Potential of ribozymes against deoxycytidine kinase to confer drug resistance to cytosine nucleoside analogs

Hematopoietic toxicity is the dose-limiting side effect produced in cancer chemotherapy with deoxycytidine nucleoside analogs. Deletion of the deoxycytidine kinase (dCK), results in a drug resistance phenotype to these analogs. An interesting gene therapy strategy to confer drug resistance to cytosine nucleoside analogs would be to specifically inactivate the dCK in normal hematopoietic stem cell. In this study, we designed hammerhead ribozymes that can specifically cut and downregulate the murine dCK mRNA. Three different ribozymes were identified and shown to cleave in vitro the dCK RNA. After introduction of ribozyme cDNA into murine L1210 leukemic cells by retroviral transfer, two of the ribozymes showed some capacity in reducing dCK activity. However, analysis of transduced L1210 clones showed that the significant reduction in the dCK mRNA was not sufficient to confer drug resistance to cytosine arabinoside. Nevertheless, these results provide a new avenue of modulating the dCK enzyme activity and with improved modifications may have the potential for use in gene therapy to confer drug resistance to deoxycytidine analogs.     

Special function of deoxycytidine kinase (dCK) in the activation of chemotherapeutic nucleoside analogs and in the inhibition of cell proliferation

Deoxycytidine kinase (dCK) plays a central role in the deoxynucleoside salvage processes, phosphorylating dC, dA, and dG to their monophosphates. In mammalian cells, the major source of dTTP comes also from dC via dCMP deaminase. Moreover, based on its broad substrate specificity, this enzyme is responsible for the activation of several nucleoside analogues of therapeutical importance, influencing the sensitivity of malignant tissues towards chemotherapy. The expression of dCK is highest in different lymphoid cells/tissues, in embryonic cells and in most malignant cells (2, 7, 13-15, 18). The activity of dCK is not cell cycle-regulated. In contrast to this, dCK activity was found to be elevated several fold upon short-term treatments of normal human lymphocytes with therapeutic nucleoside analogs, and other genotoxic agents as well as by DNA damaging agents including the DNA polymerase inhibitor aphidicolin, the topoisomerase II inhibitor etoposide and gamma-irradiation, which might be a potentially important phenomenon with respect to the clinical practice, too. These findings indicated that the main trigger of activation could be the damaged DNA itself, and the biological relevance might be to supply the dNTPs for the enhanced DNA repair. Activation of dCK was paralleled by elevated levels of intracellular dATP, raising the possibility that dCK activation is linked to the induction of apoptosis. With regard to the mechanism of enzyme activation, no changes were found in the protein and mRNA levels of dCK upon stimulation, while the activation process was calcium dependent and comprised a protein phosphorylation step. A positive correlation was found between the enzymatic activity and the native immunoreactivity of dCK, strongly arguing that dCK undergoes a conformational change during activation, which results in the formation of a catalytically more active steric structure (8-11, 22, 26, 32-34, 35, 36).     

Cytotoxic activity of 2',2'-difluorodeoxycytidine, 5-aza-2'-deoxycytidine and cytosine arabinoside in cells transduced with deoxycytidine kinase gene

Deoxycytidine nucleoside analogs must be first phosphorylated to become active anticancer drugs. The rate-limiting enzyme in this pathway is deoxycytidine kinase (dCK). Cells deficient in this enzyme are resistant to these analogs. To evaluate the potential of dCK to be used as suicide gene for deoxycytidine nucleoside analogs, we transduced both human A-549 lung carcinoma and murine NIH3T3 fibroblast cell lines with this gene. The dCK-transduced cells showed an increase in cytotoxicity to the analogs, cytosine arabinoside (ARA-C), and 5-aza-2'-deoxycytidine (5-AZA-CdR). Unexpectedly, the related analog, 2',2'-difluorodeoxycytidine (dFdC), was less cytotoxic to the dCK-transduced cells than the wild-type cells. For the A-549-dCK cells, the phosphorylation of dFdC by dCK was much greater than control cells. In accord with the elevated enzyme activity, we observed a 6-fold increased dFdC incorporation into DNA and a more pronounced inhibition of DNA synthesis in the A-549-dCK cells. In an attempt to clarify the mechanism of dFdC, we investigated its action on A549 and 3T3 cells transduced with both cytidine deaminase (CD) and dCK. We reported previously that overexpression of CD confers drug resistance to deoxycytidine analogs. In this study, when the CD-transduced cells were also transduced with dCK they became relatively more sensitive to dFdC. In addition, we observed that dFdU, the deaminated form of dFdC, was cytotoxic to the A-549-dCK cells, but not the wild-type cells. Our working hypothesis to explain these results is that the mitochondrial thymidine kinase (TK2), an enzyme reported to phosphorylate dFdC, acts as an important modulator of dFdC-induced cell toxicity. These findings may further clarify the action of dFdC and the mechanism by which it induces cell death.     

