Hybridization properties and enzymatic replication of oligonucleotides containing the photocleavable 7-nitroindole base analog.

Universal DNA base analogs having photocleavable properties would be of great interest for development of new nucleic acid fragmentation tools. The photocleavable 7-nitroindole 2'-deoxyribonucleoside d(7-Ni) was previously shown to furnish a highly efficient approach to photochemically trigger DNA backbone cleavage at preselected position when inserted in a DNA fragment. In the present report, we examine its potential use as universal DNA nucleoside, by analogy with the 5-nitroindole analog that is generally considered as universal base. The d(7-Ni) phosphoramidite was incorporated into oligonucleotides. Hybridization properties of resulting 11mer duplexes indicated a behavior close to that of the 5-nitroindole analog. Enzymatic recognition by Klenow fragment exonuclease-free using 40mers containing the unnatural bases as templates indicated notably a decrease of the polymerase activity with preferential incorporation of dAMP opposite both the 7-Ni and 5-Ni bases. Incorporation of the d(7-Ni) triphosphate was also studied indicating absence of significant differences between the incorporation kinetics opposite each natural base in the template. All the hybridization and enzymatic data indicate that 7-nitroindole can be considered as a cleavable base analog, although not strictly fulfilling, like the 5-nitro isomer, all properties required for a universal base.

Misincorporation of 2'-deoxyoxanosine into DNA: a molecular basis for NO-induced mutagenesis derived from theoretical calculations.

A wide range of theoretical methods, including high level ab initio, density functional, self-consistent reaction field, molecular dynamics and thermodynamic integration calculations, have been used to analyze the mutagenic properties of oxanosine. The major tautomeric forms in the gas phase and aqueous solution have been determined. The ability of oxanosine to recognize thymine and cytosine in the gas phase and in the DNA environment has been compared with that of guanine. A physicochemical explanation for the mutagenic properties of oxanosine is suggested.

Genotoxicity of ribo- and deoxyribonucleosides of 8-hydroxyguanine, 5-hydroxycytosine, and 2-hydroxyadenine: induction of SCE in human lymphocytes and mutagenicity in Salmonella typhimurium TA 100.

The induction of SCE by ribo- and deoxyribonucleosides of 8-hydroxyguanine, 5-hydroxycytosine, and 2-hydroxyadenine was tested using human peripheral blood lymphocytes. All of these compounds caused an increase in SCE frequency. The potency of SCE induction was as follows: 5-OH-C, 5-OH-dC > 8.OH-G > 8-OH-dG > 2-OH-A, 2-OH-dA. These results suggest that the oxidized nucleosides are incorporated into DNA with different efficiencies (or are repaired with different efficiencies) and exhibit genotoxicity in human blood cells. Ribo- and deoxyribo-derivatives of 5-OH-Cyt and 2-OH-Ade also showed mutagenic activity in Salmonella typhimurium TA 100.

Comparative mutagenicities of N6-methoxy-2,6-diaminopurine and N6-methoxyaminopurine 2'-deoxyribonucleosides and their 5'-triphosphates.

The structure of the deoxyribonucleoside derived from N 6-methoxy-2, 6-diaminopurine (dK) was examined by NMR. The methoxyamino residue was found predominantly in the imino rather than the amino tautomer (ratio: 9:1 in DMSO). The nucleoside proved to be a potent transition mutagen in Escherichia coli , in contrast to the closely related nucleoside derived from the analogue N6-methoxyaminopurine (dZ), which was only weakly mutagenic. The 5'-triphosphate derivatives, dKTP and dZTP, were synthesized; Taq polymerase incorporated dKTP opposite both T and, less well, opposite dC in template DNA. Both analogue triphosphates produced transition mutations when added to PCR reactions. In each case, there was a large excess of AT-->GC compared to GC-->AT mutations (ratios were 15:1 for dKTP and 10:1 for dZTP). Polymerase extension times in each cycle had to be extended, consistent with a decreased rate of DNA synthesis in the presence of the analogues. This and the mutagenic ratios are discussed in terms of syn-anti inversion of the methoxyl group.

