pentostatin and 2--deoxyadenosine-triphosphate

N6-Cyclopropyl-PMEDAP (cPr-PMEDAP) is a novel derivative of the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP). Its cytostatic activity was found to be 8- to 20-fold more pronounced than that of PMEDAP and equivalent to that of the guanine derivative 9-(2-phosphonylmethoxyethyl)guanine (PMEG) against a variety of tumor cell lines. Unlike PMEDAP, but like PMEG, cPr-PMEDAP was equally cytostatic to wild-type and 9-(2-phosphonylmethoxyethyl)adenine/PMEDAP-resistant variants of the human erythroleukemia K562 and the murine leukemia L1210 cell lines. Also, cPr-PMEDAP and PMEG proved to be equipotent inducers of K562 and rat choriocarcinoma RCHO cell differentiation, whereas the differentiation-inducing activity of PMEDAP was 5- to 25-fold less pronounced. Furthermore, compared to PMEDAP, cPr-PMEDAP and PMEG were 10- to 25-fold more potent in inhibiting the progression of K562 cells through the S phase of the cell cycle, resulting in a marked accumulation of the four 2'-deoxyribonucleoside 5'-triphosphate pools. The biological effects of cPr-PMEDAP, but not PMEDAP, were reversed by the adenylate deaminase inhibitor 2'-deoxycoformycin (dCF). Formation of the deaminated derivative of cPr-PMEDAP (i.e. PMEG) was demonstrated in crude extracts from K562 and L1210 cells and in metabolism studies with radiolabeled cPr-PMEDAP and PMEG. This is the very first example of an acyclic nucleoside phosphonate analogue that is susceptible to deamination. However, cPr-PMEDAP was not recognized as a substrate by purified adenosine deaminase or by adenylate deaminase. These findings might point to an as yet unidentified cellular enzyme, sensitive to dCF but different from the common adenosine and AMP deaminases. Our data demonstrate the superior antiproliferative and differentiation-inducing effects of cPr-PMEDAP on tumor cells, as compared to the parent compound PMEDAP, based on the unique metabolic properties of this novel compound.

Topics: Adenine; Adenosine Deaminase; Adenosine Triphosphate; AMP Deaminase; Animals; Antineoplastic Agents; Cell Cycle; Cell Differentiation; Cell Division; Deoxyadenine Nucleotides; Drug Interactions; Drug Screening Assays, Antitumor; Erythrocytes; Guanine; Humans; K562 Cells; Leukemia L1210; Mice; Organophosphonates; Organophosphorus Compounds; Pentostatin; Prodrugs; Rats; Tumor Cells, Cultured

Topics: Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Apoptosis; CD4-Positive T-Lymphocytes; Deoxyadenine Nucleotides; Deoxyadenosines; Deoxyribonucleotides; Flow Cytometry; Genes, p53; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Models, Biological; Pentostatin; Proto-Oncogene Proteins c-bcl-2; Thymus Gland; Transcription, Genetic; Tumor Suppressor Protein p53

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.

Topics: Apoptosis; Caspase 3; Caspases; Cytosol; Deoxyadenine Nucleotides; Deoxyadenosines; Enzyme Activation; Growth Inhibitors; HL-60 Cells; Humans; Leukemia; Leukemia, Monocytic, Acute; Lymphoma; Neoplasm Proteins; Pentostatin; Tumor Cells, Cultured; U937 Cells

The effect of the adenosine deaminase (ADA) inhibitor 2'-deoxycoformycin (dCF) on the development of insulin-dependent diabetes mellitus (IDDM) was assessed in the BB Wistar rat. Sixty-one male rats were treated from days 30 to 120 with 0, 0.5, 1.0 or 1.5 mg dCF/kg/week. The incidence of IDDM was 78% in the controls and was significantly (P < 0.01) decreased in rats receiving 1.5 mg dCF/kg/week (32%), but not in rats receiving lower doses of the drug. However, for those rats that became diabetic the mean time to the development of IDDM was unchanged in animals receiving dCF compared with control. dCF treatment did not produce significant weight loss in the animals or gross changes in the thymus, spleen or kidneys. Although the protective effect of dCF against IDDM was likely produced by immunosuppression, the different dCF dosages had similar effects on ADA suppression in spleen or thymus and on dATP accumulation in these organs.

Topics: Adenosine Deaminase Inhibitors; Animals; Deoxyadenine Nucleotides; Diabetes Mellitus, Type 1; Disease Models, Animal; Dose-Response Relationship, Drug; Male; Organ Size; Pentostatin; Rats; Rats, Inbred BB; Spleen; Thymus Gland

Conditions for labeling the dATP pool of V79 and 3T3 cells from [3H]deoxyadenosine (salvage) or [3H]adenine (via ribonucleotide reduction) were established. With deoxyadenosine the specific radioactivity of dATP reached a constant value after 60 min. In resting 3T3 cells this value was 30 times higher than in S-phase cells. Turnover of dATP and absolute rates of DNA synthesis and excretion of breakdown products of dATP were determined from the accumulation of isotope in various compartments and the specific activity of dATP. In S-phase cells the dATP pool had a half-life of 4 min, identical to that of dTTP determined earlier. Deoxyadenosine was the major breakdown product of dATP in the presence of an inhibitor of adenosine deaminase. The rate of deoxyadenosine excretion of V79 cells amounted to 4% of the rate of dATP incorporation into DNA. Inhibition of DNA replication increased deoxyadenosine excretion 5- to 10-fold, demonstrating a continued de novo synthesis of dATP, albeit at a slightly reduced rate. Our results fit a model involving a substrate cycle between dAMP and deoxyadenosine regulating the dATP pool, similar to the model of substrate cycles involved in the regulation of pyrimidine deoxyribonucleotide pools developed earlier.