Incorporation of nucleoside analogs into nuclear or mitochondrial DNA is determined by the intracellular phosphorylation site

Nucleoside analogs used in cancer chemotherapy and in treatment of virus infections are phosphorylated in cells by nucleoside and nucleotide kinases to their pharmacologically active form. The phosphorylated nucleoside analogs are incorporated into DNA and cause cell death or inhibit viral replication. Cellular DNA is replicated both in the nucleus and in the mitochondria, and nucleoside analogs may interfere with DNA replication in both these subcellular locations. In the present study we created a cell model system where nucleoside analogs were phosphorylated, and thereby pharmacologically activated, in either the nucleus, cytosol, or mitochondria of cancer cells. The system was based on the reconstitution of deoxycytidine kinase (dCK)-deficient Chinese hamster ovary cells with genetically engineered dCK targeted to the different subcellular compartments. The nucleoside analogs phosphorylated by dCK in the mitochondria were predominantly incorporated into mitochondrial DNA, whereas the nucleoside analogs phosphorylated in the nucleus or cytosol were incorporated into nuclear DNA. We further show that the nucleoside analogs phosphorylated in the mitochondria induced cell death by an apoptotic program. These data showed that the subcellular site of nucleoside analog phosphorylation is an important determinant for incorporation of nucleoside analogs into nuclear or mitochondrial DNA.     

The subcellular location of nucleoside analog phosphorylation is a determinant of synergistic effects of hydroxyurea

The ribonucleotide reductase inhibitor hydroxyurea exhibits synergistic pharmacological activity with several nucleoside analogs used in antiviral and anticancer chemotherapy. We have used a cell model system where a deoxycytidine kinase (dCK)-deficient cell line was reconstituted with genetically engineered dCK targeted to the cytosol, the nucleus, or the mitochondria to investigate how the subcellular location of nucleoside analog phosphorylation affected the synergistic effects of a ribonucleotide reductase inhibitor. Hydroxyurea showed synergistic cytotoxicity with the nucleoside analogs 1-beta-d-arabinofuranosylcytosine and 2-chloro-2'-deoxyadenosine when dCK was expressed in the cytosol or in the nucleus, but not when dCK was expressed in the mitochondria. These data indicate that the synergistic effect of ribonucleotide reductase inhibition is limited to nucleoside analogs phosphorylated in the cytosol or the cell nucleus.     

RNAi Depletion of Deoxycytidine and Deoxyguanosine Kinase in Human Leukemic CEM Cells

Resistance toward nucleoside analogues is often due to decreased activities of the activating enzymes deoxycytidine kinase (dCK) and/or deoxyguanosine kinase (dGK). With small interfering RNA (siRNA), dCK and dGK were downregulated by approximately 70% in CEM cells and tested against six nucleoside analogues using the methyl thiazol tetrazolium assay. SiRNA-transfected cells reduced in dCK activity were 3- to 6-fold less sensitive to CdA, AraC, and CAFdA. The sensitivity to AraG and FaraA was unchanged, while the sensitivity toward gemcitabine was significantly increased. dGK depletion in cells resulted in lower sensitivity to FaraA, dFdC, CAFdA, and AraG, but slightly higher sensitivity to CdA and AraC.     

RNAi depletion of deoxycytidine and deoxyguanosine kinase in human leukemic CEM cells

Resistance toward nucleoside analogues is often due to decreased activities of the activating enzymes deoxycytidine kinase (dCK) and/or deoxyguanosine kinase (dGK). With small interfering RNA (siRNA), dCK and dGK were downregulated by approximately 70% in CEM cells and tested against six nucleoside analogues using the methyl thiazol tetrazolium assay. SiRNA-transfected cells reduced in dCK activity were 3- to 6-fold less sensitive to CdA, AraC, and CAFdA. The sensitivity to AraG and FaraA was unchanged, while the sensitivity toward gemcitabine was significantly increased. dGK depletion in cells resulted in lower sensitivity to FaraA, dFdC, CAFdA, and AraG, but slightly higher sensitivity to CdA and AraC.     

