lattal, K.M.，R.M. Barrett，M.A. Wood. Systemic or intrahippocampaldelivery of histone deacetylase inhibitors facilitates fear extinction. BehavNeurosci,2007,121(5):1125-31
17Mai, A，A Perrone，A Nebbioso，et al. Novel uracil-based2-aminoanilideand2-aminoanilide-like derivatives: histone deacetylase inhibition andin-cell activities. Bioorg Med Chem Lett，2008，18(8):2530-5
18Wang, G.G.，C.D. Allis，P. Chi. Chromatin remodeling and cancer, Part II:ATP-dependent chromatin remodeling. Trends Mol Med,2007,13(9):373-80
19Mahlknecht U, Hoelzer D. Histone acetylation modifiers in thepathogenesis of malignant disease[J]. Mol Med,2000,6(8):623-644
20Hou SY, Sang MX, Geng CZ, et al.Expressions of MAGE-A9andMAGE-A11in Breast Cancer and Their Mechanism of Expression. ArchMed Res,2014,45(1):44-51
21De Smet C, Lurquin C, Lethé B, et al. DNA methylation is the primarysilencing mechanism for a set of germ line-and tumor-specific genes witha CpG-rich promoter. Mol Cell Biol,1999,19(11):7327-7335
22Schrump DS, Fischette MR, Nguyen DM, et al. Phase I study ofdecitabine-mediated gene expression in patients with cancers involving thelungs, esophagus, or pleura. Clin Cancer Res,2006,12:5777-5785
23Bestor TH. Gene silencing. Methylation meets acetylation. Nature,1998,393(6683):311-312
24Billard LM, Magdinier F, Lenoir GM, et al. MeCP2and MBD2expressionduring normal and pathological growth of the human mammary gland.Oncogene,2002,21(17):2704-2712
25Picard V, Bergeron A, Larue H, et al. MAGE-A9mRNA and proteinexpression in bladder cancer. Int J Cancer,2007,120(10):2170-2177
26Wischnewski F, Pantel K, Schwarzenbach H. Promoter demethylation andhistone acetylation mediate gene expression of MAGE-A1,-A2,-A3,and-A12in human cancer cells. Mol Cancer Res,2006,4:339-349
27Donadelli M, Costanzo C, Faggioli L, et al. Trichostatin A, an inhibitor ofhistone deacetylases, strongly suppresses growth of pancreaticadenocarcinoma cells. Mol Carcinog,2003,38(2):59-69
28Drummond DC, Noble CO, Kirpotin DB, et al. Clinical development ofhistone deacetylase inhibitors as anticancer agents. Annu Rev PharmacolToxicol,2005,45:495-528
29Sang M, Wang L, Ding C, et al. Melanoma-associated antigen genes-anupdate. Cancer Lett2011,302:85-90
30Kim KH, Choi JS, Kim IJ, Ku JL and Park JG: Promoter hypomethylationand reactivation of MAGE-A1and MAGE-A3genes in colorectal cancercell lines and cancer tissues. World J Gastroenterol12(35):5651-5657,2006
1Van der Bruggen P,Travbersari C, Chomez P, etal. A gene encoding anantigen recognized by cytolytic T lymphocytes on a human melanoma[J].Science,1991,254(5038):1643-1647
2Ohman Forslund K, Nordqvist K. The melanoma antigen genes-any clues totheir functions in normal tissues? Exp Cell Res,2001,265(2):185-194
3马鸣，俞莉章.肿瘤特异性抗原MAGE基因的表达及意义.国外医学泌尿系统分册，2003,23:526-529
4Simpson AJ, Caballero OL, Jungbluth A, et al. Cancer/testis antigens,gametogenesis and cancer. Nat Rev Cancer,2005,5(8):615-625
5Chomez P, De Backer O, Bertrand M, et al. An overview of the MAGE genefamily with the identification of all human members of the family. CancerRes,2001,61(14):5544-5551
6De Plaen E, Arden K, Traversari C, et al. Structure, chromosomallocalization, and expression of12genes of the MAGE family.Immunogenetics,1994,40(5):360-369
7Kirkin AF, Dzhandzhugazyan KN, Zeuthen J. Cancer/testis antigens:structural and immunobiological properties. Cancer Invest,2002,20(2):222-236
