2'-Deoxyadenosine causes apoptotic cell death in a human colon carcinoma cell line

The combination of 2'-deoxyadenosine and 2'-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2'-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2'-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2'-deoxyadenosine and 2'-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted.     

The effects of an induced adenosine deaminase deficiency on T-cell differentiation in the rat

Inherited deficiency of the enzyme adenosine deaminase (ADA) has been found in a significant proportion of patients with severe combined immunodeficiency disease and inherited defect generally characterized by a deficiency of both B and T cells. Two questions are central to understanding the pathophysiology of this disease: (1) at what stage or stages in lymphocyte development are the effects of the enzyme deficiency manifested; (2) what are the biochemical mechanisms responsible for the selective pathogenicity of the lymphoid system. We have examined the stage or stages of rat T-cell development in vivo which are affected by an induced adenosine deaminase deficiency using the ADA inhibitors, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and 2'-deoxycoformycin (DCF). In normal rats given daily administration of an ADA inhibitor, cortical thymocytes were markedly depleted; peripheral lymphocytes and pluripotent hemopoietic stem cells (CFU-S) all were relatively unaffected. Since a deficiency of ADA affects lymphocyte development, the regeneration of cortical and medullary thymocytes and their precursors after sublethal irradiation was used as a model of lymphoid development. By Day 5 after irradiation the thymus was reduced to 0.10-0.5% of its normal size; whereas at Days 9 and 14 the thymus was 20-40% and 60-80% regenerated, respectively. When irradiated rats were given daily parenteral injections of the ADA inhibitor plus adenosine or deoxyadenosine, thymus regeneration at Days 9 and 14 was markedly inhibited, whereas the regeneration of thymocyte precursors was essentially unaffected. Thymus regeneration was at least 40-fold lower than in rats given adenosine or deoxyadenosine alone. Virtually identical results were obtained with both ADA inhibitors, EHNA and DCF. The majority of thymocytes present at Day 9 and at Day 14 in inhibitor-treated rats had the characteristics of subcapsular cortical thymocytes which are probably the most ancestral of the thymocytes. Thus, an induced ADA deficiency blocked the proliferation and differentiation of subcapsular cortical thymocytes which are the precursors of cortical and medullary thymocytes.     

Adenosine deaminase: physical and chemical properties of partially purified mitochondrial and cytosol enzyme from rat liver.

Adenosine deaminase (ADA) was partially purified 486- and 994-fold from rat liver mitochondria and cytosol, respectively. Relative molecular mass of the enzymes from both fractions was 34,000. Km for adenosine and 2'-deoxy-adenosine were 3.08 x 10(-5) M and 3.03 x 10(-5) M for mitochondrial ADA and 3.12 x 10(-5) M and 2.87 x 10(-5) M for cytosolic ADA. The enzyme from both subcellular fractions had the maximum activity at pH 7.5-8.0, and pI 5.2 and 4.2 for mitochondrial and cytosolic enzyme, respectively. The enzyme was inhibited by erythro-9-(2-hydroxy-3-nonyl)adenine and 2'-deoxycoformycin with Ki 4.4 x 10(-7) M and 3.2 x 10(-7) M for mitochondrial ADA and 4.9 x 10(-7) M 2.8 x 10(-7) M for cytosolic ADA. Among the natural nucleoside and deoxynucleotide derivatives tested, deoxy-GTP and UTP inhibited only cytosolic adenosine deaminase by 60% and 40%, respectively.     

Adenosine deaminase from bovine brain: purification and partial characterization.

Bovine brain adenosine deaminase cytoplasmatic form was purified about 450 fold by salt fractionation, column chromatography on DEAE-cellulose, octyl-sepharose 4B and affinity chromatography on CH-sepharose 4B 9-(p-aminobenzyl)adenine. The purified enzyme was homogeneous on disc gel electrophoresis; the enzyme had a molecular mass of about 65 kDa with an isoelectric point at pH 4.87. The Km values for adenosine and 2'-deoxyadenosine were 4 x 10(-5) and 5.2 x 10(-5) M, respectively. The enzyme showed a great stability to temperature with a half life of 15 hours at 53 degrees C significantly different compared to that known for other mammalian forms of this enzyme. Aza and deaza analogs of adenosine and erythro-9-(2-hydroxy-3-nonyl) adenine were good inhibitors of the bovine brain enzyme with little difference with respect to those reported for the adenosine deaminases purified from other sources. Kinetic constants for the association and dissociation of coformycin and 2'-deoxycoformycin with the bovine brain adenosine deaminase are reported.     

