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The pathological mechanisms of radiation ulcer remain unsolved and there is currently no effective medicine. Here, we demonstrate that persistent DNA damage foci and cell senescence are involved in radiation ulcer development. Further more, we identify cordycepin, a natural nucleoside analogue, as a potent drug to block radiation ulcer (skin, intestine, tongue) in rats/mice by preventing cell senescence through the increase of NRF2 nuclear expression (the assay used is mainly on skin). Finally, cordycepin is also revealed to activate AMPK by binding with the α1 and γ1 subunit near the autoinhibitory domain of AMPK, then promotes p62-dependent autophagic degradation of Keap1, to induce NRF2 dissociate from Keap1 and translocate to the nucleus. Taken together, our findings identify cordycepin prevents radiation ulcer by inhibiting cell senescence via NRF2 and AMPK in rodents, and activation of AMPK or NRF2 may thus represent therapeutic targets for preventing cell senescence and radiation ulcer.

Oxidatively induced DNA lesions 8,5'-cyclopurine-2'-deoxynucleosides (cdPus) are prevalent and cytotoxic by impeding DNA replication and transcription. Both the 5' R - and 5' S -diastereomers of cdPu can be removed by nucleotide excision repair; however, the 5' S -cdPu is more resistant to repair than the 5' R counterpart. Here, we report the crystal structures of human polymerase (Pol) η bypassing 5' S -8,5'-cyclo-2'-deoxyadenosine (cdA) in insertion and the following two extension steps. The cdA-containing DNA structures vary in response to the protein environment. Supported by the "molecular splint" of Pol η, the structure of 5' S -cdA at 1.75-Å resolution reveals that the backbone is pinched toward the minor groove and the adenine base is tilted. In the templating position, the cdA takes up the extra space usually reserved for the thymine dimer, and dTTP is efficiently incorporated by Pol η in the presence of Mn 2+ Rigid distortions of the DNA duplex by cdA, however, prevent normal base pairing and hinder immediate primer extension by Pol η. Our results provide structural insights into the strong replication blockage effect and the mutagenic property of the cdPu lesions in cells., Competing Interests: The authors declare no conflict of interest.

The aim of this work was to develop a fast-dissolving film formulation containing EFdA for potential use as a topical vaginal microbicide for prevention of HIV sexual transmission. Solid state compatibility approaches were used to screen commonly used polymers for formulation development. Factorial design and desirability function were used to investigate the effect of two variables, the ratio of the polymers and the concentration of selected plasticizer on four mechanical responses including tensile strength, elongation at break, toughness and elastic modulus for optimization of the film formulation. Assessments of EFdA-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity, ex vivo permeability and bioactivity test. The optimal placebo film was composed of PVA, HPMC E5 and propylene glycol (7:3:3, w/w), and its mechanical characteristics were comparable to those of VCF(®) film (a commercial vaginal film product). Permeability studies using human ectocervical explants showed that there was no significant difference in cumulative permeated amount of EFdA between EFdA film and free EFdA. The results of in vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) was several orders of magnitude higher than 50% effective concentration (EC50) of EFdA. Furthermore, epithelial integrity study showed that EFdA-loaded film had a much lower toxicity to HEC-1A cell monolayers as compared to VCF(®). Therefore, EFdA-loaded vaginal film may be considered as a promising vaginal microbicide for HIV prevention., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The potent antiretroviral 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a promising experimental agent for treating HIV infection. Pre-steady-state kinetics were used to characterize the interaction of EFdA-triphosphate (EFdA-TP) with human mitochondrial DNA polymerase γ (Pol γ) to assess the potential for toxicity. Pol γ incorporated EFdA-TP 4,300-fold less efficiently than dATP, with an excision rate similar to ddATP. This strongly indicates EFdA is a poor Pol γ substrate, suggesting minimal Pol γ-mediated toxicity, although this should be examined under clinical settings.

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2'-deoxyguanosine (8-Br-dG), 8-bromo-2'-deoxyadenosine (8-Br-dA), and 5-bromo-2'-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cordyceps sinensis is a prized traditional Chinese medicine and its major component cordycepin is found to have anti-leukemia activities. However, its cytotoxicity in erythrocytes was unclear. To examine the effect of cordycepin on the induction of eryptosis (an apoptosis-like process in enucleated erythrocytes), flow cytometric assays based on membrane integrity and asymmetry were employed. For comparison, analyses were performed in parallel with two other anti-leukemia agents, indirubin 3'-monoxime (IDM) and As2O3. We found that at the IC50 against leukemia HL-60, cordycepin elicited eryptosis while IDM and As2O3 showed no erythrotoxicity in mouse erythrocytes. Mechanistically, cordycepin increased the [Ca2+]i and activated mu-calpain protease in a dose-dependent manner. Yet, no caspase-3 activation was observed in the cordycepin-treated erythrocytes. When extracellular Ca2+ was depleted, both the cordycepin-induced eryptosis and mu-calpain cleavage were suppressed. Our study therefore demonstrated for the first time that cordycepin induces eryptosis through a calcium-dependent pathway in the absence of mitochondria and caspase-3 activation.

The synthesis of the title 2'-deoxyadenosine derivatives bearing bipyridine, phenanthroline or terpyridine ligands and their corresponding RuII-complexes in position 8 linked via acetylene or phenylene tethers was accomplished through cross-coupling reactions. The Suzuki-Miyaura reactions of boronic acids or the Sonogashira reactions of terminal acetylene derivatives of oligopyridine ligands were performed either on protected 8-bromoadenosines in organic solvents or, more efficiently, directly on unprotected nucleosides in aqueous acetonitrile or DMF. Direct cross-coupling reactions of unprotected nucleosides with RuII-complexes or the oligopyridine-boronic acids or -acetylenes gave the Ru-labelled nucleosides in one step in fair to good yields. This method was also proven to be applicable for direct Ru-labelling of dATP. Terpyridine-containing 2'-deoxyadenosine exerted significant antiviral and cytostatic effects.

An idea to use 4'-C-substituted-2'-deoxynucleoside derivatives was proposed based on a working hypothesis to solve the problems of existing acquired immune deficiency syndrome chemotherapy (highly active antiretroviral therapy). Subsequent studies have successfully proved the validity of the idea and resulted in the development of 2'-deoxy-4'-C-ethynyl-2-fluoroadenosine, a nucleoside reverse transcriptase inhibitor, which is highly potent to all human immunodeficiency viruses type 1 (HIV-1s) including multidrug-resistant HIV-1 and has a low toxicity.

A working hypothesis to solve the critical problems of existing HAART was proposed. The study based on the hypothesis proved the validity of the hypothesis and resulted in the development of 2'-deoxy-4'-C-ethynyl-2-fluoro-adenosine (4'Ed2FA), a nucleoside reverse transcriptase inhibitor (NRTI) with highly potent activity against all HIV-1 strains, very favourable toxic profiles, and stability in plasma.