Topics: 3T3 Cells; Adenine; Animals; Aphidicolin; Cell Line; Cytarabine; Deoxyadenine Nucleotides; Deoxyadenosines; DNA; Hypoxanthine; Hypoxanthines; Kinetics; Mice; Pentostatin; Substrate Cycling; Thymine Nucleotides

Deoxyadenosine triphosphate (dATP) is present in adenosine deaminase (ADA)-deficient or ADA-inhibited human red cells and in the red cells of the opossum Didelphis virginiana. In order to investigate the functions of dATP in the red cell, red cells were treated with 2'-deoxycoformycin (dCf), a powerful inhibitor of ADA, and incubated with phosphate, deoxyadenosine and glucose. These red cells in which ATP was almost completely replaced by dATP, had the same shape, lactate production, nucleotide consumption, stability of reduced glutathione, osmotic fragility and cell deformability as red cells containing ATP. Cells merely depleted of ATP showed reduced viability. This indicates that dATP compensates well for the absence of ATP and acts as an energy-transferring molecule to maintain cell viability. These results indicate that the accumulation of dATP or the reduction of ATP is not the cause of the hemolysis observed after dCf administration.

Topics: Adenosine Deaminase; Adenosine Triphosphate; Deoxyadenine Nucleotides; Energy Transfer; Erythrocyte Deformability; Erythrocytes; Glycolysis; Humans; Osmotic Fragility; Pentostatin

An experimental model of adenosine deaminase deficiency was established on the human T cell line Jurkat by using 2'-deoxycoformycin, a strong specific inhibitor of the enzyme. When deoxyadenosine was added to the inhibited cells, the nucleotide profile was modified reproducing that found in lymphocytes from adenosine deaminase-deficient children. The metabolism of phosphoinositides, analyzed by either the release of [3H]inositol phosphates or the breakdown of 32P-prelabeled phosphatidyl inositides, was compared in normal and modified cells where dATP was accumulated. No modification in 32P labeling of phosphoinositides was detectable within the 32P-loading period. However, when the cells were stimulated by phytohemagglutinin or anti-CD3 monoclonal antibody, the phosphoinositide hydrolysis was strongly reduced in the dATP-containing lymphoblasts. This decrease was correlated with the intracellular dATP concentration.

Topics: Adenosine Deaminase; Adenosine Deaminase Inhibitors; Antigens, Differentiation, T-Lymphocyte; CD3 Complex; Cell Line; Deoxyadenine Nucleotides; Humans; In Vitro Techniques; Lymphocyte Activation; Nucleoside Deaminases; Pentostatin; Phosphatidylinositols; Receptors, Antigen, T-Cell; T-Lymphocytes

Deoxycoformycin (DCF) is a specific inhibitor of adenosine deaminase (ADA) and has been shown to be active in lymphoid neoplasms. Cytotoxicity is thought to be mediated by the accumulation of deoxyadenosine (AdR) and deoxyadenosine triphosphate (dATP) which inhibits ribonucleotide reductase and DNA synthesis in rapidly proliferating cells. Others suggested mechanisms leading to cell death particularly in non-dividing cells include depletion of ATP and NAD pools, inhibition of S-adenosylhomocysteine (SAH) hydrolase and induction of DNA strand breaks. In patients with high leukemic counts who were subsequently treated with DCF, we have studied (a) the levels of ADA, ecto-5'-nucleotidase (5NT), deoxyadenosine kinase (AdR-kinase) and SAH-hydrolase in the leukemic cells; [b) the in-vitro effects of DCF on dATP, ATP, NAD, SAH-hydrolase levels and on DNA strand breaks; and (c) the correlation between these parameters with clinical response to DCF. No significant difference in ADA, 5NT, AdR-kinase and SAH-hydrolase activities could be found between responders and non-responders. Incubation of the leukemic cells in vitro with DCF caused an inhibition of ADA, an accumulation of dATP, a moderate reduction in ATP and NAD levels, a suppression of SAH-hydrolase activity and an increase in DNA strand breaks in practically all the leukemic samples, irrespective of clinical response. Our results show that neither measurement of these enzymes nor studies of these biochemical sequelae of ADA inhibition in vitro predicts clinical responsiveness to DCF therapy.

