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。由于患者对治疗的反应性不同，因此治疗应针对特定亚型的疾病进行定制，例如根据耐药性进行定义。为了筛选可能与耐药有关的候选基因，我们使用市售的cDNA微阵列Human Cancer CHIP version 3.0 （TaKaRa Biotechnology）分析了耐药白血病细胞的基因表达谱。它携带630个互补dna的探针组，用于监测那些被认为参与癌症生物学过程的基因的表达。从甲氨蝶呤耐药白血病MOLT-3细胞（MOLT-3/TMQ200）和敏感细胞中分别提取RNA样品对，在逆转录反应中分别用Cy3或Cy5染料进行荧光标记。混合样品与微阵列上的目标竞争性杂交，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MDR1过表达外，参与DNA修复或基因转录的新基因如ATM也出现过表达。下调的基因有很多，包括参与细胞粘附和细胞内信号转导的基因。另一项对耐伊达柔比星MOLT-3基因表达分析的实验显示，拓扑异构酶抑制剂II α的表达减少，GS3955蛋白（假定的丝氨酸/苏氨酸激酶）的表达增加。这些结果经Northern blot分析证实。这种由上调和下调基因组成的特异性基因表达谱可能是细胞耐药的指示，其检测可以预测药物的反应。基因表达谱分析将通过筛选和注释新基因来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.