Interspecies differences in the kinetic properties of deoxycytidine kinase elucidate the poor utility of a phase I pharmacologically directed dose-escalation concept for 2-chloro-2′-deoxyadenosine

2-Chloro-2-deoxyadenosine (CdA, Cladribine), is a purine antimetabolite currently under investigation in phase II clinical trials for the treatment of lymphoid malignancies. Significant differences in CdA toxicity between mice and humans were observed during phase I clinical evaluation. For the elucidation of interspecies differences in drug toxicity the pharmacokinetics of CdA after subcutaneous injection and the kinetic properties of the CdA-phosphorylating enzyme, deoxycytidine kinase (dCK), were compared in mice and humans. The ratio of the dose lethal to 10% of mice (LD₁₀) to the maximum tolerated dose (MTD) in humans was 50 and the ratio of the area under the curve obtained at approximately one-half the LD₁₀ (AUC_{approx. one-half the LD 10})/AUC_MTD was 49. A significant interspecies difference was observed in the kinetic properties of dCK, the main CdA-activating enzyme. With CdA as a substrate, the Michaelis constant (K _m) of dCK in crude extracts of mouse thymus was 10 times higher than that in human thymus. An approximately 9-fold interspecies difference in maximum velocity (V_max)/K _m indicated a higher efficiency of dCK for CdA in humans than in mice. The peak plasma concentration was 210 times higher and exceeded theK _m in mice. Initial and terminal half-lives were approximately 7 times shorter in mice and trough levels were similar in mice and humans. Thus, the differences in AUCs at equitoxic doses are largely explained by differences in the target enzyme properties and the pharmacokinetic pattern. The observed lower tolerance for CdA in humans as compared with mice confirms the view that antimetabolites may not be good candidates for pharmacokinetically guided dose-escalation schemes unless detailed information on interspecies variability in drug bioactivation is available.     

Down-regulation of deoxycytidine kinase in human leukemic cell lines resistant to cladribine and clofarabine and increased ribonucleotide reductase activity contributes to fludarabine resistance

Mechanisms of acquired resistance to three purine analogues, 2-chloro-2'-deoxyadenosine (cladribine, CdA), 9-beta-D-arabinofuranosyl-2-fluoroadenine (fludarabine, Fara-A), and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (clofarabine, CAFdA) were investigated in a human T-lymphoblastic leukemia cell line (CCRF-CEM). These analogues are pro-drugs and must be activated by deoxycytidine kinase (dCK). The CdA and CAFdA resistant cell lines exhibited increased resistance to the other nucleoside analogues activated by dCK. This was also the case for the Fara-A resistant cells, except that they were sensitive to CAFdA and guanosine analogues. The CdA and CAFdA resistant cells displayed a deficiency in dCK activity (to <5%) while the Fara-A resistant cells showed only a minor reduction of dCK activity (20% reduction). The activity of high K(m) 5'-nucleotidase (5'-NT) (cN-II) using IMP as substrate, was 2-fold elevated in the resistant cell lines. The amount of the small subunit R2 of ribonucleotide reductase (RR) was higher in the Fara-A resistant cells, which translated into a higher RR activity, while CdA and CAFdA cells had decreased activity compared to the parental cells. Expression of the recently identified RR subunit, p53R2 full-size protein, in CAFdA cells was low compared to parental cells, but a protein of lower molecular weight was detected in CdA and CAFdA cells. Co-incubation of Fara-A with the RR inhibitor 3,4-dihydroxybenzohydroxamic acid (didox) enhanced cytotoxicity in the Fara-A resistant cells by a factors of 20. Exposure of the cells to the nucleoside analogues studied here also caused structural and numerical instability of the chromosomes; the most profound changes were recorded for CAFdA cells, as demonstrated by SKY and CGH analysis. We conclude that down-regulation of dCK in cells resistant to CdA and CAFdA and increased activity of RR in cells resistant to Fara-A contribute to resistance.     