Topics: Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Adenosylhomocysteinase; Antineoplastic Agents; Coformycin; Deoxyadenine Nucleotides; DNA Damage; Humans; Hydrolases; Leukemia; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Prolymphocytic; Leukemia, Prolymphocytic, T-Cell; NAD; Nucleoside Deaminases; Pentostatin; Ribonucleosides

Deoxyadenosine plus deoxycoformycin (dCf) causes increased DNA breaks in lymphoid cells. This study explored the possible inhibition of repair synthesis of DNA by dAdo plus dCf as a cause of DNA breakage. It was shown that DNA breaks accumulated in a human T-lymphoblast cell line, CCRF-CEM, following incubation with dAdo plus dCf and were not fully repaired 20 h after their removal. Analysis of the density distribution of radiolabeled DNA on alkaline CsCl gradient showed that incubation of CCRF-CEM cells with dAdo plus dCf caused inhibition of semiconservative, but not repair synthesis of DNA. Semiconservative synthesis of DNA was also inhibited in CCRF-CEM nuclei isolated from cells pretreated with dAdo and dCf, suggesting damage to DNA replicative machinery. However, no such inhibition was observed in the nuclei of a similarly treated CCRF-CEM mutant that was deficient in adenosine kinase and deoxycytidine kinase. This suggests that dAdo must be phosphorylated in intact cells to exert its effect. Using [3H]dTTP incorporation in isolated CCRF-CEM nuclei to measure DNA synthesis, it was found that a high concentration (greater than 100 microM) of dATP inhibits semiconservative but not repair synthesis of DNA. The present studies thus indicate that accumulation of DNA strand breaks induced by dAdo plus dCf is not the consequence of inhibition of repair DNA synthesis. This implies the mechanism may involve perturbation of DNA ligation or activation of a certain process which causes DNA strand breaks. In addition, dATP may interfere with some steps of semiconservative DNA synthesis, but not the repair synthesis of DNA.

Topics: Cell Line; Cell Nucleus; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; DNA Repair; DNA Replication; Humans; Kinetics; Pentostatin; Ribonucleosides; T-Lymphocytes

The effects of irradiation were evaluated in L5178Y lymphoblasts treated with the adenosine deaminase inhibitor, 2'-deoxycoformycin, and deoxyadenosine. A synergistic antitumor effect was observed in resting cells between irradiation and 2'-deoxycoformycin/deoxyadenosine, with the dose required to reduce the surviving cell fraction to 0.1 being 25% lower than predicted for an additive effect. Synergy was enhanced with increasing deoxyadenosine concentration or with increasing radiation dose. When cells were treated with 2'-deoxycoformycin/deoxyadenosine for 1 h prior to irradiation, synergy was increased by prolonging postirradiation drug treatment. With 4-h postirradiation exposure to drug, varying the preirradiation incubation time did not affect synergy. In contrast, only a small enhancement of antitumor activity was observed in irradiated proliferating cells treated with 2'-deoxycoformycin/deoxyadenosine. Incubation of resting cells with 2'-deoxycoformycin/deoxyadenosine resulted in inhibition of the rate and extent of repair of radiation-induced DNA single strand breaks and an increase in dATP, but had no effect on NAD or ATP. With removal of drug, the dATP level fell rapidly and DNA repair resumed. Repair of DNA single strand breaks was more rapid in proliferating cells than in resting cells and was minimally affected by 2'-deoxycoformycin/deoxyadenosine, although the accumulation of dATP in these cells was 2-fold greater than in resting cells. The repair of DNA single strand breaks in chronic lymphocytic leukemia cells was as rapid as for proliferating L5178Y cells, but repair was significantly inhibited by 2'-deoxycoformycin/deoxyadenosine. These results suggest that 2'-deoxycoformycin/deoxyadenosine can function as a radiosensitizer, and this effect is associated with the cellular accumulation of dATP and inhibition of repair of DNA single strand breaks.

Topics: Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Animals; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; DNA Damage; DNA Repair; Drug Synergism; Leukemia L5178; Leukemia, Experimental; Leukemia, Lymphoid; Mice; NAD; Pentostatin; Ribonucleosides

Four patients with refractory chronic lymphocytic leukemia were treated with the adenosine deaminase inhibitor, 2'-deoxycoformycin, and initially received 4 mg/m2 i.v. weekly. Clinical responses to therapy varied: Patient A had a minimal response; whereas Patient D showed an 85% decrease in lymphocyte count at 2 wk; and Patients B and C had intermediate responses. The pretreatment mononuclear cell adenosine deaminase activities, which ranged from 1.6 to 44.6 nmol adenosine/h/10(6) cells, decreased to approximately 1 nmol adenosine /h/10(6) cells 24 h following 2'-deoxycoformycin, and increased to 15 to 50% of the pretreatment activity prior to the second drug treatment. The clinical response to 2'-deoxycoformycin was unrelated to the pre- or posttreatment adenosine deaminase activities or to the rate of return of enzyme activities following treatment. The plasma deoxyadenosine levels and the leukemic cell dATP concentrations rose slightly with therapy, but there was no correlation between the magnitude of increase and clinical response. No significant levels of DNA strand breaks were observed in the leukemic cells following treatment, although the NAD levels decreased slightly in two patients. When peripheral mononuclear cells from the patients and two controls were incubated in vitro for 24 h with 2'-deoxycoformycin and increasing concentrations of deoxyadenosine, a concentration-dependent increase in dATP and decrease in NAD were observed in both the patients and normals. The normal cells, and cells from two patients, developed a significant number of DNA strand breaks. However, there was no relationship between the formation of DNA breaks and the degree of accumulation of dATP or depletion of NAD, or between any of these changes and subsequent clinical responses to 2'-deoxycoformycin. Based on this study, it appears that the antitumor activity of 2'-deoxycoformycin in chronic lymphocytic leukemia is unrelated to the induction of DNA strand breaks or to changes in the levels of dATP or NAD in the leukemic cells.

