Bromodeoxyuridine enhancement of 1-beta-D-arabinofuranosylcytosine metabolic activation and toxicity in HL-60 leukemic cells.
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We tested whether bromodeoxyuridine (BrdUrd), an analogue of thymidine (dThd), enhances 1-beta-D-arabinofuranosylcytosine (ara-C) metabolic activation, as does dThd. HL-60 cells were exposed to 10, 100, or 1000 nM ara-C for 3 h. Simultaneous exposure of log phase HL-60 cells to BrdUrd (1-1000 microM) and ara-C for 3 h resulted in enhancement of ara-C incorporation into DNA, with a doubling of incorporation in response to 10 nM ara-C occurring at concentrations of BrdUrd greater than 100 microM. Preexposure of cells to BrdUrd for 16 h followed by addition of ara-C for 3 h resulted in even greater ara-C incorporation into DNA. This increase was most marked at the lower concentrations of ara-C (10 and 100 nM), where approximately 3-fold enhancement of ara-C incorporation was observed in response to BrdUrd concentrations greater than 100 microM. Intracellular pools of 1-beta-D-arabinofuranosyl-CTP increased significantly (up to 3-fold) following 16-h exposure to BrdUrd (30, 100, or 300 microM) at all concentrations of ara-C tested. The ara-C phosphorylating activity of cell-free extracts obtained following 16-h exposure of cells to BrdUrd increased 1.5- to 2.3-fold over control. Intracellular dCTP pools fell to approximately 50% of control after exposure to 750 microM BrdUrd or dThd. Exposure to BrdUrd for 16 h caused a concentration-dependent increase in cells with S-phase DNA content, as assessed by flow cytometry, with a doubling of cells in S phase (to 60%) observed in response to 500 microM BrdUrd. HL-60 cells exposed to identical conditions of BrdUrd for 3 h showed no significant alteration in cell cycle phase distribution. Thus, although BrdUrd does increase cells in S phase, the increased ara-C incorporation caused by BrdUrd cannot be explained solely on a cytokinetic basis since enhancement of incorporation was observed after a 3-h exposure of cells to BrdUrd and ara-C. The combination of ara-C (100 nM) and BrdUrd (100-1000 microM) exhibited cytotoxic synergism, as measured by the fluorescein diacetate/propidium iodide method. These data demonstrate a clear potential for BrdUrd modulation of ara-C metabolism in human leukemia. Additionally, the interaction of BrdUrd and ara-C should be considered in the interpretation of studies of the effects of ara-C on DNA synthesis as measured by flow cytometric quantification of incorporated BrdUrd. (+info)
Effects of 1-beta-D-arabinofuranosylcytosine incorporation on elongation of specific DNA sequences by DNA polymerase beta.
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1-beta-D-Arabinofuranosylcytosine (ara-C) is an effective antileukemic agent which acts as an inhibitor of DNA synthesis. The precise mechanism responsible for this inhibitory effect, however, remains unclear. The present work has examined the effects of the triphosphate derivative, ara-CTP, on purified DNA polymerase beta. These studies were performed on M13 phage DNA templates of defined sequence. The results demonstrate that ara-C is incorporated into DNA by DNA polymerase beta. The results also demonstrate that the incorporated ara-C residue acts as a relative chain terminator. Moreover, the relative chain terminating effects of ara-C are sequence specific. In this regard, DNA strand elongation was progressively slowed at sequences of two, three, and four contiguous sites for cytosine incorporation. We also demonstrate that the inhibitory effects of ara-C are reversed by competition with deoxycytidine-triphosphate for incorporation into the DNA strand. Taken together, these findings are consistent with structural differences of the incorporated arabinosyl moiety which alter reactivity of the 3'-terminus and thereby inhibit chain elongation. These findings also provide new insights regarding the inhibitory effects of ara-C on elongation of specific DNA sequences. (+info)
Comparison of the cellular pharmacokinetics and toxicity of 2',2'-difluorodeoxycytidine and 1-beta-D-arabinofuranosylcytosine.
