肿瘤抗原MAGE-A4在乳腺癌组织和细胞系中的表达及功能研究
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摘要
目前以肿瘤免疫治疗为代表的肿瘤生物治疗已经成为继手术、化疗和放疗之后的第四种肿瘤治疗模式,研究和开发新型肿瘤疫苗已经成为近年国际上肿瘤免疫治疗的热点之一。在目前已知的众多肿瘤相关抗原中,癌睾丸抗原(cancer/testis Antigen, CTA)有其特异的表达模式,即在各种肿瘤组织中有不同频率的表达,而在正常组织中仅在睾丸生殖细胞中表达,偶然在胎盘中表达。但睾丸是免疫豁免器官,不引起机体特异性免疫反应。因此,CTA适于作为特异性肿瘤免疫治疗的靶抗原。
作为CTA亚家族的黑色素瘤抗原基因(melanoma antigen gene, MAGE)是从黑色素瘤细胞中分离出来的一大家族抗原基因,其编码的肿瘤排斥抗原在细胞内经加工产生抗原肽,并与HLA-I类分子结合形成复合物,能够被自体细胞毒性T细胞识别,诱导出对相应肿瘤细胞的特异性杀伤。MAGE基因在许多肿瘤组织中有较高的表达,但在正常组织(除睾丸和胚胎外)均不表达,因此可作为CTL介导肿瘤特异性免疫治疗的理想靶分子。深入研究MAGE基因的功能将会为开展肿瘤分子学诊断和肿瘤免疫治疗的临床应用奠定良好的基础。
有研究提示,MAGE-A4可能表现了与该家族中其它成员不同的分子生物学特性,但是其具体的生物学功能和分子机制到目前还不十分清楚。本研究采用逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测了MAGE-A4 mRNA在77例乳腺癌良恶性组织及乳腺癌细胞系中的表达;构建了同时表达MAGE-A4和FLAG标签的表达载体FLAG-pcDNA3-MAGE-A4,并通过MTT分析和克隆形成实验研究了MAGE-A4过表达对MAGE-A4低表达的乳腺癌细胞增殖和凋亡的影响;采用荧光素酶报告基因分析、RT-PCR、Western-blot、克隆形成实验和TUNEL染色法检测了MAGE-A4对p53转录活性的影响;采用细胞质与细胞核分离实验检测了MAGE-A4在细胞中的定位;采用免疫荧光染色技术检测了MAGE-A4与p73蛋白在细胞中的共定位;采用免疫共沉淀技术检测了MAGE-A4与p53家族成员之间的物理性结合;利用siRNA技术研究了抑制MAGE-A4基因对高表达MAGE-A4的乳腺癌细胞系凋亡的影响。主要研究内容和结果如下:
第一部分:肿瘤抗原MAGE-A4在乳腺癌组织和细胞系中的表达
目的:通过检测MAGE-A4在乳腺正常组织、癌旁组织和癌组织及六种乳腺癌细胞系中的表达,探讨其表达与乳腺癌临床指标及生物学行为的关系
方法:采用逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)技术从转录水平上检测MAGE-A4在乳腺正常组织、癌旁组织和癌组织及六种乳腺癌细胞系中的表达,并回顾性分析其表达与乳腺癌临床指标与生物学行为(包括患者年龄、肿瘤大小、TNM分期、病理类型、组织学分级、腋淋巴结转移状况、有否脉管瘤栓、ER/PR和HER-2表达)的关系。
结果:
1. 77例乳腺正常组织中均未发现MAGE-A4 mRNA的表达;77例乳腺癌旁组织中有4例MAGE-A4 mRNA表达阳性,阳性率为5.19%;77例乳腺癌组织中有33例MAGE-A4 mRNA表达阳性,阳性率为42.9%。
2. MAGE-A4 mRNA的表达与乳腺癌患者临床指标及生物学行为的关系
25例60及60岁以上的乳腺癌患者中有15例MAGE-A4 mRNA表达阳性,阳性率为60%,52例60岁以下的乳腺癌患者中有18例MAGE-A4 mRNA表达阳性,阳性率为34.6%,两者之间有统计学差异(χ~2=4.442,p=0.035);MAGE-A4 mRNA表达与其它临床指标及生物学行为包括临床分期(χ~2=0.421,p=0.517)、肿瘤大小(χ~2=2.301,p=0.316)、病理类型(χ~2=1.820,p=0.402)、组织学分级(χ~2=0.229,p=0.892)、淋巴结转移(χ~2=5.707,p=0.058)、脉管瘤栓(χ~2=0.000,p=1.000)、ER/PR表达(χ~2=0.040,p=0.980)及HER-2表达(χ~2=0.090,p=0.764)之间均没有显著相关性。
3. MAGE-A4 mRNA在六种乳腺癌细胞系中的表达
乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-157、MDA-MB-453和MDA-MB-468中MAGE-A4 mRNA表达均为阴性;乳腺癌细胞系MDA-MB-436中MAGE-A4 mRNA高表达。
结论:
1.正常乳腺组织中无MAGE-A4 mRNA表达;乳腺癌旁组织中MAGE-A4 mRNA表达阳性率为5.19%;乳腺癌组织中MAGE-A4 mRNA表达阳性率为42.9%。提示MAGE-A4可作为肿瘤特异性抗原。
2. MAGE-A4 mRNA表达与乳腺癌患者的肿瘤大小、临床分期、病理类型、组织学分级、淋巴结转移、脉管瘤栓、ER/PR及HER-2状态无明显相关性(p>0.05),但与患者的年龄存在明显的相关性。60岁以上乳腺癌患者肿瘤组织中MAGE-A4mRNA表达阳性率明显高于60岁以下乳腺癌患者(p<0.05)。
3.六种乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-157、MDA-MB-453和MDA-MB-468中均无MAGE-A4 mRNA表达;MDA-MB-436中MAGE-A4 mRNA呈高表达。
第二部分:FLAG-pcDNA3-MAGE-A4表达载体的构建及功能分析
目的:构建带有FLAG标签的MAGE-A4基因表达载体,并研究其对乳腺癌细胞增殖和凋亡的影响。
方法:利用巢式PCR及基因重组技术构建带有FLAG标签的MAGE-A4基因表达载体FLAG-pcDNA3-MAGE-A4,并通过克隆形成实验和MTT法研究MAGE-A4过表达对MAGE-A4低表达的乳腺癌细胞增殖和凋亡的影响。
结果:
1.成功构建了MAGE-A4基因表达载体FLAG-pcDNA3-MAGE-A4,并在体外获得良好的表达。
2.克隆形成实验结果显示,转染MAGE-A4基因,并经G418筛选两周后,MCF-7细胞克隆形成数为2±0,明显低于对照组(25±2)(p<0.05);MDA-MB-231细胞克隆形成数为22±2,均明显低于对照组(104±5)(p<0.05)。
3. MTT法分析结果显示,MCF-7细胞转染MAGE-A4基因后,经阿霉素(1μM)分别处理24小时和48小时,细胞的生存率分别为28.56%和14.65%;与转染空载体的对照组细胞(42.55%和29.74%)相比细胞存活细胞率显著下降;MDA-MB-231细胞转染MAGE-A4基因后,经阿霉素(1μM)分别处理24小时和48小时,细胞的生存率分别为69.49%和20.63%;与转染空载体的对照组细胞(68.57%和21.85%)相比细胞存活细胞率没有明显变化。
结论:
1.成功构建了MAGE-A4基因表达载体FLAG-pcDNA3-MAGE-A4,并且构建好的载体在体外表达阳性。
2.外源性MAGE-A4可以抑制MCF-7和MDA-MB-231细胞的增殖。
3.外源性MAGE-A4增加了阿霉素诱导的MCF-7细胞的死亡率;但对MDA-MB-231细胞没有显著影响。
第三部分:MAGE-A4与p53家族之间关系的研究
目的:研究MAGE-A4对p53转录活性的影响,MAGE-A4是否与p53家族成员之间存在直接的物理结合,进而证实MAGE-A4的功能发挥是否部分或全部通过p53通路而完成。
方法:采用荧光素酶报告基因分析、RT-PCR、Western-blot、克隆形成实验和TUNEL染色法检测MAGE-A4对p53转录活性的影响;采用细胞质与细胞核分离实验检测MAGE-A4在细胞中的定位;采用免疫荧光染色技术检测MAGE-A4与p73蛋白在细胞中的共定位;采用免疫共沉淀技术检测MAGE-A4与p53家族成员之间的物理性结合。
结果:
1. MAGE-A4对p21WAF1启动子介导的荧光素酶表达的影响
在H1299细胞系中,对照组的荧光素酶活性为1±0.00;单独转染25ng pcDNA3-p53表达质粒组的荧光素酶活性为18.83±2.16,和内参照组相比明显增高(p<0.01);共转染恒定量(25ng)pcDNA3-p53表达质粒和逐渐增加量(100ng、200ng、400ng)的FLAG-pcDNA3-MAGE-A4表达质粒之后,各组的荧光素酶活性分别为39.25±1.68、45.07±6.78和50.49±4.93,与内参照组相比均显著增高( p<0.01 );单独转染400ng FLAG-pcDNA3-MAGE-A4表达质粒组的荧光素酶活性为1.01±0.05,与内参照组相比无显著差异(p>0.05)。
2. MAGE-A4对p21WAF1mRNA表达的影响
在H1299细胞系中,单独转染p53基因之后,p21WAF1 mRNA的表达水平明显高于未转染组(p<0.01),共转染p53和MAGE-A4基因之后,p21WAF1 mRNA表达水平明显高于单独转染p53基因组(p<0.01)。
3. MAGE-A4对p21WAF1蛋白表达的影响
在H1299细胞系中,单独转染p53基因之后,p21WAF1蛋白表达水平明显高于未转染组(p<0.01),共转染p53和MAGE-A4基因之后,p21WAF1蛋白表达水平明显高于单独转染p53基因组(p<0.01)。
4. MAGE-A4对H1299细胞克隆形成的影响
克隆形成实验结果显示,在H1299细胞中,转染1μg空载体,G418抗性的细胞克隆数为96±3,转染0.5μg pcDNA3-p53表达载体之后,G418抗性的细胞克隆数为47±2,共转染0.5μg pcDNA3-p53和0.5μg FLAG-pcDNA3-MAGE-A4表达载体之后,G418抗性的细胞克隆数为15±1,三组G418抗性的细胞克隆数之间均有统计学差异(p<0.01)。
5. MAGE-A4对H1299细胞凋亡的影响
TUNEL染色结果显示,在H1299细胞系中转染1μg空载体48h后,凋亡细胞率为1%,转染0.5μg pcDNA3-p53 48h后,凋亡细胞率为6.8%,共转染0.5μg pcDNA3-p53和0.5μg FLAG-pcDNA3-MAGE-A4 48h后,凋亡细胞率为19.3%,三组均数之间均有统计学差异(p<0.01)。
6. MAGE-A4在细胞中的定位
细胞质与细胞核分离实验结果证实,在H1299细胞中,外源性MAGE-A4在细胞质和细胞核均有表达。
7. MAGE-A4与p73在细胞中的共定位
免疫荧光染色实验结果证实,在H1299细胞中,p73蛋白定位于细胞核中;部分MAGE-A4定位于细胞质,部分则定位于细胞核;p73和MAGE-A4在细胞核中存在共定位现象。
8. MAGE-A4与p53之间的物理性结合
在H1299细胞中共转染p53和MAGE-A4表达载体之后,免疫共沉淀分析结果显示,p53的免疫沉淀物中含有MAGE-A4;反之,MAGE-A4的免疫沉淀物中也含有p53。
9. MAGE-A4与p73之间的物理性结合
在H1299细胞中共转染p73和MAGE-A4表达载体之后,免疫共沉淀分析结果显示,p73的免疫沉淀物中含有MAGE-A4;反之, MAGE-A4的免疫沉淀物中也含有p73。
结论:
1. MAGE-A4可以增加p53的转录活性。
2. MAGE-A4可以通过增加p53转录活性而产生抑制细胞增殖和诱导细胞凋亡的功能。
3. MAGE-A4在细胞质和细胞核中均有表达。
4. p73和MAGE-A4在细胞核中存在共定位。
5. p53和MAGE-A4蛋白之间以及p73和MAGE-A4蛋白之间均存在物理性结合。MAGE-A4蛋白通过和p53家族成员之间的直接结合而发挥生物学功能。
第四部分:MAGE-A4 siRNA对乳腺癌细胞MDA-MB-436凋亡的影响
目的:检测靶向MAGE-A4 siRNA对乳腺癌细胞MDA-MB-436凋亡的影响。
方法:采用MAGE-A4 siRNA转染抑制乳腺癌细胞MDA-MB-436中MAGE-A4的表达,然后使用顺铂诱导细胞凋亡,利用TUNEL染色法、流式细胞分析技术及检测PARP蛋白裂解来分析MAGE-A4 siRNA对乳腺癌细胞MDA-MB-436凋亡的影响。
结果:
1.靶向MAGE-A4 siRNA转染对MDA-MB-436细胞中MAGE-A4 mRNA的抑制效率
靶向MAGE-A4 siRNA转染MDA-MB-436细胞48h后,MAGE-A4 mRNA的表达与阴性对照组相比明显下调。转染30pmol MAGE-A4 siRNA与转染60pmol MAGE-A4 siRNA48h后,与阴性对照组相比其mRNA抑制率分别达到了87.5%与97.5%。
2. MDA-MB-436细胞对化疗药物阿霉素和顺铂的敏感性
MTT分析结果显示,MDA-MB-436细胞在阿霉素(1μM)分别处理24小时和48小时后细胞生存率分别为99.20%和98.62%;在顺铂(10μM)分别处理24小时和48小时后细胞生存率分别为79.44%和68.10%。
3. TUNEL染色法分析靶向MAGE-A4 siRNA转染对顺铂处理后的MDA-MB-436细胞凋亡的影响
