dCyd Kinase Research Articles

Clofarabine: a next-generation deoxyadenosine analogue

Acute lymphoblastic leukaemia (ALL) is the most common of the paediatric leukaemias. It is estimated that the use of modern combination chemotherapy results in long-term remission in nearly 80% of children diagnosed with ALL. Despite therapy advances, approximately 20% of children with ALL, experience leukaemia relapse. Clofarabine (2-chloro-2’-fluoro-2’-deoxy-9-β-D-arabinofuranosyladenine) is a second-generation nucleoside analogue and is structurally related to fludarabine and cladribine which are widely used in the treatment of lymphoproliferative disorders. Clofarabine exhibits greater affinity to deoxycytidine kinase (dCyd kinase) and prolonged retention in leukaemic blasts compared to fludarabine and cladribine. Clofarabine inhibits both DNA polymerases and ribonucleotide reductase (RNR). This results in impaired DNA synthesis through inhibition of DNA elongation as well as depletion of deoxyribonucleotides. Accumulation of clofarabine triphosphate, in the blasts of patients with refractory leukemia has been demonstrated. Prolonged intracellular half-life of 24 hours for clofarabine triphosphate. Clofarabine is indicated for the treatment of pediatric patients 1 to 21 years old with relapsed or refractory acute lymphoblastic leukemia after at least two prior regimens.

Influence of Cytidine Deaminase on Antitumor Activity of 2′-Deoxycytidine Analogs In Vitro and In Vivo

Antitumor 2'-deoxycytidine (dCyd) analogs such as gemcitabine (dFdC), cytarabine (Ara-C), and 2'-C-cyano-2'-deoxy-1-β-d-arabinofuranosylcytosine (CNDAC) are activated by dCyd kinase, whereas cytidine deaminase (CDA) inactivates them by conversion to their uracil forms. To elucidate the relationship between the chemosensitivity to antitumor dCyd nucleosides and CDA expression, we established a stable line of human gastric carcinoma TMK-1 cells constitutively overexpressing CDA (TMK-1/CDA) and examined its chemosensitivity to antitumor dCyd analogs in vitro and in vivo. We observed comparable reactivity for dFdC and Ara-C, and the substrate reactivity of CNDAC to recombinant human CDA was more than 10 times less efficient than those of Ara-C and dFdC. Next, we examined the in vitro chemosensitivity of TMK-1/CDA and observed a marked decrease in the sensitivity of TMK-1/CDA to Ara-C, dFdC, and CNDAC compared with mock-transfected cells. In addition, we transplanted TMK-1/CDA cells into a nude mouse xenograft model and examined their in vivo chemosensitivity to CNDAC. The in vivo antitumor effect of CNDAC on TMK-1/CDA cells was substantially reduced compared with that of mice transplanted with mock-transfected cells. These results indicate that CDA could play an important role in regulating susceptibility to antitumor dCyd analogs in vitro and in vivo. In addition, the expression level of CDA was found to affect the antitumor activity of CNDAC, even though the substrate reactivity of CNDAC to CDA is relatively low.

In vivo Induction of Resistance to Gemcitabine Results in Increased Expression of Ribonucleotide Reductase Subunit M1 as the Major Determinant

Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance.

A study of the influence of newly synthesized acyclonucleosides and 1,2,3,4-tetrahydroisoquinoline derivatives on deoxythymidine and deoxycytidine kinase activities in human neurofibrosarcoma and ovarian cancer.