Topics: Adenosine Deaminase Inhibitors; Aged; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; DNA; Humans; Leukemia, Lymphoid; Male; Middle Aged; Monocytes; NAD; Pentostatin; Ribonucleosides

The in-vitro effects of deoxycoformycin (dCF) on dATP, NAD, ATP and DNA strand breaks have been evaluated in the cells from 42 patients with various types of chronic lymphoid leukemia. These included 18 with B-cell chronic lymphoid leukemias of different types (BCL); 10 with hairy cell leukemia (HCL) and 14 with T-cell chronic lymphoid leukemias of different types (TCL). The dATP concentrations in HCL, BCL and TCL increased from means of 2.9, 1.8 and 3.0 to 100.3, 68.2 and 51.3 pmol/10(6) cells respectively after 2 h with 10(-5) M dCF and 10(-4)M deoxyadenosine. After 18-24 h, the NAD levels and total double-stranded DNA decreased to 37 and 12.5% (HCL) to 36 and 21.6% (BCL) and 40 and 20.5% (TCL) of control values respectively. Similar decreases were observed in ATP levels. The results do not suggest that these measurements in vitro would predict which patients with these disorders will respond to dCF therapy. Although HCL responds particularly well to dCF in vivo, no difference in the in-vitro effects of dCF studied here could be detected between cases of HCL and the other types of chronic leukemia.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; DNA Damage; Humans; Leukemia, Lymphoid; NAD; Pentostatin; Ribonucleosides

The adenosine deaminase inhibitor 2'-deoxycoformycin and interferon are highly effective in the treatment of hairy-cell leukemia. In this study, a patient with type 2 hairy-cell leukemia was treated with one cycle of 2'-deoxycoformycin (4 mg/m2, i.v. weekly for 3 weeks), which was repeated at 9 wk. No toxicity was observed, and the hairy cell count fell from 72,000/mm3 to 5,000/mm3 in 3 mo, with a concomitant 50% decrease in the spleen size. The erythrocyte deoxyadenosine triphosphate content increased to 13.6 pmol/10(6) cells following the initial three weekly treatments, but there was no decrease in the adenosine triphosphate pool size and no evidence of hemolysis. The hairy cell adenosine deaminase activity was inhibited by greater than 95% 24 h following the first 2'-deoxycoformycin injection and returned to the pretreatment value at Day 8, although there was a linear decline in peripheral hairy cell count (50%) during this period. No ultrastructural changes were observed in the hairy cells following 2'-deoxycoformycin to suggest lymphocytotoxicity or cellular differentiation. The antitumor activity of 2'-deoxycoformycin could not be attributed to alterations in the hairy cell deoxyadenosine triphosphate/adenosine triphosphate levels or to the induction of DNA strand breaks. Additionally, the plasma levels of interferon did not change during therapy, making it unlikely that 2'-deoxycoformycin exerts its activity by inducing endogenous interferon synthesis.

Topics: Adenosine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Aged; Antineoplastic Agents; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; DNA; Humans; Interferons; Leukemia, Hairy Cell; Leukocyte Count; Male; Nucleoside Deaminases; Pentostatin; Ribonucleosides

Resting peripheral blood lymphocytes have a large number of single strand breaks and are especially sensitive to DNA damaging agents. Deoxyadenosine, an adenosine deaminase substrate, in combination with the adenosine deaminase inhibitor deoxycoformycin, causes accumulation of single strand breaks in resting peripheral blood lymphocytes. The induction of single strand breaks by deoxyadenosine is the result of the accumulation of large amounts of intracellular dATP, which creates imbalance in deoxynucleoside triphosphate levels. This imbalance in deoxynucleoside triphosphate levels interferes with the repair of single strand breaks in deoxyadenosine treated cells. Deoxyadenosine acts synergistically with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA alkylating agent, by inhibiting the repair of N-methyl-N'-nitro-N-nitrosoguanidine-induced single strand breaks. We propose that the increased sensitivity of resting peripheral blood lymphocytes to deoxyadenosine and possibly to other DNA damaging agents may be associated with impaired DNA repair ability due to imbalance in intracellular levels of deoxynucleoside triphosphate.

Topics: Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; DNA Repair; Humans; Lymphocyte Activation; Lymphocytes; Methylnitronitrosoguanidine; Pentostatin

The metabolic changes induced by the deoxycoformycin inhibition of adenosine deaminase were studied in human erythrocytes incubated with nucleosides. 1 Adenosine nucleotide levels and glycolytic rate were increased by adenosine. 2 With deoxyadenosine, the cellular ATP level was reduced when dATP increased and the glycolytic rate was similarly enhanced. 3 The hypoxanthine production was equivalent in both cases. Our data demonstrate that human red cells are able to catabolize adenine deoxynucleotides into hypoxanthine, and the control of energy metabolism is not impaired by adenosine deaminase inhibition when PO identical to 4 and NAD+ are not limiting.