TUNEL染色法结果显示,靶向MAGE-A4 siRNA转染MDA-MB-436细胞48h后,顺铂(10μmol/L)处理细胞12h和24h,凋亡细胞率分别为9.1%和2.2%,与阴性对照组(24.1%和10.0%)相比明显减少(P<0.05)。
4.流式细胞技术分析靶向MAGE-A4 siRNA转染对顺铂处理后的MDA-MB-436细胞凋亡的影响
流式细胞分析结果显示,靶向MAGE-A4 siRNA转染MDA-MB-436细胞48h后,顺铂(10μmol/L)处理细胞12h和24h,实验组subG1%((4.68±0.04)%)与对照组subG1%((10.88±0.11)%)相比显著下降(P<0.05)。
5.通过检测PARP蛋白裂解分析靶向MAGE-A4 siRNA转染对顺铂处理后的MDA-MB-436细胞凋亡的影响
Western blot结果显示,靶向MAGE-A4 siRNA转染MDA-MB-436细胞48h后,顺铂(10μmol/L)处理细胞12h和24h,细胞凋亡的核心成员caspase的酶切底物PARP蛋白均有不同程度的裂解。实验组裂解带的灰度值与actin灰度值的比值(0.129±0.014)与对照组(0.549±0.023)相比显著降低(P<0.05)。
结论:
1.靶向MAGE-A4 siRNA转染对MDA-MB-436细胞MAGE-A4表达有明显的抑制效果。
2. MDA-MB-436对阿霉素耐药,而对顺铂较敏感。
3.靶向MAGE-A4 siRNA转染可降低MDA-MB-436对CDDP诱导的凋亡,提示:MAGE-A4在一定程度上发挥肿瘤抑制因子的作用。
Currently, tumor biotherapy represented by tumor immunotherapy has become the fourth therapeutic pattern following surgery, chemotherapy and radiotherapy. In recent years, the development of new tumor vaccine has become one of the focuses in the field of tumor immunotherapy. Among the numerous known tumor-associated antigens, the cancer/testis antigens (CTA) displayed the sepicific expression pattern. They expressed in a variety of tumor tissues but not in normal tissues except the testicular germ cell, and occasionally expressed in the placenta. However, testis is immune privilege organ, which does not cause the specific immune response. Therefore, CTA was a suitable target antigen for specific tumor immunotherapy.
As a sub-family of CTA, melanoma antigen gene (MAGE) is a big family which was separated from melanoma cells. MAGE gene encodes tumor-sepicific antigenic peptides which were presented to CD8+ T lymphocytes by HLA-I class I molecules, and therefore inducing tumor sepicific killing. MAGE are highly expressed in various forms of cancer, but not in most healthy adult tissues except for the testis and embryo, therefore is an ideal CTL-mmediated target molecule for tumor specific immunotherapy. The study in-depth to MAGE gene will open a door for the molecular diagnosis of tumors and the cilinical application of tumor immunotherapy.
Some studies reported that unlike other MAGE-A family members, MAGE-A4 maybe had different biological characteristics. However, its biological functions and molecular mechanism were still unclear.
In our study, RT-PCR (reverse transcription-polymerase chain reaction) was adopted to investigate the expression of MAGE-A4 mRNA expression in 77 benign and malignant breast tumors and six breast cancer cell lines. FLAG-pcDNA3-MAGE-A4 expression plasmid was constructed. MTT assay and colony formation assay were used to detect the effect of overexpression of MAGE-A4 on the proliferation and apoptosis of breast cancer cells. Luciferase reporter assay, RT-PCR, Western blot, colony formation assay and TUNEL assay were used to investigate the effect of MAGE-A4 on the transcriptional activity of p53. Cell fractionation assay was adopted to detect the localization of MAGE-A4 in cells. Immunofluorescence was adopted to detect the co-locolization of MAGE-A4 and p73. Immunoprecipitation experiment was used to examine the physical interaction between MAGE-A4 and p53/p73. SiRNA knockdown against MAGE-A4 was used to investigate the effect of MAGE-A4 siRNA on the apoptosis of MDA-MB-436 cells.
The main research contents and results were shown as follows:
PartⅠThe expression of MAGE-A4 mRNA in breast cancer tissues and breast cell lines
Objective: To investigate the ralationship between MAGE-A4 mRNA expression and the clinical parameter/biological behaviors of breast cancer through detecting the expression of MAGE-A4 mRNA in normal breast tissues, pericancerous tissues and breast cancer tissues as well as breast cancer cell lines.
Methods: RT-PCR was adopted to detect the expression of MAGE-A4 at the transcription level in normal breast tissues, pericancerous tissues and breast cancer tissues as well as six breast cancer cell lines. Retrospectively analyze was used to analyze the relationship between MAGE-A4 mRNA expression and the clinical/biological behaviors of breast cancer including age of the patients, tumor size, TNM stages, pathological types, histology grades, metastasis of axillary lymph nodes, haemal tube tumor embolus, ER/PR status and HER-2 status.
Results:
1. Ther was no MAGE-A4 mRNA expression in 77 normal breast tissues.
MAGE-A4 mRNA expression was detected in 4 of 77 pericancerous tissues and 33 of 77 beast cancer tissues, respectively, and the positive rate were 5.19% and 42.9%, respectively.
2. The ralationship between MAGE-A4 mRNA expression and the clinical parameter/biological behaviors of breast cancer
MAGE-A4 mRNA expression was detected in 15 of 25 patients with the age more than 60 years and in 18 of 52 patients with the age less than 60 years, and the positive rate were 60% and 34.6%, with significant difference (χ~2=4.442, p=0.035). There were no significant differences between MAGE-A4 mRNA expression and other clinical parameter/biological behaviors of breast cancer including tumor size (χ~2=2.301, p=0.316), TNM stages (χ~2=0.421, p=0.517), pathological types (χ~2=1.820, p=0.402), histology grades (χ~2=0.229, p=0.892), metastasis of axillary lymph nodes (χ~2=5.707, p=0.058), haemal tube tumor embolus (χ~2=0.000, p=1.000), ER/PR status (χ~2=0.040, p=0.980) and HER-2 status (χ~2=0.090, p=0.764).
3. MAGE-A4 mRNA expression in six breast cancer cell lines
MAGE-A4 mRNA was negtively expressed in MCF-7, MDA-MB-231, MDA-MB-157, MDA-MB-453 and MDA-MB-468 cells, but highly expressed in MDA-MB-436 cells.
Conclusions:
1. MAGE-A4 mRNA was not expressed in normal breast tissues. The positive rate of MAGE-A4 mRNA in pericancerous tissues and breast cancer tissues were 5.19% and 42.9%, respectively, suggesting that MAGE-A4 was tumor specific antigen.