The influence of nine newly synthesized uracil acyclonucleosides, and 36 derivatives of 1,2,3,4-tetrahydroisoquinoline on the activity of enzymes catalysing dTMP and dGMP synthesis, on the content of dTTP and dGTP in acid soluble fraction and on the incorporation of [14C]dThd and [14C ]dGuo into DNA in tumour homogenates was studied. The influence of the compounds was studied in the cytosol from intraoperatively excised human tumours - neurofibrosarcoma and ovarian cancer. It was shown that dTMP and dGMP synthesis is inhibited competitively by 34.1+/-4.0% in both types of tumours by 0.2 mM 1-N-(3'-hydroxypropyl)-6-methyluracil (1) and 0.2 mM 1-N-(3'-hydroxypropyl)- 5,6- tetramethyleneuracil (2). The mentioned acyclonucleosides reduced the content of dTTP and dGTP in the acid soluble fraction of tumours (59.7+/-3.1% of control). 1-(4-chlorophenyl)-6,7-dihydroxy- 1,2,3,4-tetrahydroisoquinoline (3), 1-(2,3-dichlorophenyl)-6,7-dihydroxy 1,2,3,4-tetrahydroisoquinoline (4) and 1-(3-methoxyphenyl)-6,7-dihydroxy 1,2,3,4-tetrahydroisoquinoline (5) at 0.2 mM concentration caused a mixed type inhibition of the synthesis of dTMP and dGMP by, on average, 33.2+/-4.4%, and reduced the content of dTTP and dGTP in the acid soluble fraction (52.6+/-3.7% of control) but were active only in the cytosol of neurofibrosarcoma. While acyclonucleosides undergo phosphorylation in the cytosol by cellular kinases, with their triphosphates being active acyclonucleoside metabolites, active 1,3,4,5-tetrahydroisoquinoline derivatives (compounds not containing a deoxyribose moiety), cannot be phosphorylated. ACN and THI derivatives which inhibit dThd and dCyd kinase activities, inhibit also the incorporation of [14C]dThd and [14C]dGuo (ACN - 50.2+/-2.7%, THI - 53.4+/-3.9% of incorporation inhibition) into tumour DNA. The obtained results point to the mechanism of uracil acyclonucleosides and 1,2,3,4-tetrahydroisoquinoline biological activity consisting in inhibiting the synthesis of DNA components.

A study of the influence of newly synthesized acyclonucleosides and 1,2,3,4-tetrahydroisoquinoline derivatives on deoxythymidine and deoxycytidine kinase activities in human neurofibrosarcoma and ovarian cancer.

The influence of nine newly synthesized uracil acyclonucleosides, and 36 derivatives of 1,2,3,4-tetrahydroisoquinoline on the activity of enzymes catalysing dTMP and dGMP synthesis, on the content of dTTP and dGTP in acid soluble fraction and on the incorporation of [14C]dThd and [14C ]dGuo into DNA in tumour homogenates was studied. The influence of the compounds was studied in the cytosol from intraoperatively excised human tumours - neurofibrosarcoma and ovarian cancer. It was shown that dTMP and dGMP synthesis is inhibited competitively by 34.1+/-4.0% in both types of tumours by 0.2 mM 1-N-(3'-hydroxypropyl)-6-methyluracil (1) and 0.2 mM 1-N-(3'-hydroxypropyl)- 5,6- tetramethyleneuracil (2). The mentioned acyclonucleosides reduced the content of dTTP and dGTP in the acid soluble fraction of tumours (59.7+/-3.1% of control). 1-(4-chlorophenyl)-6,7-dihydroxy- 1,2,3,4-tetrahydroisoquinoline (3), 1-(2,3-dichlorophenyl)-6,7-dihydroxy 1,2,3,4-tetrahydroisoquinoline (4) and 1-(3-methoxyphenyl)-6,7-dihydroxy 1,2,3,4-tetrahydroisoquinoline (5) at 0.2 mM concentration caused a mixed type inhibition of the synthesis of dTMP and dGMP by, on average, 33.2+/-4.4%, and reduced the content of dTTP and dGTP in the acid soluble fraction (52.6+/-3.7% of control) but were active only in the cytosol of neurofibrosarcoma. While acyclonucleosides undergo phosphorylation in the cytosol by cellular kinases, with their triphosphates being active acyclonucleoside metabolites, active 1,3,4,5-tetrahydroisoquinoline derivatives (compounds not containing a deoxyribose moiety), cannot be phosphorylated. ACN and THI derivatives which inhibit dThd and dCyd kinase activities, inhibit also the incorporation of [14C]dThd and [14C]dGuo (ACN - 50.2+/-2.7%, THI - 53.4+/-3.9% of incorporation inhibition) into tumour DNA. The obtained results point to the mechanism of uracil acyclonucleosides and 1,2,3,4-tetrahydroisoquinoline biological activity consisting in inhibiting the synthesis of DNA components.

