Duan Zhenfeng, Duan Yifei, Lamendola Diana E, Yusuf Rushdia Z, Naeem Rizwan, Penson Richard T, Seiden Michael V
Department of Hematology/Oncology Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
Clin Cancer Res. 2003 Jul;9(7):2778-85.
Previous studies directed at identifying paclitaxel resistance genes in a paclitaxel-resistant subclone of the human ovarian cancer cell line SKOV-3 identified a novel cancer testis antigen, Taxol resistance-associated gene 3 (TRAG-3). Because investigation suggested that TRAG-3, located on chromosome Xq28, does not directly participate in the paclitaxel-resistant phenotype, it was hypothesized that TRAG-3 might be linked to a neighboring gene that is directly involved in the drug-resistant phenotype, or alternatively, overexpression of TRAG-3 might be attributable to coregulation with other cancer testis antigens. To distinguish between these two hypotheses, expression of the genes that flank TRAG-3 was evaluated, namely the Centrin 2 gene and several members of the MAGE gene cluster. Northern analysis demonstrates overexpression of MAGE2 but not Centrin 2. Extension of this analysis to other neighboring and non-neighboring representative cancer testis antigens reveals overexpression of MAGE3, MAGE6, MAGE11, and MAGE12, as well as GAGE-2, GAGE-4, GAGE-5, GAGE-6, and GAGE-7 (clustered on Xp11) in SKOV-3(TR), as compared with SKOV-3. In addition, Affymetrix-based analysis of gene expression in SKOV-3 subclones with variable paclitaxel resistance demonstrates MAGE gene overexpression occurs early in the development of the paclitaxel-resistant phenotype, whereas GAGE gene overexpression occurs somewhat later. Evaluation of additional breast and ovarian cancer cell lines reveals MAGE/GAGE overexpression in both paclitaxel- and doxorubicin-resistant cell lines, whereas gemcitabine-resistant subclones of several ovarian cancer cell lines, including SKOV-3(GR), reveals no change in MAGE/GAGE expression. To determine whether MAGE gene overexpression contributes directly to the drug-resistant phenotype, MAGE2 or MAGE6, cDNA was introduced into the paclitaxel-sensitive human ovarian cancer cell line OVCAR8. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity analysis of both MAGE2 and MAGE6 transfectants demonstrates a 4-fold increase in resistance to paclitaxel and 2-fold increase in resistance to doxorubicin but not to other drugs, such as topotecan and cisplatin, through a nonmultidrug resistance-1 mechanism. MAGE2 or MAGE6 overexpression also induces a growth advantage in OVCAR8-transfected cells. These studies suggest that the in vitro acquisition of paclitaxel and doxorubicin resistance can be associated with increased expression of a variety of both neighboring and non-neighboring cancer testis antigens genes. This does not appear to be a consequence of random genetic instability or genomic amplification of the X chromosome. These antigens, because of limited expression in normal tissues, may be suitable targets for immunotherapy and novel therapeutic strategies in the treatment of chemotherapy-resistant epithelial tumors.
先前针对人卵巢癌细胞系SKOV-3的紫杉醇耐药亚克隆中鉴定紫杉醇耐药基因的研究，确定了一种新的癌睾丸抗原，即紫杉醇耐药相关基因3（TRAG-3）。由于研究表明位于Xq28染色体上的TRAG-3并不直接参与紫杉醇耐药表型，因此推测TRAG-3可能与直接参与耐药表型的邻近基因相关，或者，TRAG-3的过表达可能归因于与其他癌睾丸抗原的共调节。为区分这两种假设，对TRAG-3侧翼的基因表达进行了评估，即中心体蛋白2基因和MAGE基因簇的几个成员。Northern分析表明MAGE2过表达而中心体蛋白2未过表达。将该分析扩展到其他邻近和非邻近的代表性癌睾丸抗原，结果显示与SKOV-3相比，SKOV-3(TR)中MAGE3、MAGE6、MAGE11和MAGE12以及GAGE-2、GAGE-4、GAGE-5、GAGE-6和GAGE-7（聚集在Xp11）过表达。此外，基于Affymetrix的对具有不同紫杉醇耐药性的SKOV-3亚克隆的基因表达分析表明，MAGE基因过表达在紫杉醇耐药表型发展的早期出现，而GAGE基因过表达出现得稍晚。对其他乳腺癌和卵巢癌细胞系的评估显示，在紫杉醇和阿霉素耐药的细胞系中MAGE/GAGE均过表达，而包括SKOV-3(GR)在内的几种卵巢癌细胞系的吉西他滨耐药亚克隆中，MAGE/GAGE表达无变化。为确定MAGE基因过表达是否直接导致耐药表型，将MAGE2或MAGE6的cDNA导入紫杉醇敏感的人卵巢癌细胞系OVCAR8。对MAGE2和MAGE6转染子的3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐细胞毒性分析表明，通过非多药耐药-1机制，对紫杉醇的耐药性增加了4倍，对阿霉素的耐药性增加了2倍，但对其他药物如拓扑替康和顺铂没有影响。MAGE2或MAGE6的过表达也在OVCAR8转染细胞中诱导了生长优势。这些研究表明，体外获得紫杉醇和阿霉素耐药性可能与多种邻近和非邻近的癌睾丸抗原基因表达增加有关。这似乎不是随机遗传不稳定或X染色体基因组扩增的结果。由于这些抗原在正常组织中表达有限，它们可能是免疫治疗和治疗化疗耐药上皮肿瘤的新治疗策略合适的靶点。
