Accumulation Of dATP Research Articles

A nuclear orthologue of the dNTP triphosphohydrolase SAMHD1 controls dNTP homeostasis and genomic stability in Trypanosoma brucei.

Maintenance of dNTPs pools in Trypanosoma brucei is dependent on both biosynthetic and degradation pathways that together ensure correct cellular homeostasis throughout the cell cycle which is essential for the preservation of genomic stability. Both the salvage and de novo pathways participate in the provision of pyrimidine dNTPs while purine dNTPs are made available solely through salvage. In order to identify enzymes involved in degradation here we have characterized the role of a trypanosomal SAMHD1 orthologue denominated TbHD82. Our results show that TbHD82 is a nuclear enzyme in both procyclic and bloodstream forms of T. brucei. Knockout forms exhibit a hypermutator phenotype, cell cycle perturbations and an activation of the DNA repair response. Furthermore, dNTP quantification of TbHD82 null mutant cells revealed perturbations in nucleotide metabolism with a substantial accumulation of dATP, dCTP and dTTP. We propose that this HD domain-containing protein present in kinetoplastids plays an essential role acting as a sentinel of genomic fidelity by modulating the unnecessary and detrimental accumulation of dNTPs.

Improved Synthesis of Deoxyadenosine Triphosphate by Saccharomyces cerevisiae Using an Efficient ATP Regeneration System: Optimization of Response Surface Analysis.

Deoxyadenosine triphosphate (dATP) is an important biochemical molecule. In this paper, the synthesis of dATP from deoxyadenosine monophosphate (dAMP), catalyzed by Saccharomyces cerevisiae, was studied. By adding chemical effectors, an efficient ATP regeneration and coupling system was constructed to achieve efficient synthesis of dATP. Factorial and response surface designs were used to optimize process conditions. Optimal reaction conditions were as follows: dAMP 1.40 g/L, glucose 40.97 g/L, MgCl2·6H2O 4.00 g/L, KCl 2.00 g/L, NaH2PO4 31.20 g/L, yeast 300.00 g/L, ammonium chloride 0.67 g/L, acetaldehyde 11.64 mL/L, pH 7.0, temperature 29.6 °C. Under these conditions, the substrate conversion was 93.80% and the concentration of dATP in the reaction system was 2.10 g/L, which was 63.10% higher than before optimization, and the concentration of product was 4 times higher than before optimization. The effects of glucose, acetaldehyde, and temperature on the accumulation of dATP were analyzed.

A Prospective Multicenter Phase II Trial of Pentostatin in Adult Patients with Steroid-Refractory Chronic Graft-Versus-Host Disease (cGvHD): A Cancer and Leukemia Group B/Eastern Cooperative Oncology Group Study.