Structural basis for substrate promiscuity of dCK

Deoxycytidine kinase (dCK) is an essential nucleoside kinase critical for the production of nucleotide precursors for DNA synthesis. This enzyme catalyzes the initial conversion of the nucleosides deoxyadenosine (dA), deoxyguanosine (dG), and deoxycytidine (dC) into their monophosphate forms, with subsequent phosphorylation to the triphosphate forms performed by additional enzymes. Several nucleoside analog prodrugs are dependent on dCK for their pharmacological activation, and even nucleosides of the non-physiological L-chirality are phosphorylated by dCK. In addition to accepting dC and purine nucleosides (and their analogs) as phosphoryl acceptors, dCK can utilize either ATP or UTP as phosphoryl donors. To unravel the structural basis for substrate promiscuity of dCK at both the nucleoside acceptor and nucleotide donor sites, we solved the crystal structures of the enzyme as ternary complexes with the two enantiomeric forms of dA (D-dA, or L-dA), with either UDP or ADP bound to the donor site. The complexes with UDP revealed an open state of dCK in which the nucleoside, either D-dA or L-dA, is surprisingly bound in a manner not consistent with catalysis. In contrast, the complexes with ADP, with either D-dA or L-dA, adopted a closed and catalytically competent conformation. The differential states adopted by dCK in response to the nature of the nucleotide were also detected by tryptophan fluorescence experiments. Thus, we are in the unique position to observe differential effects at the acceptor site due to the nature of the nucleotide at the donor site, allowing us to rationalize the different kinetic properties observed with UTP to those with ATP.     

Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser-74 dephosphorylation

Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn²⁺-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity.     

Structural basis for activation of the therapeutic L-nucleoside analogs 3TC and troxacitabine by human deoxycytidine kinase

L-nucleoside analogs represent an important class of small molecules for treating both viral infections and cancers. These pro-drugs achieve pharmacological activity only after enzyme-catalyzed conversion to their tri-phosphorylated forms. Herein, we report the crystal structures of human deoxycytidine kinase (dCK) in complex with the L-nucleosides (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC)--an approved anti-human immunodeficiency virus (HIV) agent--and troxacitabine (TRO)--an experimental anti-neoplastic agent. The first step in activating these agents is catalyzed by dCK. Our studies reveal how dCK, which normally catalyzes phosphorylation of the natural D-nucleosides, can efficiently phosphorylate substrates with non-physiologic chirality. The capability of dCK to phosphorylate both D- and L-nucleosides and nucleoside analogs derives from structural properties of both the enzyme and the substrates themselves. First, the nucleoside-binding site tolerates substrates with different chiral configurations by maintaining virtually all of the protein-ligand interactions responsible for productive substrate positioning. Second, the pseudo-symmetry of nucleosides and nucleoside analogs in combination with their conformational flexibility allows the L- and D-enantiomeric forms to adopt similar shapes when bound to the enzyme. This is the first analysis of the structural basis for activation of L-nucleoside analogs, providing further impetus for discovery and clinical development of new agents in this molecular class.     

Nucleoside Analog Activity in Malignant Melanoma Cell Lines

Mitochondrial deoxyguanosine kinase (dGK), is an enzyme responsible for activation of nucleoside analogs (NAs) to phosphorylated compounds which exert profound cytotoxicity, especially in hematological malignancies. Screening malignant melanoma cell lines against NAs revealed high sensitivity to several of them. This was believed to be due to the high levels of dGK expression in these cells. Downregulation of dGK in the melanoma cell line RaH5 using siRNA did not cause resistance to NAs as expected, but instead cells became more sensitive. This was probably partly due to the increased activity of another mitochondrial enzyme, thymidine kinase 2, seen in transfected cells.     

Low enantioselectivities of human deoxycytidine kinase and human deoxyguanosine kinase with respect to 2'-deoxyadenosine, 2'-deoxyguanosine and their analogs

The antiviral activity of L-nucleoside analogs depends in part on the enantioselectivity of nucleoside kinases which catalyse their monophosphorylation. The substrate properties of human recombinant deoxycytidine kinase (dCK) and human recombinant deoxyguanosine kinase (dGK) with respect to L-adenosine and L-guanosine analogs, in the presence of saturating amounts of ATP and relatively high concentrations of substrates, demonstrated a marked lack of enantioselectivity of both these enzymes. Human dCK catalysed the phosphorylation of D- and L-enantiomers of beta-dA, beta-araA, and beta-dG with enantioselectivities favoring the unnatural enantiomer for the adenosine derivatives and the natural enantiomer for 2'-deoxyguanosine. No other tested L-adenosine or L-guanosine analog was a substrate of dCK. Similarly, D- and L-enantiomers of beta-dA, beta-araA, and beta-dG were substrates of human dGK but with different enantioselectivities compared to dCK, especially concerning beta-dA. The present results indicate that human dCK and dGK have similar properties including substrate properties, relaxed enantioselectivities, and possibly catalytic cycles.     