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2',2'-Difluorodeoxycytidine (dFdC) is a new deoxycytidine analogue with good activity against human leukemic cell lines and murine solid tumors, while the activity of 1-beta-D-arabinofuranosylcytosine (ara-C) is established in experimental systems and for the treatment of human adult leukemia. This study compared the cellular metabolism and cytotoxic properties of dFdC and ara-C in Chinese hamster ovary cells. In wild-type cells, dFdC was significantly more cytotoxic than ara-C after both 4- and 18-h incubations. The 5'-triphosphate of dFdC (dFdCTP) was the major cellular metabolite (85-90%), reaching cellular concentrations up to 20-fold greater than those observed for ara-C 5'-triphosphate at equimolar concentrations of the parent drug. A deoxycytidine kinase-deficient mutant neither accumulated dFdCTP nor showed any cytotoxic response up to drug concentrations of 100 microM. The cytotoxicity of dFdC could be competitively reversed by deoxycytidine further suggesting that dFdC, like ara-C, required phosphorylation by deoxycytidine kinase for biological activity. Several explanations for the different cellular accumulation of the drug triphosphates were established: (a) nucleoside transport studies demonstrated that the membrane permeation of dFdC was 65% more rapid than that of ara-C; (b) deoxycytidine kinase had a higher affinity for dFdC (Km = 3.6 microM) than for ara-C (Km = 8.8 microM), while the Km for deoxycytidine was 1.4 microM; (c) the elimination of intracellular dFdCTP was biphasic with t1/2 alpha = 3.9 and t1/2 beta greater than 16 h while the degradation of ara-CTP was monophasic and significantly faster (t1/2 = 0.7 h). The comparatively long half-life of dFdCTP was related to the prolonged inhibition of DNA synthesis after removal of exogenous nucleoside. Together these factors contribute to the more potent cytotoxicity of dFdC compared with ara-C. (+info)
Correlation of drug-perturbed marrow cell growth kinetics and intracellular 1-B-D-arabinofuranosylcytosine metabolism with clinical response in adult acute myelogenous leukemia.
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To define the relationship between leukemic cell growth, intracellular metabolism of 1-B-D-arabinofuranosylcytosine (ara-C), and the clinical response to timed sequential induction therapy with ara-C in adult acute myelogenous leukemia (AML), growth kinetic and biochemical pharmacologic determinants were examined in AML bone marrow populations. Leukemic blasts from 45 previously untreated patients obtained prior to therapy were cultured in vitro in autologous pretreatment serum (APS) and in serum containing drug-induced humoral stimulatory activity (HSA). Cell populations cultured in HSA demonstrated both increased proliferation, as measured by both [3H]dThd incorporation into DNA and [3H]dThd leukemic blast labeling index, and greater [3H] ara-C leukemic blast labeling index relative to cells maintained in APS. HSA-cultured marrow cells from the 31 patients who achieved complete remission with ara-C-containing therapy demonstrated enhanced intracellular formation of ara-C 5'-triphosphate over three hours and retention of this active form during one subsequent hour in drug-free medium relative to cells maintained in APS. In contrast, cells from the 14 nonresponsive patients demonstrated no such HSA-induced increases in intracellular ara-C metabolism. These studies of human AML marrow cells identify behavior patterns of ara-C activation and net metabolism in the kinetically perturbed, proliferative state that may discriminate clinical sensitivity from clinical resistance to ara-C-based timed sequential therapy. Sensitive AML populations behave similarly to normal hematopoietic cohorts, with direct linkage of HSA-perturbed growth and pharmacologic parameters, while refractory cells demonstrate uncoupling of these determinants in the growth-stimulated state. These in vitro measurements may further serve as a template for prediction of clinical outcome to timed sequential therapy with ara-C, where both pharmacologic and cytokinetic determinants of response are intrinsic to the success of the designed drug scheduling. (+info)
Saturation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate accumulation in leukemia cells during high-dose 1-beta-D-arabinofuranosylcytosine therapy.
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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. (+info)
Degradation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in human leukemic myeloblasts and lymphoblasts.