Topics: Adenosine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenosine Diphosphate; Adenosine Triphosphate; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Energy Metabolism; Erythrocytes; Glycolysis; Humans; Hypoxanthine; Hypoxanthines; Nucleoside Deaminases; Pentostatin; Ribonucleosides

Topics: Adenosine Deaminase Inhibitors; B-Lymphocytes; Cells, Cultured; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Humans; Lymphocyte Activation; Nucleoside Deaminases; Pentostatin; T-Lymphocytes

Topics: 2',5'-Oligoadenylate Synthetase; Adenosine; Cell Line; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Enzyme Induction; Homocysteine; Humans; Interferon Type I; Pentostatin; Ribonucleosides; T-Lymphocytes; Vidarabine

The effect of deoxyadenosine (dAdo) with deoxycoformycin on the induction of 2',5'-oligoadenylate synthetase by interferon was investigated. After semi-purification through poly(I):poly(C) gel, the activity was similar in control and dAdo-treated cells. However, the activity in the crude extract decreased with rising concentrations of dAdo. On the other hand, the level of 2'-phosphodiesterase, which is also induced by interferon and degrades 2',5'-oligoadenylate, showed no significant change after dAdo treatment. Thus, the crude extract was speculated to contain an inhibitor of 2',5'-oligoadenylate synthetase. Further characterization of the inhibitor revealed that inhibition was not due to dATP accumulation in cells.

Topics: 2',5'-Oligoadenylate Synthetase; Adenosine Deaminase; Cell Line; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Enzyme Induction; Exoribonucleases; Humans; Immunologic Deficiency Syndromes; Interferon Type I; Pentostatin; Ribonucleosides; T-Lymphocytes

Inherited adenosine deaminase (ADA) deficiency is associated with a lymphospecific cytotoxicity affecting both dividing and non-dividing cells. The metabolic basis for this was investigated using different cell types and the potentially toxic metabolite 2'-deoxyadenosine (dAR) in short-term experiments under physiological conditions simulating ADA deficiency (1 mM Pi 8.7 microM dAR). In the uncultured cells, [8-14C] dAR alone was metabolized almost completely only by thymocytes and tonsil-derived B-lymphocytes. The greater percentage of counts (greater than 75%) were in the medium (deoxyinosine, hypoxanthine). Cellular counts were predominantly in adenine nucleotides, and to a lesser extent guanine nucleotides. Interestingly, both thymocytes and tonsil-derived B-lymphocytes, and a partially ADA deficient B lymphoblast line, accumulated detectable amounts of dATP even in the absence of ADA inhibition. Peripheral blood lymphocytes (PBMs) did not, and showed little dAR metabolism. In experiments simulating ADA deficiency varying amounts of 2'-deoxycoformycin (2'dCF) were needed to completely inhibit ADA (20-60 microM), with thymocytes requiring the highest amount. ADA inhibited thymocytes and tonsillar B-lymphocytes accumulated very high dATP levels, which were sustained to an equal extent by both over a 60-min period; PBMs accumulated the lowest values. Results in cultured cells reflected findings in previous studies. Some counts were also found in ATP by a route excluding ADA or PNP. These results again question the hypothesis that B-cells are more resistant than T-cells to the toxic effects of dAR because of an inability to accumulate and sustain elevated dATP levels and underline the lack of comparability between enzyme activity in intact as distinct from lysed cells. They cast doubt on the validity of cultured cells as a model for ADA deficiency and suggest the observed toxicity in some instances might result from altered ATP or GTP pools through inadequate ADA inhibition. They indicate that combined immunodeficiency in ADA deficiency could relate to an equal sensitivity of B-cells and T-cell precursors to the toxic effects of dATP accumulation.

Topics: Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenosine Triphosphate; B-Lymphocytes; Cell Line; Child; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Guanosine Triphosphate; Humans; Nucleoside Deaminases; Palatine Tonsil; Pentostatin; Plasma Cells; T-Lymphocytes; Thymus Gland

Both established cell lines and human leukemic cells in circulating blood which were incubated in vitro with 2'-deoxyadenosine (AdR) plus adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), showed different metabolic responses depending upon the histologic and immunologic types of leukemia. The leukemic T-cell lines in tissue culture were 200-fold more sensitive than B-cell lines to the toxic effect of deoxyadenosine. The increased sensitivity of T-cell lines to AdR plus dCF was associated with the accumulation of deoxyadenosine triphosphate (dATP) in the cells. In established cell lines, an inverse correlation was observed between ED 50 of AdR plus dCF and the relative increase of dATP levels in the cells after the incubation of the cells with AdR plus dCF. In circulating leukemic cells that had been incubated with AdR and dCF, dATP arose in all groups but the correlation was not found between the sensitivity of AdR and the relative dATP accumulation. The failure to find the correlation in patients's leukemic cells may be attributed to the heterogeneity of the response of the blasts to AdR and dCF.