Acute cytotoxicity of arabinofuranosyl nucleoside analogs is not dependent on mitochondrial DNA

The nucleoside analogs 9-β-D-arabinofuranosylguanine (araG) and 1-β-d-arabinofuranosylthymine (araT) are substrates of mitochondrial nucleoside kinases and have previously been shown to be predominantly incorporated into mtDNA of cells, but the pharmacological importance of their accumulation in mtDNA is not known. Here, we examined the role of mtDNA in the response to araG, araT and other anti-cancer and anti-viral agents in a MOLT-4 wild-type (wt) T-lymphoblastoid cell line and its petite mutant MOLT-4 ρ⁰ cells (lacking mtDNA). The mRNA levels and activities of deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), thymidine kinase 1 (TK1) and thymidine kinase 2 (TK2) were determined in the two cell lines. Compared to that in the MOLT-4 wt cells the mRNA level of the constitutively expressed TK2 was higher (p < 0.01) in the ρ⁰ cells, whereas the TK1 mRNA level was lower (p < 0.05). The enzyme activity of the S-phase restricted TK1 was also lower (p < 0.05) in the MOLT-4 ρ⁰ cells, whereas the activities of dGK, dCK and TK2 were similar in MOLT-4 wt and ρ⁰ cell lines. The sensitivities to different cytotoxic nucleoside analogs were determined and compared between the two cell lines. Interestingly, we found that the acute cytotoxicity of araG, araT and other anti-viral and anti-cancer agents is independent of the presence of mtDNA in MOLT-4 T-lymphoblastoid cells.     

Selective assays for thymidine kinase 1 and 2 and deoxycytidine kinase and their activities in extracts from human cells and tissues.

Human cells salvage pyrimidine deoxyribonucleosides via 5'-phosphorylation which is also the route of activation of many chemotherapeutically used nucleoside analogs. Key enzymes in this metabolism are the cytosolic thymidine kinase (TK1), the mitochondrial thymidine kinase (TK2) and the cytosolic deoxycytidine kinase (dCK). These enzymes are expressed differently in different tissues and cell cycle phases, and they display overlapping substrate specificities. Thymidine is phosphorylated by both thymidine kinases, and deoxycytidine is phosphorylated by both dCK and TK2. The enzymes also phosphorylate nucleoside analogs with very different efficiencies. Here we present specific radiochemical assays for the three kinase activities utilizing analogs as substrates that are by more than 90 percent phosphorylated solely by one of the kinases; i.e. 3'-azido-2',3'-dideoxythymidine (AZT) as substrate for TK1, 1-beta-D-arabinofuranosylthymidine (AraT) for TK2 and 2-chlorodeoxyadenosine (CdA) for dCK. We determined the fraction of the total deoxycytidine and thymidine phosphorylating activity that was provided by each of the three enzymes in different human cells and tissues, such as resting and proliferating lymphocytes, lymphocytic cells of leukemia patients (chronic lymphocytic, chronic myeloic and hairy cell leukemia), muscle, brain and gastrointestinal tissue. The detailed knowledge of the pyrimidine deoxyribonucleoside kinase activities and substrate specificities are of importance for studies on chemotherapeutically active nucleoside analogs, and the assays and data presented here should be valuable tools in that research.     

Low level of mitochondrial deoxyguanosine kinase is the dominant factor in acquired resistance to 9-beta-D-arabinofuranosylguanine cytotoxicity

9-beta-D-arabinofuranosylguanine (Ara-G) is an important and relatively new guanosiue analog with activity in patients with T-cell malignancies. The biochemical and molecular events leading to resistance to Ara-G are not fully understood. Therefore we generated two Ara-G-resistant human MOLT-4 leukemic cell lines with different levels of resistance. The mitochondrial enzyme deoxyguanosine kinase (dGK) and the nuclear/cytosol enzyme deoxycytidine kinase (dCK) are key enzymes in the activation of Ara-G. Decreased levels of dGK protein and mRNA were found in both resistant cell sublines. The activity of dCK was decreased in the subline with higher resistance to Ara-G and these cells were highly cross-resistant to other nucleosides activated by dCK. Increased activity of the mitochondrial enzyme thymidine kinase 2 was observed in both resistant sublines and this could be related to the dGK deficiency. In search for other resistance mechanisms it was found that the resistant cells overexpress the mdr1 gene, while no changes were detected in the levels of multidrug resistance-associated protein 1 through 6, lung resistance-associated protein or topoisomerase IIalpha or IIbeta. Taken together, our findings demonstrate that multiple mechanisms are involved in the acquired resistance to Ara-G. However, low expression of dGK is the most apparent alteration in both resistant cell lines. Partial deficiency of dCK was found in the subline cells with higher resistance to Ara-G. Furthermore, Ara-G may select for high expression of the multidrug resistance (mdr1) which could be a specific resistance mechanism but more likely part of an overall cellular stress response.     

