Development and clinical application of the molecular diagnostic tests to select anti-cancer drugs

耐药是白血病患者化疗的主要障碍。由于患者的治疗反应性不同，因此治疗应针对特定的疾病亚型，如耐药性定义的疾病亚型。为了筛选与耐药性相关的候选基因，我们使用市售的cDNA微阵列Human Cancer CHIP version 3.0（TaKaRa Biotechnology）分析了耐药性白血病细胞中的基因表达谱。它携带了630个互补DNA的探针组，这些探针组被设计用于监测基因，这些基因的表达被认为与癌症的生物过程有关。从抗苯三甲氧嘧啶的白血病MOLT-3细胞（MOLT-3/TMQ 200）和敏感细胞中提取一对RNA样品，并在逆转录反应期间用Cy 3或Cy 5染料独立地荧光标记。将样品混合物与点样到微阵列上的靶竞争性杂交，并用扫描阵列4000（通用扫描）扫描和计算原始数字图像。除了作为分子机制的已知基因如MDR 1的过表达之外，还出现了参与DNA修复或基因转录的新基因如ATM的过表达。下调的基因很多，包括细胞粘附和细胞内信号转导相关基因。另一项用于对艾达鲁肽耐药的MOLT-3的基因表达分析的实验显示，拓扑异构酶抑制剂II α的表达降低，而GS 3955蛋白（推定的丝氨酸/苏氨酸激酶）的表达增加。这些结果通过北方印迹分析证实。这种由上调和下调的基因组成的特异性基因表达谱可以指示具有药物抗性的细胞，并且其检测可以预测响应。基因表达谱分析将有助于筛选新的耐药基因并对相关基因进行注释，从而阐明耐药的分子机制。

Drug resistance is a major obstacle in chemotherapy of leukemia patients. Since the treatment responsiveness is different among patients, the therapy should be tailored to specific subtypes of diseases such as defined by drug resistance. To screen candidate genes putatively involved in the drug resistance, we analyzed gene expression profiling in drug-resistant leukemia cells using a commercially available cDNA microarray, Human Cancer CHIP version 3.0 (TaKaRa Biotechnology). This carries probe sets for 630 complementary DNAs designed to monitor genes of which expression is assumed to be involved in the biological process of cancer. A pair of RNA samples was extracted from trimetrexate-resistant leukemia MOLT-3 cells (MOLT-3/TMQ200) and sensitive cells, and independently fluorescence-labeled with Cy3 or Cy5 dye during reverse-transcription reaction. A sample mixture was competitively hybridized with the targets spotted onto the microarray, and a raw digital image was scanned and computed with Scan Array 4000 (General Scanning). In addition to overexpression of known genes as molecular mechanisms such as MDR1, there appeared overexpression of novel genes that are involved in DNA repair or gene transcription such as ATM. There were many down-regulated genes, including those involved in cell adhesion and intracellular signal transduction. Another experiment for gene expression analysis of MOLT-3 resistant to idarubicin showed decreased expression of topoisomerase inhibitor II alfa and increased expression of GS3955 protein (putative serine/threonine kinase). These results were confirmed by Northern blot analysis. This specific gene expression profiling comprised of those up-regulated and down-regulated may be indicative of cells with resistance to drugs, and its detection may predict the response. Gene expression profiling analysis will clarify molecular mechanisms of drug resistance by screening of novel genes and annotation that are involved in it.