The Molecular Targets of Antitumor 2-deoxycytidine Analogues

Most antitumor 2'-deoxycytidine (dCyd) analogues, such as Ara-C (1-beta-arabinofuranosylcytosine) and gemcitabine (2'-deoxy-2',2'-difluolo-cytidine), have common antitumor mechanisms and metabolic pathways. These nucleosides are transported into tumor cells via specific nucleoside transporters (NT), and then phosphorylated toward each monophosphate form by dCyd kinase. Finally, tri-phosphate forms are enzymatically produced and efficiently inhibit DNA synthesis. It is believed that dCyd kinase is a very important activator of antitumor 2'-dCyd analogues and an attractive molecular target for biochemical modulation. Resistant cells established by continuous exposure to 2'-dCyd analogues in vitro have extremely high resistance as compared with parental cells, and their resistance indexes are sometimes increased between several hundred to thousand times. Such high resistance is generally attributed to deficiency of dCyd kinase activity, but the clinical resistance index of Ara-C-resistant patients is estimated to be increased a maximum of 20 times compared with non-treated patients. The differences between experimental and clinical resistances may be caused by different mechanisms of resistance. To clarify such resistance mechanisms, we carried out research focused on NT and dCyd kinase. Our results show that earlier resistant cells, that exhibited a 20 times lower resistance index, had a reduced NT activity but retained dCyd kinase activity. In contrast, dCyd kinase activity was deficient in later resistant cells that showed maximum resistance. Both NT and dCyd kinase activities are important for the acquisition of resistance and are useful as molecular targets for biochemical modulation or the development of novel antitumor 2'-dCyd analogues. These results suggest that NT activity is likely to be responsible for clinical resistance.

Deletion mutants of human deoxycytidine kinase mRNA in cells resistant to antitumor cytosine nucleosides.

We studied mutational events in deoxycytidine (dCyd) kinase mRNA expression, focusing on aberrant dCyd kinase mRNA, which has been frequently observed in established cell lines resistant to antitumor dCyd nucleoside analogues such as 1‐β‐D‐arabinofuranosyl cytosine (Ara‐C), gemcita‐bine (dFdC) and 2′‐C‐cyano‐2′‐deoxy‐l‐β‐D‐arabinofuranosylcytosine (CNDAC). We describe here the expression of aberrant dCyd kinase mRNAs identified as splicing mutants. These mutants included deletions of the fifth exon in CNDAC‐resistant cells (originating from HT‐1080 cells), of the third exon in Ara‐C‐resistant cells (originating from SK‐MEL‐28 cells) and of the fourth exon in 2′‐deoxy‐2′‐methylidenecytidine (DMDC)‐resistant cells (originating from SK‐MEL‐28 cells). Various nucleoside‐resistant cells originating from the same parental HT‐1080 cells were established. The resulting cells expressed the same mRNA with deletion of the fifth exon, and the location of splicing was independent of the type of nucleosides used for the establishment of resistant cells. The deletion of the fifth exon in dCyd kinase seems to be a target for acquisition of resistance to antitumor cytosine nucleosides. However, distinct mutations in the dCyd kinase gene seem to be associated with acquisition of resistance to different antitumor cytosine nucleosides.

Determinants in chemosensitivity of oncogene-transformed NIH3T3 cells to 2'-C-cyano-2'-deoxy-1-beta-D-arabinofuranosylcytosine.

2'-C-Cyano-2'-deoxy-1-beta-D-arabinofuranosylcytosine (CNDAC) is a novel 2'-deoxycytidine (dCyd) analogue with potent antitumor activity. To elucidate the determinants of chemosensitivity to CNDAC, the intracellular accumulation of CNDAC and the activities of dCyd kinase and cytidine deaminase were investigated in transformed NIH 3T3 cells with different genetic bases. The results indicate that the primary determinants of chemosensitivity to CNDAC are different in each cell type, but membrane transportation and the enzyme activities of dCyd kinase and cytidine deaminase are critical factors underlying the antitumor action of CNDAC. Moreover, the expression or function of these factors appears to be influenced by the activation of various oncogenes.

Mechanisms responsible for resistance of sublines derived from leukemia cell lines to an antitumor agent 9-beta-D-arabinofuranosyl-2-fluoroadenine.

