Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase.

Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2'-deoxy-2'-fluoro-ß-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.

Electron transfer characteristics of 2'-deoxy-2'-fluoro-arabinonucleic acid, a nucleic acid with enhanced chemical stability.

The non-biological nucleic acid 2'-deoxy-2'-fluoro-arabinonucleic acid (2'F-ANA) may be of use because of its higher chemical stability than DNA in terms of resistance to hydrolysis and nuclease degradation. In order to investigate the charge transfer characteristics of 2'F-ANA, of relevance to applications in nucleic acid-based biosensors and chip technologies, we compare the electronic couplings for hole transfer between stacked nucleobase pairs in DNA and 2'F-ANA by carrying out density functional theory (DFT) calculations on geometries taken from molecular dynamics simulations. We find similar averages and distribution widths of the base-pair couplings in the two systems. On the basis of this result, 2'F-ANA is expected to have charge transfer properties similar to those of DNA, while offering the advantage of enhanced chemical stability. As such, 2'F-ANA may serve as a possible alternative to DNA for use in a broad range of nanobiotechnological applications. Furthermore, we show that the (experimentally observed) enhanced chemical stability resulting from the backbone modifications does not cause reduced fluctuations of the base-pair electronic couplings around the values found for "ideal" B-DNA (with standard step parameter values). Our study also supports the use of a DFT implementation, with the M11 functional, of the wave function overlap method to compute effective electronic couplings in nucleic acid systems.

Activation of guanine-ß-D-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints.

The deoxyguanosine (GdR) analog guanine-ß-d-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.

[Phase I study of nelarabine in patients with relapsed or refractory T-ALL/T-LBL].

The safety, tolerability, pharmacokinetics and efficacy of nelarabine were evaluated in adult and pediatric patients with relapsed or refractory T-ALL/T-LBL. Adult patients received nelarabine i.v. over 2 hours on days 1, 3 and 5 in every 21 days, and pediatric patients received this regimen over 1 hour for 5 consecutive days in every 21 days. Safety was evaluated in 7 adult and 6 pediatric patients. Adverse events (AEs) were reported in all patients. Most frequently reported AEs included somnolence and nausea in adult patients and leukopenia and lymphocytopenia in pediatric patients. Five grade 3/4 AEs were reported in both adult and pediatric patients, most of which were hematologic events. There were no dose-limiting toxicities. Efficacy was evaluated in 7 adult and 4 pediatric patients. Complete response was noted in 1 adult and 2 pediatric patients. Higher intracellular ara-GTP concentrations were suggested to be associated with efficacy. Japanese adult and pediatric patients with T-ALL/T-LBL well tolerated nelarabine treatment, warranting further investigation.

Studies on the hydrolytic stability of 2'-fluoroarabinonucleic acid (2'F-ANA).

The stability of 2'-deoxy-2'-fluoroarabinonucleic acid (2'F-ANA) to hydrolysis under acidic and basic conditions was compared to that of DNA, RNA and 2'F-RNA. In enzyme-free simulated gastric fluid (pH approximately 1.2), 2'F-ANA was found to have dramatically increased stability (virtually no cleavage observed after 2 days) with respect to both DNA (t(1/2) approximately 2 min) and RNA (t(1/2) approximately 3 h (PO) or 3 days (PS)). These results were observed for both phosphodiester and phosphorothioate backbones and with multiple mixed-base sequences. Under basic conditions, 2'F-ANA also showed good stability. In 1 M NaOH at 65 degrees C, 2'F-ANA had a t(1/2) of approximately 20 h, while RNA was entirely degraded in a few minutes. Furthermore, the nuclease cleavage of phosphorothioate 2'F-ANA and DNA by snake venom phosphodiesterase was studied in detail. One diastereomer of the PS-2'F-ANA linkage was found to be much more vulnerable to enzymatic cleavage than the other, which is parallel to the properties observed for PS-DNA. Additional studies of 2'F-ANA-containing oligonucleotides are warranted based on the excellent stability properties described here.