Oral lichen planus (OLP) is a chronic inflammatory disease, which has been clinically associated with development to oral cancer. A double immunofluorescence labeling study found that 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) accumulated in oral epithelium in OLP and oral squamous cell carcinoma (OSCC) biopsy specimens, whereas little or no immunoreactivity was observed in normal oral mucosa. Colocalization of 8-nitroguanine and inducible nitric oxide synthase (iNOS) was found in oral epithelium of OLP and OSCC. Immunoreactivity of 3-nitrotyrosine, which is formed by protein tyrosine nitration and is considered to be a biochemical marker for inflammation, was also observed in oral epithelial cells and colocalized with 8-nitroguanine. Accumulation of p53 was more strongly observed in oral epithelium in OSCC than OLP, whereas there was no p53 accumulation in normal oral mucosa. Our findings demonstrate that iNOS-dependent DNA damage in OLP may lead to p53 accumulation in not only OLP but also OSCC. We conclude that the formation of potentially mutagenic DNA lesions including 8-nitroguanine and 8-oxodG may contribute to the development of oral cancer from OLP., ((Cancer Sci 2005; 96: 553 -559).)

Pre-treatment with bryostatin 1 (bryo) has been shown to potentiate the efficacy of (2-chloro-2-deoxyadenosine, cladribine, 2-CdA) in B-cell chronic lymphocytic leukemia (B-CLL) by increasing the ratio of deoxycytidine kinase (dCK) to 5'-nucleotidase (5'-NT) activity. The bryo-induced increase in dCK/5'-NT activity alone has not been a conclusive indication of final clinical outcome. Therefore, we used an ex vivo assay to investigate factors which may affect the bryo-induced enhancement of 2-CdA efficacy in B-CLL patient-derived samples. Bryo-induced increase in dCK/5'-NT was inversely associated with Rai stage CLL (r=-0.86). Increased dCK/5'-NT activity was not correlated with increased efficacy (cell death) or percentage of cellular [8-3H]-2-CdA converted to [8-3H]-2-CdATP ex vivo. Bryo pre-treatment increased the cellular uptake of [8-3H]-2-CdA and incorporation of [8-3H]-2-CdA metabolites into the DNA fraction. Cell death from 2-CdA was inversely correlated with bryo-induced activity of the DNA repair enzyme, DNA-PKcs, (r=-0.77). Thus, the ability of B-CLL to repair damaged DNA may be a more important predictor of the response to bryo/2-CdA and eventual clinical outcome than dCK/5'-NT activity. Additional CLL patients under bryo-2-CdA therapy are needed to verify these important observations.

Syntheses of phosphoramidate protides of several 2',3'-dideoxy-3'-fluoroadenosine derivatives by treatment of the nucleoside with phosphorochloridates in the presence of pyridine and t-BuMgCl is described. Several of these protides showed significantly improved antiviral potency over the parent nucleoside against HIV and HBV. Especially marked was the improvement in potency of phosphoramidate protides of 2',3'-dideoxy-3'-fluoroadenosine against both HIV and HBV.

Therapeutic options for controlling autoimmune diseases are still very limited. Interleukin-10 has been reported to be a promising approach to therapeutic intervention. In the search for a drug which results in the selective upregulation of interleukin-10, we investigated the immunoregulative effects of cordycepin. We have measured interleukin-10 and interleukin-2 secretion of human peripheral blood mononuclear cells that were incubated with cordycepin and assessed the influence of cordycepin on the expression of interleukin-10 mRNA, the proliferative response and the expression of surface markers on T lymphocytes. In addition, the subsets of interleukin-10-secreting cells, the influence of anti-interleukin-10 neutralizing antibody and cytotoxicity of cordycepin were evaluated. Our results suggest that cordycepin has a significantly upregulative effect on interleukin-10 production and interleukin-10 mRNA expression. Interleukin-10-producing cells included in CD4+, CD8+, CD19+, CD56+ and CD14+ cells. At the same time, cordycepin inhibited phytohaemagglutinin-induced interleukin-2 production and proliferation of peripheral blood mononuclear cells. A restricted T lymphocyte activation was also reflected by a reduced expression of the surface markers CD25, CD45RO, CD54, CD71 and HLA DR. Anti-interleukin-10 neutralizing antibody could not completely block the suppressive effect of cordycepin on production of interleukin-2. Cordycepin in the effective concentration presented slight cytotoxicity but did not increase apoptosis. These results indicate that cordycepin exerts immunoregulative effects. Further research on it may provide an approach for the development of novel immunomodulatory drugs which directly alter the secretion of cytokines.

beta-L-2'-deoxyadenosine (beta-L-dA), beta-L-2',3'-dideoxyadenosine (beta-L-ddA) and its two bis (S-acyl-2-thioethyl; SATE) phosphotriester derivatives, beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(MeSATE) and beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(tButylSATE) have been previously shown to exhibit potent and selective anti-hepatitis B activity in vitro. None of the four compounds was mutagenic up to 100 microg in the Ames test (microtechnique) using Salmonella typhimurium strains TA 97a, TA 98, TA 100 and TA 102, with and without metabolic activation. In addition, the genotoxicity of beta-LdA and the three other compounds was evaluated in human lymphocytes using the Comet assay, at doses up to 5 microg with or without the addition of a microsomal S9 fraction. None of the four compounds induced DNA strand breakage with and without metabolic activation. In summary, the data clearly demonstrate that the purine nucleoside beta-L-dA, beta-L-ddA and the two prodrugs, beta-L-ddAMP-bis(MeSATE) and beta-L-ddAMP-bis(tButylSATE) are not mutagenic in the Ames test and do not induce DNA damage in human lymphocytes, as assessed by the Comet assay.

To study the genotoxic properties of 1,N6-ethenodeoxyadenosine (epsilondA) in human cells, a novel site-specific mutagenesis approach was developed, in which a single DNA adduct was uniquely placed in either strand of a shuttle plasmid vector. The analysis of progeny plasmid derived from the modified strand shows that epsilondA, when incorporated into the position of the second A of 5'-CAA (codon 61 of the ras gene), is mutagenic in human cells, inducing A-->T, A-->G, and A-->C mutations. The efficient induction of A-->T transversions in experiments using modified double- and singlestranded DNA substrates supports the hypothesis that A:T-->T:A transversions in human and animal tumors induced by vinyl compounds reflect misinsertion of dAMP opposite this adduct. Mutagenic events were similar when the adduct was incorporated into either the leading or the lagging strand. EpsilondA was more mutagenic than 8-oxodeoxyguanosine, which induced targeted G-->T transversions in HeLa cells. In Escherichia coli, epsilondA did not significantly miscode (<0.27%) even in the presence of induced SOS functions.

