Activation of deoxycytidine kinase by protein kinase inhibitors and okadaic acid in leukemic cells

Deoxycytidine kinase (dCK) is a key enzyme in the deoxynucleoside salvage pathway and in the activation of numerous nucleoside analogues used in cancer and antiviral chemotherapy. Recent studies indicate that dCK activity might be regulated through reversible phosphorylation. Here, we report the effects of a large panel of protein kinase inhibitors on dCK activity in the B-leukemia cell line EHEB, both in basal conditions and in the presence of the nucleoside analogue 2-chloro-2'-deoxyadenosine (CdA) which induces activation of dCK. Except staurosporine and H-7 that significantly reduced the activation of dCK by CdA, no specific protein kinase inhibitor diminished basal dCK activity or its activation by CdA. In contrast, genistein, a general protein tyrosine kinase inhibitor, and AG-490, an inhibitor of JAK2 and JAK3, increased basal dCK activity more than two-fold. Two specific inhibitors of the MAPK/ERK pathway, PD-98059 and U-0126, also enhanced dCK activity. These data suggest that the JAK/MAPK pathway could be involved in the regulation of dCK. Moreover, we show that the activity of dCK, raised by CdA, can return to its initial level by treatment with protein phosphatase-2A (PP2A). Accordingly, dCK activity in intact cells increased upon incubation with okadaic acid (OA) at concentrations that should inhibit PP2A, but not protein phosphatase-1. Activation of dCK by protein kinase inhibitors and OA was also observed in CCRF-CEM cells and in chronic lymphocytic leukemia B-lymphocytes, suggesting a general mechanism of post-translational regulation of dCK, which could be exploited to enhance the activation of antileukemic nucleoside analogues.     

Pharmacological inhibition of the MAPK/ERK pathway increases sensitivity to 2-chloro-2'-deoxyadenosine (CdA) in the B-cell leukemia cell line EHEB

EHEB leukemic cells, which are derived from a patient suffering B-cell chronic lymphocytic leukemia (B-CLL), display intermediate sensitivity to the purine analogue 2-chloro-2'-deoxyadenosine (CdA). Because the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway can rescue cancer cells from apoptotic signals, we investigated MAPK/ERK signaling in EHEB cells in response to CdA. We observed that CdA, at concentrations around its IC50, dose- and time-dependently increased the phosphorylation state of ERK 1/2 (p-ERK), indicating an activation of the MAPK/ERK pathway. This activation required CdA metabolism and de novo protein synthesis, and was independent on caspase activation. Interruption of ERK signaling, using the specific MEK inhibitors U-0126 and PD-98059, significantly enhanced CdA cytotoxicity, evaluated by the MTT assay. Drug interaction analysis showed synergism in the majority of combinations between CdA and MEK inhibitors tested. MEK inhibitors also dramatically increased apoptosis induced by CdA alone, evaluated by caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage. Collectively, these observations show that ERK 1/2 activation elicited by CdA serves as a cytoprotective function and suggest that inhibitors of this pathway could be combined with CdA in the treatment of selected hematological malignancies.     

Identification of residues involved in the substrate specificity of human and murine dCK

Deoxycytidine kinase (dCK) is a salvage pathway enzyme that can phosphorylate both pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. Earlier studies showed that there are differences in kinetic properties between human and murine dCK, which may explain differences in toxic effects of nucleoside analogs. To determine if certain substitutions in amino acid sequences between human and mouse dCK give these differences in substrate specificity the 14 mutants and hybrid forms of human dCK were studied. All variants were characterised with dCyd, dAdo and dGuo as phosphate acceptors and ATP and UTP as phosphate donor. The relative activities with dCyd, dAdo and dGuo were about 70, 20, 30%, respectively, with UTP as compared to ATP for human dCK and 40, 60, 70% for mouse dCK. Among all tested mutants only the triple combination of substitutions Q179R-T184K-H187N (RKN) had a kinetic behaviour very similar to mouse dCK. The kinetic patterns with several important nucleoside analogs, such as AraC, CdA, ddC and AraG have also been studied. Results demonstrated 50-70% low relative capacities of the recombinant mouse and triple mutant RKN to phosphorylate this nucleoside analogs compare with human dCK. A model for dCK was used to try to explain the functional role of these amino acid substitutions. According to this model the triple mutant RKN have altered amino acids in a region necessary for conformational changes during catalyses. This may affects the substrate selectivity both for the nucleosides and the phosphate donors.     