Casein kinase 1δ activates human recombinant deoxycytidine kinase by Ser-74 phosphorylation, but is not involved in the in vivo regulation of its activity

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI δ. We showed that recombinant CKI δ phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI δ correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI δ, strengthening the key role of this residue in the control of dCK activity. However, neither CKI δ inhibitors nor CKI δ siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI δ in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI δ could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme.     

Activation of deoxycytidine kinase by deoxyadenosine: implications in deoxyadenosine-mediated cytotoxicity

The inborn deficiency of adenosine deaminase is characterised by accumulation of excess amounts of cytotoxic deoxyadenine nucleotides in lymphocytes. Formation of dATP requires phosphorylation of deoxyadenosine by deoxycytidine kinase (dCK), the main nucleoside salvage enzyme in lymphoid cells. Activation of dCK by a number of genotoxic agents including 2-chlorodeoxyadenosine, a deamination-resistant deoxyadenosine analogue, was found previously. Here, we show that deoxyadenosine itself is also a potent activator of dCK if its deamination was prevented by the adenosine deaminase inhibitor deoxycoformycin. In contrast, deoxycytidine was found to prevent stimulation of dCK by various drugs. The activated form of dCK was more resistant to tryptic digestion, indicating that dCK undergoes a substrate-independent conformational change upon activation. Elevated dCK activities were accompanied by decreased pyrimidine nucleotide levels whereas cytotoxic dATP pools were selectively enhanced. dCK activity was found to be downregulated by growth factor and MAP kinase signalling, providing a potential tool to slow the rate of dATP accumulation in adenosine deaminase deficiency.     

Identification of phosphorylation sites on human deoxycytidine kinase after overexpression in eucaryotic cells

Compelling evidence suggests that deoxycytidine kinase (dCK), a key enzyme in the salvage of deoxyribonucleosides and in the activation of clinically relevant nucleoside analogues, can be regulated by reversible phosphorylation. In this study, we show that dCK overexpressed in HEK-293T cells was labelled after incubation of the cells with [32P]orthophosphate. Tandem mass spectrometry allowed the identification of 4 in vivo phosphorylation sites, Thr3, Ser11, Ser15, and Ser74. These results provide the first evidence that dCK is constitutively multiphosphorylated in intact cells. In addition, site-directed mutagenesis demonstrated that phosphorylation of Ser74, the major in vivo phosphorylation site, is crucial for dCK activity.     

Identification of Phosphorylation Sites on Human Deoxycytidine Kinase After Overexpression in Eucaryotic Cells

Compelling evidence suggests that deoxycytidine kinase (dCK), a key enzyme in the salvage of deoxyribonucleosides and in the activation of clinically relevant nucleoside analogues, can be regulated by reversible phosphorylation. In this study, we show that dCK overexpressed in HEK-293T cells was labelled after incubation of the cells with [³²P]orthophosphate. Tandem mass spectrometry allowed the identification of 4 in vivo phosphorylation sites, Thr3, Ser11, Ser15, and Ser74. These results provide the first evidence that dCK is constitutively multiphosphorylated in intact cells. In addition, site-directed mutagenesis demonstrated that phosphorylation of Ser74, the major in vivo phosphorylation site, is crucial for dCK activity.     

Down-regulation of mitochondrial thymidine kinase 2 and deoxyguanosine kinase by didanosine: Implication for mitochondrial toxicities of anti-HIV nucleoside analogs

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial rate limiting phosphorylation of deoxynucleosides and are essential enzymes for mitochondrial function. Chemotherapy using nucleoside analogs is often associated with mitochondrial toxicities. Here we showed that incubation of U2OS cells with didanosine (ddI, 2′,3′-dideoxyinosine), a purine nucleoside analog used in the highly active antiretroviral therapy (HAART), led to selective degradation of both mitochondrial TK2 and dGK while the cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) were not affected. Addition of guanosine to the ddI-treated cells prevented the degradation of mitochondrial TK2 and dGK. The levels of intracellular reactive oxygen species and protein oxidation in ddI-treated and control cells were also measured. The results suggest that down-regulation of mitochondrial TK2 and dGK may be a mechanism of mitochondrial toxicity caused by antiviral and anticancer nucleoside analogs.