8Salehi AH, Roux PP, Kubu CJ, et al. NRAGE, a novel MAGE protein,interacts with the p75neurotrophin receptor and facilitates nerve growthfactor-dependent apoptosis. Neuron,2000,27(2):279-288
9付伟，陈广生.黑色素瘤抗原家族.国际肿瘤学杂志，2006,33:173-175
10Brasseur F, Rimoldi D, Liénard D, et al. Expression of MAGE genes inprimary and metastatic cutaneous melanoma. Int J Cancer1995,63:375-80
11Otte M, Zafrakas M, Riethdorf L, et al. MAGE-A gene expression patternin primary breast cancer. Cancer Res,2001,61(18):6682-6687
12Chen H, Cai S, Wang Y, et al. Expression of the MAGE-1gene in humanhepatocellular carcinomas. Clin Med J (Engl),2000,113(12):1112-1118
13Bergeron A, Picard V, LaRue H, et al. High frequency of MAGE-A4andMAGE-A9expression in high-risk bladder cancer. Int J Cancer,2009,125(6):1365-1371
14Peikert T, Specks U, Farver C, et al. Melanoma antigen A4is expressed innon-small cell lung cancers and promotes apoptosis. Cancer Res,2006,66(9):4693-4700
15Kim KH, Choi JS, Kim IJ, et al. Promoter hypomethylation andreactivation of MAGE-A1and MAGE-A3genes in colorectal cancer celllines and cancer tissues. World J Gastroenterol,2006,12(35):5651-5657
16Liu J, Wang G, Okutomi T, et al. Expression of MAGE-A1and MAGE-A3genes in human salivary gland carcinomas. Clin Med J(Engl),2003,116(6):897-900
17Napoletano C, Bellati F, Tarquini E, et al. MAGE-A and NY-ESO-1expression in cervical cancer: prognostic factors and effects ofchemotherapy. Am J Obstat Gynecol,2008,198(1):99.e1-7
18Yakirevich E, Sabo E, Lavie O, et al. Expression of the MAGE-A4andNY-ESO-1cancer-testis antigens in serous ovarian neoplasms. Clin CancerRes,2003,9(17):6453-6460
19Chambost H, Van Baren N, Brasseur F, et al. Expression of geneMAGE-A4in Reed-Sternberg cells. Blood,2000,95(11):3530-3533
20Kim YD, Park HR, Song MH, et al. Pattern of cancer/testis antigenexpression in lung cancer patients. Int J Mol Med,2012,29(4):656-62
21Sang M, Wang L, Ding C, et al. Melanoma-associated antigen genes-anupdate. Cancer Lett,2011,302(2):85-90
22Atanackovic D, Blum I, Cao Y, et al. Expression of cancer-testis antigens aspossible targets for antigen-specific immunotherapy in head and necksquamous cell carcinoma. Cancer Biol Ther,2006,5(9):1218-1225
23Lucas S, De Smet C, Arden KC, et al. Identification of a new MAGE genewith tumor-specific expression by representational difference analysis.Cancer Res,1998,58(4):743-752
24Rimoldi D, Salvi S, Schultz-Thater E,et al. Anti-MAGE-3antibody57Band anti-MAGE-1antibody6C1can be used to study different proteins ofthe MAGE-A family. Int J Cancer,2000,86(5):749-751
25Sang M, Lian Y, Zhou X, et al. MAGE-A family: Attractive targets forcancer immunotherapy [J]. Vaccine,2011,29(47):8496-8500
26Lian Y, Sang M, Ding C, et al. Expressions of MAGE-A10andMAGE-A11in breast cancers and their prognostic significance: aretrospective clinical study. J Cancer Res Clin Oncol,2012,138(3):519-527
27Montoro JR, Mamede RC, Neder Serafini L, et al. Expression ofcancer-testis antigens MAGE-A4and MAGE-C1in oral squamous cellcarcinoma. Head Neck,2012,34(8):1123-1128
28Picard V, Bergeron A, Larue H, et al. MAGE-A9mRNA and proteinexpression in bladder cancer. Int J Cancer,2007,120:2170-2177
