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

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。为了阐明甲氨蝶呤（MTX）耐药机制是高剂量MTX联合亚叶酸蛋白（LV）抢救治疗失败的可能原因，我们建立了过量亚叶酸蛋白存在下MTX耐药的人t细胞白血病细胞系CCRF-CEM细胞，并对其特性进行了表征。在1000 nM亚叶酸钙的存在下，将细胞持续暴露于MTX浓度上升至20μM的环境中，形成了3个MTX抗性亚系，其抗性程度分别在4个对数范围内相差约40倍、900倍和44000倍。MTX在这些亚管中的跨膜转运分别减少了52%，35%和12%。细胞内MTX潴留率与亲本细胞无明显差异。一项不同浓度亚叶酸钙的细胞生长研究表明，对MTX具有更高抗性的细胞需要更多的亚叶酸钙供应以实现最佳生长。northern blot检测结果显示，与抗性水平一致，二氢叶酸还原酶基因mRNA表达量增加，胸苷酸合成酶基因mRNA表达量减少，而还原性叶酸载体（RFC1）基因mRNA表达量没有变化。对3个亚系的RFC1基因测序结果显示，在第47个密码子（TCC→TTC）发生点突变，导致Phe取代Ser残基，并在抗性最高的亚系中额外缺失了第112个密码子的CTG。综上所述，MTX暴露于1000 nM亚叶酸钙存在的CCRF-CEM细胞导致异质细胞群的建立，这些细胞群具有广泛的运输介导的MTX抗性，这与RFC基因的差异改变有关。结合mtx耐药分子机制中涉及的基因表达和突变，可以为mtx耐药的检测和评价提供靶点。少

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