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.     

Adenosine deaminase activity of chicken erythrocyte and heart cytosols

1. The adenosine deaminase (ADA) activities of chicken erythrocyte and heart cytosols had pH optima of 6.5. The temperature optima for erythrocyte and heart ADA were 30 and 35 degrees C, respectively. 2. The deoxyadenosine/adenosine deamination ratios ranged from 0.75 to 0.84 for both ADA activities. 3. For erythrocyte ADA, Km values were 8.9-12.9 microM adenosine (range) and 8.3 microM 2'-deoxyadenosine. For heart ADA, Km values were 6.7-12.0 microM adenosine (range) and 5.3 microM 2'-deoxyadenosine. 4. Inosine was a competitive inhibitor of both erythrocyte (Ki = 73 microM) and heart (Ki = 109 microM) ADA.     

Establishment and characterization of adenosine deaminase-deficient human T cell lines

We have established long term cell lines from a patient with adenosine deaminase (ADA)-deficient severe combined immunodeficiency by stimulation of blood and bone marrow cells with PHA and IL-2 followed by transformation of the activated cells with the human retrovirus HTLV-I. Despite the absence of detectable T cells in the patients blood, cell lines grew that carried the phenotype of mature activated T cells. TJF-2, the line established from blood, was characterized in detail. The concentration of ADA in TJF-2 cells was less than 1% of normal (3.2 U vs 413.0 U). Studies with pharmacologic inhibitors of ADA suggest that the residual adenosine deaminating activity of TJF-2 is from an enzyme distinct from true ADA, a nonspecific aminohydrolyase. Growth of TJF-2 cells was hypersensitive to inhibition by 2'-deoxyadenosine compared to normal T cells (ID50, 55 microM vs greater than 1000 microM). Analysis of 2'-deoxyadenosine-challenged cells showed that TJF-2 cells accumulated significant levels of deoxyadenosine triphosphate, whereas normal T cells did not unless they were also incubated with the ADA inhibitor deoxycoformycin. Southern and Northern blot analysis of these cells revealed a grossly intact ADA gene that produced a normal size ADA mRNA. Yet, despite ADA deficiency, cells of the TJF-2 line were otherwise indistinguishable from HTLV-I-transformed T cells derived from normal donors with respect to dependence on exogenous IL-2 for growth, clonal rearrangement patterns of TCR beta-chain genes, response to PHA, and rapid restoration of cellular volume after hypotonic challenge. The TJF-2 line thus represents a unique HTLV-I-transformed human T cell line exhibiting ADA deficiency and its expected metabolic consequences.     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Early postimplantation embryolethality in mice following in utero inhibition of adenosine deaminase with 2'-deoxycoformycin

Adenosine deaminase (ADA) catalyzes the hydrolytic deamination of adenosine (or 2'-deoxyadenosine) to inosine (or 2'-deoxyinosine). Previously, we have shown that ADA activity is subject to strong cell-specific developmental regulation in placental tissues of mice between days 6 and 11 of gestation (Knudsen et al.:Biology of Reproduction 39:937-951, 1988). In the present study, we examined the effects of intrauterine exposure to 2'-deoxycoformycin (dCF; pentostatin), a potent irreversible inhibitor of ADA, on early postimplantation development. Deoxycoformycin was administered to pregnant ICR mice as a single intraperitoneal injection at a dose of 5 mg/kg on one of days 6 through 11 of gestation (plug day 0). A marked increase in the incidence of implantation site resorptions was observed following treatment specifically on days 7 (61% resorbed) or 8 (78% resorbed). No effect was observed following treatment on days 6, 9, 10, or 11. ADA-immunoreactive protein was shown, by ABC-immunoperoxidase staining on days 7 or 8 of gestation, to be present at high levels in decidual cells of the antimesometrial region but at below-detectable levels in the embryo. Treatment of pregnant dams with dCF on day 7 produced a complete (greater than 99%) inhibition of ADA activity in the antimesometrial decidua by 30 min, induced excessive cell death in the prospective neural plate and primary mesenchyme of the trilaminar disc by 6 h, and arrested embryonic development at an early somite stage. These results suggest that the antimesometrial decidua plays a protective role in preventing an inappropriate accumulation of endogenous ADA substrates in the implantation site.     