Cytosolic and mitochondrial deoxyribonucleotidases: activity with substrate analogs,inhibitors and implications for therapy

Nucleoside analogs act as prodrugs that must be converted to 5'-phosphates by intracellular kinases to become active in the treatment of viral and oncological diseases. Activation may be reversed by dephosphorylation if the 5'-phosphates are substrates for 5'-nucleotidases. Dephosphorylation by cytosolic enzymes decreases the efficacy of the analogs, whereas dephosphorylation by mitochondrial enzymes may decrease mitochondrial toxicity. Both effects may influence the outcome of therapy. We investigated the dephosphorylation of the 5'-phosphates of commonly used nucleoside analogs by two cytosolic (cN-II and dNT-1) and one mitochondrial (dNT-2) nucleotidase. Most uracil/thymine nucleotide analogs were dephosphorylated by all three human enzymes but cytosine-containing nucleotide analogs were inactive. Only cN-II showed some activity with the monophosphates of the two purine analogs 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosylguanine. We conclude that overproduction of any of the three 5'-nucleotidases cannot explain development of resistance against cytosine analogs but that overproduction of cN-II could lead to resistance against purine analogs. Of the tested analogs, only (E)-5-(2-bromovinyl)-2'-deoxyuridine was preferentially dephosphorylated by mitochondrial dNT-2. We propose that in future developments of analogs this aspect be considered in order to reduce mitochondrial toxicity. We tested inhibition of dNT-1 and dNT-2 by a large variety of synthetic metabolically stable nucleoside phosphonate analogs and found one (PMcP-U) that inhibited dNT-1 and dNT-2 competitively and a second (DPB-T) that inhibited dNT-2 by mixed inhibition. Both inhibitors are useful for specific 5'-nucleotidase assays and structural studies and may open up possibilities for therapy.     

The pattern of deoxycytidine- and deoxyguanosine kinase activity in relation to messenger RNA expression in blood cells from untreated patients with B-cell chronic lymphocytic leukemia

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).     

Phosphorylation of deoxycytidine kinase on Ser-74: impact on kinetic properties and nucleoside analog activation in cancer cells

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme in the activation of several therapeutic nucleoside analogs (NA). Its activity can be increased in vivo by Ser-74 phosphorylation, a property that could be used for enhancing NA activation and clinical efficacy. In line with this, studies with recombinant dCK showed that mimicking Ser-74 phosphorylation by a S74E mutation increases its activity toward pyrimidine analogs. However, purine analogs had not been investigated. Here, we show that the S74E mutation increased the k(cat) for cladribine (CdA) by 8- or 3-fold, depending on whether the phosphoryl donor was ATP or UTP, for clofarabine (CAFdA) by about 2-fold with both ATP and UTP, and for fludarabine (F-Ara-A) by 2-fold, but only with UTP. However, the catalytic efficiencies (k(cat)/Km) were not, or slightly, increased. The S74E mutation also sensitized dCK to feed-back inhibition by dCTP, regardless of the phosphoryl donor. Importantly, we did not observe an increase of endogenous dCK activity toward purine analogs after in vivo-induced increase of Ser-74 phosphorylation. Accordingly, treatment of CLL cells with aphidicolin, which enhances dCK activity through Ser-74 phosphorylation, did not modify the conversion of CdA or F-Ara-A into their active triphosphate form. Nevertheless, the same treatment enhanced activation of gemcitabine (dFdC) into dFdCTP in CLL as well as in HCT-116 cells and produced synergistic cytotoxicity. We conclude that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of some NA, like dFdC, but not of CdA or F-Ara-A.     