2. There were no significant differences between MAGE-A4 mRNA expression and tumor size, TNM stages, pathological types, histology grades, metastasis of axillary lymph nodes, haemal tube tumor embolus, ER/PR status and HER-2 status of breast cancer patients. However, MAGE-A4 mRNA expression was positively correlated with patient’s age. The positive rate of MAGE-A4 mRNA in the patients older than 60 years was significantly higher than the patients younger than 60 years (p<0.05).
3. Among six breast cancer cell lines, MAGE-A4 mRNA was negtively expressed in MCF-7, MDA-MB-231, MDA-MB-157, MDA-MB-453 and MDA-MB-468 cells, but highly expressed in MDA-MB-436 cells.
PartⅡThe construction of FLAG-pcDNA3-MAGE-A4 expression plasmid and its function
Objective: To construct the MAGE-A4 expression plasmid with FLAG tag, and investigate its effects on the proliferation and apoptosis of breast cancer cells.
Methods: Nested PCR and gene recombination technique were used to construct the MAGE-A4 expression plasmid with FLAG tag, and MTT assay and colony formation assay were used to investigate the effects of MAGE-A4 overexpression on the proliferation and apoptosis of breast cancer cells
Results:
1. FLAG-pcDNA3-MAGE-A4 expression plasmid was successfully constructed and expressed well in cells.
2. Colony formation assay showed that after MAGE-A4 transfection and G418 selection, the number of colonies of MCF-7 cells was 2±0, which was significant lower than the control group 25±2 (p<0.05), and the number of colonies of MDA-MB-231 cells was 22±2, which was significant lower than the control group 104±5 (p<0.05).
3. MTT assay showed that after MAGE-A4 transfection and ADR (1μM) treatment for 24h and 48h, the cell survival rates of MCF-7 cells were 28.56% and 14.65%, which was significantly lower than the control group (42.55% and 29.74%). After MAGE-A4 transfection and ADR (1μM) treatment for 24h and 48h, the cell survival rates of MDA-MB-231 cells were 69.49% and 20.63%, which was significantly lower than the control group (68.57% and 21.85%).
Conclusions
1. FLAG-pcDNA3-MAGE-A4 expression plasmid was successfully constructed and positively expressed in cells.
2. Exogenous MAGE-A4 could inhibit the proliferation of MCF-7 and MDA-MB-231 cells.
3. Exogenous MAGE-A4 could increase the ADR-induced cell death of MCF-7 cells but not MDA-MB-231 cells.
PartⅢThe relationship between MAGE-A4 and p53 family members
Objective: To investigate the effect of MAGE-A4 on the transcriptional activity of p53 and the physical interaction between MAGE-A4 and p53 family members, and confirm whether MAGE-A4 plays the role partly or completely via p53 pathway.
Methods : Luciferase reporter assay, RT-PCR, Western-blot, colony formation assay and TUNEL assay were used to investigate the effects of MAGE-A4 on the transcriptional activity of p53. Cell fractionation assay and immunofluorescence were used to detect the co-localization of MAGE-A4 and p73 in cells. Immunoprecipitation was used to detect the physical interaction between MAGE-A4 and p53 family members.
Results:
1. The effect of MAGE-A4 on the luciferase activity of p21WAF1 promotor induced by p53
In H1299 cells, the luciferase activity in the control group was 1±0.00; the luciferase activity in the group with 25ng pcDNA3-p53 transfection alone was 18.83±2.16 (p<0.01 as compared with the control); the luciferase activity in the group with constant (25ng) pcDNA3-p53 and guadually increased (100ng, 200ng and 400ng) FLAG-pcDNA3-MAGE-A4 trasfection were 39.25±1.68, 45.07±6.78 and 50.49±4.93, respectively (p<0.01 as compared with the control); the luciferase activity in the group with 400ng FLAG-pcDNA3-MAGE-A4 transfection alone was 1.01±0.05 (p<0.01 as compared with the control).
2. The effect of MAGE-A4 on p21WAF1 mRNA expression
In H1299 cells, p21WAF1 mRNA expression in the group with p53 transfection alon was significantly higher than the control group (p<0.01), and p21WAF1 mRNA expression in the group with p53 and MAGE-A4 cotransfection was significantly higher than the group with p53 transfection alone (p<0.01).
3. The effect of MAGE-A4 on p21WAF1 protein expression
In H1299 cells, p21WAF1 protein expression in the group with p53 transfection alon was significantly higher than the control group (p<0.01), and p21WAF1 protein expression in the group with p53 and MAGE-A4 cotransfection was significantly higher than the group with p53 transfection alone (p<0.01).
4. The effect of MAGE-A4 on the colony formation in H1299 cells
In H1299 cells, the colony numbers with G418 resistance were 96±3, 47±2 and 15±1 in the groups transfected with empty vector, 0.5μg pcDNA3-p53 and 0.5μg pcDNA3-p53/0.5μg FLAG-pcDNA3-MAGE-A4, respectively. There were significant difference among these three groups (p<0.01).
5. The effect of MAGE-A4 on the apoptosis of H1299 cells
TUNEL staining experiment showed that the apoptotic rate of H1299 cells was 1%, 6.8% and 19.3%, respectively after transfection with 1μg pcDNA3, 0.5μg pcDNA3-p53 and 0.5μg pcDNA3-p53 plus 0.5μg FLAG-pcDNA3-MAGE-A4, respectively. There were significant difference among these three groups (p<0.01).
6. The localization of MAGE-A4 in cells
Cell fractionation assay confiemed that the exogenous MAGE-A4 was expressed in both cytoplasm and cell nucleus in H1299 cells.
7. The colocolization of MAGE-A4 and p73
Immunofluorescence showed that in H1299 cells p73 was located in cell nucleus and part of MAGE-A4 was located in cytoplasm and another part of MAGE-A4 was located in cell nucleus, suggestiong that p73 and MAGE-A4 co-locolized in cell nucleus.
8. The physical interaction between MAGE-A4 and p53
Immunoprecipitation experiment showed that after co-transfected with MAGE-A4 and p53 plasimids, the immunoprecipitates of p53 contained MAGE-A4 and the immunoprecipitates of MAGE-A4 contained p53.
9. The physical interaction between MAGE-A4 and p73
Immunoprecipitation experiment showed that after co-transfected with MAGE-A4 and p73 plasimids, the immunoprecipitates of p73 contained MAGE-A4 and the immunoprecipitates of MAGE-A4 contained p73.
Conclusions:
1. MAGE-A4 could enhance the transcriptional activity of p53.
2. MAGE-A4 could enhance the proliferation inhibition role and apoptosis activity induced by p53.
3. MAGE-A4 was expressed in both cytoplasm and cell nucleus in H1299 cells.
4. p73 and MAGE-A4 co-locolized in cell nucleus.
5. MAGE-A4 physically interacted with p53 and p73.
Part IV The effect of MAGE-A4 siRNA on the apoptosis of MDA-MB-436 cells
Objective: To investigate the effect of MAGE-A4 siRNA on the apoptosis of MDA-MB-436 cells.
Methods : SiRNA knockdown experiment was used to inhibit the expression of MAGE-A4. TUNEL assay and flow cytometry (FCM) and PARP cleavage detection were used to investigate the effect of MAGE siRNA on the apoptosis of MDA-MB-436 cells in response to CDDP.
Results:
1. The inhibitory efficancy of MAGE-A4 siRNA on the expression of MAGE-A4 in MDA-MB-436 cells
After the 48h transfection of MAGE-A4 siRNA, the mRNA expression of MAGE-A4 was significantly inhibited. The inhibitory efficancy were 87.5% and 97.5% after 30pmol and 60pmol MAGE-A4 siRNA transfection.
2. The result of MTT assay
The cell survivals of MDA-MB-436 cells were 99.20% and 98.62% after ADR (1μM) treatment for 24h and 48h, respectively. Whereas the cell survivals of MDA-MB-436 cells were 79.44% and 68.10% after CDDP (10μM) treatment for 24h and 48h, respectively.
3. The result of TUNEL staining
TUNEL staining showed that after MAGE-A4 siRNA transfection for 48h and CDDP (10μM) treatment for 12h and 24h, the apoptotic rate of MDA-MB-436 cells were 9.1% and 2.2%,which were significantly decresed as compared with the control group (24.1% and 10.0%) (P<0.05).
4. The result of flow cytometry assay
After MAGE-A4 siRNA transfection for 48h and CDDP (10μM) treatment for 24h, the subG1% were (4.68±0.04)%,which were significantly decresed as compared with the control group (10.88±0.119)% (P<0.05).
5. The cleavage of PARP protein
Western blot result showed that after MAGE-A4 siRNA transfection for 48h and CDDP (10μM) treatment for 24h, the caspase substrate PARP were cleaved. The tario of gray scale was 0.129±0.014, which was significantly decreased as compared with the control (0.549±0.023) (P<0.05).
Conclusions:
1. MAGE-A4 siRNA has obvious inhibitory effects on MAGE-A4 expression.
2. MDA-MB-436 was resistant to ADR but sensitive to CDDP.
3. MAGE-A4 siRNA could reduce the apoptosis induced by CDDP in MDA-MB-436 cells, suggesting that MAGE-A4 played a role of tumor suppressor under certain conditions.