The comparative mutagenicity of 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was explored using simian kidney (COS-7) cells. Oligodeoxynucleotides ¿5'-TCCTCCT- G(1)X(2)CCTCTC or 5'-TCCTCCTX(1)G(2)CCTCTC (X = dA, dG, 8-oxodA or 8-oxodG) containing 8-oxodA or 8-oxodG positioned within codon 60 or 61 of the non-coding strand of human c-Ha-ras1 gene were inserted into a single-stranded phagemid shuttle vector. The vector was replicated in COS-7 cells and the progeny plasmids were used to transform Escherichia coli DH10B. The transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequence analysis to establish the mutation frequency and specificity. When 8-oxodA was positioned at X(1), targeted A(oxo)-->C transversions were detected; the mutation frequency was 1.2%. When 8-oxodA was positioned at X(2), one targeted mutant among 416 colonies screened (an A(oxo)-->G transition) was detected. Thus, the mutation frequency and spectrum of 8-oxodA depend on the sequence context of the lesion. The mutation frequency of 8-oxodG at X(1) and X(2) was 5.2 and 6.8%, respectively. G(oxo)-->T transversions dominated the spectrum, accompanied by small numbers of G(oxo)-->A transitions and G(oxo)-->C transversions. We conclude that 8-oxodA has mutagenic potential in mammalian cells, generating A-->C transversions. However, when tested under similar conditions, the mutation frequency of 8-oxodA is at least four times lower than that of 8-oxodG.

Adenosine deaminase deficiency is an inborn error resulting in immunodeficiency. The pathogenesis of the lymphopenia is not fully understood. Intracellular increases in dATP in the absence of deamination retard DNA repair in human resting lymphocytes and results in the slow accumulation of DNA strand breaks. We focused on the relationship between DNA damage and DNA precursor pools in cultures of deoxycoformycin-treated, ADA-inhibited resting lymphocytes. The addition of 10 microM deoxyadenosine led to a substantial number of DNA strand breaks within 12 h, breaks equivalent to those which occur with about 190 rad irradiation. Addition of any of the other deoxynucleosides used partially prevented this dAdo-induced DNA damage and promoted DNA repair. However, the preventive effects did not correlate inversely with intracellular dATP levels. Resting lymphocytes have very small dNTP pools. Treatment with dAdo slightly reduced dTTP and dCTP. Three kinds of deoxynucleosides, other than dAdo, restored or raised the corresponding dNTP level but the pool imbalance was only minimally corrected. Regarding the toxic effects of dAdo in ADA deficiency, not only dATP levels but also dNTP pool balance has a crucial role in the pathogenesis. Pool sizes of dTTP, dCTP, and possibly dGTP must be maintained at normal levels, if dAdo-induced DNA damage is to be avoided., (Copyright 1999 Academic Press.)

Previous work has shown that nucleosides produce apoptosis in sympathetic ganglion (SG) cells in vitro. The present study examined the effects of nucleosides on the development of the chick embryo in vivo with special attention to the SG and the optic tectum of the central nervous system. In the presence of an adenosine deaminase inhibitor, adenosine and 2'-deoxyadenosine (2'-dAdo) produced different toxicity patterns: both adenosine and 2'-dAdo were toxic to E3 embryos, but only 2'-dAdo was toxic at later stages (E6 1/2, E11). Dosage experiments on E6 1/2 embryos showed that adenosine was less toxic than 2'-dAdo and that 2'-dAdo in sublethal doses was teratogenic. We also examined the effects of 2'-dAdo on embryonic chicken SG and optic tectum in vivo to determine whether sublethal doses of 2'-dAdo produced cell death in these centers on E6 1/2 and 10. In the E6 1/2 SG, 2'-dAdo produced significant neuron loss (83%) and a decrease in SG volume (65%); however, at E10, there was only minor cell loss (7%) and no significant change in SG volume. In the optic tectum at E6 1/2, cell loss was confined mainly to the tectal ventricular zone, but there was little sign of cell loss in this organ at E10. Since cell production is vigorous in the SG and optic tectum at E6 1/2 but relatively low at E10, 2'-dAdo appears to work by stopping cell proliferation. The ineffectiveness of 2'-dAdo at E10 may result from the lethality of 2'-dAdo to the embryo at low concentrations (30 microM) in vivo, well below the apoptosis-inducing concentrations employed in vitro (100-300 microM). These data extend previous findings showing that purine and pyrimidine metabolism plays an important role in development.

Our past work on nucleoside toxicity in sympathetic neurons has clearly revealed that adenosine and 2'-deoxyadenosine (dAdo) have different mechanisms of action in inducing apoptotic death. For example, adenosine is toxic to neurons only during early phase of growth whereas dAdo kills even mature neurons. In this study, we hypothesize that dAdo-induced apoptosis is initiated when ATP concentration of sympathetic neurons decreases below a critical level. To prove our hypothesis we used adenosine as a tool to replenish ATP levels of sympathetic neurons. We demonstrate that dAdo toxicity in mature sympathetic neurons was fully prevented by adenosine treatment. Furthermore, we demonstrate that depletion of ATP caused by dAdo was prevented by pretreatment with adenosine. These data suggest that intracellular accumulation of adenosine could play a neuroprotective role in preventing death associated with reduction in neuronal ATP concentration.

Recently, we have demonstrated that adenosine and 2'-deoxyadenosine are toxic to embryonic sympathetic neurons and proposed that purine and pyrimidine metabolism may play a critical role in the growth and development of sympathetic neurons. To extend this hypothesis further, we examined the effects of these nucleosides on two other neuronal populations in the chick embryo, sensory dorsal root ganglion neurons and parasympathetic ciliary ganglion neurons. Now, we show that 2'-deoxyadenosine and adenosine have no visible adverse effect on the viability of either sensory or parasympathetic neurons. Instead, 2'-deoxyadenosine proved to be highly toxic to the nonneuronal cells. The toxic effects of 2'-deoxyadenosine were markedly enhanced by inhibition of adenosine deaminase. In contrast, adenosine was much less toxic to nonneuronal cells than 2'-deoxyadenosine and its effect was not potentiated by inhibition of adenosine deaminase. Priming of pyrimidine pools by exogenous uridine and the specific inhibitor of the nucleoside transporter, nitrobenzylthioinosine, did not protect nonneuronal cells from 2'-deoxyadenosine toxicity. Since phosphorylation of internalized nucleosides was a key step in the initiation of toxicity in sympathetic neurons, adenosine kinase activity was compared in sensory and sympathetic neuronal cultures. The adenosine kinase activity in dorsal root ganglion cultures was only 20% of that in sympathetic ganglion cultures. Furthermore, inhibition of phosphorylation by blocking 2'-deoxyadenosine kinase with iodotubercidin and 5'-amino-5'-deoxyadenosine had no protective effect against 2'-deoxyadenosine toxicity. [3H]-thymidine incorporation was inhibited over 90% by 2'-deoxyadenosine as early as 6 h following its addition and for up to 4 days, suggesting inhibition of proliferation of nonneuronal cells by 2'-deoxyadenosine. The nucleoside was also able to wipe out already well established nonneuronal cells, leaving behind an enriched population of sensory neurons. The selective vulnerability of nonneuronal cells to 2'-deoxyadenosine offers a convenient and effective tool for removing nonneuronal cells from neuronal cultures as well as providing a new model for studying the mechanisms of nucleoside toxicity., (Copyright 1998 Elsevier Science B.V.)