An agent 9-beta-D-arabinofuranosyl-2-fluoroadenine (2-F-Ara-A) is a main metabolite of fludarabine, a fluorinated purine analogue with antitumor activity in lymphoproliferative malignancies. In this study, the mechanism responsible for the resistance of cancer cells to fludarabine was examined using the 2-F-Ara-A-resistant sublines JOK-1/F-Ara-A and L1210/F-Ara-A from a human hairy leukemic cell line (JOK-1) and a mouse leukemic cell line (L1210) respectively, which were established by continuous treatment of the parental cell lines with 2-F-AraA. JOK-1/F-Ara-A and L1210/F-Ara-A cells were more than 55 and 29 times more resistant to 2-F-Ara-A than were their parent cell lines, and showed a high cross-resistance to 1-beta-D-arabinofuranosylcytosine but not to doxorubicin or vincristine. These resistant sublines intracellularly accumulated almost the same amount of 2-F-Ara-A as did their parent cell lines. However, the amount of 2-F-Ara-ATP, a cytotoxic metabolite of 2-F-Ara-A, decreased by 2.6% (JOK-1/F-Ara-A C3), 6% (L1210/F-Ara-A C1) and 3.7% (L1210/F-Ara-A C7) relative to the levels in the parent cell lines. Enzymatically, these resistant cells hardly activated deoxycytidine (dCyd) and 2-F-Ara-A. In addition, the abilities to phosphorylate deoxyadenosine and deoxyguanosine were also decreased in the resistant cells in comparison with the parent cells. These findings suggest that the deficiency in activity of dCyd kinase may contribute to the resistance of 2-F-Ara-A.

Development and biochemical characterization of a 2′- C-cyano-2′-deoxy-1- β- d-arabino-pentofuranosylcytosine (CNDAC)-resistant variant of the human fibrosarcoma cell line HT-1080

2′- C-Cyano-2′-deoxy-1- β- d-arabino-pentofuranosylcytosine (CNDAC) is an antitumor nucleoside with a novel chemical structure that exerts potent antitumor activity against various human tumor cells in vitro and in vivo. In order to be active it needs to be phosphorylated by deoxycytidine (dCyd) kinase. We induced resistance to CNDAC in the human fibrosarcoma cell line HT-1080 by exposure to increasing concentrations of CNDAC. The resistant cells showed over 560 times higher resistance as compared to that of the parental HT-1080 cells and were cross-resistant to the other 2′-deoxycytidine derivatives. The dCyd kinase mRNA expression of the resistant cells decreased and there was the expression of aberrant mRNA of dCyd kinase which contained a 116-nucleotide deletion within the coding region, corresponding to the fifth exon of the gene. The dCyd kinase enzymatic activity of the resistant cells was deficient. The initial uptake of CNDAC into the resistant cells was similar to that of the parental cells. However, the incorporation of CNDAC into the DNA fraction of the resistant cells was significantly less than that of the parent cells. These results led us to conclude that the acquired resistance to CNDAC can be attributed to a deficiency of dCyd kinase activity, which should be based on a remarkable decrease in mRNA expression and genetic mutation of the dCyd kinase gene, but not on cellular CNDAC accumulation.

Regulation of deoxycytidine kinase by deoxycytidine and deoxycytidine 5' triphosphate in whole leukemia and tumor cells.