Topics: Acute Disease; B-Lymphocytes; Cell Line; Cells, Cultured; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Growth Inhibitors; Humans; Leukemia; Leukemia, Experimental; Leukemia, Myeloid; Pentostatin; Ribonucleosides; T-Lymphocytes; Thymidine

The biochemical and metabolic effects of deoxycoformycin, a potent inhibitor of adenosine deaminase, were investigated using two human T lymphoblastoid cell lines. A dose-response analysis demonstrated that the concentration of deoxycoformycin at which there was 50% inhibition of growth was greater than 1 X 10(-3) M in lymphoblastoid cells. Uptake of deoxycoformycin was biphasic and occurred much more slowly than for natural nucleosides, and lower saturation levels were reached. The intracellular concentration of deoxycoformycin achieved was 0.4 to 0.5 microM when the extracellular concentration was 1 microM. At 10 microM extracellular concentration, the intracellular concentration was 3-4 microM. Although deoxycoformycin at very low concentrations (1 or 10 microM) did not have any detectable effects on the growth of these cells, the nucleoside was found to be metabolized, and was phosphorylated to give the mono-, di-, and triphosphate derivatives. The triphosphate derivative was incorporated into cellular DNA with little incorporation into cellular RNA. Metabolism of deoxycoformycin in several mutant lymphoblastoid cells deficient in adenosine kinase and/or deoxycytidine kinase was found to be unchanged from wild-type cells, indicating that these major nucleoside kinases do not play a significant role in the phosphorylation of deoxycoformycin. These results may account, at least in part, for the differences that are observed between the pharmacologic inhibition of adenosine deaminase, and the inherited deficiency of adenosine deaminase.

Topics: Adenosine Deaminase Inhibitors; Adenosine Kinase; Chromatography, High Pressure Liquid; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Deoxycytidine Kinase; DNA; Humans; Nucleoside Deaminases; Pentostatin; Phosphorylation; Ribonucleosides; RNA; T-Lymphocytes; Vidarabine

Topics: Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Erythrocytes; Glycolysis; Humans; In Vitro Techniques; Pentostatin

The catabolisms of deoxy-ATP and ATP were compared in cultured human T lymphoblastoid cells incubated under various conditions. It was found that in the presence of deoxycoformycin, an inhibitor of adenosine deaminase, deoxyadenosine was the only product of deoxy-ATP catabolism. In contrast, the main products of ATP catabolism in either the presence or the absence of deoxycoformycin were inosine and hypoxanthine. These results demonstrate that deoxy-ATP catabolism proceeds exclusively via deoxyadenosine deamination, whereas ATP catabolism proceeds mainly via adenylate deamination. These findings thus provide an explanation for the selective deoxynucleotide metabolic abnormalities associated with adenosine deaminase deficiency in humans.

Topics: Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Cell Line; Coformycin; Deoxyadenine Nucleotides; Humans; Hypoxanthine; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Inosine; Kinetics; Pentostatin; Structure-Activity Relationship; T-Lymphocytes

High levels of dATP and dADP, accompanied by ATP depletion, were found in the platelets of two ADA-deficient children with severe combined immunodeficiency (SCID). In vitro studies demonstrated that even normal platelets had the ability to make dATP from deoxyadenosine (dAR) under physiological conditions. This capability was greatly enhanced by conditions simulating ADA deficiency. These results question whether the platelet has a specific role in the normal immune response.

Topics: Adenosine Deaminase; Adenosine Triphosphate; Blood Platelets; Coformycin; Deoxyadenine Nucleotides; Humans; Immunologic Deficiency Syndromes; Nucleoside Deaminases; Nucleotides; Pentostatin

The combination of 2'-deoxyadenosine and deoxycoformycin is known to be markedly toxic to T-lymphocyte cell lines relative to B-cell lines, and this difference appears to be related to the capacity of the cells to accumulate deoxyadenosine triphosphate (dATP). In the presence of dipyridamole and 2'-deoxyadenosine and when adenosine deaminase was inhibited with deoxycoformycin, the L1210 leukemia cell which is a non-T-, non-B-cell type accumulated dATP like a T-cell type. The intracellular L1210 concentration of dATP using the triple combination (1.1 microM deoxycoformycin-40 microM deoxyadenosine-10 microM dipyridamole) reached 400 microM at which concentration ribonucleotide reductase specific activity was reduced by 80%. While this degree of enzyme may be significant, complete inhibition might have been expected, since 400 microM dATP is approximately 40 times the concentration to give 50% inhibition in some purified systems.