Anabolism of amdoxovir: phosphorylation of dioxolane guanosine and its 5'-phosphates by mammalian phosphotransferases

Amdoxovir [(-)-beta-D-2,6-diaminopurine dioxolane, DAPD], the prodrug of dioxolane guanosine (DXG), is currently in Phase I/II clinical development for the treatment of HIV-1 infection. In this study, we examined the phosphorylation pathway of DXG using 15 purified enzymes from human (8), animal (6), and yeast (1) sources, including deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), high Km 5'-nucleotidase (5'-NT), guanylate (GMP) kinase, nucleoside monophosphate (NMP) kinase, adenylate (AMP) kinase, nucleoside diphosphate (NDP) kinase, 3-phosphoglycerate (3-PG) kinase, creatine kinase, and pyruvate kinase. In addition, the metabolism of 14C-labeled DXG was studied in CEM cells. DXG was not phosphorylated by human dCK, and was a poor substrate for human dGK with a high Km (7 mM). Human 5'-NT phosphorylated DXG with relatively high efficiency (4.2% of deoxyguanosine). DXG-MP was a substrate for porcine brain GMP kinase with a substrate specificity that was 1% of dGMP. DXG-DP was phosphorylated by all of the enzymes tested, including NDP kinase, 3-PG kinase, creatine kinase, and pyruvate kinase. The BB-isoform of human creatine kinase showed the highest relative substrate specificity (47% of dGDP) for DXG-DP. In CEM cells incubated with 5 microM DXG for 24 h, 0.015 pmole/10(6) cells (approximately 7.5 nM) of DXG-TP was detected as the primary metabolite. Our study demonstrated that 5'-nucleotidase, GMP kinase, creatine kinase, and NDP kinase could be responsible for the activation of DXG in vivo.     

Activation of deoxycytidine kinase by UV-C-irradiation in chronic lymphocytic leukemia B-lymphocytes

Deoxycytidine kinase (dCK), a key enzyme of the deoxynucleoside salvage pathway, might have a preponderant role in DNA synthesis in resting chronic lymphocytic leukemia B-lymphocytes. In these cells, two important enzymes in deoxynucleoside triphosphate production, ribonucleotide reductase and thymidine kinase (TK), both cell-cycle regulated, are indeed very weakly expressed. This study investigated the regulation of dCK activity in response to UV-C light, a condition which causes DNA lesions and DNA repair synthesis. We observed that activity of dCK in B-CLL cells was upregulated up to 3-fold, 30 min after irradiation with 30 J/m(2) UV-C, whereas TK activity was unchanged. Activation of dCK by UV-C light was caused neither by a change in concentration of a low molecular weight metabolite nor by an increase in the amount of dCK protein. Activation of dCK by UV-C was mimicked by H(2)O(2), markedly counteracted by N-acetylcysteine, a general antioxidant, and completely abolished by the growth factor receptor inhibitor suramin. Taken together, these results indicate that dCK activity is upregulated by UV-C light through a postranslational modification that may be initiated at the cell surface through oxidative mechanisms. Suramin also suppressed the increase in DNA repair synthesis elicited by UV-C irradiation, suggesting that upregulation of dCK activity could contribute to the normal completion of DNA repair synthesis elicited by UV light.     

Expression of deoxynucleoside kinases and 5'-nucleotidases in mouse tissues: implications for mitochondrial toxicity