Abstract 2238Poster Board II-215Chronic graft-versus-host disease is the most common late complication occurring following allogeneic transplantation, and is the principal cause of late morbidity and non-relapse mortality. Corticosteroids remain the cornerstone of primary treatment for cGvHD with about half of patients responding. Patients who fail to respond to initial corticosteroid therapy, however, have a poor outcome with no standard approach uniformly accepted. Pentostatin is a purine nucleoside analogue that irreversibly inhibits adenosine deaminase, with accumulation of dATP, causing apoptosis of lymphocytes, and holds promise for the management of acute and cGvHD. We conducted a multi-institutional, prospective phase II trial to evaluate the response rate (RR) of pentostatin in patients with steroid-refractory cGvHD. In a two-stage design with both types I and II errors set at 0.1, we hypothesized that pentostatin would produce a response rate (complete response + partial response) of 40% or more, with less than 20% being unaccpetable. Patients were eligible if they were >18 years old, had a Karnofsky performance status (KPS) of >30%, histologically-proven extensive stage cGvHD that was refractory to corticosteroids defined as progression of disease despite at least 2 weeks of treatment with 1 mg/kg/day of prednisone or equivalent, no response or minor response to at least 4 weeks of 1 mg/kg/day prednisone (or equivalent), or who achieved only a partial response after 8 weeks of corticosteroid therapy. Response was assessed using the Hopkins scoring system as previously reported (Jacobsohn et al. J Clin Oncol, 25:4255-61, 2007). Pentostatin was administered at a dose of 4 mg/m2 IV every 2 weeks for a maximum of 12 doses. From March 2004 to March 2007, 38 patients with median age 46.5 (27-66) years and median KPS 70% (40%-90%) were treated with pentostatin. cGvHD followed transplantation from matched related donors in 29 and unrelated donors in 9 patients. Thirty-three patients had progressive onset cGvHD, while onset was quiescent in 2, and de novo in 3 patients. The median time from onset of cGvHD to start of study treatment was 416 (26-2,813) days, and the median number of prior lines of therapy for cGvHD was 3 (range, 1-6). cGvHD involved skin (n=35) with scleroderma present in 21 patients, oral mucosa (n=24), liver (n=9), gut (n=4), and lung (n=2). Thirty-five patients were evaluable for response; 2 patients died after 6 and 7 days of treatment, respectively, due to sepsis, and data was incomplete in 1 patient who committed suicide after 3 cycles. Of 35 patients evaluable for response, there were 2 complete responses (CR), 5 partial responses (PR) for a total response rate of 20%. If minor responses (in 7 patients) are also considered, then 14 of 35 (40%) had an objective improvement in cGvHD. Six patients had stable disease, 1 a mixed response, and 14 patients progressed on therapy. Of 34 patients evaluable for toxicity, grade 3-4 hematological toxicity included neutropenia (n=4), thrombocytopenia (n=3), and anemia (n=1). Grade 3-4 clinically significant non-hematological toxicity that was at least possibly related to pentostatin included fatigue (n=3), renal failure (n=3), anorexia (n=2), infection (n=9), CNS hemorrhage (n=1), and rash (n=1). Thirteen patients died on study; causes of death were infection (n=6), cGvHD progression (n=3), and unrelated causes (n=3). The 1-year and 2-year progression-free survival (for GvHD) for all patients treated on study were 32% (95% CI: 18%-46%) and 24% (95% CI: 12%-38%), respectively. The 1-year and 2-year overall survival was 53% (95% CI: 38%-60%) and 50% (95% CI: 33%-65%), respectively. We conclude that pentostatin is an active agent in patients with steroid-refractory chronic GvHD but should be investigated further in cGvHD patients in an earlier course of their disease where a greater impact on long-term outcome may be observed. Disclosures:No relevant conflicts of interest to declare.

Restoration of Adenosine Deaminase-Deficient Human Thymocyte Development In Vitro by Inhibition of Deoxynucleoside Kinases

Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an "in vivo-like" environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.

Inhibition of Deoxynucleoside Kinases in Human Thymocytes Prevents dATP Accumulation and Induction of Apoptosis

Thymocytes lacking adenosine deaminase (ADA) activity, a purine metabolism enzyme, accumulate intracellular dATP and consequently undergo apoptosis during development. We have analyzed the effect of ADA enzyme inhibition in human thymocyte suspension cultures with regard to accumulation of intracellular dATP and induction of apoptosis. We demonstrate that while inhibition of deoxycytidine kinase will prevent the accumulation of dATP and induction of apoptosis to a large degree, inhibition of both deoxycytidine kinase and adenosine kinase completely abrogates the accumulation of dATP and significantly reduces the induction of apoptosis. Thus, both deoxynucleoside kinases are involved in this model of ADA deficiency.

Further Differentiation of Murine Double-Positive Thymocytes Is Inhibited in Adenosine Deaminase-Deficient Murine Fetal Thymic Organ Culture

Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4(-)CD8(-)CD25(+)CD44(low) stage in ADA-deficient FTOC initiated at day 15 of gestation when essentially all thymocytes are CD4(-)CD8(-). In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOC when substantial numbers of CD4(+)CD8(+) thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of alphabeta TCR(+) thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or by the use of apoptotic protease-activating factor-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4(+)CD8(+) cells, when FTOC were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positive/negative selection checkpoint.