For 3 consecutive days, the nucleoside cordycepin (3'-deoxyadenosine) was administered as 1-hr iv infusions (0, 1, 4, 8, 10, or 20 mg/kg/day) to dogs. These doses were given 1 hr after a bolus iv injection (0.25 mg/kg/day) of 2'-deoxycoformycin (dCF), a potent inhibitor of adenosine deaminase. The hypothesis was that dCF would affect the toxicity of cordycepin. Plasma adenosine deaminase activity was strongly inhibited during the dose period and for 5 days following the final dose of dCF. Dogs given cordycepin alone showed no drug-related toxicities. In dogs given only dCF, drug-related toxicity to lymphoid tissue (lymphopenia and thymus lymphoid depletion), thrombocytopenia, and decreases in food consumption were observed. Cordycepin in combination with dCF produced symptoms associated with severe gastrointestinal toxicity (decreased body weights, emesis, diarrhea, decreased food consumption, and necrosis of the gastrointestinal tract) and bone marrow toxicity (lymphopenia, thrombocytopenia, and depletion of hematopoietic cells). The gastrointestinal tract and bone marrow were sites associated with dose-limiting toxicities. In surviving dogs, most of the effects were reversible by Day 30. The maximum tolerated dose of cordycepin administered in combination with dCF was 8 mg/kg/day (160 mg/m2/day) given daily for 3 days., (Copyright 1997 Academic Press.)

We show here that 2'-deoxyadenosine (2'-dAdo) but not adenosine was toxic to chromaffin cells of 3-4-week-old rat adrenal glands. More than 75% of the cells plated in culture gradually died over a 3-day period in the presence of 100 microM 2'-dAdo plus 3 microM deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and terminal deoxynucleotidyl transferase-mediated nick and labeling showed membrane blebbing, shrinkage of cell bodies, chromatin condensation, and DNA fragmentation, suggesting apoptosis-like cell death by 2'-dAdo. Lethal effects of 2'-dAdo were potentiated by DCF, a drug that inhibits adenosine deaminase. 2'-dAdo-prompted cell death was not prevented by inhibitors of nucleoside transporter (3 microM dilazep or 1 microM nitrobenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 microM uridine or 100 microM 2'-deoxycytidine), or 5 mM nicotinamide. Cells incubated with 2'-dAdo (100 and 300 microM) showed a three- and ninefold, respectively, increase in content of dATP, a product known to be an inhibitor of ribonucleotide reductase, an enzyme essential for DNA synthesis. Formation of dATP was completely prevented by iodotubercidin (ITu), a drug that inhibits phosphorylation of 2'-dAdo to dATP by nucleoside kinase. It is interesting that nanomolar concentrations of ITu also completely protected chromaffin cells from 2'-dAdo lethality. Our study demonstrates for the first time that mammalian adrenal chromaffin cells undergo apoptotic cell death by a natural nucleoside and suggests that this model could be used to study apoptosis and cell function.

These experiments characterize the nucleoside transport and quantify the neurotoxicity of adenosine and 2'-deoxyadenosine (dAdo) in chick sympathetic neurons. We show that [3H]adenosine transport was sensitive to low temperature, specific inhibitors of nucleoside transport, and an excess concentration of adenosine. However, many of these treatments had a marginal effect on [3H]dAdo transport. Total retention of [3H]dAdo over short and long periods was approximately 10 times less than that of [3H]adenosine. These data suggest that adenosine and dAdo enter sympathetic neurons by different routes. Uptake of (3H]norepinephrine ([3H]NE) decreased in neurons damaged by nucleosides and increased to control levels when neurons were protected by various agents against adenosine or dAdo toxicity. These results indicate that [3H]NE uptake serves as a quantitative index of toxicity by the nucleosides. Using this approach we demonstrate that phosphorylation of both nucleosides is essential for their lethal action. For example, iodotubercidin prevented nucleoside-induced neuronal death, but the effect was much more pronounced in the case of dAdo toxicity (IC50 of 0.83 +/- 0.4 vs. 30 +/- 1.6 nM). Another kinase inhibitor, 5'-amino 5'-deoxyadenosine, was effective in protecting neurons against dAdo but had no effect against adenosine toxicity. These results suggest that specific kinases are associated with the phosphorylation of adenosine and dAdo in sympathetic neurons to produce toxic metabolic products. Finally, neurons were susceptible to dAdo toxicity from the time of plating to 4 weeks in culture but were resistant to adenosine toxicity 8 h after plating. In conclusion, our results highlight major differences in the mechanism of neurotoxicity by adenosine and dAdo and provide insights for identification of biochemical pathways leading to neuronal death.

We have shown previously that a low concentration of tritiated deoxyadenosine, i.e., 1 microCi/ml, selectively kills wild-type S49 murine lymphoma cells. Mutant cells resistant to [3H] deoxyadenosine lacked adenosine kinase completely but retained a significant level of deoxyadenosine phosphorylating activity. To study further the specificity of [3H] deoxyadenosine selection, lymphoma cell clones resistant to 15 microCi/ml [3H] deoxyadenosine have been derived. The resistant line, S49-dA15, is also resistant to high levels of nonradioactive deoxyadenosine and to deoxyguanosine but remains sensitive to thymidine. The thymidine inhibition of the growth of the mutant, in contrast to that of the wild-type cells, cannot be prevented by deoxycytidine. The mutant line lacks deoxycytidine kinase that also phosphorylates deoxyadenosine. In addition, the mutant cells excrete a large amount of deoxycytidine into culture medium, consistent with a failure of salvage of the nucleoside in the absence of an appropriate kinase, i.e., deoxycytidine kinase. In contrast, a deoxycytidine kinase-deficient cell line that was selected with arabinosylcytosine does not excrete deoxycytidine and contains high deoxycytidine deaminase activity. [3H] Deoxyadenosine can be used as a selective agent for specific selection of deoxycytidine kinase-negative mutants.