Anti-HIV nucleoside therapy can result in mitochondrial toxicity affecting muscles, peripheral nerves, pancreas and adipose tissue. The cytosolic deoxycytidine kinase (dCK; EC 2.7.1.74) and thymidine kinase (TK1; EC 2.7.1.21), the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK; EC 2.7.1.113) as well as 5'-deoxynucleotidases (5'-dNT; EC 3.1.3.5) are enzymes that control rate-limiting steps in formation of intracellular and intra-mitochondrial nucleotides. The mRNA levels and activities of these enzymes were determined in mouse tissues, using real-time PCR and selective enzyme assays. The expression of mRNA for all these enzymes and the mitochondrial deoxynucleotide carrier was detected in all tissues with a 5-10-fold variation. TK1 activities were only clearly detected in spleen and testis, while TK2, dGK and dCK activities were found in all tissues. dGK activities were higher than any other dNK in all tissues, except spleen and testis. In skeletal muscle dGK activity was 5-fold lower, TK2 and dCK levels were 10-fold lower as compared with other tissues. The variation in 5'-dNT activities was about eight-fold with the highest levels in brain and lowest in brown fat. Thus, the salvage of deoxynucleosides in muscles is 5-10-fold lower as compared to other non-proliferating tissues and 100-fold lower compared to spleen. These results may help to explain tissue specific toxicity observed with nucleoside analogs used in HIV treatment as well as symptoms in inherited mitochondrial TK2 deficiencies.     

Modifications of deoxycytidine kinase and deaminase activities by docosahexaenoic acid in normal and transformed rat fibroblasts

Deoxycytidine kinase (dCK) and deoxycytidine deaminase (dCDA) are two key enzymes in the activation and inactivation, respectively, of deoxycytidine (dCyd) and several chemotherapeutically important nucleoside analogues. To investigate whether supplementation of docosahexaenoic acid, an n-3 fatty acid found mainly in fish oil, can modulate the activity of both enzymes, normal (Rat-2) and transformed (NW-16) rat fibroblasts were cultured in medium supplemented with or without DHA. DHA supplementation increased the phosphorylation efficiency (V(max)/K(m)) of dCK but decreased the deamination efficacy of dCDA in the transformed cells as compared with those in the normal fibroblasts. Enzyme activity of dCK was decreased by DHA in Rat-2 cells and increased in NW-16 cells. In contrast, dCDA activity was elevated in the normal fibroblasts in response to DHA. As a result, the activity ratio of dCK/dCDA (a potential indicator of chemosensitivity) was decreased in the normal fibroblasts but increased in the transformed cells by DHA. We have observed previously that the toxicity of nucleoside drugs (particularly arabinosylcytosine) was increased in tumor cells and decreased in normal cells in response to DHA and proposed a mechanism of changes in drug activation/inactivation. The present data support this hypothesis and suggest that DHA has the potential to selectively target chemotherapeutic drugs toward tumor cells while at the same time reducing host toxicity.     

3'-Azido-2',3'-dideoxythymidine induced deficiency of thymidine kinases 1, 2 and deoxycytidine kinase in H9 T-lymphoid cells

Continuous cultivation of T-lymphoid H9 cells in the presence of 3′-azido-2′,3′-dideoxythymidine (AZT) resulted in a cell variant cross-resistant to both thymidine and deoxycytidine analogs. Cytotoxic effects of AZT, 2′,3′-didehydro-3′-deoxythymidine as well as different deoxycytidine analogs such as 2′,3′-dideoxycytidine, 2′,2′-difluoro-2′-deoxycytidine (dFdC) and 1-ß-D-arabinofuranosylcytosine (Ara-C) were strongly reduced in H9 cells continuously exposed to AZT when compared to parental cells (>8.3-, >6.6-, >9.1-, 5×10⁴-, 5×10³-fold, respectively). Moreover, anti-HIV-1 effects of AZT, d4T, ddC and 2′,3′-dideoxy-3′-thiacytidine (3TC) were significantly diminished (>222-, >25-, >400-, >200-fold, respectively) in AZT-resistant H9 cells. Study of cellular mechanisms responsible for cross-resistance to pyrimidine analogs in AZT-resistant H9 cells revealed decreased mRNA levels of thymidine kinase 1 (TK1) and lack of deoxycytidine kinase (dCK) mRNA expression. The loss of dCK gene expression was confirmed by western blot analysis of dCK protein as well as dCK enzyme activity assay. Moreover, enzyme activity of TK1 and TK2 was reduced in AZT-resistant cells. In order to determine whether lack of dCK affected the formation of the active triphosphate of the deoxycytidine analog dFdC, dFdCTP accumulation and retention was measured in H9 parental and AZT-resistant cells after exposure to 1 and 10 μM dFdC. Parental H9 cells accumulated about 30 and 100 pmol dFdCTP/10⁶ cells after 4 hr, whereas in AZT-resistant cells no dFdCTP accumulation was detected. These results demonstrate that continuous treatment of H9 cells in the presence of AZT selected for a thymidine analog resistant cell variant with cross-resistance to deoxycytidine analogs, due to deficiency in TK1, TK2, and dCK.     