Activation of deoxycytidine kinase by deoxyadenosine: Implications in deoxyadenosine-mediated cytotoxicity

The inborn deficiency of adenosine deaminase is characterised by accumulation of excess amounts of cytotoxic deoxyadenine nucleotides in lymphocytes. Formation of dATP requires phosphorylation of deoxyadenosine by deoxycytidine kinase (dCK), the main nucleoside salvage enzyme in lymphoid cells. Activation of dCK by a number of genotoxic agents including 2-chlorodeoxyadenosine, a deamination-resistant deoxyadenosine analogue, was found previously. Here, we show that deoxyadenosine itself is also a potent activator of dCK if its deamination was prevented by the adenosine deaminase inhibitor deoxycoformycin. In contrast, deoxycytidine was found to prevent stimulation of dCK by various drugs. The activated form of dCK was more resistant to tryptic digestion, indicating that dCK undergoes a substrate-independent conformational change upon activation. Elevated dCK activities were accompanied by decreased pyrimidine nucleotide levels whereas cytotoxic dATP pools were selectively enhanced. dCK activity was found to be downregulated by growth factor and MAP kinase signalling, providing a potential tool to slow the rate of dATP accumulation in adenosine deaminase deficiency.

Metabolic response patterns of nucleotides in B-cell chronic lymphocytic leukaemias to cladribine, fludarabine and deoxycoformycin

Nucleotides (NTPs and dNTPs) have been measured in patient-derived chronic lymphocytic leukaemia (CLL) cells stimulated with TPA plus ionomycin and then exposed to cladribine, fludarabine or deoxycoformycin (1 μM, 16 h). dNTPs were not measurable in 10 8 unstimulated CLL cells which are quiescent. Time-dependent changes in nucleotides in CLL cells showed that the mechanism of action changed as the drug triphosphate accumulated. dCf induced substantial accumulation of dATP in CLL cells in culture, and similar levels of dATP in the same patient during subsequent treatment with dCf. Determination of “ metabolic response patterns” of nucleotides in CLL cells treated with drugs might distinguish patients with susceptible and refractory CLL prior to chemotherapy.

Impaired germinal center maturation in adenosine deaminase deficiency.

Mice deficient in the enzyme adenosine deaminase (ADA) have small lymphoid organs that contain reduced numbers of peripheral lymphocytes, and they are immunodeficient. We investigated B cell deficiency in ADA-deficient mice and found that B cell development in the bone marrow was normal. However, spleens were markedly smaller, their architecture was dramatically altered, and splenic B lymphocytes showed defects in proliferation and activation. ADA-deficient B cells exhibited a higher propensity to undergo B cell receptor-mediated apoptosis than their wild-type counterparts, suggesting that ADA plays a role in the survival of cells during Ag-dependent responses. In keeping with this finding, IgM production by extrafollicular plasmablast cells was higher in ADA-deficient than in wild-type mice, thus indicating that activated B cells accumulate extrafollicularly as a result of a poor or nonexistent germinal center formation. This hypothesis was subsequently confirmed by the profound loss of germinal center architecture. A comparison of levels of the ADA substrates, adenosine and 2'-deoxyadenosine, as well resulting dATP levels and S-adenosylhomocysteine hydrolase inhibition in bone marrow and spleen suggested that dATP accumulation in ADA-deficient spleens may be responsible for impaired B cell development. The altered splenic environment and signaling abnormalities may concurrently contribute to a block in B cell Ag-dependent maturation in ADA-deficient mouse spleens.

Mechanisms of apoptosis in developing thymocytes as revealed by adenosine deaminase-deficient fetal thymic organ cultures

Adenosine deaminase (ADA) catalyzes the conversion of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. ADA-deficient individuals suffer from severe combined immunodeficiency and are unable to produce significant numbers of mature T or B lymphocytes. This occurs as a consequence of the accumulation of ADA substrates or their metabolites. dATP is a candidate toxic metabolite because its concentration in RBCs of ADA-deficient patients correlates with the severity of disease. Murine fetal thymic organ culture (FTOC) under ADA-deficient conditions can be used as a model system to investigate the biochemical mechanism responsible for the inhibition of thymopoiesis. In ADA-deficient FTOCs initiated at day 15 of gestation, thymocyte development was arrested at the CD4 −CD8 −CD44 loCD25 + to CD4 −CD8 −CD44 loCD25 − transition. Apoptosis appeared to be involved because the cultures could be rescued by the pan-caspase inhibitor zVADfmk, a Bcl-2 transgene, or deletion of apoptotic protease activating factor-1. As in ADA-deficient patients, dATP was also elevated in ADA-deficient FTOCs. dATP levels were normalized and thymocyte development was rescued in cultures treated with an inhibitor of adenosine kinase, the enzyme that phosphorylates deoxyadenosine to dAMP. zVADfmk also prevented the accumulation of dATP in ADA-deficient FTOCs, suggesting that deoxyadenosine was derived from thymocytes undergoing apoptosis as a consequence of failing the β selection checkpoint. In contrast, dATP levels remained elevated in ADA-deficient FTOCs with fetal thymuses from Bcl-2 transgenic mice. These data suggest that thymocyte apoptosis as a consequence of failing developmental checkpoints involves one or more caspases that are not regulated by Bcl-2.