作为CTA亚家族的黑色素瘤抗原基因(melanoma antigen gene, MAGE)是从黑色素瘤细胞中分离出来的一大家族抗原基因,其编码的肿瘤排斥抗原在细胞内经加工产生抗原肽,并与HLA-I类分子结合形成复合物,能够被自体细胞毒性T细胞识别,诱导出对相应肿瘤细胞的特异性杀伤。MAGE基因在许多肿瘤组织中有较高的表达,但在正常组织(除睾丸和胚胎外)均不表达,因此可作为CTL介导肿瘤特异性免疫治疗的理想靶分子。深入研究MAGE基因的功能将会为开展肿瘤分子学诊断和肿瘤免疫治疗的临床应用奠定良好的基础。
有研究提示,MAGE-A4可能表现了与该家族中其它成员不同的分子生物学特性,但是其具体的生物学功能和分子机制到目前还不十分清楚。本研究采用逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测了MAGE-A4 mRNA在77例乳腺癌良恶性组织及乳腺癌细胞系中的表达;构建了同时表达MAGE-A4和FLAG标签的表达载体FLAG-pcDNA3-MAGE-A4,并通过MTT分析和克隆形成实验研究了MAGE-A4过表达对MAGE-A4低表达的乳腺癌细胞增殖和凋亡的影响;采用荧光素酶报告基因分析、RT-PCR、Western-blot、克隆形成实验和TUNEL染色法检测了MAGE-A4对p53转录活性的影响;采用细胞质与细胞核分离实验检测了MAGE-A4在细胞中的定位;采用免疫荧光染色技术检测了MAGE-A4与p73蛋白在细胞中的共定位;采用免疫共沉淀技术检测了MAGE-A4与p53家族成员之间的物理性结合;利用siRNA技术研究了抑制MAGE-A4基因对高表达MAGE-A4的乳腺癌细胞系凋亡的影响。主要研究内容和结果如下:
第一部分:肿瘤抗原MAGE-A4在乳腺癌组织和细胞系中的表达
目的:通过检测MAGE-A4在乳腺正常组织、癌旁组织和癌组织及六种乳腺癌细胞系中的表达,探讨其表达与乳腺癌临床指标及生物学行为的关系
方法:采用逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)技术从转录水平上检测MAGE-A4在乳腺正常组织、癌旁组织和癌组织及六种乳腺癌细胞系中的表达,并回顾性分析其表达与乳腺癌临床指标与生物学行为(包括患者年龄、肿瘤大小、TNM分期、病理类型、组织学分级、腋淋巴结转移状况、有否脉管瘤栓、ER/PR和HER-2表达)的关系。
结果:
1. 77例乳腺正常组织中均未发现MAGE-A4 mRNA的表达;77例乳腺癌旁组织中有4例MAGE-A4 mRNA表达阳性,阳性率为5.19%;77例乳腺癌组织中有33例MAGE-A4 mRNA表达阳性,阳性率为42.9%。
2. MAGE-A4 mRNA的表达与乳腺癌患者临床指标及生物学行为的关系
25例60及60岁以上的乳腺癌患者中有15例MAGE-A4 mRNA表达阳性,阳性率为60%,52例60岁以下的乳腺癌患者中有18例MAGE-A4 mRNA表达阳性,阳性率为34.6%,两者之间有统计学差异(χ~2=4.442,p=0.035);MAGE-A4 mRNA表达与其它临床指标及生物学行为包括临床分期(χ~2=0.421,p=0.517)、肿瘤大小(χ~2=2.301,p=0.316)、病理类型(χ~2=1.820,p=0.402)、组织学分级(χ~2=0.229,p=0.892)、淋巴结转移(χ~2=5.707,p=0.058)、脉管瘤栓(χ~2=0.000,p=1.000)、ER/PR表达(χ~2=0.040,p=0.980)及HER-2表达(χ~2=0.090,p=0.764)之间均没有显著相关性。
3. MAGE-A4 mRNA在六种乳腺癌细胞系中的表达
乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-157、MDA-MB-453和MDA-MB-468中MAGE-A4 mRNA表达均为阴性;乳腺癌细胞系MDA-MB-436中MAGE-A4 mRNA高表达。
结论:
1.正常乳腺组织中无MAGE-A4 mRNA表达;乳腺癌旁组织中MAGE-A4 mRNA表达阳性率为5.19%;乳腺癌组织中MAGE-A4 mRNA表达阳性率为42.9%。提示MAGE-A4可作为肿瘤特异性抗原。
2. MAGE-A4 mRNA表达与乳腺癌患者的肿瘤大小、临床分期、病理类型、组织学分级、淋巴结转移、脉管瘤栓、ER/PR及HER-2状态无明显相关性(p>0.05),但与患者的年龄存在明显的相关性。60岁以上乳腺癌患者肿瘤组织中MAGE-A4mRNA表达阳性率明显高于60岁以下乳腺癌患者(p<0.05)。
3.六种乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-157、MDA-MB-453和MDA-MB-468中均无MAGE-A4 mRNA表达;MDA-MB-436中MAGE-A4 mRNA呈高表达。
第二部分:FLAG-pcDNA3-MAGE-A4表达载体的构建及功能分析
目的:构建带有FLAG标签的MAGE-A4基因表达载体,并研究其对乳腺癌细胞增殖和凋亡的影响。
方法:利用巢式PCR及基因重组技术构建带有FLAG标签的MAGE-A4基因表达载体FLAG-pcDNA3-MAGE-A4,并通过克隆形成实验和MTT法研究MAGE-A4过表达对MAGE-A4低表达的乳腺癌细胞增殖和凋亡的影响。
结果:
1.成功构建了MAGE-A4基因表达载体FLAG-pcDNA3-MAGE-A4,并在体外获得良好的表达。
2.克隆形成实验结果显示,转染MAGE-A4基因,并经G418筛选两周后,MCF-7细胞克隆形成数为2±0,明显低于对照组(25±2)(p<0.05);MDA-MB-231细胞克隆形成数为22±2,均明显低于对照组(104±5)(p<0.05)。
3. MTT法分析结果显示,MCF-7细胞转染MAGE-A4基因后,经阿霉素(1μM)分别处理24小时和48小时,细胞的生存率分别为28.56%和14.65%;与转染空载体的对照组细胞(42.55%和29.74%)相比细胞存活细胞率显著下降;MDA-MB-231细胞转染MAGE-A4基因后,经阿霉素(1μM)分别处理24小时和48小时,细胞的生存率分别为69.49%和20.63%;与转染空载体的对照组细胞(68.57%和21.85%)相比细胞存活细胞率没有明显变化。
结论:
1.成功构建了MAGE-A4基因表达载体FLAG-pcDNA3-MAGE-A4,并且构建好的载体在体外表达阳性。
2.外源性MAGE-A4可以抑制MCF-7和MDA-MB-231细胞的增殖。
3.外源性MAGE-A4增加了阿霉素诱导的MCF-7细胞的死亡率;但对MDA-MB-231细胞没有显著影响。
第三部分:MAGE-A4与p53家族之间关系的研究
目的:研究MAGE-A4对p53转录活性的影响,MAGE-A4是否与p53家族成员之间存在直接的物理结合,进而证实MAGE-A4的功能发挥是否部分或全部通过p53通路而完成。
方法:采用荧光素酶报告基因分析、RT-PCR、Western-blot、克隆形成实验和TUNEL染色法检测MAGE-A4对p53转录活性的影响;采用细胞质与细胞核分离实验检测MAGE-A4在细胞中的定位;采用免疫荧光染色技术检测MAGE-A4与p73蛋白在细胞中的共定位;采用免疫共沉淀技术检测MAGE-A4与p53家族成员之间的物理性结合。
结果:
1. MAGE-A4对p21WAF1启动子介导的荧光素酶表达的影响
在H1299细胞系中,对照组的荧光素酶活性为1±0.00;单独转染25ng pcDNA3-p53表达质粒组的荧光素酶活性为18.83±2.16,和内参照组相比明显增高(p<0.01);共转染恒定量(25ng)pcDNA3-p53表达质粒和逐渐增加量(100ng、200ng、400ng)的FLAG-pcDNA3-MAGE-A4表达质粒之后,各组的荧光素酶活性分别为39.25±1.68、45.07±6.78和50.49±4.93,与内参照组相比均显著增高( p<0.01 );单独转染400ng FLAG-pcDNA3-MAGE-A4表达质粒组的荧光素酶活性为1.01±0.05,与内参照组相比无显著差异(p>0.05)。
2. MAGE-A4对p21WAF1mRNA表达的影响
在H1299细胞系中,单独转染p53基因之后,p21WAF1 mRNA的表达水平明显高于未转染组(p<0.01),共转染p53和MAGE-A4基因之后,p21WAF1 mRNA表达水平明显高于单独转染p53基因组(p<0.01)。
3. MAGE-A4对p21WAF1蛋白表达的影响
在H1299细胞系中,单独转染p53基因之后,p21WAF1蛋白表达水平明显高于未转染组(p<0.01),共转染p53和MAGE-A4基因之后,p21WAF1蛋白表达水平明显高于单独转染p53基因组(p<0.01)。
4. MAGE-A4对H1299细胞克隆形成的影响
克隆形成实验结果显示,在H1299细胞中,转染1μg空载体,G418抗性的细胞克隆数为96±3,转染0.5μg pcDNA3-p53表达载体之后,G418抗性的细胞克隆数为47±2,共转染0.5μg pcDNA3-p53和0.5μg FLAG-pcDNA3-MAGE-A4表达载体之后,G418抗性的细胞克隆数为15±1,三组G418抗性的细胞克隆数之间均有统计学差异(p<0.01)。
5. MAGE-A4对H1299细胞凋亡的影响
TUNEL染色结果显示,在H1299细胞系中转染1μg空载体48h后,凋亡细胞率为1%,转染0.5μg pcDNA3-p53 48h后,凋亡细胞率为6.8%,共转染0.5μg pcDNA3-p53和0.5μg FLAG-pcDNA3-MAGE-A4 48h后,凋亡细胞率为19.3%,三组均数之间均有统计学差异(p<0.01)。
6. MAGE-A4在细胞中的定位
细胞质与细胞核分离实验结果证实,在H1299细胞中,外源性MAGE-A4在细胞质和细胞核均有表达。
7. MAGE-A4与p73在细胞中的共定位
免疫荧光染色实验结果证实,在H1299细胞中,p73蛋白定位于细胞核中;部分MAGE-A4定位于细胞质,部分则定位于细胞核;p73和MAGE-A4在细胞核中存在共定位现象。
8. MAGE-A4与p53之间的物理性结合
在H1299细胞中共转染p53和MAGE-A4表达载体之后,免疫共沉淀分析结果显示,p53的免疫沉淀物中含有MAGE-A4;反之,MAGE-A4的免疫沉淀物中也含有p53。
9. MAGE-A4与p73之间的物理性结合
在H1299细胞中共转染p73和MAGE-A4表达载体之后,免疫共沉淀分析结果显示,p73的免疫沉淀物中含有MAGE-A4;反之, MAGE-A4的免疫沉淀物中也含有p73。
结论:
1. MAGE-A4可以增加p53的转录活性。
2. MAGE-A4可以通过增加p53转录活性而产生抑制细胞增殖和诱导细胞凋亡的功能。
3. MAGE-A4在细胞质和细胞核中均有表达。
4. p73和MAGE-A4在细胞核中存在共定位。
5. p53和MAGE-A4蛋白之间以及p73和MAGE-A4蛋白之间均存在物理性结合。MAGE-A4蛋白通过和p53家族成员之间的直接结合而发挥生物学功能。
第四部分:MAGE-A4 siRNA对乳腺癌细胞MDA-MB-436凋亡的影响
目的:检测靶向MAGE-A4 siRNA对乳腺癌细胞MDA-MB-436凋亡的影响。
方法:采用MAGE-A4 siRNA转染抑制乳腺癌细胞MDA-MB-436中MAGE-A4的表达,然后使用顺铂诱导细胞凋亡,利用TUNEL染色法、流式细胞分析技术及检测PARP蛋白裂解来分析MAGE-A4 siRNA对乳腺癌细胞MDA-MB-436凋亡的影响。
结果:
1.靶向MAGE-A4 siRNA转染对MDA-MB-436细胞中MAGE-A4 mRNA的抑制效率
靶向MAGE-A4 siRNA转染MDA-MB-436细胞48h后,MAGE-A4 mRNA的表达与阴性对照组相比明显下调。转染30pmol MAGE-A4 siRNA与转染60pmol MAGE-A4 siRNA48h后,与阴性对照组相比其mRNA抑制率分别达到了87.5%与97.5%。
2. MDA-MB-436细胞对化疗药物阿霉素和顺铂的敏感性
MTT分析结果显示,MDA-MB-436细胞在阿霉素(1μM)分别处理24小时和48小时后细胞生存率分别为99.20%和98.62%;在顺铂(10μM)分别处理24小时和48小时后细胞生存率分别为79.44%和68.10%。
3. TUNEL染色法分析靶向MAGE-A4 siRNA转染对顺铂处理后的MDA-MB-436细胞凋亡的影响
TUNEL染色法结果显示,靶向MAGE-A4 siRNA转染MDA-MB-436细胞48h后,顺铂(10μmol/L)处理细胞12h和24h,凋亡细胞率分别为9.1%和2.2%,与阴性对照组(24.1%和10.0%)相比明显减少(P<0.05)。
4.流式细胞技术分析靶向MAGE-A4 siRNA转染对顺铂处理后的MDA-MB-436细胞凋亡的影响
流式细胞分析结果显示,靶向MAGE-A4 siRNA转染MDA-MB-436细胞48h后,顺铂(10μmol/L)处理细胞12h和24h,实验组subG1%((4.68±0.04)%)与对照组subG1%((10.88±0.11)%)相比显著下降(P<0.05)。
5.通过检测PARP蛋白裂解分析靶向MAGE-A4 siRNA转染对顺铂处理后的MDA-MB-436细胞凋亡的影响
Western blot结果显示,靶向MAGE-A4 siRNA转染MDA-MB-436细胞48h后,顺铂(10μmol/L)处理细胞12h和24h,细胞凋亡的核心成员caspase的酶切底物PARP蛋白均有不同程度的裂解。实验组裂解带的灰度值与actin灰度值的比值(0.129±0.014)与对照组(0.549±0.023)相比显著降低(P<0.05)。
结论:
1.靶向MAGE-A4 siRNA转染对MDA-MB-436细胞MAGE-A4表达有明显的抑制效果。
2. MDA-MB-436对阿霉素耐药,而对顺铂较敏感。
3.靶向MAGE-A4 siRNA转染可降低MDA-MB-436对CDDP诱导的凋亡,提示:MAGE-A4在一定程度上发挥肿瘤抑制因子的作用。
Currently, tumor biotherapy represented by tumor immunotherapy has become the fourth therapeutic pattern following surgery, chemotherapy and radiotherapy. In recent years, the development of new tumor vaccine has become one of the focuses in the field of tumor immunotherapy. Among the numerous known tumor-associated antigens, the cancer/testis antigens (CTA) displayed the sepicific expression pattern. They expressed in a variety of tumor tissues but not in normal tissues except the testicular germ cell, and occasionally expressed in the placenta. However, testis is immune privilege organ, which does not cause the specific immune response. Therefore, CTA was a suitable target antigen for specific tumor immunotherapy.
