Lotfi Kourosh, Karlsson Karin, Fyrberg Anna, Juliusson Gunnar, Jonsson Viggo, Peterson Curt, Eriksson Staffan, Albertioni Freidoun
Department of Medicine and Care, Clinical Pharmacology, Faculty of Health Sciences, Linköping University, Linköping, Sweden.
Biochem Pharmacol. 2006 Mar 14;71(6):882-90. doi: 10.1016/j.bcp.2005.12.007. Epub 2006 Jan 24.
Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) catalyze the first step in the intracellular cascade of fludarabine (2-fluoroadenine-beta-D-arabinofuranoside) and cladribine (2-chlorodeoxyadenosine) phosphorylation, which leads to activation of these prodrugs, commonly used for treatment of chronic lymphocytic leukemia (CLL). Thus, resistance to nucleoside analogues may primarily be due to low levels of deoxynucleoside kinase activity. The purpose of this study was to investigate the activity profiles of dCK and dGK and characterize the possible relationship between the levels of dCK enzymatic activities and mRNA levels in B-CLL cells from untreated patient samples in an attempt to determine the best approach for predicting sensitivity to nucleoside analogues and thereby optimizing treatment of CLL. For this purpose, dCK and dGK analyses were done in blood cells from 59 untreated symptomatic patients with CLL. The dGK activity towards 2-chlorodeoxyadenosine was significantly lower than of dCK (median 73 pmol/mg protein/min (85-121, 95% CI) versus 353 pmol/mg protein/min (331-421)). The median dCK mRNA level was 0.107 (0.096-0.120, 95% CI). There was a lack of correlation between the activities of dCK and dGK, which indicates that these proteins are regulated independently. We also found that the dCK and dGK activity measurement towards their endogenous substrates were comparable to the nucleoside analogues tested. Such variations in enzyme activities and mRNA levels may well explain differences in clinical responses to treatment. There was no correlation between the levels of dCK mRNAs and enzymatic activities using a quantitative real-time PCR procedure. Sequencing of dCK mRNA did not reveal alternate splicing or mutations in the coding region. The relation between activity and mRNA levels was studied by short interfering RNA (siRNA) method, which showed that in the siRNA treated cells the down-regulation of dCK expression, and activity followed each other. However, in control cells the mRNA levels remained stable but the protein activity markedly decreased. These data demonstrate that the dCK activity is not reflected by dCK mRNA expression that indicates a post-translational mechanism(s).
脱氧胞苷激酶（dCK）和脱氧鸟苷激酶（dGK）催化氟达拉滨（2-氟腺嘌呤-β-D-阿拉伯呋喃糖苷）和克拉屈滨（2-氯脱氧腺苷）细胞内磷酸化级联反应的第一步，这会导致这些常用于治疗慢性淋巴细胞白血病（CLL）的前体药物被激活。因此，对核苷类似物的耐药性可能主要归因于脱氧核苷激酶活性水平较低。本研究的目的是调查dCK和dGK的活性谱，并确定未治疗患者样本中B-CLL细胞中dCK酶活性水平与mRNA水平之间的可能关系，以试图确定预测对核苷类似物敏感性的最佳方法，从而优化CLL的治疗。为此，对59例未治疗的有症状CLL患者的血细胞进行了dCK和dGK分析。dGK对2-氯脱氧腺苷的活性显著低于dCK（中位数分别为73 pmol/mg蛋白/分钟（85 - 121，95%CI）和353 pmol/mg蛋白/分钟（331 - 421））。dCK mRNA水平的中位数为0.107（0.096 - 0.120，95%CI）。dCK和dGK的活性之间缺乏相关性，这表明这些蛋白质是独立调节的。我们还发现，dCK和dGK对内源性底物的活性测量结果与所测试的核苷类似物相当。酶活性和mRNA水平的这种变化很可能解释了临床治疗反应的差异。使用定量实时PCR方法，dCK mRNA水平与酶活性之间没有相关性。dCK mRNA测序未发现编码区的可变剪接或突变。通过短干扰RNA（siRNA）方法研究了活性与mRNA水平之间的关系，结果表明在siRNA处理的细胞中，dCK表达和活性的下调相互伴随。然而，在对照细胞中，mRNA水平保持稳定，但蛋白质活性显著下降。这些数据表明，dCK活性不能由dCK mRNA表达反映，这表明存在翻译后机制。
