权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Development and clinical application of the molecular diagnostic tests for individualization of cancer chemotherapy: detection of gene polymorphism of metabolizing enzyme and expression pattern

癌症化疗个体化分子诊断检测的开发及临床应用：代谢酶基因多态性及表达模式检测

基本信息

批准号：
12672254
负责人：
MIYACHI Hayato
金额：
$ 1.54万
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：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

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 elucidate mechanism(s) of methotrexate (MTX)-resistance as a possible reason behind treatment failure in high-dose MTX regimens combined with leucovorin (LV) rescue, we established MTX-resistant human T-cell leukemia cell line CCRF-CEM cells in the presence of excessive leucovorin, and characterized their property. Continuous exposure of the cells to escalating concentrations of MTX up to 20μM in the presence of 1000 nM leucovorin resulted in establishment of three MTX-resistant sublines with a wide disparity of resistance degree over 4 logarithmic range by approximately 40-, 900- and 44000-fold, respectively. Transraembrane transport of MTX in these sublines was diminished to 52%, 35% and 12%, respectively. Intracellular retention of MTX in these s … More ublines was not different from the parent cells. A cell growth study in various concentrations of leucovorin showed that cells with higher resistance to MTX required more leucovorin supply for their optimal growth. In parallel with the resistance levels, there was increase in mRNA expression of dihydrofolate reductase gene and decrease in that of thymidylate synthase gene, but no change in that of reduced folate carrier (RFC1) gene, as assessed by northern blot analysis. Sequencing of RFC1 gene in all of the 3 sublines revealed a point mutation in codon 47 (TCC→TTC) resulting in substitution of Phe for Ser residue, and additional deletion of CTG of codon 112 in the subline with the highest resistance. In summary, MTX exposure to CCRF-CEM cells in the presence of 1000 nM leucovorin resulted in an establishment of heterogeneous cell populations with a wide range of transport-mediated MTX-resistance, which was associated with differential alterations of RFC gene. Combination of gene expression and mutations involved in the molecular mechanisms of MTX-resistance may give targets for detection and evaluation of it. Less

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。为了阐明甲氨蝶呤（MTX）耐药的可能机制，即高剂量MTX方案联合甲酰四氢叶酸（LV）挽救治疗失败的可能原因，我们在过量甲酰四氢叶酸存在下建立了MTX耐药的人T细胞白血病细胞系CCRF-CEM细胞，并表征其性质。在1000 nM亚叶酸存在下，将细胞连续暴露于浓度递增至20μM的MTX，导致建立了三种MTX耐药亚系，其耐药程度在4个对数范围内分别相差约40、900和44000倍。MTX的跨膜转运分别减少52%、35%和12%。MTX在这些细胞中的细胞内滞留 ...更多信息亚系与亲本细胞没有不同。在不同浓度的亚叶酸中进行的细胞生长研究表明，对甲氨蝶呤耐药性较高的细胞需要更多的亚叶酸供应才能达到最佳生长。北方印迹分析表明，随着抗性水平的提高，二氢叶酸还原酶基因mRNA表达量增加，胸苷酸合成酶基因mRNA表达量下降，而还原型叶酸载体（RFC 1）基因mRNA表达量无明显变化。3个亚系的RFC 1基因测序结果均显示47位密码子发生点突变（TCC→TTC），导致Ser残基被Phe取代，其中抗性最高的亚系还缺失了112位密码子的CTG。总之，MTX暴露于CCRF-CEM细胞在1000 nM甲酰四氢叶酸的存在下，导致建立了具有广泛的转运介导的MTX耐药性的异质性细胞群，这与RFC基因的差异性改变有关。MTX耐药分子机制中基因表达和突变的结合可能为检测和评估MTX提供靶点。