Development of the molecular diagnostic tests for drug-resistant cancer cells based on gene expression profiling using microarray

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。我们试图利用微阵列技术开发基于基因表达谱的耐药癌细胞分子诊断测试。本研究的目的是通过分析候选基因在耐药中的功能，选择一个用于诊断阵列的基因面板。ATM基因被选为一个新的候选基因。利用市售的cDNA微阵列（TaKaRa Biotechnology, Human Cancer CHIP version 3.0）进行基因表达谱分析，发现ATM在耐甲氨蝶呤白血病MOLT-3细胞（MOLT-3/TMQ200）中过表达。经Northern blot分析证实。将插入ATM siRNA的质粒转染到MOLT-3/TMQ200细胞后，RT-PCR结果显示ATM表达降低。从MOLT-3/TMQ细胞中获得的三个亚系中，实时PCR检测到ATM的表达水平与耐药程度平行升高。通过琼脂糖克隆测定中的氧化石墨烯值评估的辐照抗性在三个亚系中与ATM水平平行增加。该基因已被证实在耐药机制中发挥作用，将为基于基因表达谱的耐药癌细胞分子诊断测试提供一个有用的标记。

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. We attempted to develop the molecular diagnostic tests for drug-resistant cancer cells based on gene expression profiling using a microarray method. The aim of this study was to select a gene panel for a diagnostic array by analyzing function of candidate genes in the drug resistance. The ATM gene was chosen as a novel and candidate gene. ATM was found overexpressed in trimetrexate-resistant leukemia MOLT-3 cells (MOLT-3/TMQ200) as analyzed by gene expression profiling using a commercially available cDNA microarray (Human Cancer CHIP version 3.0, TaKaRa Biotechnology). It was confirmed by Northern blot analysis. When the plasmid with insertion of the siRNA for ATM was transfected to MOLT-3/TMQ200 cells, ATM expression was decreased as analyzed by RT-PCR. Expression levels of ATM as assessed by real-time PCR was increased in parallel with drug resistance degree in three sublines obtained from MOLT-3/TMQ cells. Resistance to irradiation as assessed by GO values in agarose clonogenic assay was increased in parallel with the ATM levels in three sublines. The novel gene which was demonstrated as functioning in the mechanisms of drug resistance would be a useful marker for molecular diagnostic tests for drug-resistant cancer cells based on gene expression profiling using a microarray method.