As a sub-family of CTA, melanoma antigen gene (MAGE) is a big family which was separated from melanoma cells. MAGE gene encodes tumor-sepicific antigenic peptides which were presented to CD8+ T lymphocytes by HLA-I class I molecules, and therefore inducing tumor sepicific killing. MAGE are highly expressed in various forms of cancer, but not in most healthy adult tissues except for the testis and embryo, therefore is an ideal CTL-mmediated target molecule for tumor specific immunotherapy. The study in-depth to MAGE gene will open a door for the molecular diagnosis of tumors and the cilinical application of tumor immunotherapy.
Some studies reported that unlike other MAGE-A family members, MAGE-A4 maybe had different biological characteristics. However, its biological functions and molecular mechanism were still unclear.
In our study, RT-PCR (reverse transcription-polymerase chain reaction) was adopted to investigate the expression of MAGE-A4 mRNA expression in 77 benign and malignant breast tumors and six breast cancer cell lines. FLAG-pcDNA3-MAGE-A4 expression plasmid was constructed. MTT assay and colony formation assay were used to detect the effect of overexpression of MAGE-A4 on the proliferation and apoptosis of breast cancer cells. Luciferase reporter assay, RT-PCR, Western blot, colony formation assay and TUNEL assay were used to investigate the effect of MAGE-A4 on the transcriptional activity of p53. Cell fractionation assay was adopted to detect the localization of MAGE-A4 in cells. Immunofluorescence was adopted to detect the co-locolization of MAGE-A4 and p73. Immunoprecipitation experiment was used to examine the physical interaction between MAGE-A4 and p53/p73. SiRNA knockdown against MAGE-A4 was used to investigate the effect of MAGE-A4 siRNA on the apoptosis of MDA-MB-436 cells.
The main research contents and results were shown as follows:
PartⅠThe expression of MAGE-A4 mRNA in breast cancer tissues and breast cell lines
Objective: To investigate the ralationship between MAGE-A4 mRNA expression and the clinical parameter/biological behaviors of breast cancer through detecting the expression of MAGE-A4 mRNA in normal breast tissues, pericancerous tissues and breast cancer tissues as well as breast cancer cell lines.
Methods: RT-PCR was adopted to detect the expression of MAGE-A4 at the transcription level in normal breast tissues, pericancerous tissues and breast cancer tissues as well as six breast cancer cell lines. Retrospectively analyze was used to analyze the relationship between MAGE-A4 mRNA expression and the clinical/biological behaviors of breast cancer including age of the patients, tumor size, TNM stages, pathological types, histology grades, metastasis of axillary lymph nodes, haemal tube tumor embolus, ER/PR status and HER-2 status.
Results:
1. Ther was no MAGE-A4 mRNA expression in 77 normal breast tissues.
MAGE-A4 mRNA expression was detected in 4 of 77 pericancerous tissues and 33 of 77 beast cancer tissues, respectively, and the positive rate were 5.19% and 42.9%, respectively.
2. The ralationship between MAGE-A4 mRNA expression and the clinical parameter/biological behaviors of breast cancer
MAGE-A4 mRNA expression was detected in 15 of 25 patients with the age more than 60 years and in 18 of 52 patients with the age less than 60 years, and the positive rate were 60% and 34.6%, with significant difference (χ~2=4.442, p=0.035). There were no significant differences between MAGE-A4 mRNA expression and other clinical parameter/biological behaviors of breast cancer including tumor size (χ~2=2.301, p=0.316), TNM stages (χ~2=0.421, p=0.517), pathological types (χ~2=1.820, p=0.402), histology grades (χ~2=0.229, p=0.892), metastasis of axillary lymph nodes (χ~2=5.707, p=0.058), haemal tube tumor embolus (χ~2=0.000, p=1.000), ER/PR status (χ~2=0.040, p=0.980) and HER-2 status (χ~2=0.090, p=0.764).
3. MAGE-A4 mRNA expression in six breast cancer cell lines
MAGE-A4 mRNA was negtively expressed in MCF-7, MDA-MB-231, MDA-MB-157, MDA-MB-453 and MDA-MB-468 cells, but highly expressed in MDA-MB-436 cells.
Conclusions:
1. MAGE-A4 mRNA was not expressed in normal breast tissues. The positive rate of MAGE-A4 mRNA in pericancerous tissues and breast cancer tissues were 5.19% and 42.9%, respectively, suggesting that MAGE-A4 was tumor specific antigen.
2. There were no significant differences between MAGE-A4 mRNA expression and tumor size, TNM stages, pathological types, histology grades, metastasis of axillary lymph nodes, haemal tube tumor embolus, ER/PR status and HER-2 status of breast cancer patients. However, MAGE-A4 mRNA expression was positively correlated with patient’s age. The positive rate of MAGE-A4 mRNA in the patients older than 60 years was significantly higher than the patients younger than 60 years (p<0.05).
3. Among six breast cancer cell lines, MAGE-A4 mRNA was negtively expressed in MCF-7, MDA-MB-231, MDA-MB-157, MDA-MB-453 and MDA-MB-468 cells, but highly expressed in MDA-MB-436 cells.
PartⅡThe construction of FLAG-pcDNA3-MAGE-A4 expression plasmid and its function
Objective: To construct the MAGE-A4 expression plasmid with FLAG tag, and investigate its effects on the proliferation and apoptosis of breast cancer cells.
Methods: Nested PCR and gene recombination technique were used to construct the MAGE-A4 expression plasmid with FLAG tag, and MTT assay and colony formation assay were used to investigate the effects of MAGE-A4 overexpression on the proliferation and apoptosis of breast cancer cells
Results:
1. FLAG-pcDNA3-MAGE-A4 expression plasmid was successfully constructed and expressed well in cells.
2. Colony formation assay showed that after MAGE-A4 transfection and G418 selection, the number of colonies of MCF-7 cells was 2±0, which was significant lower than the control group 25±2 (p<0.05), and the number of colonies of MDA-MB-231 cells was 22±2, which was significant lower than the control group 104±5 (p<0.05).
3. MTT assay showed that after MAGE-A4 transfection and ADR (1μM) treatment for 24h and 48h, the cell survival rates of MCF-7 cells were 28.56% and 14.65%, which was significantly lower than the control group (42.55% and 29.74%). After MAGE-A4 transfection and ADR (1μM) treatment for 24h and 48h, the cell survival rates of MDA-MB-231 cells were 69.49% and 20.63%, which was significantly lower than the control group (68.57% and 21.85%).