Deoxyadenosine modulates human suppressor T cell function and B cell differentiation stimulated by Staphylococcus aureus protein A

Adenosine deaminase (ADA) deficiency and the resultant accumulation of deoxyadenosine (AdR) are associated with profound T cell dysfunction and variable B cell dysfunction in vivo. We examined the effects of AdR on the in vitro function of normal human peripheral blood B and T lymphocytes whose ADA activity was inhibited by 2'-deoxycoformycin. We found that OKT8+ T cell-mediated suppression of SPA-induced Ig production was markedly reduced by concentrations of AdR (3 to 10 microM) that did not affect helper T cell function. Because the lectin-induced proliferative responses of OKT8+ T cells and OKT8- T cells were equally susceptible to AdR, modulation of in vitro immune responses by low-dose AdR probably reflected different proliferative requirements for the expression of T cell helper or suppressor functions. Although low doses of AdR did not inhibit Ig production in SPA-stimulated cultures, we found that T cell-dependent, SPA-stimulated B cell proliferation was blocked by 3 to 10 microM AdR. Therefore, it appeared that B cell proliferation was not required for the induction of Ig synthesis in this system. Higher doses (30 to 100 microM) of AdR did block the induction of Ig synthesis, presumably by interfering with T-helper functions via a mechanism other than inhibition of proliferation and/or by inhibiting B cell differentiation events.     

Characterization of a cell culture model for the study of adenosine deaminase- and purine nucleoside phosphorylase-deficient immunologic disease.

The absence of erythrocytic adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) has been associated with severe immunodeficiency disease in children. We have developed a cell culture model to study the possible relationships between purine salvage enzymes and immunologic function using an established T cell lymphosarcoma (S49) and a potent inhibitor of ADA, erythro-9(2-hydroxy-3-nonyl) adenine (EHNA). Wild-type S49 cells are killed by dexamethasone or dbc AMP, and adenosine (5 muM) in the presence of an ADA inhibitor (6 muM EHNA) also prevents the growth of and kills these S49 cells. It has been proposed that adenosine is toxic to lymphoid cells by virtue of its ability to increase the intracellular concentrations of cyclic AMP. We examined the sensitivity of three mutants of S49 cells, with distinctive defects in some component of cyclic AMP metabolism or action, to killing by adenosine and EHNA. All three mutants are resistant to killing by isoproterenol or cholera toxin and two are resistant to dbc AMP itself, but all are sensitive to killing by adenosine and EHNA. Similarly, two dexamethasone-resistant S49 mutants are as sensitive to adenosine and EHNA as are the wildtype cells. We have also simulated the purine nucleoside phosphorylase deficiency in S49 cells by adding inosine and adenosine to the growth medium. In the presence of EHNA or inosine, the toxic effects of adenosine can be partially reversed by addition of (10-20 muM) uridine, an observation suggesting that adenosine is toxic as the result of its inducing pyrimidine starvation.     

Effects of (R)-deoxycoformycin (pentostatin) on intrauterine nucleoside catabolism and embryo viability in the pregnant mouse.