DNA polymerase recognition of 2'-deoxy-2'-fluoroarabinonucleoside 5'-triphosphates (2'F-araNTPs).

We examined the ability of 2'-deoxy-2'-fluroarabinonucleoside 5'-triphosphates (2'F-araNTPs) to serve as substrates of various DNA polymerases. In addition, we also examined the ability of these polymerases to accept DNA-FANA (2'-deoxy-2'-fluoroarabinonucleic acids) chimeras as template strands while synthesizing a DNA or FANA-DNA complementary strand. We provide preliminary data demonstrating that 2'F-araNTPs are indeed substrates of several DNA polymerases, and that FANA-DNA chimeric templates are generally well recognized by these polymerase enzymes.

Long intracellular retention of 4'-thio-arabinofuranosylcytosine 5'-triphosphate as a critical factor for the anti-solid tumor activity of 4'-thio-arabinofuranosylcytosine.

4'-Thio-arabinofuranosylcytosine (T-araC) is a new cytosine analog, which exhibits excellent antitumor activity against various solid tumor xenografts in mice. T-araC is a structural analog of arabinofuranosylcytosine (araC), which is known to be marginally active against solid tumors. We have continued to study the biochemical pharmacology of T-araC in solid tumor cells to further characterize the mechanism of action of this new agent and to elucidate why these compounds show a profound difference in antitumor activity against solid tumors. AraC was a slightly more potent inhibitor of cell growth than T-araC when cells were continuously exposed to the drugs. However, T-araC was markedly more cytotoxic than araC when high concentrations of the compounds were given for short periods of time. Despite the fact that T-araC is a much poorer substrate, as compared to araC, for deoxycytidine kinase (the rate-limiting step in the formation of the triphosphates), similar intracellular concentrations of T-araC-5'-triphosphate (T-araCTP) and araCTP were formed in cells at these high, pharmacologically relevant concentrations due to similar Vmax's. The major difference in the metabolism of araC and T-araC was that the half-life of T-araCTP was tenfold longer than that of araCTP and much higher levels of T-araCTP were sustained in cells for long durations after exposure to T-araC. Inhibition of cytidine deaminase, deoxycytidylate deaminase, or DNA replication did not affect the half-life of either araCTP or T-araCTP. In addition, the rates of disappearance of the mono- and tri-phosphates of araC and T-araC in crude cell extracts were similar. These results indicated that these enzymes were not rate-limiting in the degradation of the respective triphosphates. However, the rate of phosphorylation of T-araC-5'-monophosphate (T-araCMP) in crude cell extracts was about tenfold greater than that of araCMP. The results of this work suggested that the longer intracellular retention of T-araCTP was responsible for the superior activity of T-araC against solid tumors in vivo, and that the greater activity of T-araCMP as a substrate of UMP/CMP kinase was responsible for the long intracellular half-life of T-araCTP.

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2'-fluoro-ANA/RNA and DNA/RNA hybrids.

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'-fluoro-ANA analog (2'F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2'F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3'-endo (north, A-form) conformation, whereas those of the ANA strand adopt a 'rigid' O4'-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2'F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2'F-ANA/RNA and DNA/RNA helices is 9.0 +/- 0.5 A, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2'F-ANA/RNA hybrids to elicit RNase H activity.

Pharmacokinetics of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in leukemic cells after intravenous and subcutaneous administration of 1-beta-D-arabinofuranosylcytosine.