Mechanism of inhibition of T-acute lymphoblastic leukemia cells by PNP inhibitor—BCX-1777

Purine nucleoside phosphorylase (PNP) deficiency in humans produces a relatively selective depletion of T cells. BCX-1777 is a potent inhibitor of PNP. BCX-1777 in the presence of deoxyguanosine (dGuo) inhibits the proliferation of CEM-SS [T-acute lymphoblastic leukemia (T-ALL)] cells with an IC 50=0.015 μM. This inhibition by BCX-1777 and dGuo is accompanied by elevation of dGTP (154-fold) and dATP (8-fold). Deoxycytidine (dCyt) completely and lamivudine (3TC) partially reverse this inhibition caused by BCX-1777 and dGuo. dNTP analysis of these samples indicates that, in the presence of dCyt, where complete reversal of inhibition is observed, dGTP and dATP pools revert back to the control levels. In samples containing 3TC, where partial reversal of inhibition was observed, dGTP decreased from 154-fold to 38-fold and dATP levels further increased from 8-fold to 30-fold compared to the control sample. In CEM-SS cells, inhibition of proliferation by BCX-1777 and dGuo is not due to accumulation of dATP because in the presence of 3TC, where reversal of inhibition is observed, dATP levels are further increased. These studies clearly indicate that inhibition of T cells is due to accumulation of dGTP resulting in cell death with characteristics of apoptosis. The half-life of dGTP in CEM-SS cells is 18 h, which is longer than that observed in human lymphocytes (4 h), suggesting that the nucleotidase level in CEM-SS cells is lower than in human lymphocytes. A 154-fold accumulation of dGTP in CEM-SS cells in the presence of BCX-1777 and dGuo compared to a 15-fold accumulation of dGTP in human lymphocytes suggests that kinase level is higher in CEM-SS cells compared to human lymphocytes. High kinase and low nucleotidase levels make CEM-SS cells more sensitive to inhibition by BCX-1777 and dGuo than human lymphocytes. Currently, BCX-1777 is in phase I/II clinical trial for the treatment of T cell malignancies.

Adenosine kinase inhibition promotes survival of fetal adenosine deaminase-deficient thymocytes by blocking dATP accumulation.

Thymocyte development past the CD4(-)CD8(-) stage is markedly inhibited in adenosine deaminase-deficient (ADA-deficient) murine fetal thymic organ cultures (FTOCs) due to the accumulation of ADA substrates derived from thymocytes failing developmental checkpoints. Such cultures can be rescued by overexpression of Bcl-2, suggesting that apoptosis is an important component of the mechanism by which ADA deficiency impairs thymocyte development. Consistent with this conclusion, ADA-deficient FTOCs were partially rescued by a rearranged T cell receptor beta transgene that permits virtually all thymocytes to pass the beta-selection checkpoint. ADA-deficient cultures were also rescued by the adenosine kinase inhibitor 5'-amino-5'-deoxyadenosine (5'A5'dAdo), indicating that the metabolite responsible for the inhibition of thymocyte development is not adenosine or deoxyadenosine, but a phosphorylated derivative of an ADA substrate. Correction of ADA-deficient FTOCs by 5'A5'dAdo correlated with reduced accumulation of dATP, implicating this compound as the toxic metabolite. In ADA-inhibited FTOCs rescued with a Bcl-2 transgene, however, dATP levels were superelevated, suggesting that cells failing positive and negative selection continued to contribute to the accumulation of ADA substrates. Our data are consistent with dATP-induced mitochondrial cytochrome c release followed by apoptosis as the mechanism by which ADA deficiency leads to reduced thymic T cell production.