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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. (+info)
Biochemical pharmacology of high dose 1-beta-D-arabinofuranosylcytosine in childhood acute leukemia.
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The pharmacodynamic parameters of 1-beta-D-arabinofuranosylcytosine (ara-C) in patient plasma and its active anabolite 1-beta-D-arabinofuranosylcytosine-5-triphosphate (ara-CTP) in circulating and bone marrow blast cells were studied in 20 pediatric patients with acute leukemia. ara-C (3 g/m2) was administered as a short-term infusion over 3 h every 12 h for a total of eight doses. The peak plasma concentration of ara-C ranged from 0.02 to 5.6 microM after the first dose of ara-C. The area under the concentration-time curve (AUC) of ara-C in plasma ranged from 302 to 20,298 microMh after the first dose of ara-C. The half-life of elimination (t1/2,el) of ara-C from plasma was 2.4 +/- 1.5 h in three patients with acute nonlymphoblastic leukemia (ANLL) and 4.78 +/- 4.1 h in 9 patients with acute lymphoblastic leukemia (ALL). The intracellular peak concentration of ara-CTP in circulating blast cells averaged 432.2 +/- 14.5 microM and 544.3 +/- 330 microM in patients with ANLL and ALL, respectively. The elimination kinetics of ara-CTP was monoexponential with t1/2,el of 3.30 +/- 0.8 h and 6.9 +/- 2.8 h in patients with ANLL and ALL. DNA synthetic capacity (DSC) of the blast cells was inhibited to between 24 and 64% of control after the first dose of ara-C and it declined further to between 1 and 32% after four doses of ara-C. The AUC of ara-CTP in leukemic cells ranged from 1,073 to 14,751 microMh and it was not related to the AUC of ara-C in plasma. The pharmacodynamic parameters of ara-CTP in circulating blast cells were more homogeneous in patients with ANLL than in patients with ALL. Four of six patients (67%) with ANLL and six of 14 patients (43%) with ALL achieved either complete remission or partial remission with high dose ara-C. We conclude that treatment of pediatric patients with leukemia in relapse with high dose ara-C is tolerable and moderately successful. Inhibition of DSC is positively correlated with the probability of having zero nadir peripheral blast cells. In turn there is a trend for a zero nadir peripheral blast cell count to be related to achievement of a response to therapy. This latter result is consistent with the results of larger studies in adults with leukemia. (+info)
Regulation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate accumulation in human leukemia cells by deoxycytidine 5'-triphosphate.
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Cell cycle-specific fluctuations in the ability of human leukemic cells to phosphorylate 1-beta-D-arabinofuranosylcytosine (ara-C) to the toxic metabolite 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) was investigated in whole cells and in cell extracts. Exponentially growing CCRF-CEM cells were fractionated into populations enriched for G1 phase cells and S phase cells by centrifugal elutriation. The accumulation of ara-CTP by S phase-enriched cells was 50% greater than in G1-enriched cells. However, the ability of extracts of S phase-enriched cells to phosphorylate ara-C was twice that of G1 phase-enriched cell extracts. As cells passed from G1 to S phase, this disproportionality was significant. As demonstrated in other cell types, deoxycytidine 5'-triphosphate (dCTP) also potently inhibited ara-C phosphorylation in CCRF-CEM cell extracts (Ki = 5.9 microM). Deoxynucleotide pool levels determined by high pressure liquid chromatography showed a 5 microM dCTP concentration in G1-enriched cells, whereas S phase-enriched cells contained 15 microM dCTP. These findings suggest that the lack of proportionality between the accumulation of ara-CTP in whole cells and the increase of ara-C phosphorylation in extracts during the G1 to S phase transition may be caused by more stringent regulation of ara-C phosphorylation in whole cells by the concomitant increase in cellular dCTP concentrations. Because such regulation is unlikely to be observed in cell extracts, these results indicated that assays of ara-C phosphorylating activity in cell extracts represent upper limits for that function in whole cells. Such determinations may not reflect the regulated nature of the metabolic pathway. (+info)