The concentration of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate was determined in leukemic cells from 5 patients with acute nonlymphoblastic leukemia during treatment with 1-beta-D-arabinofuranosylcytosine (50 mg/sq m every 12 h). The drug was administered both s.c. and i.v. (bolus injection) to all patients. After various periods of time, venous blood samples were collected and leukemic cells were isolated by density gradient centrifugation. The intracellular concentration of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate was assayed by high-performance liquid chromatography. The peak concentration of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate was significantly higher after s.c. injection than after i.v. injection (P less than 0.05). The area under the concentration versus time curve was twice as large after s.c. injection as it was after i.v. injection (P less than 0.01). The results are consistent with clinical findings indicating that the therapeutic effect of 1-beta-D-arabinofuranosylcytosine is better when it is administered s.c. than when given as i.v. bolus injections.

Modulation of arabinosylnucleoside metabolism by arabinosylnucleotides in human leukemia cells.

Previous studies have indicated that deoxycytidine kinase (dCK) is requisite and rate limiting in the phosphorylation of 1-beta-D-arabinofuranosylcytosine (ara-C) and 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on the pathway to their respective cytotoxic 5'-triphosphates. In K562 cells, the rate of triphosphate accumulation was maximal during incubation with 10 microM ara-C (35 microM/h) and 300 microM F-ara-A (102 microM/h). Under these conditions, accumulation of cellular ara-CTP plateaued at about 110 microM after 3 h, whereas in separate cultures, F-ara-ATP continued to accumulate at a linear rate to cellular concentrations greater than 500 microM after 5 h. Other laboratories have demonstrated that dCK activity in cell-free extracts was inhibited by ara-CTP. To determine whether ara-CTP exhibited the same activity in whole cells, K562 cells were preincubated with ara-C to accumulate 110 microM ara-CTP. After washing into medium containing F-ara-A, the rate of F-ara-ATP accumulation was significantly decreased (37 microM/h). However, cells loaded with F-ara-ATP exhibited an increased rate of ara-CTP accumulation (110 microM/h) that resulted in cellular ara-CTP concentrations in excess of 400 microM after 5 h. This stimulation was proportional to the cellular concentration of F-ara-ATP, achieving a maximum effect between 75 and 100 microM. Phosphorylation of ara-C by cell-free extracts supplemented with physiological levels of ribo- and deoxyribonucleoside 5'-triphosphates was stimulated by addition of F-ara-ATP. The decreased rate of accumulation of products of dCK in intact cells containing 110 microM ara-CTP suggests that this active triphosphate may limit its own synthesis and phosphorylation of other substrates. In contrast, stimulation of the accumulation of ara-CTP in cells containing F-ara-ATP suggests new possibilities for the design of combination chemotherapy regimens.

Cellular retention of 9-beta-D-arabinofuranosyladenine 5'-triphosphate and the pattern of recovery of DNA synthesis in Chinese hamster ovary cells.

The factors associated with the recovery of DNA synthesis in Chinese hamster ovary cells after inhibition with 9-beta-D-arabinofuranosyladenine have been investigated. Maintenance of cellular concentrations of the active metabolite 9-beta-D-arabinofuranosyladenine 5'-triphosphate was dependent upon a constant exogenous source of 9-beta-D-arabinofuranosyladenine, without which the triphosphate rapidly degraded. DNA synthesis was inhibited maximally at cellular concentrations of 9-beta-D-arabinofuranosyladenine 5'-triphosphate in excess of 100 microM, but it recovered substantially as the triphosphate concentrations declined below 30 microM. Autoradiographic studies indicated that 9-beta-D-arabinofuranosyladenine had a synchronizing effect on the cells and that recovery of DNA synthesis occurred initially at rates much reduced from those seen in uninhibited cells. A greater portion of an inhibited population recovered DNA synthesis than was able to retain clonogenic potential. These results are consistent with a mechanism of toxicity that postulates recovery of DNA synthesis as a requisite for the loss of viability.

Saturation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate accumulation in leukemia cells during high-dose 1-beta-D-arabinofuranosylcytosine therapy.