Glucagon, dibutyryl cAMP (Bt2cAMP) and 8-(4-chlorophenylthio) cAMP (CptcAMP), singly or when combined, stimulated tyrosine aminotransferase (TAT) activity in 17-day-old chick embryos in ovo. Maximal induction was produced within 4 hr of injection of the inducers. The effects of glucagon and the cAMP analogues were not additive. Glucagon administration was accompanied by a rapid increase in hepatic cAMP concentration which remained elevated for at least 4 hr. The stimulated increase in TAT activity elicited by the hormone or cyclic nucleotide was prevented by injection of cycloheximide or cordycepin. These results are discussed vis-à-vis the possible regulation of TAT in ovo by physiological concentrations of glucagon and the likely role of cAMP as a second messenger in this process during chick embryogenesis.

Adenosine deaminase (ADA) catalyzes the irreversible hydrolytic deamination of adenosine and deoxyadenosine to nontoxic derivatives. The importance of this reaction in the female reproductive tract of mice is suggested by pronounced utero-placental expression of ADA, and by embryolethality of the potent ADA-inhibitor deoxycoformycin (dCF) on day 7-8 of gestation. The present study investigated the effects of dCF, adenosine, and deoxyadenosine on the mouse neurula. Morphological cell death was monitored by the acridine orange reaction (AOR), and biochemical cell death by internucleosomal DNA cleavage (IDC). A strong AOR appeared in day 7-8 embryos between 3 and 4.5 hr post-exposure to dCF in utero; there was no apparent effect on day 6 or day 9 embryos. Most embryonic tissues were responsive, although the heart and extraembryonic membranes were resistant. Up to 75% of the embryonic chromatin was degraded in a regular pattern in concert with the AOR. Immediate activation of "whole-body" apoptosis was reproduced in short-term whole embryo culture with 0.1 mM deoxyadenosine in the presence of 0.01 mM dCF. This was not activated by exposure to dCF alone nor to adenosine; however, high concentrations of adenosine completely blocked the response to deoxyadenosine, whereas niacinamide inhibited the AOR without changing IDC. The cytotoxic effect of deoxyadenosine was correlated with an expansion of embryonic dATP pools determined by high-performance liquid chromatography analysis. The results suggest that deoxyadenosine is the embryotoxic metabolite which accumulates in the antimesometrium of pregnant mice treated with dCF. Exposure to this metabolic toxin activates apoptosis in day 7-8 embryos through an adenosine-sensitive, NAD-dependent mechanism.

The potential diadenosine polyphosphate analogue precursor, bis(adenosin-N6-yl)dodecane (A[CH2]12A) (Chen, H. & McLennan, A. G. (1993) Eur. J. Biochem. 213, 935-944.) is equally toxic to both wild-type and adenosine-kinase-deficient BHK cells at concentrations up to 100 microM; at higher concentrations, wild-type cells are more sensitive, as are cells over-expressing adenosine kinase. Thus both the nucleoside and its nucleotide products are toxic. In contrast to adenosine toxicity, the toxicity of A[CH2]12A to S-49 T-lymphoma cells could not be reversed by uridine or by L-homocysteine thiolactone. A[CH2]12A and all its shorter chain bis(adenosin-N6-yl)alkane homologues could relieve the toxicity of low adenosine concentrations (< 20 microM) to S-49 cells, mainly through inhibition of adenosine kinase, while relief of the toxicity of high adenosine concentrations (> 20 microM) required the longer chain homologues. A[CH2]12A at 10 microM completely eliminated adenosine toxicity. Deoxyadenosine toxicity could also be relieved, but only that due to low concentrations (< 4 microM). A[CH2]12A had only a slight stimulatory effect on S-adenosylhomocysteine-hydrolase activity.

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.

2-Chlorodeoxyadenosine (CdA) is active in chronic lymphocytic leukemia, hairy-cell leukemia, and low-grade lymphomas. In part, this spectrum of activity may be attributable to the selective toxicity of CdA to nondividing lymphocytes and monocytes. However, CdA is unstable at acidic pH and is degraded by bacterial nucleoside phosphorylases. The present experiments demonstrate that the 2'-arabino-fluoro derivative of CdA, designated CAFdA, is also directly toxic to quiescent lymphocytes and macrophages. Unlike CdA, CAFdA was stable at pH 2 and resisted degradation by Escherichia coli nucleoside phosphorylase. Cell killing was preceded by the formation of DNA strand breaks and could be prevented by supplementation of the medium with deoxycytidine. The initial DNA damage initiated the pattern of oligonucleosomal DNA fragmentation characteristic of apoptosis. Mutant lymphoblasts, deficient in deoxycytidine kinase, with elevated cytoplasmic 5'-nucleotidase, or with expanded deoxynucleotide pools secondary to increased ribonucleotide reductase activity, were cross-resistant to both CAFdA and CdA toxicity. One-week oral treatment with CAFdA (1 mg/ml in drinking water) achieved an average plasma concentration of 0.56 microM and eliminated 90% of chronic lymphocytic leukemia cells transplanted into severe combined immunodeficiency (scid) mice. Under the same conditions, CdA was much less active. Collectively, these results suggest that CAFdA could be effective as an oral agent in indolent lymphoproliferative diseases and in autoimmune diseases where lymphocyte and monocyte depletion is desirable.

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.

The toxic effect of 3'-deoxyadenosine N1-oxide (3'-dANO) on mice, on their different organs, and on Ehrlich ascites tumor cells was studied. In both healthy and tumour-bearing animals, the lethal dose for 10% of the mice receiving i.p. injections (LD10) of 3'-dANO was estimated to be about 300 mg/kg x 4 days in one mouse strain (Theiller). In another mouse strain (NMRI), we obtained a markedly higher LD10 value (675 mg/kg x 5 days). At nonlethal doses (250 mg/kg x 4 days), we observed reversible neurological symptoms on days 4-12 after treatment, but no macroscopical or microscopical changes was detected in the brain, heart, thymus, lung, lymph node, spleen, liver, kidney, bone marrow, or gastrointestinal tract. At doses of 450 mg/kg x 4 days, severe neurological symptoms were observed, and atony of the gastrointestinal canal and damage to the kidney and liver were registered. Even at doses that were lethal to the mice, no histopathological change was observed in the bone marrow or in the gastrointestinal canal. Pharmacokinetics studies showed that after the i.p. injection of 3'-dANO, the maximal plasma concentration was reached after 10 min, after which it declined showing a half-life of about 40 min. A transient accumulation of 3'-deoxyadenosine triphosphate (3'-dATP) was observed within 24 h in the liver and kidney, with the maximal concentration being reached after about 2-3 h. 3'-dANO was excreted partly as the unchanged substance and partly as the metabolite 3'-deoxyinosine within 24 h. Flow-cytometric DNA analysis of Ehrlich tumor cells treated either in vitro or in vivo with 3'-dANO revealed no therapy-induced change in the cell-cycle perturbations, which indicates that cells were randomly killed during all phases of the cycle.

