Månsson E, Liliemark E, Söderhäll S, Gustafsson G, Eriksson S, Albertioni F
Department of Medicine, Division of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden.
Leukemia. 2002 Mar;16(3):386-92. doi: 10.1038/sj.leu.2402388.
The relative levels of the deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK), and the 5'-nucleotidase (5'-NT) are of importance for the effect of many nucleoside analogues used in the treatment of hematological malignancies. To elucidate dCK, dGK and 5'-NT gene expressions in cell lines and in samples from patients with leukemia, we have established a real-time quantitative PCR (RQ-PCR) method. From the available dCK, dGK and 5'-NT cDNA sequences we designed specific primers and fluorogenic probes for the respective genes. The mRNA of dCK, dGK and 5'-NT was also measured by semi-quantitative RT-PCR, the enzyme activities by a radioactive substrate-based technique and Western blot was used to measure the amount of dCK and dGK protein. A MOLT-4 wild-type and its 9-beta-D-arabinofuranosylguanine (Ara-G)-resistant subline was used for the methods comparisons and the RQ-PCR assay was used in 35 samples from pediatric patients with ALL and AML. The results from RQ-PCR for the cell lines were in agreement with the semi-quantitative RT-PCR. The mRNA expression for dCK, dGK and 5'-NT (expressed as the ratio of the respective gene and the reference gene) in pediatric ALL and AML patients showed a large interindividual variability from 0.06 to 2.34, non-detectable to 0.06 and 0.04 to 0.30, respectively. These results show that the quantitative evaluation by RQ-PCR is a valuable tool in the determination of dCK, dGK and 5'-NT mRNA levels in cell lines and in clinical samples which were expressed at various levels. This rapid, convenient and specific method is suitable for further studies of these genes in clinical samples.
脱氧胞苷激酶（dCK）、脱氧鸟苷激酶（dGK）和5'-核苷酸酶（5'-NT）的相对水平对于许多用于治疗血液系统恶性肿瘤的核苷类似物的疗效至关重要。为了阐明白血病细胞系和患者样本中dCK、dGK和5'-NT基因的表达情况，我们建立了一种实时定量PCR（RQ-PCR）方法。根据现有的dCK、dGK和5'-NT cDNA序列，我们为各个基因设计了特异性引物和荧光探针。dCK、dGK和5'-NT的mRNA也通过半定量RT-PCR进行检测，酶活性通过基于放射性底物的技术进行测定，蛋白质印迹法用于检测dCK和dGK蛋白的含量。使用MOLT-4野生型及其9-β-D-阿拉伯呋喃糖基鸟嘌呤（Ara-G）抗性亚系进行方法比较，并将RQ-PCR检测应用于35例小儿急性淋巴细胞白血病（ALL）和急性髓细胞白血病（AML）患者的样本。细胞系的RQ-PCR结果与半定量RT-PCR结果一致。小儿ALL和AML患者中dCK、dGK和5'-NT的mRNA表达（以各自基因与参照基因的比值表示）显示个体间差异很大，分别为0.06至2.34、未检测到至0.06以及0.04至0.30。这些结果表明，RQ-PCR定量评估是测定细胞系和临床样本中dCK、dGK和5'-NT mRNA水平的有价值工具，这些样本中这些基因的表达水平各不相同。这种快速、便捷且特异的方法适用于在临床样本中对这些基因进行进一步研究。