29Kariyama K, Higashi T, Kobayashi Y, et al. Expression of MAGE-1and-3genes and gene products in human hepatocellular carcinoma. Br J Cancer,1999,81(6):1080-1087
30桑梅香，单保恩，耿翠芝,等.人黑素瘤抗原MAGE-A4对p53转录活性的影响[J].肿瘤，2009，29(5)：428-432
31Atanackovic D, Luetkens T, Hildebrandt Y, et al. Longitudinal analysis andprognostic effect of cancer-testis antigen expression in multiple myeloma.Clin Cancer Res,2009,15(4):1343-1352
32Perez D, Hauswirth F, J ger D, et al. Protein expression of cancer testisantigens predicts tumor recurrence and treatment response to imatinib ingastrointestinal stromal tumors. Int J Cancer,2011,128(12):2947-2952
33Riener MO, Wild PJ, Soll C, et al. Frequent expression of the novel cancertestis antigen MAGE-C2/CT-10in hepatocellular carcinoma. Int J Cancer,2009,124(2):352-357
34Lian, Y.，M. Sang，C. Ding，et al. Expressions of MAGE-A10andMAGE-A11in breast cancers and their prognostic significance: aretrospective clinical study. J Cancer Res Clin Oncol,2012,138(3):519-527
35Yanagawa N, Tamura G, Oizumi H, et al. MAGE expressions mediated bydemethylation of MAGE promoters induce progression of non-small celllung cancer.Anticancer Res,2011,31(1):171-175
36Kufer P, Zippelius A, Lutterbüse R, et al. Heterogeneous expression ofMAGE-A genes in occult disseminated tumor cells: a novel multimarkerreverse transcription-polymerase chain reaction for diagnosis ofmicrometastatic disease. Cancer Res,2002,62(1):251-261
37Krüger S, Ola V, Feller AC, et al. Expression of cancer-testis antigen ct7(mage-c1) in breast cancer: an immunohistochemical study with emphasison prognostic utility. Pathol Oncol Res,2007;13:91-96
38Prasad ML, Jungbluth AA, Patel SG, et al. Expression and significance ofcancer testis antigens in primary mucosal melanoma of the head and neck.Head Neck,2004,26(12):1053-1057
39WolffeA P, MatzkeM A. Epigenetics: regulation through repression.Science,1999,286(5439):481-486
40Weber J, Salgaller M, Samid D, et al. Expression of the MAGE-1tumorantigen is up-regulated by the demethylating agent5-aza-2'-deoxycytidine.Cancer Res,1994,54(7):1766-1771
41Weiser TS, Ohnmacht GA, Guo ZS, et al. Induction of MAGE-3expressionin lung and esophageal cancer cells. Ann Thorac Surg,2001,71(1):295-301
42De Smet C, Martelange V, Lucas S, et al. Identification of humantestis-specific transcripts and analysis of their expression in tumor cells.Biochem Biophys Res Commun,1997,29,241(3):653-657
43De Smet C, Lurquin C, Lethé B, et al. DNA methylation is the primarysilencing mechanism for a set of germ line-and tumor-specific genes witha CpG-rich promoter. Mol Cell Biol,1999,19(11):7327-7335
44Schrump DS, Fischette MR, Nguyen DM, et al. Phase I study ofdecitabine-mediated gene expression in patients with cancers involving thelungs, esophagus, or pleura. Clin Cancer Res,2006,12:5777-5785
45Suyama T, Ohashi H, Nagai H, et al. The MAGE-A1gene expression is notdetermined solely by methylation status of the promoter region inhematological malignancies. Leuk Res,2002,26:1113-1118
46Bert T, Lubomierski N, Gangsauge S, et al. Expression spectrum andmethylation-dependent regulation of melanoma antigen-encoding genefamily members in pancreatic cancer cells. Pancreatology,2002,2:146-154
47Karpf AR, Lasek AW, Ririe TO, et al. Limited gene activation in tumor andnormal epithelial cells treated with the DNA methyltransferase inhibitor5-aza-2-deoxycytidine. Mol Pharmacol,2004,65:18-27