We compared deoxyadenosine (AdR)- and cyclic AMP (cAMP)-induced cell cycle arrest and cytotoxicity in wild type and mutant S49 cells to determine whether they resulted from the same or different mechanisms. Cyclic AMP and deoxyadenosine are synergistic rather than additive in cytotoxicity assays, suggesting different mechanisms of toxicity. Although cyclic AMP causes cell death after 72 h, in concentrations sufficient to result in cell cycle arrest it is reversible with virtually no cytotoxicity for at least 24 h, whereas AdR-induced cell cycle arrest is lethal and irreversible. AdR-induced G1 cell cycle arrest results in diminished ribonucleotide reductase activity but the kinetics of this inhibition differ from cyclic AMP-induced cell cycle arrest. Cyclic AMP arrest and cytotoxicity depend on cyclic AMP-dependent protein kinase (PKA) activity, whereas AdR toxicity does not differ between cell lines with or without PKA activity. Furthermore, deoxycytidine prevents AdR cell cycle arrest and cytotoxicity but has no effect on cyclic AMP G1 arrest. Finally, comparison of cytofluorographic patterns of G1-arrested cells suggests that the AdR block is later in G1 than cyclic AMP-induced cell cycle arrest. In summary, these data show that while the mechanisms of cell cycle arrest and cytotoxicity of cyclic AMP and deoxyadenosine are uncertain, they do appear to involve different pathways.

Although several different enzymes with 5'-nucleotidase activity have been described in mammalian cells, their functions in nucleotide metabolism have not been clearly distinguished. In the present experiments, a mutant human T lymphoblastoid cell line (CEM-dAdoR) was selected specifically for resistance to deoxyadenosine toxicity. Compared to parental CEM cells, the variant had 4-fold elevated ATP-activated cytosolic 5'-nucleotidase activity. Other enzymes of potential importance for deoxyadenosine metabolism were indistinguishable in the two cell types. In medium supplemented with the adenosine deaminase inhibitor deoxycoformycin, the T cells with increased 5'-nucleotidase accumulated less nucleotides from exogenously added deoxyadenosine, or 9-beta-D-arabinofuranosyladenine, than did parental T lymphocytes. These metabolic changes were associated with resistance to the growth inhibitory effects of these nucleosides, and also to deoxyguanosine and to 9-beta-D-arabinofuranosylguanine. The T cells with elevated 5'-nucleotidase activity formed more 2',3'-dideoxyadenosine than did parental cells, in deoxycoformycin-supplemented medium. The accumulation of 2',3'-dideoxyadenosine 5'-triphosphate from 2',3'-dideoxyinosine was similarly augmented in the mutant. These data establish the importance of the cytosolic 5'-nucleotidase for the metabolism of purine 2'-deoxyribonucleosides, arabinonucleosides and 2',3'-dideoxyribonucleosides in T lymphoblasts.

Extracorporeal photochemotherapy is an effective treatment for cutaneous T cell lymphoma but its mode of action is uncertain. The reduction in viability of patients' photoirradiated buffy coat lymphocytes was correlated with a 35% increase in DNA single-strand breaks and marked decreases in cellular ATP and NAD levels (to 58 and 34% of control, respectively) immediately after photoirradiation. Complementary in vitro studies were conducted with normal human peripheral blood lymphocytes using a Therakos ultraviolet A (UVA) light box. UVA light was cytotoxic on its own but was potentiated by 8-methoxysporalen. 3-aminobenzamide, a poly (ADP-ribose) synthetase inhibitor, mitigated the cytotoxic effect of ultraviolet A light in the presence of 8-methoxypsoralen in lymphocytes and reduced the amount of nucleotide depletion they caused. 10 J/cm2 of UVA light in the presence of 300 ng/ml 8-methoxypsoralen increased the poly (ADP-ribose) synthetase activity of peripheral blood lymphocytes. Exposing lymphocytes to deoxycoformycin and deoxyadenosine was found to induce biochemical and physical effects similar to those of photochemotherapy. In summary, we have shown that the lymphocytotoxic effect of extracorporeal photochemotherapy for cutaneous T cell lymphoma is apparently mediated by DNA damage, subsequent poly (ADP-ribosyl)ation and adenine nucleotide depletion. It is not known how the DNA damage and resultant biochemical effects relate to the possible immunological mechanism of extracorporeal photochemotherapy; however, it is possible that its effects can be mimicked by other DNA-damaging agents.

Lymphoid cells were thought to be uniquely susceptible to excess 2'-deoxyadenosine (dAdo), when exposed to inhibitors of adenosine deaminase (ADA). However, we now find that human monocytes are as sensitive as lymphocytes to dAdo or to the ADA-resistant congener 2-chloro-2'-deoxyadenosine (CldAdo). Monocytes exposed in vitro to CldAdo, or to dAdo plus deoxycoformycin rapidly developed DNA strand breaks. Both the DNA damage and the toxicity of CldAdo or dAdo toward monocytes were blocked by deoxycytidine, but not by inhibitors of poly(ADP-ribose) polymerase. A partial decrease in RNA synthesis and a gradual decline of cellular NAD were early biochemical events associated with monocyte DNA damage. Low CldAdo concentrations (5-20 nM) inhibited monocyte phagocytosis and reduced the release of interleukin 6. Higher CldAdo concentrations led to a dose- and time-dependent loss of monocyte viability. Circulating monocytes disappeared within 1 wk in patients with cutaneous T cell lymphoma or with rheumatoid arthritis during continuous CldAdo infusion. The marked sensitivity of human monocyte function and survival to CldAdo in vitro, together with the monocyte depletion in patients receiving CldAdo chemotherapy, suggests that CldAdo or other dAdo analogues offer a novel therapeutic strategy for chronic inflammatory and autoimmune diseases characterized by inappropriate monocyte deployment or function.

2',3'-Dideoxyadenosine (DDA) was shown not only to possess antibacterial activity in vitro against a variety of Enterobacteriaceae, but also to be effective in vivo, DDA was active in experimental mouse infections by the oral route against 5 Salmonella strains, 2 of 3 Arizona strains, 5 of 7 Citrobacter strains, 3 of 8 Klebsiella strains, 3 of 5 Escherichia strains, 1 of 3 Shigella strains, and 3 of 15 Serratia strains at concentrations generally well below the toxic level. Closely related compounds, with the exception of 2',3'-dideoxyinosine, were found to be inactive in vivo, indicating that a high degree of structural specificity was required for activity. The synthesis of deoxyribonucleic acid was inhibited by DDA in those strains susceptible in vitro to DDA, whereas ribonucleic acid and protein syntheses were not affected. The concentration of DDA which inhibited bacterial deoxyribonucleic acid synthesis by 50% was calculated based on the relative rates of deoxyribonucleic acid synthesis in ;the absence and in the presence of DDA. This value correlated well with the minimal inhibitory concentration determined by the in vitro broth dilution assay but not always with in vivo activity determined by the mouse protection test.