The viability of early mouse embryos is acutely sensitive to (R)-deoxycoformycin (pentostatin), a tight-binding inhibitor of adenosine deaminase (ADA). Previous studies have shown that a single 5-mg/kg dose on day 7 (plug = day 0) of gestation fully inhibits uteroplacental ADA activity within 0.5 h; causes massive cell death in the neural plate and primary mesenchyme by 6 h, major craniofacial anomalies by day 10, and resorption by day 12 (Knudsen et al., '89; Airhart et al., '91). The present study has examined further the developmental toxicity and early effects of this inhibitor on ADA metabolism. (R)-Deoxycoformycin was administered to pregnant CD-1 (ICR) mice as a single intraperitoneal dose of 0.5-10 mg/kg total body weight on days 6-11 of gestation. The major adverse effect, early resorption, was dose dependent and specific to day 7-8 exposure. Treatment with 5 mg/kg on day 7 resulted in 85% resorptions, 15% malformations, and a 24% reduction in mean fetal weight, whereas the same dose of (S)-deoxycoformycin had no effect. Levels of adenosine and 2'-deoxyadenosine, which are the endogenous substrates of ADA, were monitored in the embryo/decidual unit (E/D) by reversed-phase high-performance liquid chromatography (RP-HPLC). In response to the inhibitor, both nucleosides increased transiently in the antimesometrial compartment (antimesometrial decidua + embryo). Peak levels (Cmax) of adenosine and 2'-deoxyadenosine were dose dependent over the range tested (0.05-10 mg/kg). Exposure to 5 mg/kg on day 7 raised adenosine levels within 0.5 h to 42-fold over the basal level of 0.06 nmol/mg protein. There was an even stronger effect on 2'-deoxyadenosine levels, which were elevated 674-fold over the detection limit of 0.0005 nmol/mg protein. Direct exposure to the inhibitor in serum-free E/D culture produced similar results: 50 microM (R)-deoxycoformycin within 1 h raised adenosine levels 26-fold and 2'-deoxyadenosine levels 410-fold. In vivo studies also showed a general correlation between embryolethality and the length of adenine nucleoside pool expansion, apparent for exposure on day 7, 8, or 9 but not on day 6, suggesting that the embryo becomes sensitive to adenosine or 2'-deoxyadenosine once the neural plate has formed.     

Influence of dipeptidyl peptidase IV on enzymatic properties of adenosine deaminase

The importance of ADA (adenosine deaminase) in the immune system and the role of its interaction with an ADA-binding cell membrane protein dipeptidyl peptidase IV (DPPIV), identical to the activated immune cell antigen, CD26, has attracted the interest of researchers for many years. To investigate the specific properties in the structure-function relationship of the ADA/DPPIV-CD26 complex, its soluble form, identical to large ADA (LADA), was isolated from human blood serum, human pleural fluid and bovine kidney cortex. The kinetic constants (Km and Vmax) of LADA and of small ADA (SADA), purified from bovine lung and spleen, were compared using adenosine (Ado) and 2'-deoxyadenosine (2'-dAdo) as substrates. The Michaelis constant, Km, evidences a higher affinity of both substrates (in particular of more toxic 2'-dAdo) for LADA and proves the modulation of toxic nucleoside neutralization in the extracellular medium due to complex formation between ADA and DPPIV-CD26. The values of Vmax are significantly higher for SADA, but the efficiency, Vmax/Km, in LADA-catalyzed 2'-dAdo deamination is higher than that in Ado deamination. The interaction of all enzyme preparations with derivatives of adenosine and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was studied. 1-DeazaEHNA and 3-deazaEHNA demonstrate stronger inhibiting activity towards LADA, the DPPIV-CD26-bound form of ADA. The observed differences between the properties of the two ADA isoforms may be considered as a consequence of SADA binding with DPPIV-CD26. Both SADA and LADA indicated a similar pH-profile of adenosine deamination reaction with the optimum at pHs 6.5-7.5, while the pH-profile of dipeptidyl peptidase activity of the ADA/DPPIV-CD26 complex appeared in a more alkaline region.     

Deoxyadenosine toxicity in an adenosine deaminase-inhibited human CCRF-CEM T-lymphoblastoid cell line causes cell swelling.

The human T-lymphoblastoid cell line CCRF-CEM, pre-treated with 2'-deoxycoformycin, was used as a model for adenosine deaminase deficiency to investigate how 2'-deoxyadenosine exerts its cytotoxic effects. Incubation of these cells with 1 microM or 5 microM deoxyadenosine for 24 and 48 h caused an increase of up to 50% in their modal cell volume as measured by a Coulter Size Distribution Analyzer and this increase in cell volume was accompanied by an increase in their fragility and deformability. The swelling of cells was concomitant with the phosphorylation of deoxyadenosine and its intracellular accumulation as dATP. There was no evidence of osmotic imbalance or of inhibition of the Na+/K(+)-dependent ATPase activity as the intracellular concentrations (and the intracellular:extracellular ratios) of Na+, K+ and Ca2+ were essentially unchanged. Cytochalasin B (20 microM) also caused lymphoblasts to swell over a 6-h period and its effect on cell size was similar to that of either 1 microM or 5 microM deoxyadenosine over 24 or 48 h. Longer time-courses of incubation with cytochalasin B caused severe toxicity leading to the death and lysis of a significant proportion of the cells. Other drugs, such as colchicine, vincristine and vinblastine that are known to affect various components of the cytoskeleton also caused swelling of cells in a concentration- and time-dependent manner but there was no evidence that these effects were additive or synergistic with those of deoxyadenosine. Inhibition of DNA synthesis, either directly by aphidicolin or indirectly by hydroxyurea, was less cytotoxic than the effect caused by deoxyadenosine. We conclude that one of the toxic effects resulting from the excessive phosphorylation of deoxyadenosine and its accumulation as dATP in human T-lymphoblasts is not dependent on inhibition of DNA synthesis but may be caused by the disruption of the cytoskeleton in these cells.     