48Takai D, Jones PA. Comprehensive analysis of CpG islands in humanchromosomes21and22. Proc Natl Acad Sci USA,2002,19,99(6):3740-3745
49Bestor TH. Gene silencing. Methylation meets acetylation. Nature,1998,393(6683):311-312
50Billard LM, Magdinier F, Lenoir GM, et al. MeCP2and MBD2expressionduring normal and pathological growth of the human mammary gland.Oncogene,2002,21(17):2704-2712
51Wischnewski F, Pantel K, Schwarzenbach H. Promoter demethylation andhistone acetylation mediate gene expression of MAGE-A1,-A2,-A3,and-A12in human cancer cells. Mol Cancer Res,2006,4:339-349
52Sang M, Wang L, Ding C, et al. Melanoma-associated antigen genes-anupdate. Cancer Lett,2011,302(2):85-90
53Pascolo S, Schirle M, Guckel B, et al. A MAGE-A1HLA-A A*0201epitope identified by mass spectrometry. Cancer Res,2001,61(10):4072-4077
54Chinnasamy N, Wargo JA, Yu Z, et al. A TCR targeting theHLA-A*0201-restricted epitope of MAGE-A3recognizes multipleepitopes of the MAGE-A antigen superfamily in several types of cancer[J].J Immunol,2011,186(2):685-696
55Zhang Y, Stroobant V, Russo V, et al. A MAGE-A4peptide presented byHLA-B37is recognized on human tumors by cytolytic T lymphocytes.Tissue Antigens,2002,60(5):365-371
56Jia ZC, Tian Y, Huang ZM, et al. Identification of a new MAGE-A10antigenic peptide presented by HLA-A*0201on tumor cells [J]. CancerBiol Ther,2011,11(4):395-400
57Heidecker L, Brasseur F, Probst-Kepper M, et al. Cytolytic T lymphocytesraised against a human bladder carcinoma recognize an antigen encodedby gene MAGE-A12. J Immunol,2000,164(11):6041-6045
58Scanlan MJ, Gure AO, Jungbluth AA, et al. Cancer/testis antigens: anexpanding family of targets for cancer immunotherapy. Immunol Rev,2002,188:22-32
59Marchand M, Van Baren N, Weynants P, et al. Tumor regressions observedin patients with metastatic melanoma treated with an antigenic peptideencoded by gene MAGE-3and presented by HLA-A1[J]. Int J Cancer,1999,80(2):219-230
60Sadanaga N, Nagashima H, Mashino K, et al. Dendritic cell vaccinationwith MAGE peptide is a novel therapeutic approach for gastrointestinalcarcinomas. Clin Cancer Res,2001,7(8):2277-2284
61Russo V, Tanzarella S, Dalerba P, et al. Dendritic cells acquire the MAGE-3human tumor antigen from apoptotic cells and induce a class I-restricted Tcell response. Proc Natl Acad Sci USA,2000,97(5):2185-2190
62Goodyear O, Agathanggelou A, Novitzky-Basso I, et al. Induction of aCD8+T-cell response to the MAGE cancer testis antigen by combinedtreatment with azacitidine and sodium valproate in patients with acutemyeloid leukemia and myelodysplasia. Blood,2010,16;116(11):1908-1918
63江继发，魏海明.检测肿瘤转移指标的临床意义.国外医学肿瘤学分册,1998,25(2):82-84
64Park JW, Kwon TK, Kim IH, et al. A new strategy for the diagnosis ofMAGE-expressing cancers [J]. J Immunol Methods,2002,266(1-2):79-86
65Kufer P, Zippelius A, Lutterbüse R, et al. Heterogeneous expression ofMAGE-A genes in occult disseminated tumor cells: a novel multimarkerreverse transcription-polymerase chain reaction for diagnosis ofmicrometastatic disease. Cancer Res,2002,62(1):251-61
66Sienel W, Varwerk C, Linder A, et al. Melanoma associated antigen(MAGE)-A3expression in Stages I and II non-small cell lung cancer:results of a multi-center study. Eur J Cardiothorac Surg,2004,25(1):131-
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