采用标准化的基于竞争模板的逆转录聚合酶链反应（RT-PCR）方法测定儿童白血病中氨甲蝶呤耐药相关蛋白的mRNA表达水平。

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method.

作者信息

Rots M G, Willey J C, Jansen G, Van Zantwijk C H, Noordhuis P, DeMuth J P, Kuiper E, Veerman A J, Pieters R, Peters G J

机构信息

Department of Pediatric Hematology/Oncology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.

出版信息

Leukemia. 2000 Dec;14(12):2166-75. doi: 10.1038/sj.leu.2401943.

DOI:10.1038/sj.leu.2401943

PMID:11187907

Abstract

Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of beta-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three- and four-fold higher, respectively; P < 0.001), while FPGS expression was lower (three-fold, P = 0.006) compared to common/preB-ALL. The ratio of (DHFR x FPGH)/(RFC x FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P < 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P = 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (four-fold higher; P = 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r = 0.80; P < 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance.

摘要

白血病原始细胞的耐药性与无事件生存期相关，并且可能通过耐药相关蛋白的mRNA表达来预测。甲氨蝶呤（MTX）是儿童白血病治疗中的重要组成部分。MTX耐药的机制包括：（1）通过还原型叶酸载体（RFC）的转运减少；（2）靶酶水平改变，如二氢叶酸还原酶（DHFR）和胸苷酸合成酶（TS）；（3）叶酰聚谷氨酸合成酶（FPGS）/叶酰聚谷氨酸水解酶（FPGH）的比例降低。我们为这些基因中的每一个设计了竞争性模板，通过定量RT-PCR测量mRNA表达，并将表达量标准化为β-肌动蛋白的表达量。与普通/前B细胞急性淋巴细胞白血病（common/preB-ALL）相比，T系急性淋巴细胞白血病（T-ALL）对MTX相对耐药，其DHFR和TS的mRNA水平更高（分别高3倍和4倍；P<0.001），而与common/preB-ALL相比，FPGS表达更低（3倍，P = 0.006）。（DHFR×FPGH）/（RFC×FPGS）的比值在T-ALL和c/preB-ALL之间的区分度（高8倍；P<0.001）比单独的任何一个靶标都更高。急性髓系白血病（AML）细胞被认为对MTX耐药，与c/preB-ALL相比，其FPGS mRNA水平低两倍（P = 0.04）。FPGH/FPGS的比值在AML和c/preB-ALL之间的区分度（高4倍；P = 0.001）比单独的任何一个靶标都更高。对于79个白血病样本的总体，DHFR的mRNA表达变化了549倍，并且与TS mRNA表达平行（r = 0.80；P<0.001）。尽管mRNA表达的变化类似于功能活性的变化，但未发现RFC（mRNA表达变化58倍）、FPGS（95倍）和FPGH（178倍）有直接相关性。总之，通过竞争性RT-PCR检测到的白血病亚型之间MTX耐药参数的mRNA表达差异与已知的MTX耐药差异一致。