Twenty-seven patients with refractory leukemia were treated with 1-beta-D-arabinofuranosylcytosine (ara-C), 0.3 to 3.0 g/m2 as i.v. infusions over 1, 2, 4, or 24 h. The pharmacokinetics of ara-C in plasma and its 5'-triphosphate (ara-CTP) in leukemic cells from peripheral blood were studied after a single infusion of 3 g/m2 over 2 h in 13 patients. Accumulation of ara-CTP in leukemic cells remained linear until 1 to 2 h after the infusion. At the time when the rate of ara-CTP accumulation deviated from linearity, the plasma concentration of ara-C was 5- to 20-fold lower [8.1 +/- 4.4 (SD) microM] than the steady-state level during the infusion. Plasma ara-C and cellular ara-CTP pharmacokinetics were studied after two serial infusions in 14 additional patients. Varying the duration of infusion of an ara-C dose between 1, 2, and 4 h (corresponding to infusion rates of 3000, 1500, and 750 mg/m2/h) did not substantially change the rate of ara-CTP accumulation by leukemic cells. The peak ara-CTP concentration and the area under the concentration times time curve (AUC) of ara-CTP in leukemic cells increased with prolongation of the infusion. Although steady-state concentration of ara-C and AUC of ara-C in plasma were proportionally reduced by 1.0 or 0.5 g/m2 infusion over 2 h, ara-CTP accumulation rate and AUC in leukemic cells did not change compared with administration of 3 g/m2 over 2 h. However, when the infusion rate was further reduced to 0.4 or 0.3 g/m2 over 2 h, resulting in steady-state plasma ara-C concentrations of less than 7 microM, the accumulation rate of ara-CTP was substantially reduced as was the ara-CTP intracellular AUC. The cellular elimination rate of ara-CTP remained constant under all infusion conditions. These findings support the conclusion that high-dose ara-C therapy, as currently administered, results in plasma ara-C concentrations that saturate the accumulation of ara-CTP by circulating leukemic cells. We recommend that intermediate dose rates, 200 to 250 mg/m2/h, be evaluated in future studies as an alternative to the substantially higher ara-C dose rates currently in use.

Degradation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in human leukemic myeloblasts and lymphoblasts.

The intracellular half-life for retention of the active triphosphate metabolite 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP) of 1-beta-D-arabinofuranosylcytosine was measured in vitro in blast cells from patients with acute myeloblastic leukemia, acute lymphoblastic leukemia, and T-cell lymphoblastic lymphoma. araCTP accumulation from 1 microM 1-beta-D-arabinofuranosylcytosine in leukemic blast cells was closely correlated with the nucleoside transport capacity as measured by equilibrium binding of [3H]nitrobenzylthioinosine. The half-life of araCTP retention was related to araCTP accumulation only when the level of araCTP was expressed as a percentage of total intracellular 1-beta-D-arabinofuranosylcytosine metabolites. Accumulation of 1-beta-D-arabinofuranosyluracil 5'-monophosphate was inversely related to the half-life of araCTP retention and directly related to dCMP deaminase activity in cell free extracts. No conversion of 1-beta-D-arabinofuranosyluracil to 1-beta-D-arabinofuranosyluracil 5'-monophosphate was detectable in intact cells. The end product of araCTP degradation was 1-beta-D-arabinofuranosyluracil and it is proposed that conversion of 1-beta-D-arabinofuranosylcytosine 5'-monophosphate to 1-beta-D-arabinofuranosyluracil 5'-monophosphate is a step in the degradative pathway of araCTP. However, it is the cells' nucleoside transport capacity which primarily determines the level of intracellular araCTP accumulation.

Methotrexate-induced changes in the levels of 1-beta-D-arabinofuranosylcytosine triphosphate in L1210 cells.