The clinical importance of the dCTP salvage pathway is given by the fact that deoxycytidine (dCyd) analogs like cytosine arabinoside (ara-C) are activated via the salvage pathway. The non-toxic prodrug ara-C gains its cytotoxic potential by phosphorylation to the 5′-triphosphate ara-CTP. Not only cellular accumulation, but also retention of ara-CTP are known determinants of response to antileukemic treatment. Deoxycytdine kinase is the rate-limiting enzyme for intracellular ara-CTP formation. Experiments performed with purified dCK (dCK) demonstrated that dCTP, the final product of phosphorylation, acts as a potent feed-back inhibitor of dCyd kinase.1,2 In fact, depletion of intracellular dCTP as induced by inhibition of the de novo pathway increases of ara-C phosphorylation.3 Therefore it was concluded that the release of dCTP-mediated feed-back inhibition was responsible for enhanced dCK activity. The present study investigates the role of dCTP for regulation of dCK activity in the cell lines CCRF-CEM (T-cell line), Raji (B-cell line), HL-60 (promyelocytic), and CHO (Chinese hamster ovary). Compared to T-cells, B-cells are characterised by a fast turnover of dNTP and a short retention of ara-CTP. CHO cells were included because of their deficiency in deoxycytidylate (dCMP) deaminase activity. Cellular accumulation of ara-CTP was evaluated as a measure of salvage pathway activity. Inhibition of ribonucleotide reductase as induced by hydroxyurea may cause an activation of the salvage pathway to maintain adequate dCTP levels for DNA synthesis. Cells with a fast dCTP turnover and a greater dependence on the de novo pathway should respond with a greater enhancement of the salvage pathway than cells with long retention of deoxynucleotides and a correspondingly lower dependence on the de novo pathway.4 KeywordsChinese Hamster Ovary CellRibonucleotide ReductaseRaji CellSalvage PathwayDeoxycytidine KinaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Kinetic analysis of human deoxycytidine kinase with the true phosphate donor uridine triphosphate.

Deoxycytidine kinase is the rate-limiting process in the activation for several clinically important antitumor agents. Previous studies have focused on deoxycytidine (dCyd) and adenosine triphosphate (ATP) as substrates for this enzyme. In view of recent data indicating that uridine triphosphate (UTP) is the physiologic phosphate donor for this enzyme, a study of the kinetic properties of dCyd kinase with dCyd and UTP was undertaken. The results presented here demonstrate that UTP and ATP produce kinetically distinguishable differences in nucleoside phosphorylation by dCyd kinase. At high dCyd concentrations, dCyd kinase exhibited substrate activation with ATP. In contrast, in the presence of UTP, substrate inhibition was observed at concentrations of dCyd greater than 3 microM. Inhibition by dCyd was noncompetitive with respect to UTP and could not be reversed by a 200-fold increase in UTP concentration, indicating that the inhibition was not due to dCyd binding at the nucleotide binding site. The kinetic mechanism for dCyd kinase was determined with dCyd and UTP as substrates. UTP was the preferred phosphate donor with a true Km value of 1 microM compared to 54 microM with ATP, resulting in a 50-fold greater substrate efficiency for UTP. Although the double-reciprocal plots with UTP produced parallel lines, initial velocity plots with other phosphate donors and product inhibition studies indicated that dCyd kinase formed a ternary complex with its substrates. The parallel lines with UTP were apparently due to a low dissociation constant for UTP, which was calculated as more than 13-fold lower than its Km value. Analysis of product inhibition studies indicated that dCyd kinase followed an ordered A-B random P-Q reaction sequence, with UTP as the first substrate to bind. In contrast, previous results demonstrated a random bi-bi sequence for dCyd kinase in the presence of ATP. The combined results indicate that the enzyme can follow a random bi-bi reaction sequence, but with UTP as the phosphate donor, the addition of nucleotide prior to dCyd is strongly preferred. The noncompetitive substrate inhibition, which was independent of UTP concentration, indicates that high concentrations of dCyd promote addition of the nucleoside prior to UTP, resulting in a lower velocity.

Deoxycytidine kinase mRNA Levels in leukemia cells with competitive polymerase chain reaction assay

Deoxycytidine kinase (dCyd kinase) is important for the phosphorylation of several different nucleoside antimetabolites. To understand the significance of dCyd kinase levels in chemotherapy, dCyd kinase mRNA levels were evaluated in several cells with a quantitative competitive polymerase chain reaction (PCR) assay. dCyd kinase catalytic activity and intracellular ara-CTP production were also compared with the levels of dCyd kinase mRNA. The assay was able to show: (i) that dCyd kinase catalytic activity and dCyd kinase mRNA levels were correlated in cells; (ii) that dCyd kinase mRNA levels were more sensitive in lower levels of 10 amol/μg of total RNA; and (iii) in cytosine arabinoside (ara-C)-resistant cells, both dCyd kinase mRNA levels and intracellular ara-CTP levels were lower compared with levels in sensitive cells. The PCR assay for dCyd kinase mRNA could be useful in the selection and monitoring of patients treated with nucleosides that are activated by this enzyme.