Molecular and biochemical mechanisms of fludarabine and cladribine resistance in a human promyelocytic cell line

2F-Adenine arabinoside (fludarabine, Fara-A) and 2-chloro-2'-deoxyadenosine (cladribine, CdA) are nucleoside analogues with antineoplastic activity in vitro and in vivo. Lack of clinical resistance between CdA and Fara-A has been demonstrated in patients with chronic lymphocytic leukemia (G. Juliusson et al., N. Engl. J. Med., 327: 1056-1061, 1992). To clarify the differences in mechanism of resistance to CdA and Fara-A in vitro, we developed two stable, resistant cell lines, HL60/CdA and HL60/ Fara-A, by exposure to increasing concentrations of analogues over a period of 8 months. Resistant cells tolerated >8,000 and 5-fold higher concentrations of CdA and Fara-A, respectively. The specific activity of the nucleoside phosphorylating enzyme (using deoxycytidine as substrate) in cell extracts from HL60/CdA and HL60/Fara-A mutants was about 10 and 60%, respectively, compared with the parental cell line. Western blot analysis using a polyclonal antibody showed no detectable deoxycytidine kinase (dCK) protein in CdA-resistant cells, whereas in Fara-A-resistant cells, it was at the same level as in the parental cells. The mitochondrial enzyme deoxyguanosine kinase was not altered in resistant cell lines. The HL60/CdA cells showed cross-resistance to 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, Fara-A, arabinofuranosyl cytosine, difluorodeoxyguanosine, and difluorodeoxycytidine toxicity, most likely because of the decreased phosphorylation of these analogues by dCK. Using real-time quantitative PCR, the mRNA levels of dCK and cytosolic 5'-nucleotidase (5'-NT), a major nucleoside dephosphorylating enzyme, were measured. It was shown that the dCK mRNA levels in both CdA- and Fara-A resistant cells were decreased in parallel with the activity. The expression of 5'-NT mRNA was not significantly elevated in CdA- and Fara-A resistant cells, as compared with the parental cells. Ribonucleotide reductase maintains a balanced supply of deoxynucleotide triphosphate pools in the cell and may also be a major cellular target for CdA and Fara-A nucleotides. Except for the deoxycytidine triphosphate level, the intracellular deoxynucleotide triphosphate pools were significantly higher in Fara-A-resistant cells compared with the parental cell line. This might be a consequence of mutation or altered regulation of ribonucleotide reductase activity and may explain the 2-5-fold cross-resistance to several nucleoside analogues observed with HL60/Fara-A cells. It is likely that the resistance for CdA was mainly attributable to a dCK deficiency, and Fara-A-resistant cells might have another contributing factor to the resistance beyond the dCK deficiency.     

Pharmacological basis for cladribine resistance in a human acute T lymphoblastic leukaemia cell line selected for resistance to etoposide

Cross-resistance between different classes of anti-neoplastic agents can jeopardize successful combination cancer chemotherapy. In this study, we observed an unexpected cross-resistance between the podophyllotoxine derivative etoposide (VP) and the nucleoside analogue cladribine (CdA) in CCRF-CEM cells developed for resistance to VP. The resistant cells also displayed 14- and twofold resistance to cytarabine (ara-C) and gemcitabine respectively. Closer analysis of these cells showed that they contained lower amounts of topoisomerase (topo) IIalpha (P < 0.001) and beta protein (P < 0.026), formed substantially lower amounts of the topo II-DNA complex, and had a markedly decreased level of Fas (CD95/APO-1)-ligand mRNA expression. Interestingly, Fas expression in the resistant cells did not differ from that in the parental cell line. No differences were observed in the accumulation/efflux of daunorubicin or in the gene expressions of P-glycoprotein, multidrug resistance-associated protein and the lung resistance-related protein. The activity of deoxycytidine kinase (dCK), responsible for activation of CdA and ara-C, was the same for resistant and wild-type cells. However, there was an increase in the activity of the cytosolic 5'-nucleotidases (5'-NT), responsible for deactivation of nucleotides, amounting to 206% (P < 0.001) for the high Km and 134% (P < 0.331) for the low Km 5'-NT in resistant cells. The high Km 5'-NT is probably responsible for the decreased amount of the active metabolite CdA 5'-triphosphate [40% decreased (P < 0.045)], as well as for other purine ribonucleosides and deoxyribonucleosides triphosphates in the resistant cells. In contrast, a significantly higher deoxycytidine triphosphate (dCTP) level (167%, P < 0.001) was observed in the resistant cells. Thus, this study suggests that the major cause of resistance to the nucleoside analogues CdA and ara-C in cells selected for resistance to VP is a result of metabolic alterations producing increased activity of 5'-NT and higher dCTP levels. Furthermore, these results indicate that there is a common factor in the regulation of nucleotide-degrading enzymes and DNA topoisomerases, which may be altered in cross-resistant cells.     