Treatment with methotrexate (MTX) plus 1-beta-D-arabinofuranosylcytosine (ara-C) on Days 1, 4, and 7 after i.p. inoculation of L1210 ascites cells was more effective than treatment with one drug on Days 1, 4, and 7 followed by the second drug on Days 2, 5, and 8. Simultaneous treatment with both drugs was associated with a retention of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) but no increase in the activity of deoxycytidine kinase in L1210 cells, whereas pretreatment with MTX 24 hr before the administration of ara-C was associated with approximately 2-fold increases in the level of ara-CTP and of deoxycytidine kinase in L1210 cells. However, from 5 hr after treatment with ara-C, higher levels of ara-CTP were observed in L1210 cells treated simultaneously with both drugs than in cells from animals pretreated with MTX 24 hr before treatment with ara-C. The superiority of simultaneous treatment over sequential treatment and the synergism between MTX and ara-C, previously reported for this schedule of simultaneous treatment, are attributed in part to the MTX-induced retention of ara-CTP and the increased exposure of L1210 cells to ara-CTP that results from the slower clearance of ara-CTP.

Role of dephosphorylation in accumulation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in human lymphoblastic cell lines with reference to their drug sensitivity.

After a 3-day exposure to 0.1 microM 1-beta-D-arabinofuranosylcytosine (ara-C) in culture, growth was inhibited to 5.6% in MOLT-4F, 25% in Raji, and 91% in Daudi cells compared with control. Growth inhibition was more profound when exposure time was extended up to 7 days. Inhibition of DNA synthesis varied with sensitivity to ara-C. Plateau levels of intracellular 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) were 35.5, 13.4, and 3.6 nmol/10(9) cells exposed to 0.1 microM ara-C in MOLT-4F, Raji, and Daudi cells, respectively, corresponding to the sensitivity to ara-C. The nucleotide levels at the plateau, however, did not correspond to the initial levels of the ara-CTP or to the calculated rate of ara-CTP synthesis, which decreased from Raji to MOLT-4F to Daudi cells. ara-C deamination had negligible effect on the differential accumulation of ara-CTP. ara-CTP degradation due to dephosphorylation was marked in Raji and Daudi cells but slight in MOLT-4F cells. The half-life of intracellular ara-CTP was 204, 26.4, 31.1 min in MOLT-4F, Raji, and Daudi cells, respectively. The ara-CTP level was considered to be maintained bimodally by synthesis and degradation of the nucleotide. This conclusion was supported by the fact that, in Raji and Daudi cells exposed to 0.1 microM ara-C in the presence of 1 mM hydroxyurea, the plateau levels of ara-CTP increased 3-fold through inhibition of the nucleotide degradation. Thus, not only ara-C phosphorylation but also subsequent ara-CTP dephosphorylation was important in the accumulation and maintenance of ara-CTP and in the sensitivity to ara-C.

Modulatory activity of 2',2'-difluorodeoxycytidine on the phosphorylation and cytotoxicity of arabinosyl nucleosides.

This investigation analyzed the metabolism of 2',2'-difluorodeoxycytidine (dFdC) in K562 human leukemia cells and evaluated it as a biochemical modulator for the phosphorylation of several arabinosyl nucleosides. The rate of accumulation of dFdC triphosphate was linear up to 3 h and maximal during incubation with 10 microM dFdC (92 microM/h). Deoxynucleotides analyzed at this time showed a decrease in dCTP, dATP, and dGTP levels, indicating an inhibitory role of dFdC nucleotides in ribonucleotide reduction. We evaluated the hypothesis that dFdC-mediated deoxyribonucleoside triphosphate perturbation enhances the phosphorylation of substrates that use deoxycytidine kinase or deoxyguanosine kinase, because these enzymes are inhibited by dCTP or dGTP, respectively. When the activity of these nucleoside kinases was rate limiting to triphosphate formation, the accumulation of triphosphates of deoxycytidine, 1-beta-D-arabinofuranosylcytosine, and 1-beta-D-arabinofuranosylguanine was potentiated in cells pretreated with dFdC. In contrast, the phosphorylation of 9-beta-D-arabinofuranosyladenine was not affected, since it is mainly phosphorylated by adenosine kinase, which is not influenced by deoxyribonucleoside triphosphates. Treatment of cells with dFdC followed by 1-beta-D-arabinofuranosylcytosine resulted in greater cytotoxicity than sum effects of each drug alone. The data indicate that an enhanced cytotoxicity could be obtained by administering dFdC as a modulator followed by 1-beta-D-arabinofuranosylcytosine or 1-beta-D-arabinofuranosylguanine in optimal sequence, suggesting that these results should be considered in the design of combination clinical protocols.