Conclusions
1. FLAG-pcDNA3-MAGE-A4 expression plasmid was successfully constructed and positively expressed in cells.
2. Exogenous MAGE-A4 could inhibit the proliferation of MCF-7 and MDA-MB-231 cells.
3. Exogenous MAGE-A4 could increase the ADR-induced cell death of MCF-7 cells but not MDA-MB-231 cells.
PartⅢThe relationship between MAGE-A4 and p53 family members
Objective: To investigate the effect of MAGE-A4 on the transcriptional activity of p53 and the physical interaction between MAGE-A4 and p53 family members, and confirm whether MAGE-A4 plays the role partly or completely via p53 pathway.
Methods : Luciferase reporter assay, RT-PCR, Western-blot, colony formation assay and TUNEL assay were used to investigate the effects of MAGE-A4 on the transcriptional activity of p53. Cell fractionation assay and immunofluorescence were used to detect the co-localization of MAGE-A4 and p73 in cells. Immunoprecipitation was used to detect the physical interaction between MAGE-A4 and p53 family members.
Results:
1. The effect of MAGE-A4 on the luciferase activity of p21WAF1 promotor induced by p53
In H1299 cells, the luciferase activity in the control group was 1±0.00; the luciferase activity in the group with 25ng pcDNA3-p53 transfection alone was 18.83±2.16 (p<0.01 as compared with the control); the luciferase activity in the group with constant (25ng) pcDNA3-p53 and guadually increased (100ng, 200ng and 400ng) FLAG-pcDNA3-MAGE-A4 trasfection were 39.25±1.68, 45.07±6.78 and 50.49±4.93, respectively (p<0.01 as compared with the control); the luciferase activity in the group with 400ng FLAG-pcDNA3-MAGE-A4 transfection alone was 1.01±0.05 (p<0.01 as compared with the control).
2. The effect of MAGE-A4 on p21WAF1 mRNA expression
In H1299 cells, p21WAF1 mRNA expression in the group with p53 transfection alon was significantly higher than the control group (p<0.01), and p21WAF1 mRNA expression in the group with p53 and MAGE-A4 cotransfection was significantly higher than the group with p53 transfection alone (p<0.01).
3. The effect of MAGE-A4 on p21WAF1 protein expression
In H1299 cells, p21WAF1 protein expression in the group with p53 transfection alon was significantly higher than the control group (p<0.01), and p21WAF1 protein expression in the group with p53 and MAGE-A4 cotransfection was significantly higher than the group with p53 transfection alone (p<0.01).
4. The effect of MAGE-A4 on the colony formation in H1299 cells
In H1299 cells, the colony numbers with G418 resistance were 96±3, 47±2 and 15±1 in the groups transfected with empty vector, 0.5μg pcDNA3-p53 and 0.5μg pcDNA3-p53/0.5μg FLAG-pcDNA3-MAGE-A4, respectively. There were significant difference among these three groups (p<0.01).
5. The effect of MAGE-A4 on the apoptosis of H1299 cells
TUNEL staining experiment showed that the apoptotic rate of H1299 cells was 1%, 6.8% and 19.3%, respectively after transfection with 1μg pcDNA3, 0.5μg pcDNA3-p53 and 0.5μg pcDNA3-p53 plus 0.5μg FLAG-pcDNA3-MAGE-A4, respectively. There were significant difference among these three groups (p<0.01).
6. The localization of MAGE-A4 in cells
Cell fractionation assay confiemed that the exogenous MAGE-A4 was expressed in both cytoplasm and cell nucleus in H1299 cells.
7. The colocolization of MAGE-A4 and p73
Immunofluorescence showed that in H1299 cells p73 was located in cell nucleus and part of MAGE-A4 was located in cytoplasm and another part of MAGE-A4 was located in cell nucleus, suggestiong that p73 and MAGE-A4 co-locolized in cell nucleus.
8. The physical interaction between MAGE-A4 and p53
Immunoprecipitation experiment showed that after co-transfected with MAGE-A4 and p53 plasimids, the immunoprecipitates of p53 contained MAGE-A4 and the immunoprecipitates of MAGE-A4 contained p53.
9. The physical interaction between MAGE-A4 and p73
Immunoprecipitation experiment showed that after co-transfected with MAGE-A4 and p73 plasimids, the immunoprecipitates of p73 contained MAGE-A4 and the immunoprecipitates of MAGE-A4 contained p73.
Conclusions:
1. MAGE-A4 could enhance the transcriptional activity of p53.
2. MAGE-A4 could enhance the proliferation inhibition role and apoptosis activity induced by p53.
3. MAGE-A4 was expressed in both cytoplasm and cell nucleus in H1299 cells.
4. p73 and MAGE-A4 co-locolized in cell nucleus.
5. MAGE-A4 physically interacted with p53 and p73.
Part IV The effect of MAGE-A4 siRNA on the apoptosis of MDA-MB-436 cells
Objective: To investigate the effect of MAGE-A4 siRNA on the apoptosis of MDA-MB-436 cells.
Methods : SiRNA knockdown experiment was used to inhibit the expression of MAGE-A4. TUNEL assay and flow cytometry (FCM) and PARP cleavage detection were used to investigate the effect of MAGE siRNA on the apoptosis of MDA-MB-436 cells in response to CDDP.
Results:
1. The inhibitory efficancy of MAGE-A4 siRNA on the expression of MAGE-A4 in MDA-MB-436 cells
After the 48h transfection of MAGE-A4 siRNA, the mRNA expression of MAGE-A4 was significantly inhibited. The inhibitory efficancy were 87.5% and 97.5% after 30pmol and 60pmol MAGE-A4 siRNA transfection.
2. The result of MTT assay
The cell survivals of MDA-MB-436 cells were 99.20% and 98.62% after ADR (1μM) treatment for 24h and 48h, respectively. Whereas the cell survivals of MDA-MB-436 cells were 79.44% and 68.10% after CDDP (10μM) treatment for 24h and 48h, respectively.
3. The result of TUNEL staining
TUNEL staining showed that after MAGE-A4 siRNA transfection for 48h and CDDP (10μM) treatment for 12h and 24h, the apoptotic rate of MDA-MB-436 cells were 9.1% and 2.2%,which were significantly decresed as compared with the control group (24.1% and 10.0%) (P<0.05).
4. The result of flow cytometry assay
After MAGE-A4 siRNA transfection for 48h and CDDP (10μM) treatment for 24h, the subG1% were (4.68±0.04)%,which were significantly decresed as compared with the control group (10.88±0.119)% (P<0.05).
5. The cleavage of PARP protein
Western blot result showed that after MAGE-A4 siRNA transfection for 48h and CDDP (10μM) treatment for 24h, the caspase substrate PARP were cleaved. The tario of gray scale was 0.129±0.014, which was significantly decreased as compared with the control (0.549±0.023) (P<0.05).
Conclusions:
1. MAGE-A4 siRNA has obvious inhibitory effects on MAGE-A4 expression.
2. MDA-MB-436 was resistant to ADR but sensitive to CDDP.
3. MAGE-A4 siRNA could reduce the apoptosis induced by CDDP in MDA-MB-436 cells, suggesting that MAGE-A4 played a role of tumor suppressor under certain conditions.
引文
1 Kirkin AF, Dzhandahugazyan KN, Zeuthen J. Cancer/testis antigens: structural and immunobiological properties. Cancer invest, 2002, 20(2): 222-236
2 Scanlan M, Gure AO, Jungblutn AA, et al. Cancer/testis antigens: an expanding family of targets for cancer immunotherapy. Immumo, 2002, 188: 22-32
3 Chomez P, De Backer O, Bertrand M, et al. An overview of the MAGE gene family with the identification of all human members of the family. Cancer Res, 2001, 61(14):5544–5551
4 Ohman Forslund K, Nordqvist K. The melanoma antigen genes--any clues to their functions in normal tissues? Exp Cell Res, 2001, 265(2):185-194
5 Peikert T, Specks U, Farver C, et al. Melanoma antigen A4 is expressed in non-small cell lung cancers and promotes apoptosis. Cancer Res, 2006, 66(9):4693-4700
6 Chen H, Cai S, Wang Y, et al. Expression of the MAGE-1 gene in human hepatocellular carcinomas. Clin Med J (Engl), 2000, 113(12):1112-1118
7 Lin J, Lin L, Thomas DG, et al. Melanoma-associated antigens in esophageal adenocarcinoma: identification of novel MAGE-A10 splice variants. Clin Cancer Res, 2004, 10(17):5708-5716
8 Liu J, Wang G, Okutomi T, et al. Expression of MAGE-A1 and MAGE-A3 genes in human salivary gland carcinomas. Clin Med J (Engl), 2003, 116(6):897-900
9 Picard V, Bergeron A, Larue H, et al. MAGE-A9 mRNA and protein expression in bladder cancer. Int J Cancer, 2007, 120(10):2170-2177
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13 Napoletano C, Bellati F, Tarquini E, et al. MAGE-A and NY-ESO-1 expression in cervical cancer: prognostic factors and effects of chemotherapy. Am J Obstat Gynecol, 2008, 198(1):99.e1-7
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9 Peikert T, Specks U, Farver C, et al. Melanoma antigen A4 is expressed in non-small cell lung cancers and promotes apoptosis. Cancer Res, 2006, 66(9):4693-4700.