Activation of deoxycytidine kinase by inhibition of DNA synthesis in human lymphocytes

Deoxycytidine kinase (dCK, EC.2.7.1.74) is a key enzyme in the intracellular metabolism of 2-chlorodeoxyadenosine, 1-beta-D-arabinofuranosylcytosine, difluorodeoxycytidine, and other drugs used in chemotherapy of different leukaemias and solid tumours. Recently, stimulation of dCK activity was shown by these analogues and by other genotoxic agents such as etoposide and NaF, all of which cause severe inhibition of DNA synthesis in cell cultures. Here we describe that direct inhibition of DNA polymerases by aphidicolin stimulated dCK activity in normal lymphocytes and acute myeloid leukaemic cells, as well as in HL 60 promyelocytic cell cultures. Increased dCK activity was not due to new protein synthesis under our conditions, as measured by immunoblotting. Partial purification by diethylaminoethyl-Sephadex chromatography revealed that the activated form of dCK survived purification procedure. Moreover, it was possible to inactivate purified dCK preparations by recombinant protein phosphatase with Ser/Thr/Tyr dephosphorylating activity. These data suggest that the activation of dCK may be due to phosphorylation, and that deoxynucleoside salvage is promoted during inhibition of DNA synthesis in human lymphocytes.     

Treatment of normal and malignant cells with nucleoside analogues and etoposide enhances deoxycytidine kinase activity

Deoxycytidine kinase (dCK), one of the rate-limiting enzymes in the intracellular metabolism of many antileukaemic drugs, was shown to be stimulated after treatment of human tonsillar lymphocytes by 2-chloro-2'-deoxyadenosine (cladribine, CdA) (Sasvári-Székely, et al., Biochem Pharmacol 1998, 56, 1175-1179). Here we present a comparative study of different normal and malignant cells in respect to the activation of dCK by CdA. G-phase lymphocytes showed a higher sensitivity for dCK stimulation than S-phase cells. Normal and leukaemic peripheral blood mononuclear cells, as well as the promyelocytic cell line HL60 responded to CdA treatment by a 2-5-fold increase in activity of dCK. However, no significant stimulation was detected either in CCRF-CEM T-lymphoblastoid cells, or in K562 myeloid cells. Thymidine kinase (TK) activity was not stimulated in any cases. Treatment of these cells with several other analogues beside CdA, such as 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA), 2-fluoro-1-beta-D-arabinosyladenine (Fludarabine, FaraA) and 1-beta-D-arabinosylcytosine (cytarabine, araC) gave similar results to CdA treatment. Enhancement of dCK activity could also be achieved with the topoisomerase II inhibitor, etoposide. In contrast, 2-chloro-riboadenosine (CrA) had no effect on the dCK at concentrations of 10 microM or less, while dCyd and 5-aza-dCyd caused slight inhibition. These results indicate that treatment of cells with several inhibitors of DNA synthesis potentiates the dCK activity. The drugs widely differ in their stimulatory effect on dCK, and there are also 'responsive' and 'non-responsive' cells with respect to dCK activation. Thus, enhancement of the dCK activity by specific drugs in 'responsive' cells might give a rationale for combination chemotherapy.     

Activation of deoxycytidine kinase by gamma-irradiation and inactivation by hyperosmotic shock in human lymphocytes

Deoxycytidine kinase (dCK) is a key enzyme in the intracellular metabolism of deoxynucleosides and their analogues, phosphorylating a wide range of drugs used in the chemotherapy of leukaemia and solid tumours. Previously, we found that activity of dCK can be enhanced by incubating primary cultures of lymphocytes with substrate analogues of the enzyme, as well as with various genotoxic agents. Here we present evidence that exposure of human lymphocytes to 0.5-2 Gy dosage of gamma-radiation as well as incubation of cells with calyculin A, a potent inhibitor of protein phosphatase 1 and 2A, both elevate dCK activity without changing the level of dCK protein. When cells were gamma-irradiated in the presence of calyculin A, a more pronounced activation of dCK was observed. In contrast, both basal and stimulated dCK activities were reduced by hyperosmotic treatment of the cells. DNA repair determined by the Comet assay and by thymidine incorporation was induced by irradiation. Complete repair of gamma-irradiated DNA was detected within 1 hr following the irradiation along with dCK activation, but the rate of repair was not accelerated by calyculin A. These data provide evidence for the activation of dCK upon DNA damage and repair that seems to be mediated by phosphorylation of the enzyme, suggesting the role of dCK in DNA repair processes.     