Adenosine deaminase and immunodeficiency: An in vitro model

We have examined the effect of adenosine and EHNA, a competitive inhibitor of adenosine deaminase (ADA), upon the ability of human peripheral blood lymphocytes to respond to mitogen. Addition of adenosine at concentrations greater than 10 μm (10^?5m) resulted in inhibition of lymphocyte proliferation at 48 hr of culture, provided that the culture medium was relatively free of ADA activity. The actual concentrations of adenosine remaining in inhibited cultures at the time of harvest were considerably lower than those added initially. EHNA alone also inhibited PHA response (and to a lesser extent PWM and Con A responses), but only at high concentrations. Noninhibitory concentrations of EHNA and adenosine together acted synergistically to produce profound inhibition of lymphocyte proliferation. This may provide an in vitro model to explore further the mechanism of the immunodeficiency associated with deficiency of ADA. Adenosine deaminase activity in stimulated cultures did not differ significantly from that found in unstimulated cultures, and the activity per protein or per DNA actually decreased in stimulated versus unstimulated cultures.     

Rat Brain Adenosine Deaminase After 2''-Deoxycoformycin Administration: Biochemical Properties and Evidence for Reduced Enzyme Levels Detected by 2''-[3H]Deoxycoformycin Ligand Binding

Near total inhibition of brain adenosine deaminase (ADA) activity in rats injected with the potent ADA inhibitor 2'-deoxycoformycin (DCF) was previously shown to reduce enzyme activity for up to 50 days during which time the enzyme exhibited reduced sensitivity to in vivo inhibition by DCF. Here, we investigated the biochemical properties of ADA and the basis for its reduced activity after DCF treatment. It was found that much higher doses of DCF were required to inhibit ADA in DCF-treated compared with drug-naive animals. Fourteen days after DCF administration, reduced ADA activity in brain homogenates was due to a decrease in Vmax, rather than to an altered Km of ADA for adenosine. DCF treatment had no effect on Ki values for erythro-9-(2-hydroxy-3-nonyl)adenine inhibition of ADA. The IC50 value for DCF inhibition of ADA in hypothalamus was unchanged. However, the Ki for DCF inhibition of ADA in whole brain increased by fivefold. Sucrose gradient analysis of brain ADA revealed only one corresponding peak of activity and [3H]DCF-labeled ADA in DCF-treated and control rats. A radioligand filtration assay with [3H]DCF was developed to assess the effects of DCF on ADA protein levels. Over a roughly 200-fold range of ADA activities the binding of [3H]DCF was highly correlated with deaminase activity (r = 0.99). In brain tissues taken 8 and 33 days after treatment of rats with DCF, [3H]DCF binding was reduced to 27% and 48% of control levels, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient expression of human adenosine deaminase cDNAs: identification of a nonfunctional clone resulting from a single amino acid substitution.

Human adenosine deaminase (ADA) is an important purine catabolic enzyme which irreversibly deaminates adenosine and deoxyadenosine. Severe genetic deficiency of ADA leads to an immunological deficiency state in which T-lymphoid cells are selectively destroyed by the accumulation of toxic levels of deoxyadenosine and deoxy-ATP. In preparation for transfer of ADA sequences into a variety of cell types, we explored expression of ADA cDNAs transfected into cultured cells within a simian virus 40-based expression vector. After transfection into monkey kidney (COS) cells, ADA cDNA encompassing the entire coding region of the protein generated human ADA activity. An unexpected finding, however, was the identification of a cDNA clone that failed to produce either human enzyme activity or immunoreactive ADA protein. As this pattern is typical of many naturally occurring mutant ADA alleles, we characterized the molecular defect in this clone. DNA sequence analysis revealed a single nucleotide substitution in amino acid position 50 (glycine-valine). Northern blotting with a unique 17-mer oligonucleotide demonstrated the absence of the mutant sequence in the mRNA from which the cDNA library giving rise to the mutant cDNA was constructed. Therefore, the substitution in the variant cDNA was created during cloning. These data define one critical region of the human ADA protein molecule and suggest a convenient strategy for characterization of the phenotypes associated with naturally occurring mutant alleles.     