1 Kirkin AF, Dzhandahugazyan KN, Zeuthen J. Cancer/testis antigens: structural and immunobiological properties. Cancer invest, 2002, 20(2): 222-236
2 Scanlan M, Gure AO, Jungblutn AA, et al. Cancer/testis antigens: an expanding family of targets for cancer immunotherapy. Immumo, 2002, 188: 22-32
3 Ohman Forslund K, Nordqvist K. The melanoma antigen genes--any clues to their functions in normal tissues? Exp Cell Res, 2001, 265(2):185-194
4 Chomez P, De Backer O, Bertrand M, et al. An overview of the MAGE gene family with the identification of all human members of the family. Cancer Res, 2001, 61(14):5544–5551
5 Van der Bruggen P, Traversari C, Chomez P, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science, 1991, 254(5038):1643-1647
6 Chen H, Cai S, Wang Y, et al. Expression of the MAGE-1 gene in human hepatocellular carcinomas. Clin Med J(Engl), 2000, 113(12):1112-1118
7 Picard V, Bergeron A, Larue H, et al. MAGE-A9 mRNA and protein expression in bladder cancer. Int J Cancer, 2007, 120(10):2170-2177
8 Jang SJ, Soria JC, Wang L, et al. Activation of melanoma antigen tumor antigens occurs early in lung carcinogenesis. Cancer Res, 2001; 61:7959–7963
9 Peikert T, Specks U, Farver C, et al. Melanoma antigen A4 is expressed in non-small cell lung cancers and promotes apoptosis. Cancer Res, 2006, 66(9):4693-4700
10 Otte M, Zafrakas M, Riethdorf L, et al. MAGE-A gene expression pattern in primary breast cancer. Cancer Res, 2001, 61(18):6682-6687
11 Kim KH, Choi JS, Kim IJ, et al. Promoter hypomethylation and reactivation of MAGE-A1 and MAGE-A3 genes in colorectal cancer cell lines and cancer tissues. World J Gastroenterol, 2006, 12(35):5651-5657
12 Liu J, Wang G, Okutomi T, et al. Expression of MAGE-A1 and MAGE-A3 genes in human salivary gland carcinomas. Clin Med J (Engl), 2003,116(6):897-900
13 Napoletano C, Bellati F, Tarquini E, et al. MAGE-A and NY-ESO-1 expression in cervical cancer: prognostic factors and effects of chemotherapy. Am J Obstat Gynecol, 2008, 198(1):99.e1-7
14 Lin J, Lin L, Thomas DG, et al. Melanoma-associated antigens in esophageal adenocarcinoma: identification of novel MAGE-A10 splice variants. Clin Cancer Res, 2004, 10(17):5708-5716
15 Yakirevich E, Sabo E, Lavie O, et al. Expression of the MAGE-A4 and NY-ESO-1 cancer-testis antigens in serous ovarian neoplasms. Clin Cancer Res, 2003, 9(17):6453-6460
16 Chambost H, Van Baren N, Brasseur F, et al. Expression of gene MAGE-A4 in Reed-Sternberg cells. Blood, 2000, 95(11):3530-3533
17 Weber J, Salgaller M, Samid D, et al. Expression of the MAGE-1 tumor antigen is up-regulated by the demethylating agent 5-aza-2'-deoxycytidine.Cancer Res, 1994, 54(7):1766-1771
18 Mori M, Inoue H, Mimori K, et al. Expression of MAGE genes in human colorectal carcinoma. Ann Surg, 1996, 224(2):183-188
19 Fujie T, Mori M, Ueo H, et al. Expression of MAGE and BAGE genes in Japanese breast cancers. Ann Oncol, 1997, 8(4):369-372
20 De Smet C, Lurquin C, LethéB, et al. DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter. Mol Cell Biol, 1999, 19(11):7327-7335
21 Kufer P. zippelius A, Lutterbuse R, et al. Heterogeneous expression of MAGE-A genes in occult disseminated tumor cells: a novel multimarker reverse transcription-polymerase chain reaction for diagnosis of micrometastatic disease. Cancer Res, 2002, 62(1):251-261
22 Mou DC, Cai SL, Peng JR, et al. Evaluation of MAGE-1 and MAGE-3 as tumour-specific markers to detect blood dissemination of hepatocellular carcinoma cells. Br J Cancer, 2002, 86(1):110-116
23 Kwon S, Kang SH, Ro J, et al. The melanoma antigen gene as a surveillance marker for the detection of circulating tumor cells in patients with breast carcinoma. Cancer, 2005, 104(2):251-256
24 Bandic D, Juretic A, Sarcevic B, et al. Expression and possible prognostic role of MAGE-A4, NY-ESO-1, and HER-2 antigens in women with relapsing invasive ductal breast cancer: retrospective immunohistochemical study. Croat Med J, 2006, 47(1):32-41
25 Gillespie AM, Coleman RE. The potential of melanoma antigen expression in cancer therapy. Cancer Treat Rev, 1999, 25(4):219-227
26 Monte M, Simonatto M, Peche LY, et al. MAGE-A tumor antigens target p53 transactivation function through histone deacetylase recruitment and confer resistance to chemotherapeutic agents. Proc Natl Acad Sci USA, 2006, 103(30):11160-11165
27 Morishima N, Nakanishi K, Takenouchi H, et al. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12. J Biol Chem, 2002,277:34287-34294
28 Nagao T, Higashitsuji H, Nonoguchi K, et al. MAGE-A4 interacts with the liver oncoprotein gankyrin and suppresses its tumorigenic activity. J Bio Chem, 2003, 278(12):10668-10674
29 Sakurai T, Itoh K, Higashitsuji H, et al. A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. J Bio Chem, 2004, 279(15):15505-15514
30 Pascolo S, Schirle M, Guckel B, et al. A MAGE-A1 HLA-A A*0201 epitope identified by mass spectrometry. Cancer Res, 2001, 61(10):4072-4077
31 Zhang Y, Stroobant V, Russo V, et al. A MAGE-A4 peptide presented by HLA-B37 is recognized on human tumors by cytolytic T lymphocytes. Tissue Antigens, 2002, 60(5):365-371
32 Hilling RC, Coulie PG, Stroobant V, et al. High-resolution structure of HLA-A*0201 in complex with a tumour-specific antigenic peptide encoded by the MAGE-A4 gene. J Mol Biol, 2001, 310(5):1167-1176
33 Heidecker L, Brasseur F, Probst-Kepper M, et al. Cytolytic T lymphocytes raised against a human bladder carcinoma recognize an antigen encoded by gene MAGE-A12. J Immunol, 2000, 164(11):6041-6045
34 Marchand M, Van Baren N, Weynants P, et al. Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int J Cancer, 1999, 80(2):219-230
35 Karanikas V, Lurquin C, Colau D, et al. Monoclonal anti-MAGE-3 CTL responses in melanoma patients displaying tumor regression after vaccination with a recombinant canarypox virus.J Immunol, 2003, 171(9):4898-4904
36 Lonchay C, Van der Bruggen P, Connerotte T, et al. Correlation between tumor regression and T cell responses in melanoma patients vaccinated with a MAGE antigen. Proc Natl Acad Sci USA, 2004, 101(Suppl 2):14631-14638
37 Marchand M, Punt CJ, Aamdal S, et al. Immunisation of metastatic cancer patients with MAGE-3 protein combined with adjuvant SBAS-2: a clinical report. Eur J Cancer, 2003, 39(1):70-77
38 Thurner B, Haendle I, Roder C, et al. Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med, 1999, 190(11):1669-1678
39 Van pel A, De plaen E, Dufferour MT, et al Induction of cytolytic T lymphocytes by immunization of mice with an adenovirus containing a mouse homolog of the human MAGE-A genes. Cancer Immunol Immunother, 2001, 49(11):593-602
2 Scanlan M, Gure AO, Jungblutn AA, et al. Cancer/testis antigens: an expanding family of targets for cancer immunotherapy. Immumo, 2002, 188: 22-32
3 Chomez P, De Backer O, Bertrand M, et al. An overview of the MAGE gene family with the identification of all human members of the family. Cancer Res, 2001, 61(14):5544–5551
4 Ohman Forslund K, Nordqvist K. The melanoma antigen genes--any clues to their functions in normal tissues? Exp Cell Res, 2001, 265(2):185-194
5 Peikert T, Specks U, Farver C, et al. Melanoma antigen A4 is expressed in non-small cell lung cancers and promotes apoptosis. Cancer Res, 2006, 66(9):4693-4700
6 Chen H, Cai S, Wang Y, et al. Expression of the MAGE-1 gene in human hepatocellular carcinomas. Clin Med J (Engl), 2000, 113(12):1112-1118
7 Lin J, Lin L, Thomas DG, et al. Melanoma-associated antigens in esophageal adenocarcinoma: identification of novel MAGE-A10 splice variants. Clin Cancer Res, 2004, 10(17):5708-5716
8 Liu J, Wang G, Okutomi T, et al. Expression of MAGE-A1 and MAGE-A3 genes in human salivary gland carcinomas. Clin Med J (Engl), 2003, 116(6):897-900
9 Picard V, Bergeron A, Larue H, et al. MAGE-A9 mRNA and protein expression in bladder cancer. Int J Cancer, 2007, 120(10):2170-2177
10 Kufer P, zippeliusA, Lutterbuse R, et al. Heterogeneous expression of MAGE-A genes in occult disseminated tumor cells: a novel multimarkerreverse transcription-polymerase chain reaction for diagnosis of micrometastatic disease. Cancer Res, 2002, 62(1):251-261
11 Yakirevich E, Sabo E, Lavie O, et al. Expression of the MAGE-A4 and NY-ESO-1 cancer-testis antigens in serous ovarian neoplasms. Clin Cancer Res, 2003, 9(17):6453-6460
12 Chambost H, Van Baren N, Brasseur F , et al. Expression of gene MAGE-A4 in Reed-Sternberg cells. Blood, 2000, 95(11):3530-3533
13 Napoletano C, Bellati F, Tarquini E, et al. MAGE-A and NY-ESO-1 expression in cervical cancer: prognostic factors and effects of chemotherapy. Am J Obstat Gynecol, 2008, 198(1):99.e1-7
14 Van der Bruggen P, Traversari C, Chomez P, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science, 1991, 254(5038):1643-1647
15 Otte M, Zafrakas M, Riethdorf L, et al. MAGE-A gene expression pattern in primary breast cancer. Cancer Res, 2001, 61(18):6682-6687
16 Bandic D, Juretic A, Sarcevic B, et al. Expression and possible prognostic role of MAGE-A4, NY-ESO-1, and HER-2 antigens in women with relapsing invasive ductal breast cancer: retrospective immunohistochemical study. Croat Med J, 2006, 47(1):32-41
17 Jang SJ, Soria JC, Wang L, et al. Activation of melanoma antigen tumor antigens occurs early in lung carcinogenesis. Cancer Res, 2001; 61:7959–7963
18 Gillespie AM, Coleman RE. The potential of melanoma antigen expression in cancer therapy. Cancer Treat Rey, 1999, 25(4):219-227
19 De Smet C, Lurquin C, LethéB, et al. DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter. Mol Cell Biol, 1999, 19(11):7327-35
20 Nagao T, Higashitsuji H, Nonoguchi K, et al. MAGE-A4 interacts with the liver oncoprotein gankyrin and suppresses its tumorigenic activity. J Bio Chem, 2003, 278(12):10668-10674
21 Sakurai T, Itoh K, Higashitsuji H, et al. A cleaved form of MAGE-A4binds to Miz-1 and induces apoptosis in human cells. J Bio Chem, 2004, 279(15): 15505-15514
1 Bandic D, Juretic A, Sarcevic B, et al. Expression and possible prognostic role of MAGE-A4, NY-ESO-1, and HER-2 antigens in women with relapsing invasive ductal breast cancer: retrospective immunohistochemical study. Croat Med J, 2006, 47(1):32-41
2 Peikert T, Specks U, Farver C, et al. Melanoma antigen A4 is expressed innon-small cell lung cancers and promotes apoptosis. Cancer Res, 2006, 66(9):4693-4700
3 Nagao T, Higashitsuji H, Nonoguchi K, et al. MAGE-A4 interacts with the liver oncoprotein gankyrin and suppresses its tumorigenic activity. J Bio Chem, 2003, 278(12):10668-10674
4 Sakurai T, Itoh K, Higashitsuji H, et al. A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. J Bio Chem, 2004, 279(15):15505-15514
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10 Nagao T, Higashitsuji H, Nonoguchi H, et al. MAGE-A4 interacts with the liver oncoprotein gankyrin and suppresses its tumorigenic activity. J Bio Chem, 2003, 278(12):10668-10674
11 Sakurai T, Itoh K, Higasgistuji H, et al. A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. J Biol Chem, 2004, 279(15): 15505-15514
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6 Bandic D, Juretic A, Sarcevic B, et al. Expression and possible prognosticrole of MAGE-A4, NY-ESO-1, and HER-2 antigens in women with relapsing invasive ductal breast cancer: retrospective immunohistochemical study. Croat Med J, 2006, 47(1):32-41.