Effects of 1-beta-D-arabinofuranosylcytosine on DNA replication intermediates monitored by pH-step alkaline elution.

The pH-step alkaline elution method enables the isolation and quantification of nascent DNA (nDNA) replication intermediates, including Okazaki fragments, short length nDNA from replicon origins, longer lengths of nascent but subgenomic length nDNA (molecular weight, 20-30 x 10(6)), and full (or genomic) length nDNA (L. C. Erickson et al., Chromosoma, 74: 125-139, 1979). We utilized this technique to study, in HL-60 cells, the effects of 1-beta-D-arabinofuranosylcytosine (ara-C) on the formation of these replication intermediates and the kinetics of transit of radiolabel from [3H]thymidine ([3H]dThd) or [3H]-ara-C through these nDNA fragments and into full length nDNA. In the continuous presence of [3H]-ara-C (4 microM), the majority of radiolabel (greater than 85%) remained in the nascent subgenomic fractions, with 30-50% remaining in Okazaki fragments. These proportions did not change substantially with increasing time of exposure to [3H]-ara-C (from 1 to 24 h), although the total amount of [3H]-ara-C incorporated into DNA continued to increase with increasing time of exposure. In contrast, when cells were exposed to [3H]-ara-C for 1 h, placed in drug-free medium, and studied by the pH-step method at various times thereafter, the transit of radiolabel through progressively larger nDNA intermediates and into full length nDNA was rapid and equal to that of [3H]dThd in cells not exposed to drug. The observed elution of [3H]-ara-C in the subgenomic-length DNA fragments was not due to ara-C-induced breaks in template (parental) DNA and subsequent incorporation of [3H]-ara-C into the template strand, since ara-C treatment of cells prelabeled with [14C]dThd failed to cause substantial elution of the 14C label at the various pH steps used. In studies of the effects of ara-C on [3H]dThd incorporation into nDNA, concentrations of 1 to 10 microM ara-C inhibited total incorporation of radiolabel into DNA by greater than 90% and incorporation into full length nDNA by greater than 97%. In contrast, these concentrations of ara-C failed to decrease the amount of [3H]dThd incorporated into Okazaki fragments or other non-mitochondrial low molecular weight nDNA, compared to control. These studies demonstrate that, in HL-60 cells, ara-C causes profound inhibition of nascent chain elongation, does not cause chain termination, and does not inhibit initiation. In fact, ara-C may stimulate initiation, leading credence to recent theories proposing endoreduplication or reinitiation as consequences of ara-C incorporation into DNA.

Modulation of 9-beta-D-arabinofuranosyladenine 5'-triphosphate and deoxyadenosine triphosphate in leukemic cells by 2'-deoxycoformycin during therapy with 9-beta-D-arabinofuranosyladenine.