Adenosine kinase inhibition promotes survival of fetal adenosine deaminase–deficient thymocytes by blocking dATP accumulation

Adenosine kinase inhibition promotes survival of fetal adenosine deaminase–deficient thymocytes by blocking dATP accumulation

Adenosine kinase inhibition promotes survival of fetal adenosine deaminase–deficient thymocytes by blocking dATP accumulation

Adenosine kinase inhibition promotes survival of fetal adenosine deaminase–deficient thymocytes by blocking dATP accumulation

Metabolites from apoptotic thymocytes inhibit thymopoiesis in adenosine deaminase-deficient fetal thymic organ cultures.

Murine fetal thymic organ culture was used to investigate the mechanism by which adenosine deaminase (ADA) deficiency causes T-cell immunodeficiency. C57BL/6 fetal thymuses treated with the specific ADA inhibitor 2'-deoxycoformycin exhibited features of the human disease, including accumulation of dATP and inhibition of S-adenosylhomocysteine hydrolase enzyme activity. Although T-cell receptor (TCR) Vbeta gene rearrangements and pre-TCR-alpha expression were normal in ADA-deficient cultures, the production of alphabeta TCR(+) thymocytes was inhibited by 95%, and differentiation was blocked beginning at the time of beta selection. In contrast, the production of gammadelta TCR(+) thymocytes was unaffected. Similar results were obtained using fetal thymuses from ADA gene-targeted mice. Differentiation and proliferation were preserved by the introduction of a bcl-2 transgene or disruption of the gene encoding apoptotic protease activating factor-1. The pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone also significantly lessened the effects of ADA deficiency and prevented the accumulation of dATP. Thus, ADA substrates accumulate and disrupt thymocyte development in ADA deficiency. These substrates derive from thymocytes that undergo apoptosis as a consequence of failing to pass developmental checkpoints, such as beta selection.

INTERLEUKIN-7 RECEPTOR α CHAIN–DEPENDENT SIGNALING IS REQUIRED FOR T-CELL DEVELOPMENT

Severe combined immunodeficiencies (SCIDs) are inherited diseases that represent the most severe forms of primary immunodeficiencies, affecting approximately 1 child out of every 80,000 live births. These diseases are characterized by profound defects in cellular and humoral immunity. As a result, affected individuals typically die within the first year of life because of recurrent opportunistic infections, unless they receive successful bone marrow transplants. SCID is most appropriately described as a syndrome with diverse genetic origins. Because of advances made in genetics and molecular biology, it has become possible to identify the genes responsible for many forms of SCID, although approximately 30% of cases have unknown causes.6, 20, 45 In this article, SCID refers to cases of inherited immunodeficiency characterized by a marked decrease in T cells, as previously defined.20 Patients with other inherited immunodeficiencies, such as those resulting from mutations in the T-cell receptor (TCR)–associated chains, CD3γ or CD3ε, or in the TCR-associated tyrosine kinase ZAP-70, may have normal or decreased numbers of T cells, but typically do not develop life-threatening infections in the first months of life.20 Patients with SCID then can be classified into four groups according to their basic immunologic phenotypes (Table 1). In all four groups, T cells are almost uniformly decreased in numbers; however, the levels of B and natural killer (NK) cells are more variable. In the first group, T, B, and NK cells are all diminished (T−B−NK− SCID). The most severe form in this group is reticular dysgenesis, a disease in which the lymphoid and myeloid systems are affected.18 The responsible gene for this form of SCID has not been identified. Adenosine deaminase (ADA) deficiency is less severe, but still also is characterized by a profound block in lymphocyte maturation.30 Mutations in the ADA gene result in the accumulation of dATP in cells, therefore inhibiting cell division. The major impact of ADA deficiency is in the immune system. In the second group, T and B cells are diminished but NK cells are not (T−B−NK+ SCID). Approximately 20% of all patients with SCID have such a block in T- and B-lymphocyte differentiation because of a defect in the recombination process.83 This group can be divided in two subgroups: one with a normal cell radiosensitivity, resulting from mutations in the RAG1 or RAG2 genes, which are responsible for the initiation of the V(D)J rearrangement process,1, 80 and the other with an increased cell radiosensitivity, in which the involved gene(s) have not been identified.11 The third and fourth groups are characterized by the presence of B cells (T−B+ SCID); however, the B cells are usually nonfunctional, in part because of an absence of T-cell help.13 The third group is characterized by the absence of NK cells (T−B+NK− SCID). The most frequent form of SCID in this group—and indeed the most common overall form of SCID is X-linked SCID (XSCID). In XSCID, which accounts for almost 50% of cases of SCID, patients have profoundly diminished numbers of T cells and NK cells, but normal or increased numbers of B cells.45, 47 XSCID was found to result from mutations in the gene encoding the γ chain of the interleukin-2 (IL-2) receptor.61 As discussed below, IL-2Rγ is now denoted as the common cytokine receptor γ chain, γc, because it is shared by the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15.24, 36, 38, 39, 60, 61, 75, 76, 87 The XSCID phenotype also has been found in patients with an autosomal recessive form of SCID, and it was hypothesized that this form of SCID could result from mutations in the gene coding for the tyrosine kinase Jak3,75 which associates with γc and is activated by IL-2, IL-4, IL-7, IL-9, and IL-15.48 Finally, the fourth group of SCID consists of patients with a selective T-cell defect (T−B+NK+ SCID). One of the genetic causes for this syndrome is defective expression of IL-7Rα,70 which is the principal focus of this article.