Accumulation of dATP derived from 2'-deoxyadenosine (dAdo), causing inhibition of ribonucleotide reductase and depletion of the other deoxynucleotide substrates required for DNA synthesis, has been suggested as the cause of the lymphopenia and immune defect in inheritable deficiency of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4). dAdo also inactivates the enzyme S-adenosylhomocysteine hydrolase (AdoHcyase; S-adenosyl-L-homocystein hydrolase EC 3.3.1.1) which is involved in the catabolism of S-adenosyl-L-homocysteine (AdoHcy), both a product and a potent inhibitor of S-adenosylmethionine-dependent transmethylation. We have tried to determine whether inactivation of AdoHcyase might also contribute to dAdo toxicity to adenosine deaminase-inhibited cells. dAdo rapidly inactivates intracellular AdoHcyase and causes the accumulation of AdoHcy in WI-L2 human B lymphoblastoid cells. Low concentrations of adenosine (Ado), which block binding of dAdo to purified AdoHcyase, prevented inactivation of intracellular AdoHcyase and also lessened the growth-inhibitory effect of dAdo. A mutant of this cell line which lacks Ado kinase and accumulated endogenously synthesized Ado was resistant to the effects of dAdo on both growth and AdoHcyase activity. The mutant also accumulated far less dATP from dAdo than did its parent and was resistant to the inhibitory effect of dAdo on DNA synthesis, indicating the Ado kinase is involved in dAdo phosphorylation in these cells. Combinations of deoxycytidine, thymidine, and deoxyguanosine that could prevent dATP-mediated depletion of deoxynucleotide pools but not AdoHcyase inactivation were less effective than Ado in preventing dAdo toxicity to normal lymphoblasts. Our results suggest that inactivation of AdoHcyase, as well as dATP accumulation, contributes to dAdo toxicity.

The toxicity of low concentrations of 2'-deoxyadenosine for T-lymphoblasts and certain null lymphoblasts has been attributed to the decreased degradation of the deoxynucleotides formed from deoxyadenosine in these cells. Low activities of the ectoenzymes ecto-5'-nucleotidase and ecto-ATPase have each been associated with deoxyadenosine sensitivity and dATP accumulation in human T-lymphoblasts. We studied a B-lymphoblast cell line, NC-37, which lacks detectable ecto-5'-nucleotidase and ecto-ATPase activities, but which is otherwise easily distinguishable from T-lymphoblasts by its low adenosine deaminase activity and its pattern of reactivity with monoclonal antibodies to cell surface antigens (Bl and IgM positive). The NC-37 B cells were completely analogous to other B-lymphoblast lines with high ectonucleotidase activities in their relative resistance to deoxyadenosine toxicity and low rates of dATP accumulation. This resistance could not be accounted for by lower rates of deoxyadenosine phosphorylating activity. Cytoplasmic nucleotidase activity in crude extracts from the NC-37 line was similar to that in other B-lymphoblasts with regard to both substrate specificity and optimal pH. We conclude that low ectonucleotidase activities are not etiologically associated with the accumulation of deoxynucleotides by human lymphoblasts, although they may serve as markers of deoxyadenosine sensitivity in certain malignant lymphoid cells.

Treatment of mice with 2'-deoxycoformycin (dCf) for 5 days produced inhibition of spleen and lymph node adenosine deaminase (E. C. 3.5.4.4) activity but no hematologic toxicity or weight loss. A 64-fold elevation of erythrocyte dATP was observed. However, if mice were injected with 2'-deoxyadenosine (AdR) in combination with dCf, weight loss, hematologic toxicity, and liver cell necrosis occurred. These mice had a severe blood coagulation defect and a 73-fold elevation of plasma alanine transaminase activity, plasma prealbumin became undetectable, and erythrocyte dATP levels were elevated 1500-fold. Death during treatment appeared to be from acute liver failure since bone marrow toxicity was only detected following termination of treatment. These effects were not seen in mice receiving adenosine in combination with dCf. dCf, either alone or in combination with AdR, inhibited the contact sensitization to oxazalone in mice. The inhibition was associated with signs of systemic toxicity which were more pronounced in the combination-treated groups. If dATP is the toxic metabolic accumulated in the malignant cells of patients treated with dCf, we propose that AdR supplementation of treatment should be considered with extreme caution since severe damage to normal tissues might result.

Amino acid analogs, like other effectors of the stress response, induce in mammalian cells the same gene products that are induced upon heat shock; incorporation of the analog into protein is required for induction. We show here that induction by analogs involves controls operating at the levels of both transcription and translation. The electrophoretic patterns of newly made mRNAs simplify with time such that the putative stress protein mRNAs are the only species transported from the nucleus. Concomitantly, the patterns of protein synthesis simplify such that the stress proteins become nearly exclusive polypeptide products. Although the normal mRNAs are either not used or used with greatly reduced efficiency, they are not degraded and retain translatability when transferred to cell-free systems. Soon after the stress response has been induced, there follows a defect in the initiation of polypeptide chains, as evidenced by examination of polysome profiles. Upon prolonged exposure, polysomes are recovered, and although they give rise to stress proteins almost exclusively, the normal mRNAs are still present in these structures. Thus, in addition to the initiation defect, a lesion in elongation may also be involved. The extreme sensitivity of protein synthesis to the inhibition of RNA synthesis, together with the parallel simplifications in the patterns of newly made mRNAs and polypeptides, may imply that only newly made mRNAs are efficiently translated in analog-treated cells.