Point mutations in human guanylate kinase account for acquired resistance to anticancer nucleotide analogue PMEG

Acyclic nucleotide analogue PMEG represents promising drug candidate against lymphomas. In the present work we describe the ability of PMEG to induce resistance and we elucidate the mechanisms involved in this process. CCRF-CEM T-lymphoblastic cells resistant to either PMEG or its 6-amino congener PMEDAP were prepared and assayed for the expression of membrane transporters, PMEG and PMEDAP uptake and intracellular metabolism. Genes for guanylate kinase (GUK) and adenylate kinase (AK) isolated from PMEG- and PMEDAP-resistant cells were sequenced and cloned into mammalian expression vectors. PMEG-resistant cells were transfected with GUK vectors and catalytic activities of GUKs isolated from PMEG-sensitive and resistant cells were compared. PMEG phosphorylation to PMEG mono- and diphosphate was completely impaired in resistant cells. GUK obtained from PMEG-resistant cells revealed two point mutations S³⁵N V¹⁶⁸F that significantly suppressed its catalytic activity. Transfection of resistant cells with wtGUK led to the recovery of phosphorylating activity as well as sensitivity towards PMEG cytotoxicity. No differences in PMEG uptake have been found between sensitive and resistant cells. In contrast to GUK no changes in primary sequence of AK isolated from PMEDAP resistant cells were identified. Therefore, resistance induced by PMEDAP appears to be conferred by other mechanisms. In conclusion, we have identified GUK as the sole molecular target for the development of acquired resistance to the cytotoxic nucleotide PMEG. Therefore, PMEG is unlikely to cause cross-resistance in combination therapeutic protocols with most other commonly used anticancer drugs.     

The effects of cladribine and fludarabine on DNA methylation in K562 cells

The effects of the antileukemic adenosine analogues, 2-chloro-2'-deoxyadenosine (cladribine) and 9-beta-D-arabinosyl-2-fluoroadenine (fludarabine), on DNA methylation were studied in a cell line K562. It was previously found that both drugs inactivated SAH hydrolase, an enzyme which participates in the "active methyl" cycle. The study examined the effects of these drugs on three aspects of DNA methylation: (i) activity of endogenous C-5 DNA methyltransferase; (ii) capacity of genomic DNA (gDNA) to accept methyl groups, transferred from S-adenosylmethionine by the bacterial methyltransferase, SssI; (iii) estimation of changes of methylated cytosine levels in gDNA, using methylation-dependent restriction analysis. Cladribine and fludarabine inhibited C-5 DNA methyltransferase, with ED(50) values of 3.5 and 47.0 microM, respectively, after 24hr cell growth in the presence of the drugs. After 48 hr growth of cells with cladribine (0.1 microM) or fludarabine (3 microM), the capacity of DNA to accept methyl groups, in the presence of exogenous bacterial SssI methylase, increased by approximately 1.8 and 1.6 times, respectively, compared to control DNA. Digestion of gDNA with endonucleases HpaII and BssHII followed by SssI DNA methylation, indicated that cladribine (0.1 microM) reduced the level of methylated cytosines in both CpG islands and CCGG sequences, sensitive to HpaII restriction enzyme. Inhibition of DNA methylation by fludarabine was observed mainly in CpG dinucleotide located within sequences sensitive to HpaII. The perturbation of DNA methylation was considered as a complex process. Our findings for cladribine and fludarabine should be regarded as an extra element of their antileukemic efficacy.     