Human Monocytoid Leukemia Cells Are Highly Sensitive to Apoptosis Induced by 2′-Deoxycoformycin and 2′-Deoxyadenosine: Association With dATP-Dependent Activation of Caspase-3

The adenosine deaminase (ADA) inhibitor 2′-deoxycoformycin (dCF) significantly inhibits the proliferation of leukemia and lymphoma cell lines. When cells were incubated in the presence of both dCF and 2′-deoxyadenosine (dAd), the concentration of dCF required to induce apoptosis of monocytoid leukemia cells was much lower than that required for myeloid, erythroid, or lymphoma cell lines. Among the cell lines tested, U937 cells were the most sensitive to this treatment. The concentration of dCF that effectively inhibited the proliferation of U937 cells was 1/1,000 of that required for lymphoma cell lines, on a molar basis. However, the uptake of dCF or dAd in U937 cells was comparable with that in other leukemia and lymphoma cell lines. The intracellular accumulation of dATP in U937 cells was only slightly higher than that in other leukemia cells in dCF-treated culture. Treatment with dCF plus dAd induced apoptosis in U937 cells at low concentrations, and this apoptosis was reduced by treatment with caspase inhibitors. Induction of caspase-3 (CPP32) activity accompanied the apoptosis induced by dCF plus dAd. No activation of CPP32 was observed in cytosol prepared from exponentially growing leukemia and lymphoma cells. However, dATP effectively induced CPP32 activation in cytosol from monocytoid cells, but not in that from nonmonocytoid cells, suggesting that dATP-dependent CPP32 activation is at least partly involved in the preferential induction of apoptosis in monocytoid leukemia cells. The combination of dCF and dAd may be useful for the clinical treatment of acute monocytic leukemia.© 1998 by The American Society of Hematology.

Human Monocytoid Leukemia Cells Are Highly Sensitive to Apoptosis Induced by 2′-Deoxycoformycin and 2′-Deoxyadenosine: Association With dATP-Dependent Activation of Caspase-3