Regulation of thymidine kinase and thymidylate synthase in intact human lymphoblast CCRF-CEM cells.

It has been reported that thymidylate synthase (TS) is a component of a multienzyme complex associated with DNA replication based on observations that enzyme activity was decreased when cells were treated with various DNA synthesis inhibitors (Plucinski, T. M., Fager, R. S., and Reddy, G. P. V. (1990) Mol. Pharmacol. 38, 114-120). The veracity of the TS assay (known as the tritium release assay) employed in these experiments may be compromised, however, because it requires the S phase-specific enzyme thymidine kinase (TK) to phosphorylate the substrate [5-3H]dUrd. In our study, this problem was further illustrated as the phosphorylated products of [14C]dCyd and [6-3H]dUrd were simultaneously quantitated to determine the activities of TS, TK, and dCyd kinase in intact CCRF-CEM cells. TS and dCyd kinase were unaffected by aphidicolin, hydroxyurea, and 1-beta-D-arabinofuranosylcytosine, whereas TK was strongly inhibited by these agents. Elevation of the cellular dTTP pool that accompanied drug treatment was not the primary mechanism affecting TK activity because incubation of cells with dCyd elevated the dTTP pool to similar levels, but did not inhibit TK to the same extent as did the drugs. Furthermore, after cells were washed from aphidicolin, [6-3H]dCyd incorporation, which primarily labels dTMP in DNA, proceeded at a linear rate, whereas a lag period of 15 min was observed before [3H]dThd was incorporated at a linear rate. These results suggest that TK activity is affected by more than one mechanism in intact cells. Because the activities of dCyd kinase and dCMP deaminase do not fluctuate as much as that of TK in response to changes in DNA synthesis activity and cell cycle, dCyd incorporation appears to be a more reliable assay of TS in intact cells than does dUrd incorporation. Our findings also imply that [3H]dThd incorporation assays may overestimate inhibition of DNA synthesis.

Substrate specificity of mitochondrial 2'-deoxyguanosine kinase. Efficient phosphorylation of 2-chlorodeoxyadenosine.

Mitochondrial deoxyguanosine kinase (dGK) (EC 2.7.1.113) was purified to apparent homogeneity from bovine brain. The molecular mass of the native protein was 56 kDa, as judged by gel filtration, and one single band of 28 kDa was seen in sodium dodecyl sulfate-gel electrophoresis. 2'-Deoxyguanosine (dGuo) (Km, 7.6 microM), 2'-deoxyinosine, and 2'-deoxyadenosine (Km, 60 microM) were substrates for the enzyme as well as several dGuo analogs containing a lipophilic substituent at C-2'. Carbocyclic dGuo, 9-beta-D-arabinofuranosylguanine, 9-beta-D-arabinofuranosylhypoxanthine, and 9-beta-D-arabinofuranosyladenine were substrates for the enzyme, whereas no 3'-modified dGuo analogs were effective. Interestingly, 2-chloro-2'-deoxyadenosine (CdA) was found to be an efficient substrate for dGK (Km, 85 microM). Subcellular fractionation of human CEM lymphoblasts showed that extracts of mitochondria contain significant CdA phosphorylating activity (71.5 pmol/mg/min) that is not inhibited by excess of 2'-deoxycytidine (dCyd). This contrasts with the CdA phosphorylating activity found in cytosolic extracts, which is carried out by dCyd kinase and strongly inhibited by excess of dCyd. The efficient CdA phosphorylation by mitochondrial dGK is a novel finding that may have far reaching implications for the clinical use of this potent cytostatic drug.

The role of cytoplasmic deoxycytidine kinase in the mitochondrial effects of the anti-human immunodeficiency virus compound, 2‘,3‘-dideoxycytidine.