Kinetic characterization of adenosine deaminase activity in zebrafish (Danio rerio) brain

Adenosine deaminase (ADA; EC 3.5.4.4) activity is responsible for cleaving adenosine to inosine. In this study we described the biochemical properties of adenosine deamination in soluble and membrane fractions of zebrafish (Danio rerio) brain. The optimum pH for ADA activity was in the range of 6.0-7.0 in soluble fraction and reached 5.0 in brain membranes. A decrease of 31.3% on adenosine deamination in membranes was observed in the presence of 5 mM Zn(2+), which was prevented by 5 mM EDTA. The apparent K(m) values for adenosine deamination were 0.22+/-0.03 and 0.19+/-0.04 mM for soluble and membrane fractions, respectively. The apparent V(max) value for soluble ADA activity was 12.3+/-0.73 nmol NH(3) min(-1) mg(-1) of protein whereas V(max) value in brain membranes was 17.5+/-0.51 nmol NH(3) min(-1) mg(-1) of protein. Adenosine and 2'-deoxyadenosine were deaminated in higher rates when compared to guanine nucleosides in both fractions. Furthermore, a significant inhibition on adenosine deamination in both soluble and membrane fractions was observed in the presence of 0.1 mM of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The presence of ADA activity in zebrafish brain may be important to regulate the adenosine/inosine levels in the CNS of this species.     

Transport of a nonphosphorylated nucleoside, 5'-deoxyadenosine, by murine leukemia L1210 cells.

The mode of transport of a nonphosphorylated adenosine analog, 5'-deoxyadenosine, was studied in murine leukemia L1210 cells. This compound is not subject to the action of intracellular nucleoside-trapping kinases, and its transport can be examined without regard for effects of experimental conditions on kinase activity. Accumulation of 5'-deoxyadenosine was rapid, and nonconcentrative, with equilibrium attained within 12 s at 37 degrees. Kinetic studies were carried out at 20 degrees. We found both a nonmediated (diffusion) and a mediated transport process. The latter had an apparent Km fo 115 micrometer, Vmax = 105 pmol/10(6) cells/min. Uptake of 5'-deoxyadenosine was inhibited by several heterologous nucleosides including adenosine, 2'-deoxyadenosine, thymine riboside, and inosine. Like 2'-deoxyadenosine, 5'-deoxyadenosine was more lipid-soluble than adenosine (from octanol/water partition studies). Compared with 5'-deoxyadenosine, adenosine had a much lower apparent Km (5 micrometer) and a higher Q10 over the 27-37 degrees range (3.0 versus 1.3). Data obtained with adenosine might, however, reflect properties of intracellular adenosine kinase interacting with a transport process.     

The mechanism of inhibition and "reversal" of mitogen-induced lymphocyte activation in a model of adenosine deaminase deficiency

The biochemical mechanism of lymphocyte dysfunction with adenosine deaminase deficiency has been investigated using cultured phytohemagglutinin stimulated normal peripheral blood lymphocytes and the adenosine deaminase (ADA) inhibitor 2'-deoxycoformycin. The addition of deoxyadenosine to ADA-inhibited (but not to uninhibited) cells generated increased dATP pools (up to 50-fold greater than controls) and depressed the mitogen response. dATP Accumulation was accompanied by depletion of the other three deoxynucleoside triphosphate (dNTP) pools (dTTP, dCTP, and dGTP). Suppression of the mitogen response could be prevented ("reversed") to 90% of control levels by the addition of deoxynucleoside precursors for the depleted dNTPs at the initiation of mitogen stimulation. "Reversal" restored the dTTP and possibly the dGTP pools. Thus the mechanism of toxicity in this model appears to be inhibition of ribonucleotide reductase by massive accumulation of dATP, resulting in starvation for the other three deoxyribonucleoside triphosphates. "Reversibility" of this toxicity by providing sources for the missing three deoxynucleoside triphosphates argues for ribonucleotide reductase inhibition rather than other mechanisms of deoxyadenosine toxicity in this model.