Activation of deoxycytidine kinase in lymphocytes is calcium dependent and involves a conformational change detectable by native immunostaining

Deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, plays a seminal role in the bioactivation of a wide array of cytotoxic nucleoside analogues. Recently, activation of dCK has been considered as a protective cellular response to a number of DNA-damaging agents in lymphocytes. Regarding the molecular mechanism of the enzyme activation, a post-translational modification by protein phosphorylation has been suggested. Here we provide evidence that both the activation process and the maintenance of the activated state require free cytosolic calcium. BAPTA-AM, a cell-permeable calcium chelator selectively inhibited the activation of dCK in a time- and concentration-dependent manner while extracellular calcium depletion had no effect. On the other hand, elevation of cytoplasmic calcium levels by thapsigargin did not potentiate the enzyme, referring to the permissive function of calcium in the activation process. Denaturing Western blots of extracts from lymphocytes incubated with 2-chlorodeoxyadenosine, aphidicolin and/or BAPTA-AM clearly demonstrated that dCK protein levels were unchanged during these treatments. However, a striking correlation was found between enzyme activity and the intensity of dCK-specific signals in native Western blots. Extracts from CdA-treated cells were much better recognized by the antibody raised against the C-terminal peptide of dCK than the BAPTA-AM-treated samples. These results indicate that the calcium-dependent activation of dCK is accompanied by a conformational change that renders the C-terminal epitope more accessible to the antibody.     

Biochemical pharmacology and resistance to 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, a novel analogue of cladribine in human leukemic cells.

The objective of the present study was to investigate the biochemical pharmacology of 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA)--a fluorinated analogue of cladribine [2-chloro-2'-deoxyadenosine, Leustatin (CdA)] with improved acid and metabolic stability--in human leukemic cell lines and in mononuclear cells isolated from patients with chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). We have also made and characterized two cell lines that are not sensitive to the growth inhibitory and cytotoxic effects of CAFdA. Incubation of cells isolated from the blood of CLL and AML patients with various concentrations of CdA or of CAFdA accumulated CdA and CAFdA nucleotides in a dose-dependent manner. A significantly higher rate of phosphorylation to monophosphates was observed for CAFdA than for CdA in cells from CLL patients (n = 14; P = 0.04). The differences in the phosphorylation were even more pronounced for the respective triphosphates in both CLL (n = 14; P = 0.001) and AML (n = 4; P = 0.04) cells. Retention of CAFdA 5'-triphosphate (CAFdATP) was also longer than that for CdA 5'-triphosphate (CdATP) in cells from leukemic patients. The relative efficacy of CAFdA as a substrate for purified recombinant deoxycytidine kinase (dCK), the key enzyme in the activation of nucleoside analogues, was very high and exceeded that of CdA as well as the natural substrate, deoxycytidine, by a factor of 2 and 8, respectively. The Km for CAFdA with dCK was also lower than that for CdA, as measured in crude extracts from the human acute lymphoblastic leukemia cell line CCRF-CEM and the promyelocytic leukemia cell line HL60. Acquired resistance to CAFdA in HL60 and in CCRF-CEM cell lines was directly correlated to the decreased activity of the nucleoside phosphorylating enzyme, dCK. Resistant cells also showed a considerable degree of cross-resistance to analogues that were activated by dCK. These observations demonstrated that dCK phosphorylates CAFdA more efficiently than CdA. Furthermore, CAFdATP is apparently more stable than CdATP and the mechanisms of resistance to CAFdA are similar to those leading to CdA resistance. These results encourage studies on the clinical effect of CAFdA in lymphoproliferative diseases.     

Characterization of a hairy cell leukemia-associated 5q13.3 inversion breakpoint

Previous cytogenetic analysis has indicated that chromosome anomalies involving the 5q13 band are common in hairy cell leukemia (HCL), occurring in approximately $frac13; of the patients. The data suggest that 5q13.3 is likely to harbor a gene involved in the transformational event of this disease. We selected a constitutional inv(5)(p13.1q13.3) in a patient with HCL as the starting point in an attempt to identify the relevant gene in 5q13.3. By using double color interphase fluorescence in situ hybridization (FISH) techniques, we have identified two cosmid probes from a chromosome 5-specific library that flank the 5q13.3 inversion breakpoint proximally and distally. Pulsed field gel electrophoresis (PFGE) and interphase FISH experiments suggest that the two markers are at a distance of no more than 300 kb. YAC probes covering a 21 Mb region at 5q13 were used to map the 5q13.3 inversion breakpoint and the breakpoint is located within the D5S646–D5S620 region. Two non-chimeric YACs have been identified that span the breakpoint. FISH analysis revealed that four other patients with cytogenetic aberrations of 5q carried inversions/deletions that involved the same 5q13.3 breakpoint region. The identification of a gene involved in hairy cell leukemogenesis in this region will be of major importance in the elucidation of the transformational events of HCL. Genes Chromosomes Cancer 20:337–346, 1997. © 1997 Wiley-Liss, Inc.