2',3'-Dideoxycytidine (ddC) is a potent inhibitor of human immunodeficiency virus replication in vitro and shows beneficial effects in AIDS therapy. The compound inhibits mitochondrial DNA (mtDNA) synthesis at a clinically relevant concentration, which could be responsible for the side effects of ddC observed in the clinic. Thymidine (dThd), one of the substrates of mitochondrial deoxypyrimidine kinase (dPyd kinase), was not able to reverse the mitochondrial toxicity of ddC in CEM cells. Furthermore, the cytoplasmic deoxycytidine kinase (dCyd kinase)-deficient CEM cells were highly resistant to the mitochondrial toxicity of ddC. These data suggest a critical role for cytoplasmic dCyd kinase in the mitochondrial toxicity of ddC. The metabolites of ddC, but not ddC itself, were able to inhibit mtDNA synthesis in isolated mitochondria. The potency of the inhibitory effect was in the order of ddCTP greater than ddCDP greater than ddCMP greater than ddC. The lack of inhibition by ddC of mtDNA synthesis could be due to the inefficient ddC phosphorylation in mitochondria. Although the mitochondrial dPyd kinase was reported to phosphorylate ddC, the phosphorylation of ddC in isolated mitochondria was not detectable. The data suggest that ddC is phosphorylated to ddCTP in the cytoplasm and then transported into mitochondria to exert its inhibitory effect on mtDNA synthesis.

Substrate specificity of human deoxycytidine kinase toward antiviral 2prime;,3′-dideoxynucleoside analogs

Deoxycytidine (dCyd) kinase has been purified to homogeneity from human leukemic spleen, and the capacity of the enzyme to phosphorylate 2',3 '-dideoxynucleoside (ddN) analogs that are clinically effective inhibitors of human immunodeficiency virus (HIV) replication was evaluated. Cytosine-containing ddN analogs, such as 2',3'-dideoxycytidine, 2',3'-dideoxy-2',3'-dehydrocytidine, and cytallene, were efficiently phosphorylated by dCyd kinase, while no phosphorylation of purine-containing ddN analogs was detected. dCyd kinase was completely inactive toward 2',3'-dideoxyadenosine (ddAdo), 2',3'-dideoxyinosine, 2',3'-dideoxyguanosine, and adenallene, although it was capable of phosphorylating both 2'-deoxyadenosine (dAdo) and 2'-deoxyguanosine (dGuo). The abilities of wild type and mutant human T lymphoblastoid CEM cells to accumulate ddAdo in situ and in vitro were also ascertained. Comparison of the abilities of intact wild type CEM cells and derivatives deficient in nucleoside transport, dCyd kinase, and/or adenosine (Ado) kinase to accumulate [ 3H]ddAdo-derived radioactivity revealed no significant differences among the wild type and mutant strains. However, ddAdo phosphorylating activity was decreased in extracts from Ado kinase-deficient cells but not in lysates prepared from cells genetically deficient in dCyd kinase activity. In comparative growth rate experiments, wild type, nucleoside transport-deficient, and dCyd kinase-deficient CEM cells were equally sensitive to ddAdo toxicity, while, interestingly, a deficiency in Ado kinase correlated with a 5-fold decreased growth sensitivity to the purine ddN. Insertion of an adenine phosphoribosyltransferase deficiency into the CEM cell lines did not influence ddAdo toxicity or incorporation rate. These results imply that Ado kinase may be an important factor in ddAdo phosphorylation by CEM cells. Furthermore, these studies demonstrate that cytosine- and purine-containing ddNs are transported and activated by independent pathways and, therefore, have important implications for anti-HIV therapy in that pyrimidine and purine ddNs might be used in combination for the treatment of acquired immunodeficiency syndrome.

Cloning and expression of human deoxycytidine kinase cDNA.

Deoxycytidine (dCyd) kinase is required for the phosphorylation of several deoxyribonucleosides and certain nucleoside analogs widely employed as antiviral and chemotherapeutic agents. Detailed analysis of this enzyme has been limited, however, by its low abundance and instability. Using oligonucleotides based on primary amino acid sequence derived from purified dCyd kinase, we have screened T-lymphoblast cDNA libraries and identified a cDNA sequence that encodes a 30.5-kDa protein corresponding to the subunit molecular mass of the purified protein. Expression of the cDNA in Escherichia coli results in a 40-fold increase in dCyd kinase activity over control levels. In dCyd kinase-deficient murine L cells, transfection with dCyd kinase cDNA in a mammalian expression vector produces a 400-fold increase over control in dCyd phosphorylating activity. The expressed enzyme has an apparent Km of 1.0 microM for dCyd and is also capable of phosphorylating dAdo and dGuo. Northern blot analysis reveals a single 2.8-kilobase mRNA expressed in T lymphoblasts at 5- to 10-fold higher levels than in B lymphoblasts, and decreased dCyd kinase mRNA levels are present in T-lymphoblast cell lines resistant to arabinofuranosylcytosine and dideoxycytidine. These findings document that this cDNA encodes the T-lymphoblast dCyd kinase responsible for the phosphorylation of dAdo and dGuo as well as dCyd and arabinofuranosylcytosine.