AbstractThe adenosine deaminase (ADA) inhibitor 2′-deoxycoformycin (dCF) significantly inhibits the proliferation of leukemia and lymphoma cell lines. When cells were incubated in the presence of both dCF and 2′-deoxyadenosine (dAd), the concentration of dCF required to induce apoptosis of monocytoid leukemia cells was much lower than that required for myeloid, erythroid, or lymphoma cell lines. Among the cell lines tested, U937 cells were the most sensitive to this treatment. The concentration of dCF that effectively inhibited the proliferation of U937 cells was 1/1,000 of that required for lymphoma cell lines, on a molar basis. However, the uptake of dCF or dAd in U937 cells was comparable with that in other leukemia and lymphoma cell lines. The intracellular accumulation of dATP in U937 cells was only slightly higher than that in other leukemia cells in dCF-treated culture. Treatment with dCF plus dAd induced apoptosis in U937 cells at low concentrations, and this apoptosis was reduced by treatment with caspase inhibitors. Induction of caspase-3 (CPP32) activity accompanied the apoptosis induced by dCF plus dAd. No activation of CPP32 was observed in cytosol prepared from exponentially growing leukemia and lymphoma cells. However, dATP effectively induced CPP32 activation in cytosol from monocytoid cells, but not in that from nonmonocytoid cells, suggesting that dATP-dependent CPP32 activation is at least partly involved in the preferential induction of apoptosis in monocytoid leukemia cells. The combination of dCF and dAd may be useful for the clinical treatment of acute monocytic leukemia.© 1998 by The American Society of Hematology.

Comparative effects of cladribine, fludarabine and pentostatin on nucleotide metabolism in T- and B-cell lines

Background and aims: the purine nucleoside analogues cladribine (CdA), fludarabine (F-Ara-AMP) and pentostatin (dCf), are effective therapy for a range of T- and B-cell lymphoid malignancies. The effects upon nucleotide metabolism in human CCRF-CEM T-cell leukaemia and Raji B-cell lymphoma cell lines of these drugs have been compared to assess possible mechanisms of cytotoxicity. Methods: leukaemia cells were exposed to a purine nucleoside analogue and perchloric acid extracts were analysed by HPLC for 2′-deoxynucleoside-5′-triphosphates (dNTPs), nucleoside-5′-triphosphates (NTPs) and drug metabolites. Results: after addition of a purine nucleoside analogue, CdA-TP and F-Ara-ATP accumulate in cells while the levels of dCf-TP formed were not detectable by ultra-violet absorbance. In response to accumulating concentrations of drug triphosphate, the cellular levels of dNTPs initially decrease (0–4 h), then accumulate above their initial levels (4–10 h) before slowly declining beyond 10 h. NTPs also accumulate during the period 4–10 h before declining at later times. Conclusion: the temporal effects on the levels of dNTPs and NTPs of the 3 purine nucleoside analogues are similar against CCRF-CEM and Raji cells. However, CdA induces major depletions of dTTP, dGTP and dATP in CCRF-CEM cells and F-Ara-A induces a major accumulation of dATP in Raji cells.

Preclinical cellular pharmacology of LY231514 (MTA): a comparison with methotrexate, LY309887 and raltitrexed for their effects on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells.

LY231514 (N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethy l]-benzoyl]-L-glutamic acid) is a new folate-based antimetabolite currently in broad phase II clinical evaluation. Previous in vitro studies (C. Shih et al, CancerRes 57: 1116-1123, 1997) have suggested that LY231514 could be a multitargeted antifolate (MTA) capable of inhibiting thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study compared LY231514 with methotrexate, raltitrexed and a glycinamide ribonucleotide formyltransferase inhibitor, LY309887, at 300, 100, 30 and 100 nM, respectively, for their effects on intracellular folate and at 100, 66, 20 and 30 nM respectively, for their effects on nucleoside triphosphate pools in CCRF-CEM cells. Methotrexate induced an accumulation of dihydrofolate species, together with a rapid depletion of ATP, GTP and all of the deoxynucleoside triphosphates. LY309887 caused an accumulation of 10-formyltetrahydrofolate, a rapid loss of ATP, GTP and dATP, but a slower loss in dCTP, dTTP and dGTP. Both LY231514 and raltitrexed had minimal effects on folate pools. In contrast, they caused rapid depletion of dTTP, dCTP and dGTP, but induced an accumulation of dATP at different rates, with raltitrexed doing so about 2.5 times faster. Most of the observed metabolic changes could be understood on the basis of current knowledge of folate and nucleotide metabolism. We concluded that LY231514 was distinct from methotrexate, LY309887 and raltitrexed based on their metabolic effects in CCRF-CEM cells, and that in this cell line the inhibitory effects of LY231514 were exerted primarily against the thymidylate cycle and secondarily against de novo purine biosynthesis.