Selective activation of deoxycytidine kinase by thymidine-5'-thiosulphate and release by deoxycytidine in human lymphocytes

Deoxycytidine kinase (dCK) catalyses the rate-limiting step of the salvage of three natural deoxyribonucleosides as well as several therapeutic nucleoside analogues, which in turn can enhance its enzymatic activity [Biochem Pharmacol 56 (1998) 1175], improving the efficacy of the cytostatic therapy. Here, we measured the effect of the 5'-thiosulphate (5'-TS) derivatives of four deoxyribonucleosides (deoxyadenosine, deoxycytidine (dCyd), azidothymidine, thymidine) and two ribonucleosides (ribopurine, ribouridine (Urd)) on the activity of the two main salvage deoxynucleoside kinases, and on the salvage of dCyd and deoxythymidine (dThd). It turned out that only 2'-deoxythymidine-5'-thiosulphate (dThd-5'-TS) can potentiate the dCK activity, without influencing the thymidine kinase isoenzymes during short-time treatments of human peripheral blood and tonsillar lymphocytes. The enhancement of dCK activity by dThd-5'-TS can be reversed by dCyd, but dThd had no effect on the enzyme activation in cells. Neither dThd-5'-TS nor Urd-5'-TS had any effect on the dCK and thymidine kinase activities tested in cell-free extracts. The stimulation of dCK activity in cells was accompanied by an imbalance in the dThd and dCyd metabolism. The incorporation of 3H-dThd into DNA was suppressed by 90% in cells by dThd-5'-TS, while Urd-5'-TS only slightly influenced the same process. The 3H-dCyd incorporation into DNA was inhibited only to 50% of the control, while the 3H-dCyd labelling of the nucleotide fraction was enlarged in dThd-5'-TS-treated cells, as a consequence of the increased dCK activity. We suggest that the enhancement of dCK activity is a compensatory mechanism in cells that might be induced by different "inhibitors" of DNA synthesis leading to damage of DNA. The increased dCK activity is able to supply the repair of DNA with dNTPs in quiescent cells; this suggestion seems to be supported by the counteracting effect of extracellular dCyd, too.     

Two novel nucleoside ester derivatives of chlorambucil as potential antileukemic prodrugs: a preliminary study

2-Chloro-2'-deoxyadenosine (cladribine) and chlorambucil are two drugs used in the treatment of lymphoid malignancies. We have synthesized 5'-O-esters of cladribine and its parental nucleoside 2'-deoxyadenosine with chlorambucil (2-chloro-2'-deoxyadenosine-chlorambucil and 2'-deoxyadenosine-chlorambucil, respectively) and compared some properties of the esters with regard to their potential use as antileukemic prodrugs. The 5'-O-ester bond showed no spontaneous hydrolysis at pH 7.4, but was susceptible to hydrolysis by porcine liver esterase and enzymes present in human lymphocyte lysate and blood plasma. Both 2-chloro-2'-deoxyadenosine-chlorambucil and 2'-deoxyadenosine-chlorambucil were taken up more avidly than their parental nucleosides by normal and malignant human lymphoid cells. 2-Chloro-2'-deoxyadenosine-chlorambucil was by an order of magnitude more toxic than 2'-deoxyadenosine-chlorambucil to human leukemic MOLT4 cells in culture. On the other hand, 2-chloro-2'-deoxyadenosine-chlorambucil cytotoxicity did not exceed that of its parental 2-chloro-2'-deoxyadenosine in MOLT4 cells, whereas 2'-deoxyadenosine-chlorambucil was considerably more cytotoxic than free chlorambucil in a variety of myeloid and lymphoid human malignant cell lines. Moreover, acute toxicity of 2'-deoxyadenosine-chlorambucil was lower than that of chlorambucil in mice. In summary, 2'-deoxyadenosine-chlorambucil, but not 2-chloro-2'-deoxyadenosine-chlorambucil, shows promise for clinical utility as a chlorambucil prodrug and thus warrants a more detailed study in vivo.