Metabolism in Human Leukocytes of Anti‐HIV Dideoxypurine Nucleosidesa

Of the dideoxynucleosides described to date, the purine analogues ddA and ddI have exhibited very favorable therapeutic ratios in vitro. ddI is presently undergoing extensive phase I-II clinical trials. Whereas the action of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) is usually to convert a given analogue of Ado to an inactive or less active form, ddI appears to retain the same biological activity as that of the parent ddA. An explanation for these observations was possible when we found that ddI (1) underwent only a slow cleavage to hypoxanthine through the action of PNP and (2) accumulated the same active antiviral metabolite (i.e., ddATP) as ddA in human lymphoid cells. The use of human lymphoid cells with deficiencies in cellular nucleoside kinases and of inhibitors of pathways of nucleotide metabolism have also revealed new aspects of dideoxypurine metabolism in human lymphoid cells, including the identification of a salvage pathway (phosphotransferase/5'-nucleotide pathway) by which ddA/ddI may be metabolized preferentially to the active nucleotide. The effectiveness of ddA and ddI as orally administered antiviral agents may be limited by their susceptibility to acid hydrolysis and the low efficiency for nucleotide conversion in human lymphoid cells. The presence of a fluorine atom in the arabinose configuration on C-2 confers resistance to solvolysis and renders the analogue less susceptible to enzymatic deamination and resistant to phosphorylytic cleavage by PNP. In addition, human lymphoid cells accumulated several fold higher levels of the putative active triphosphate, 2'-F-dd-ara-ATP, than those of ddA or ddI. This increased accumulation of the analogue triphosphate could be accounted for by a more direct conversion of 2'-F-dd-ara-A by a direct phosphorylation through dCyd kinase than ddA. Thus, a single substitution with fluorine at the 2' "up" position of the sugar moiety of ddA markedly improves several biochemical properties relating to dideoxynucleotide accumulation in human lymphoid cells. Whether there are significant alterations of other biochemical properties, such as the ability of the analogue triphosphate to interact with the target enzyme reverse transcriptase, has not yet been determined. Thus, a definitive resolution of the relative merit of ddA/ddI and its 2'-fluoro-arabinosyl analogue is not yet possible on the basis of the studies described here.

Direct photoaffinity-labelling of human deoxycytidine kinase with the feedback inhibitor dCTP

Deoxycytidine kinase (dCyd kinase, EC 2.7.1.74) is a key enzyme in the salvage pathway of deoxyribonucleosides, and the human enzyme is a dimer of two 30 kDa polypeptides with a broad substrate specificity, phosphorylating both purine and pyrimidine nucleosides and using various nucleoside triphosphates as phosphate donors. The enzyme is efficiently feedback-inhibited by dCTP, which presumably is the main regulator of its activity in vivo. Submicromolar concentrations of [32P]dCTP could be used for direct photoaffinity labelling of pure dCyd kinase isolated from leukaemic spleen. A clearcut saturation of photoincorporation occurred with half-maximal incorporation at 0.07 microM-dCTP. However, the total molar incorporation of dCTP was very low (approx. 0.1%), in part due to a substantial u.v. inactivation of the enzyme. Proteinase digestion of labelled enzyme showed that dCTP was incorporated predominantly into a single peptide. Addition of equimolar concentrations of dCyd or dCMP as compared with dCTP inhibited photoincorporation approx. 50%. The presence of other nucleoside substrates, as well as phosphate donors, also inhibited photolabelling of the enzyme. Thus photoincorporation of dCTP seems to occur at a site which can bind both the phosphate donors and acceptors of dCyd kinase, which strongly support the hypothesis that dCTP functions as a multi-substrate analogue, binding and bridging both substrate sites of the enzyme.