Terminal deoxynucleotidyl transferase (TdT) was used to prepare copolymers of dA and 1,N6-ethenodeoxyadenosine (epsilon dA). When used as templates for Escherichia coli DNA polymerase I (Pol I) and compared with poly (dA), normal dTTP incorporation was not significantly affected by the presence of 7% epsilon dA. dGTP misincorporation was only slightly increased and occurred about once for every 500 epsilon dA residues. The error-prone polymerase from avian myeloblastosis virus (AMV reverse transcriptase) increased this error rate 5- to 20-fold to a maximum of 1 dG/25 epsilon dA. No dCTP misincorporation was detected with either polymerase. In transcription with E. coli DNA-dependent RNA polymerase, no errors were revealed by nearest neighbor analysis. Poly (dA) treated with chloroacetaldehyde under conditions producing the same proportion of epsilon dA (without the hydrated form) as the synthesized template behaved in the same manner with a similar low level of misincorporation of dG. Such treatment of alternating poly d(A-T) caused structural changes indicative of crosslinks but did not alter its template properties. Increasing the amount of epsilon dA in either synthesized or modified polymers greatly decreased the template activity without increasing the error rate. It is suggested that epsilon dA generally does not prevent dT incorporation but behaves as a bulky lesion which is bypassed. In contrast to the low mutagenic efficiency of epsilon dA, O4-methyldeoxythymidine (m4dT), in copolymers with dA, directed the misincorporation of 1 dG/12 m4dT with Pol I and 1 dG/3 m4dT with reverse transcriptase. Nearest neighbor analysis of transcripts showed the incorporation of 1 dG/12 m4dT. These data are in agreement with the previous reported mutagenicity of m4dT in alternating poly d(A-T, m4T).

Deoxyadenosine toxicity toward lymphocytes may produce immune dysfunction in patients with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. The relationship between endogenous deoxynucleoside synthesis in adenosine deaminase-deficient cells and sensitivity to adenosine and deoxyadenosine toxicity is unclear. The human histiocytic lymphoma cell line (DHL-9) naturally lacks adenosine deaminase, and has minimal levels of thymidine kinase. Dividing DHL-9 cells excrete deoxyadenosine and thymidine into the extracellular space. The present experiments have analyzed nucleoside synthesis and excretion in a mutagenized clone of DHL-9 cells, selected for increased resistance to deoxyadenosine toxicity. The deoxyadenosine-resistant cells excreted both deoxyadenosine and thymidine at a 6-7-fold higher rate than wild-type lymphoma cells. The deoxyadenosine overproduction was accompanied by a reduced ability to form dATP from exogenous deoxyadenosine, and a 2.5-fold increase in ribonucleotide reductase activity. The pace of adenosine excretion, the growth rate, and the levels of multiple other enzymes involved in deoxyadenosine and adenosine metabolism were equivalent in the two cell types. These results suggest that the excretion of deoxyadenosine and thymidine, but not adenosine, is exquisitely sensitive to alterations in the rate of endogenous deoxynucleotide synthesis. Apparently, small changes in deoxynucleotide synthesis can significantly influence cellular sensitivity to deoxyadenosine toxicity.

The cytotoxic effects of the adenosine deaminase resistant analogues 2-bromo-2'-deoxyadenosine (2-BrdAdo) and 2-chloro-2'-deoxyadenosine (2-CldAdo) have been compared with those of deoxyadenosine (dAdo). Like 2-CldAdo, 2-BrdAdo is highly effective in inhibiting the growth of many T-lymphoblastoid, B-lymphoblastoid, and myeloid cell lines in culture. Concentrations required to inhibit growth of CCRF-CEM human T-lymphoblastoid cells by 50% (IC50) are: 2-CldAdo, 0.045 microM; 2-BrdAdo, 0.068 microM; dAdo, 0.9 microM in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl)adenine. Like dAdo, 2-BrdAdo causes a much greater decrease in DNA synthesis than in RNA and protein synthesis. For each of the nucleosides the concentration required to cause 50% inhibition of DNA synthesis (as measured by thymidine incorporation) in an 18-h exposure is very similar to the IC50 for growth and to the concentration required to decrease viability (clonogenicity) over 18 h by 50% (EC50). A fraction of CCRF-CEM cells (approximately equal to 30%) is resistant to killing by exposure to 2-BrdAdo or 2-CldAdo for 4 h at concentrations 100 times the EC50, but 3% of cells are resistant to exposure for 4 h to a concentration of dAdo 3 times the EC50. Each of the three nucleosides causes accumulation of cells in S phase, the accumulation becoming more marked with longer periods of exposure and with higher concentrations of nucleoside. During exposures for 18-24 h at a concentration of nucleoside near the EC50 most cells accumulate in S, with most in early S, whereas exposure to concentrations greater than EC95 accumulates cells at the G1/S border. This suggests that loss of viability is associated with a blockade of some process specifically occurring at the initiation of S phase. At an optimum dosage schedule, 2-BrdAdo and 2-CldAdo have similar therapeutic effects against L1210 in vivo, both producing over 99% cell kill, but the optimum dosage of 2-CldAdo is lower.

2-Chlorodeoxyadenosine (CdA), an adenosine-deaminase-resistant purine deoxynucleoside, is markedly toxic toward human T-lymphoblastoid cell lines in vitro and is an effective agent against L1210 leukemia in vivo. The present studies have examined the toxicity, and in some cases, metabolism, of CdA in (1) multiple established human cell lines of varying phenotype, (2) leukemia and lymphoma cells taken directly from patients, (3) normal bone marrow cells, and (4) normal peripheral blood lymphocytes. Nanomolar concentrations of CdA blocked the proliferation of lymphoblastoid cell lines with a high ratio of deoxycytidine kinase to deoxynucleotidase. The drug had virtually no effect on the growth of cell lines derived from solid tissues. The CdA inhibited the spontaneous uptake of tritiated thymidine by many T and non-T, non-B acute lymphoblastic leukemia cell specimens at concentrations less than or equal to 5 nM. The same concentrations did not impair either thymidine uptake or granulocyte-monocyte colony formation by normal bone marrow cells. In common with deoxyadenosine, but unlike several other agents affecting purine and purine metabolism, CdA was lethal to resting normal T lymphocytes and to slowly dividing malignant T cells. In both resting and proliferating lymphocytes, the CdA was phosphorylated by deoxycytidine kinase and entered a rapidly turning over nucleotide pool. Dividing lymphocytes also incorporated abundant CdA into DNA. The selective toxicity of CdA toward both dividing and resting lymphocytes may render the drug useful as an immunosuppressive or antileukemic agent.

Deoxyadenosine metabolism was investigated in cultured human cells to elucidate the biochemical basis for the sensitivity of T lymphoblasts and the resistance of B lymphoblasts to deoxyadenosine toxicity. T lymphoblasts have a 20-to 45-fold greater capacity to synthesize deoxyadenosine nucleotides than B lymphoblasts at deoxyadenosine concentrations of 50--300 micron. During the synthesis of dATP, T lymphoblasts accumulate large quantities of dADP, whereas B lymphoblasts do not accumulate dADP. Enzymes affecting deoxyadenosine nucleotide synthesis were assayed in these cells. No substantial differences were evident in activities of deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76) or deoxyadenylate kinase [ATP:(d)AMP phosphotransferase, EC 2.7.4.11]. The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was increased 44-fold for AMP and 7-fold for dAMP in B lymphoblasts. A model for the regulation of deoxyadenosine nucleotide synthesis by 5'-nucleotidase activity is proposed on the basis of the observations.