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

抗癌药物分子诊断检测技术的开发及临床应用

基本信息

批准号：
15590498
负责人：
MIYACHI Hayato
金额：
$ 2.3万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590498/
关键词：
anti-cancer agents DNA microarray nucleic acid test gene expression profiling leukemia 白血病細胞抗癌剤耐性 DNA マイクロアレイ個別化治療遺伝子

项目摘要

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.

耐药是白血病患者化疗的主要障碍。由于不同患者的治疗反应不同，治疗应针对特定的亚型疾病，如耐药定义。为了筛选可能与耐药相关的候选基因，我们使用商用的基因芯片芯片3.0版(Takara Biotech)分析了耐药白血病细胞的基因表达谱。它携带了630个互补DNA的探针组，旨在监测那些被认为参与癌症生物过程的基因的表达。从耐曲霉胺白血病MOLT-3细胞(MOLT-3/TMQ200)和敏感细胞中提取一对RNA，在逆转录反应中分别用Cy3或Cy5染料进行荧光标记。样品混合物与微阵列上斑点的目标竞争性杂交，原始数字图像用扫描阵列4000(通用扫描)扫描和计算。除了作为分子机制的已知基因的过度表达，如mdr1，还出现了与DNA修复或基因转录有关的新基因的过度表达，如ATM。有许多基因表达下调，包括与细胞黏附和细胞内信号转导有关的基因。另一项对伊达比星耐药的MOLT-3进行的基因表达分析显示，拓扑异构酶抑制剂IIα的表达降低，而GS3955蛋白(可能的丝氨酸/苏氨酸激酶)的表达增加。Northern印迹分析证实了上述结果。这种由上调和下调的基因表达谱组成的特异性基因表达谱可能表明细胞对药物具有耐药性，其检测可能预测反应。基因表达谱分析将通过筛选与耐药相关的新基因和注释来阐明耐药的分子机制。