Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) catalyze the first step in the intracellular cascade of fludarabine (2-fluoroadenine-β- 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). © 2005 Elsevier Inc. All rights reserved.

Screening malignant melanoma cell lines against nucleoside analogs revealed as high sensitivity to fludarabine, clofarabine, and gemcitabine as to leukemic cells, and especially in those cells expressing high levels of the mitochondrial enzyme deoxuguanosine kinase (dGK). This enzyme, together with the cytosolic deoxycytidine kinase (dCK), and the mitochondrial thymidine kinase 2 (TK2) contributes to the activation of natural deoxyribonucleosides and nucleoside analogs to phosphorylated compounds. dCK is the most prominent enzyme in hematopoietic cells, while dGK may be high in cells harbouring many mitochondria, such as neurons and melanocytes. We found that dGK mRNA and protein expression was considerably higher in melanoma cells than in a leukemic cell line, while the difference at the activity level was less profound.

Downregulation of dGK in the melanoma cell line RaH5 using siRNA led to a compensatory increase in TK2 activity, which led to significantly increased sensitivity of the cells to gemcitabine. In contrast, downregulation of dGK in the human leukemic CEM cell line decreased TK2 activity, and rendered the cells more resistant to the drugs. The compensatory regulation of deoxynucleoside kinases with over-lapping substrate specificity differed in leukemic and melanoma cell lines probably because they preferably rely on different deoxynucleoside kinases for nucleoside and nucleoside analog activation. dGK and TK2 that are both located in the mitochondria, seems to be able to compensate for each other to a higher extent in the dGK-dependent melanoma cells compared to CEM cells that possess high dCK activity. Solid tumors, such as melanoma, expressing high levels of dGK should be considered for nucleoside analog therapy preferably in combination with their standard treatment.

PURPOSE: To characterize resistance mechanisms to the nucleoside analog 9-β-D-arabinofuranosylguanine (AraG) in the T-cell acute lymphoblastic leukemia cell line MOLT-4 and its AraG-resistant variant.

METHODS: A gene expression microarray analysis was performed, as well as gene expression and enzyme activity measurements of key enzymes in the activation of AraG. Cytotoxicity of AraG and cross-resistance to other compounds were evaluated using a standard cytotoxicity assay.

RESULTS: Gene expression microarray analysis revealed that fetal hemoglobin genes and the multidrug resistance ABCB1 gene, encoding the drug efflux pump P-gp, were the most highly upregulated genes in the resistant cells, while genes traditionally associated with nucleoside analog resistance were not. Fetal hemoglobin and ABCB1 induction can be due to global DNA hypomethylation. This phenomenon was studied using AraG during a period of 4 weeks in MOLT-4 cells and the lung adenocarcinoma cell line A549, leading to up-regulation of hemoglobin gamma and ABCB1 as well as DNA hypomethylation. Inhibiting P-gp in the AraG-resistant MOLT-4 cells led to decreased proliferation, reduced hemoglobin expression, and highly induced ABCB1 expression.

CONCLUSIONS: We show that AraG can cause hypomethylation of DNA and induce the expression of the fetal hemoglobin gamma gene and the ABCB1 gene. We speculate that the induction of ABCB1/P-gp may occur in order to help with excretion of hemoglobin degradation products that would otherwise be toxic to the cells, and we present data supporting our theory that P-gp may be linked to the induction of hemoglobin.