Topics: Animals; Antineoplastic Agents; Cell Survival; Chromatography, High Pressure Liquid; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Dipyridamole; Drug Therapy, Combination; Leukemia L1210; Mice; Pentostatin; Ribonucleosides; Ribonucleotide Reductases

Enzyme inhibitors used to simulate the inherited immunodeficiency diseases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency, have been assessed in cultured human lymphocytes. Only 2'-deoxycoformycin (dCF) completely inhibited ADA in T and B cells at concentrations in excess of 5 microM. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and 8-amino guanosine (8-NH2GR) did not inhibit ADA or PNP completely at any concentration. Detailed metabolic experiments comparing viability and deoxynucleotide accumulation showed that B cell lines of malignant origin also accumulated high levels of dATP from 2'-deoxyadenosine (dAR), and dGTP from 2'-deoxyguanosine (dGR) as effectively as T cells--even without inhibitors, however, dAR reduced cell viability only when ADA was inhibited by dCF, whilst dGR was equally toxic with or without inhibitor, even to a line which accumulated no dGTP. These experiments indicate that cultured lymphocytes, using either EHNA or 8-NH2GR as enzyme inhibitor, are not valid models of the toxicity to the immune system in inherited ADA or PNP deficiency. They demonstrate that the ability to accumulate high levels of dATP or dGTP is not exclusive to T cells and that the in vitro toxicity of dAR or dGR could relate to the use of excess substrate and/or accumulation in different nucleotide, not deoxynucleotide pools.

Topics: Adenine; Adenosine Deaminase Inhibitors; B-Lymphocytes; Cell Line; Cell Survival; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Deoxyguanine Nucleotides; Deoxyguanosine; Dose-Response Relationship, Drug; Guanosine; Humans; Pentostatin; Purine-Nucleoside Phosphorylase; T-Lymphocytes

The mechanisms for cell toxicity with adenosine deaminase inhibition by 2'-deoxycoformycin (dCF) in non replicating lymphoid cells include S-adenosylhomocysteine (SAH) hydrolase inactivation and reduction of cellular ATP content. These postulates were explored in a patient with T-CLL receiving dCF with a resultant fall in peripheral blood lymphocytes from 740 X 10(9)/1 to 90 X 10(9)/1 over 15 d. In red cells there was complete inhibition of adenosine deaminase and SAH hydrolase activities, progressive deoxyadenosine triphosphate (dATP) accumulation and ATP depletion but no significant alteration in adenosine monophosphate (AMP) deaminase activity or distribution in purine intermediates from radioactive adenosine. In T-CLL lymphocytes, there was incomplete lymphoid SAH hydrolase inactivation, reduced AMP deaminase activity and progressive dATP accumulation. The limited decrease in lymphocyte ATP content was related more to dCF administration than dATP accumulation, nor accompanied by significant changes in the distribution of purine intermediates from adenosine. These findings suggest that ATP depletion with dCF therapy does not reflect AMP deaminase activity modulation nor is of critical importance for cell toxicity. The exact role for elevated cellular dATP content and SAH hydrolase inactivation in this toxicity remains to be established.

Topics: Adenosine Deaminase; Adenosine Triphosphate; Adenosylhomocysteinase; AMP Deaminase; Coformycin; Cytotoxicity, Immunologic; Deoxyadenine Nucleotides; Deoxyadenosines; Humans; Hydrolases; Leukemia, Lymphoid; Male; Middle Aged; Pentostatin; Ribonucleosides; T-Lymphocytes

Topics: Adenosine Triphosphate; Antineoplastic Agents; Coformycin; Deoxyadenine Nucleotides; DNA Replication; Humans; Leukemia; Pentostatin; Ribonucleosides

Low ATP/ADP ratios have been reported consistently for nucleotide levels of mononuclear cells separated from peripheral blood by conventional techniques. We have established that these low values (mean 2.3:1) were not due to cell damage or poor viability, but resulted from heavy platelet contamination, which is unavoidable when heparinized blood is used. The results reflect the low ATP/ADP ratios (mean 1.6:1) characteristic of platelets. Platelet-free extracts from defibrinated blood had very high ATP/ADP ratios (mean 17.4:1). The initial finding of detectable amounts of deoxy-ATP and deoxy-GTP in mononuclear cells from children with two distinct inherited immunodeficiency disorders [adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency respectively] many have been due to contamination by nucleated erythrocytes as well as platelets in non-defibrinated preparations. Defibrination before nucleotide extraction of mononuclear cells from a patient with T-cell leukaemic/lymphoma treated with the ADA inhibitor deoxycoformycin enabled the demonstration of grossly raised deoxy-ATP levels relative to deoxy-ADP levels (ratio 16.1:1), associated with severe ATP depletion. This reciprocal relationship between ATP and dATP was found by us previously in the erythrocytes in inherited ADA deficiency. These findings underline the importance of extracts uncontaminated by platelets, or nucleated erythrocytes, in the evaluation of lymphocyte nucleotide levels in inherited or acquired immunodeficiency syndromes.

Topics: Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenosine Diphosphate; Adenosine Triphosphate; Blood Platelets; Cell Separation; Child; Coformycin; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Humans; Immunologic Deficiency Syndromes; Lymphocytes; Pentostatin; Purine-Nucleoside Phosphorylase

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.