7 Sakurai T, Itoh K, Higashitsuji H, et al. A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. J Bio Chem, 2004, 279(15): 15505-15514.
8 Nagao T, Higashitsuji H, Nonoguchi K, et al. MAGE-A4 interacts with the liver oncoprotein gankyrin and suppresses its tumorigenic activity. J Bio Chem, 2003, 278(12):10668-10674.
9 Peikert T, Specks U, Farver C, et al. Melanoma antigen A4 is expressed in non-small cell lung cancers and promotes apoptosis. Cancer Res, 2006, 66(9):4693-4700.
1 Kirkin AF, Dzhandahugazyan KN, Zeuthen J. Cancer/testis antigens: structural and immunobiological properties. Cancer invest, 2002, 20(2): 222-236
2 Scanlan M, Gure AO, Jungblutn AA, et al. Cancer/testis antigens: an expanding family of targets for cancer immunotherapy. Immumo, 2002, 188: 22-32
3 Ohman Forslund K, Nordqvist K. The melanoma antigen genes--any clues to their functions in normal tissues? Exp Cell Res, 2001, 265(2):185-194
4 Chomez P, De Backer O, Bertrand M, et al. An overview of the MAGE gene family with the identification of all human members of the family. Cancer Res, 2001, 61(14):5544–5551
5 Van der Bruggen P, Traversari C, Chomez P, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science, 1991, 254(5038):1643-1647
6 Chen H, Cai S, Wang Y, et al. Expression of the MAGE-1 gene in human hepatocellular carcinomas. Clin Med J(Engl), 2000, 113(12):1112-1118
7 Picard V, Bergeron A, Larue H, et al. MAGE-A9 mRNA and protein expression in bladder cancer. Int J Cancer, 2007, 120(10):2170-2177
8 Jang SJ, Soria JC, Wang L, et al. Activation of melanoma antigen tumor antigens occurs early in lung carcinogenesis. Cancer Res, 2001; 61:7959–7963
9 Peikert T, Specks U, Farver C, et al. Melanoma antigen A4 is expressed in non-small cell lung cancers and promotes apoptosis. Cancer Res, 2006, 66(9):4693-4700
10 Otte M, Zafrakas M, Riethdorf L, et al. MAGE-A gene expression pattern in primary breast cancer. Cancer Res, 2001, 61(18):6682-6687
11 Kim KH, Choi JS, Kim IJ, et al. Promoter hypomethylation and reactivation of MAGE-A1 and MAGE-A3 genes in colorectal cancer cell lines and cancer tissues. World J Gastroenterol, 2006, 12(35):5651-5657
12 Liu J, Wang G, Okutomi T, et al. Expression of MAGE-A1 and MAGE-A3 genes in human salivary gland carcinomas. Clin Med J (Engl), 2003,116(6):897-900
13 Napoletano C, Bellati F, Tarquini E, et al. MAGE-A and NY-ESO-1 expression in cervical cancer: prognostic factors and effects of chemotherapy. Am J Obstat Gynecol, 2008, 198(1):99.e1-7
14 Lin J, Lin L, Thomas DG, et al. Melanoma-associated antigens in esophageal adenocarcinoma: identification of novel MAGE-A10 splice variants. Clin Cancer Res, 2004, 10(17):5708-5716
15 Yakirevich E, Sabo E, Lavie O, et al. Expression of the MAGE-A4 and NY-ESO-1 cancer-testis antigens in serous ovarian neoplasms. Clin Cancer Res, 2003, 9(17):6453-6460
16 Chambost H, Van Baren N, Brasseur F, et al. Expression of gene MAGE-A4 in Reed-Sternberg cells. Blood, 2000, 95(11):3530-3533
17 Weber J, Salgaller M, Samid D, et al. Expression of the MAGE-1 tumor antigen is up-regulated by the demethylating agent 5-aza-2'-deoxycytidine.Cancer Res, 1994, 54(7):1766-1771
18 Mori M, Inoue H, Mimori K, et al. Expression of MAGE genes in human colorectal carcinoma. Ann Surg, 1996, 224(2):183-188
19 Fujie T, Mori M, Ueo H, et al. Expression of MAGE and BAGE genes in Japanese breast cancers. Ann Oncol, 1997, 8(4):369-372
20 De Smet C, Lurquin C, LethéB, et al. DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter. Mol Cell Biol, 1999, 19(11):7327-7335
21 Kufer P. zippelius A, Lutterbuse R, et al. Heterogeneous expression of MAGE-A genes in occult disseminated tumor cells: a novel multimarker reverse transcription-polymerase chain reaction for diagnosis of micrometastatic disease. Cancer Res, 2002, 62(1):251-261
22 Mou DC, Cai SL, Peng JR, et al. Evaluation of MAGE-1 and MAGE-3 as tumour-specific markers to detect blood dissemination of hepatocellular carcinoma cells. Br J Cancer, 2002, 86(1):110-116
23 Kwon S, Kang SH, Ro J, et al. The melanoma antigen gene as a surveillance marker for the detection of circulating tumor cells in patients with breast carcinoma. Cancer, 2005, 104(2):251-256
24 Bandic D, Juretic A, Sarcevic B, et al. Expression and possible prognostic role of MAGE-A4, NY-ESO-1, and HER-2 antigens in women with relapsing invasive ductal breast cancer: retrospective immunohistochemical study. Croat Med J, 2006, 47(1):32-41
25 Gillespie AM, Coleman RE. The potential of melanoma antigen expression in cancer therapy. Cancer Treat Rev, 1999, 25(4):219-227
26 Monte M, Simonatto M, Peche LY, et al. MAGE-A tumor antigens target p53 transactivation function through histone deacetylase recruitment and confer resistance to chemotherapeutic agents. Proc Natl Acad Sci USA, 2006, 103(30):11160-11165
27 Morishima N, Nakanishi K, Takenouchi H, et al. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12. J Biol Chem, 2002,277:34287-34294
28 Nagao T, Higashitsuji H, Nonoguchi K, et al. MAGE-A4 interacts with the liver oncoprotein gankyrin and suppresses its tumorigenic activity. J Bio Chem, 2003, 278(12):10668-10674
29 Sakurai T, Itoh K, Higashitsuji H, et al. A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. J Bio Chem, 2004, 279(15):15505-15514
30 Pascolo S, Schirle M, Guckel B, et al. A MAGE-A1 HLA-A A*0201 epitope identified by mass spectrometry. Cancer Res, 2001, 61(10):4072-4077
31 Zhang Y, Stroobant V, Russo V, et al. A MAGE-A4 peptide presented by HLA-B37 is recognized on human tumors by cytolytic T lymphocytes. Tissue Antigens, 2002, 60(5):365-371
32 Hilling RC, Coulie PG, Stroobant V, et al. High-resolution structure of HLA-A*0201 in complex with a tumour-specific antigenic peptide encoded by the MAGE-A4 gene. J Mol Biol, 2001, 310(5):1167-1176
33 Heidecker L, Brasseur F, Probst-Kepper M, et al. Cytolytic T lymphocytes raised against a human bladder carcinoma recognize an antigen encoded by gene MAGE-A12. J Immunol, 2000, 164(11):6041-6045
34 Marchand M, Van Baren N, Weynants P, et al. Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int J Cancer, 1999, 80(2):219-230
35 Karanikas V, Lurquin C, Colau D, et al. Monoclonal anti-MAGE-3 CTL responses in melanoma patients displaying tumor regression after vaccination with a recombinant canarypox virus.J Immunol, 2003, 171(9):4898-4904
36 Lonchay C, Van der Bruggen P, Connerotte T, et al. Correlation between tumor regression and T cell responses in melanoma patients vaccinated with a MAGE antigen. Proc Natl Acad Sci USA, 2004, 101(Suppl 2):14631-14638
37 Marchand M, Punt CJ, Aamdal S, et al. Immunisation of metastatic cancer patients with MAGE-3 protein combined with adjuvant SBAS-2: a clinical report. Eur J Cancer, 2003, 39(1):70-77
38 Thurner B, Haendle I, Roder C, et al. Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med, 1999, 190(11):1669-1678
39 Van pel A, De plaen E, Dufferour MT, et al Induction of cytolytic T lymphocytes by immunization of mice with an adenovirus containing a mouse homolog of the human MAGE-A genes. Cancer Immunol Immunother, 2001, 49(11):593-602