The effect of the adenosine deaminase inhibitor, 2'-deoxycoformycin, on cellular nucleotides during therapy with continuous infusion of 9-beta-D-arabinofuranosyladenine (ara-A) has been investigated. In three courses of treatment using increasing doses, the active 5'-triphosphate of ara-A, 9-beta-D-arabinofuranosyladenine 5'-triphosphate (ara-ATP) accumulated in leukemic cells and erythrocytes from a patient treated for acute lymphocytic leukemia in proportion to the dose of ara-A. The cellular ara-ATP concentration increased more than 5-fold after the injection of a single, nontoxic, but pharmacologically active dose of 2'-deoxycoformycin 24 hr after initiation of ara-A infusion. However, this response was associated with a concomitant increase in the cellular deoxyadenosine triphosphate concentrations to levels equal to or greater than those of ara-ATP throughout the three treatment courses studied. Consistent with previous results using cell-free systems, it was demonstrated that a competitive relationship exists between deoxyadenosine triphosphate and ara-ATP for the inhibition of DNA synthesis in cultured human lymphoblastoid cells and that the ratio of the cellular concentrations of these nucleotides could predict the extent of inhibition of DNA synthesis. Application of this rationale to the nucleotides in the leukemic cells of the patient suggested that administration of 2'-deoxycoformycin may create a cellular biochemical milieu that could be antagonistic to the inhibition of DNA synthesis by ara-ATP.

Quantitation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in the leukemic cells from bone marrow and peripheral blood of patients receiving 1-beta-D-arabinofuranosylcytosine therapy.

A method for the detection and quantitation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), the active metabolite cells and leukemic cells of the peripheral blood from patients receiving ara-C therapy is described. ara-CTP is separated from normal cellular nucleotides by high-pressure liquid chromatography and is quantitated by its absorbance of ultraviolet light at 280 nm with a lower limit of sensitivity of 25 pmol/2 x 10(7) cell equivalents. During separate courses of continuous infusion of different therapeutic doses of ara-C, ara-CTP accumulated in the leukemic bone marrow cells of a patient with acute myelogenous leukemia in proportion to the dose of ara-C. Continuous infusion of ara-C (90 mg/sq m/day) resulted in plateau levels of ara-CPT in peripheral blast cells after 24 hr (115 pmol/1 x 10(7) cell equivalents). A priming dose of ara-C(125 to 250 mg/sq m) followed by a 1-hr infusion of an equal dose of ara-C to patients with acute myelogenous leukemia facilitated the determination of ara-CTP retention in bone marrow and peripheral blood leukemic cells in vivo. This procedure should be useful for extended studies of the biochemical pharmacology of ara-CTP in vivo.

Differential metabolism of 9-beta-D-arabinofuranosylguanine in human leukemic cells.

9-beta-D-Arabinofuranosylguanine (araG) is a nucleoside analogue that elicits cytotoxicity through the intracellular accumulation of its 5'-triphosphate, araGTP, araG is selectively toxic to cultured T-lymphoblasts due to their ability to accumulate higher levels of the cytotoxic metabolite, araGTP, relative to B- and null lymphoblastoid cells. In an effort to determine whether this selectivity may occur in leukemic cells in vivo, we have investigated the metabolism of araG in MOLT-4 T-lymphoblasts. MGL-8 B-lymphoblasts, HL-60 promyelocytes, and HUT-102 mature T-cells and compared it to that in freshly isolated leukemic cells from patients. MOLT-4 T-lymphoblasts were 50- to 380-fold more sensitive to growth inhibition with araG and accumulated 80-fold higher levels of araGTP than any of the other cell lines studied. Incubation of peripheral blood from patients with leukemia with araG for 4 h demonstrated that T-acute lymphocytic leukemia cells accumulated significantly higher median levels of araGTP than did acute myelogenous leukemia or chronic lymphocytic leukemia cells (187 versus 72 and 31 pmol of araGTP per 10(7) cells, respectively), araGTP accumulation was not dependent on the rate of degradation of araG during the incubation. In contrast, araG did not exhibit similar selective growth inhibition, nor did the accumulation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in the freshly isolated leukemic cells differ significantly among T-acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-T-, non-B-cell acute lymphocytic leukemia cells. These results demonstrate that the selective metabolism of araG observed in cultured cell lines was representative of the metabolism in freshly isolated leukemic cells. Furthermore, degradation of araG did not limit the accumulation of araGTP in the leukemic cells. These results indicate that araG may be valuable as a selectively acting chemotherapeutic agent in T-lymphoblastic malignancies.