Topics: Adult; Arabinonucleotides; Cell Line; Chromatography, High Pressure Liquid; Coformycin; Deoxyadenine Nucleotides; DNA; Drug Therapy, Combination; Humans; Leukemia, Lymphoid; Male; Pentostatin; Ribonucleosides; Vidarabine; Vidarabine Phosphate

The occurrence of severe immunodeficiency disease in children with inherited adenosine deaminase deficiency, and reports of remission induction in T-cell acute lymphoblastic leukaemia with the adenosine deaminase inhibitor deoxycoformycin, prompted a study of the effects of deoxyadenosine on resting peripheral blood lymphocytes (PBL) and chronic lymphocytic leukaemic (CLL) lymphocytes in short-term culture. In the presence of an inhibitor of adenosine deaminase, micromolar concentrations of dAdo caused elevation of deoxyadenosine-5'-triphosphate (dATP) pools and in vitro lysis of non-dividing PBL and CLL lymphocytes. This death of non-replicating cells indicates a mechanism of deoxyadenosine toxicity independent of DNA replication and ribonucleotide reductase inhibition. Similar changes occurred in vivo in a patient with advanced CLL who responded to treatment with deoxycoformycin, 0.1 mg/kg, days 1-5, with a fall in the WCC from 102.0 x 10(9)/1 to 6.8 x 10(9)/l over 21 d. Therapeutic blockade of deoxyadenosine catabolism deserves further investigation both in the treatment of lymphoproliferative disease and as a method lympholytic immunosuppression.

Topics: Adenosine Deaminase Inhibitors; Cell Survival; Cells, Cultured; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; DNA Replication; Humans; Leukemia, Lymphoid; Lymphocytes; Male; Middle Aged; Pentostatin; Ribonucleosides

The accumulation of deoxyadenosine triphosphate (dATP) in erythrocytes of mice treated with the adenosine deaminase inhibitor deoxycoformycin was studied in an attempt to establish and evaluate a model system for the study of at least some biochemical aspects of hereditary adenosine deaminase deficiency. Mouse erythrocytes in vitro readily phosphorylated deoxyadenosine to dATP, and this nucleotide was relatively stable once formed. dATP accumulated in vivo in mice treated with deoxycoformycin both as a function of dose from 0.25 to 10 mg/kg, and with time after administration. Major sources of the deoxyadenosine used for dATP formation in vivo appear to be normoblast nuclei produced during erythropoiesis, and dying cells; minor sources would appear to include dietary DNA, overproduction of deoxyribonucleotides, and DNA repair.

Topics: Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Coformycin; Deoxyadenine Nucleotides; Disease Models, Animal; Erythrocytes; Mice; Nucleoside Deaminases; Pentostatin; Ribonucleosides

Intracellular deoxynucleoside triphosphate pools of normal bone marrow, thymocytes and cells from patients with ALL and AML were measured after 2 h incubation with deoxycoformycin (dCF) 10(-5) M and deoxyadenosine (AdR) 10(-4) M in vitro and after another 30 min incubation in the absence of dCF and AdR ('chase' experiment). Incubation with dCF and AdR resulted in a significant rise of dATP concentrations in all groups (the highest rises occurring in the leukaemic groups particularly in AML and Thy-ALL). The concentrations of the other three deoxyribonucleoside triphosphates fell in all groups. The dATP level fell during the 'chase' period but in Thy-ALL and thymocytes this fall was insignificant and slower than in the other groups. This suggests that not only intracellular build-up of dATP but also the capacity of the cell to degrade dATP is important for in vivo cytotoxicity of dCF treatment. These results help to explain the differences in response to dCF of the different leukaemias.

Topics: Bone Marrow; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Deoxyribonucleotides; Humans; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid, Acute; Pentostatin; Ribonucleosides; T-Lymphocytes

Topics: Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenosylhomocysteinase; Animals; Body Weight; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Dose-Response Relationship, Drug; Humans; Hydrolases; Mice; Mice, Inbred Strains; Nucleoside Deaminases; Organ Size; Pentostatin; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Ribonucleosides; Thymus Gland

In four patients with Thy-acute lymphoblastic leukaemia changes in blast cell deoxynucleoside triphosphate concentrations and, in three, changes in blast cell S-adenosyl homocysteine hydrolase activity were measured during treatment with 2' deoxycoformycin, a potent inhibitor of adenosine deaminase. These studies were aimed at identifying the molecular basis of cell killing by this drug. In three patients an increase in blast deoxyadenosine triphosphate (dATP) concentration occurred which was found to be temporally related to cell killing and was accompanied by decreased concentrations of the other three deoxyribonucleoside triphosphates. In the one patient with Thy-ALL who responded poorly to treatment, the increase in dATP concentration was delayed and was not accompanied by a fall in the concentrations of the other deoxyribonucleoside triphosphates. Progressive inactivation of blast cell S-adenosyl homocysteine hydrolase was found to occur in the three patients tested but was maximal only after a substantial reduction of peripheral blast cell count. These results show that 2' deoxycoformycin has a potent cytoreductive effect in Thy-ALL and suggest that the molecular basis of this toxicity is related both to the intracellular accumulation of dATP with inhibition of ribonucleotide reductase. Inactivation of S-adenosyl homocysteine hydrolase may be of importance as an additional mechanism.

Topics: Adenosine Deaminase; Adenosylhomocysteinase; Adolescent; Adult; Bone Marrow; Coformycin; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Humans; Hydrolases; Leukemia, Lymphoid; Leukocyte Count; Male; Pentostatin; Ribonucleosides; Thymine Nucleotides