Synthesis and antineoplastic properties of 3'-deoxy-3'-fluoroketonucleoside derivatives. Correlations between structure and biological activity.

Three fluoroketonucleosides (6, 8, and 11) have been synthesized by direct oxidation of the fluoro precursors. The presence of the highly electronegative fluorine atom in the alpha position to the carbonyl group favours hydration leading to the gem-diol form so that the beta-elimination process to afford 6 and 8 was made difficult and failed in the case of the difluoro compound 11. The biological activity of compounds 6, 8, and 11 was tested on human peripherical blood lymphocytes stimulated by PHA, and on RAJI and DAUDI cells. The IC50 values showed that, surprisingly, the 3'-enopyranosyl-2'-uloses 6 and 8 have much better antineoplastic activities than their 2'-enopyranosyl-4'-ulose analogues 14 and 15 obtained previously. Moreover, compound 11, which is difluorinated at C-3' and C-6' but does not have a C = C-C = O group in its structure, is also very active. These results emphasize the important biological role played by the fluorine atom in this family of compounds and suggest a peculiar mechanism of action which is until now unspecified.

Inhibitory effect of azidothymidine, 2'-3'-dideoxyadenosine, and 2'-3'-dideoxycytidine on in vitro growth of hematopoietic progenitor cells from normal persons and from patients with AIDS.

Therapy of patients with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) with azidothymidine (AZT) and 2'-3'-dideoxycytidine (ddC) is complicated by severe anemia, neutropenia, and thrombocytopenia, the cause of which is unknown. We therefore tested the effect of AZT, ddC, and an additional 2'-3'-dideoxynucleoside analogue, 2'-3'-dideoxyadenosine (ddA), on the hematopoietic progenitor cells derived from the bone marrow of normal persons and patients with AIDS/ARC. All three substances dose-dependently inhibited the in vitro colony formation of the pluripotent (CFU-GEMM), as well as the erythroid (BFU-E) and granulocyte-macrophage progenitor cells (CFU-GM). The 50% inhibition of normal progenitors by AZT occurred at 0.13 microM for CFU-GEMM, 0.32 microM for BFU-E, and 1.9 microM for CFU-GM, by ddA at 15 microM for CFU-GEMM, 40 microM for BFU-E, and 140 microM for CFU-GM. ddC was the most toxic agent and already inhibited 71% +/- 16% (mean +/- standard error of the mean [SEM]) of CFU-GEMM and 52% +/- 22% of BFU-E at 0.1 microM, whereas the 50% inhibition of CFU-GM was reached at 0.3 microM. Hematotoxicity occurred at concentrations lower than necessary to inhibit the human immunodeficiency virus (HIV), except for ddA, which is 100 times less toxic than AZT whereas its antiviral effect is only 10 times less. The inhibition of progenitor cells from AIDS patients by the 2'-3'-dideoxynucleosides was comparable to normal progenitors, except for a higher sensitivity of AIDS-derived CFU-GEMM and BFU-E to AZT.

The anti-HTLV-III (anti-HIV) and cytotoxic activity of 2',3'-didehydro-2',3'-dideoxyribonucleosides: a comparison with their parental 2',3'-dideoxyribonucleosides.

A series of 2',3'-didehydro-2',3'-dideoxyribonucleosides (ddeNs) [i.e., 2',3'-dideoxythymidinene (ddeThd), 2',3'-dideoxyuridinene (ddeUrd), 2',3'-dideoxycytidinene (ddeCyd), and 2',3'-dideoxyadenosinene (ddeAdo)] has been synthesized and the individual members compared in terms of their in vitro antiviral, antimetabolic, and cytostatic properties to their 2',3'-saturated counterparts (ddNs) (i.e., ddThd, ddUrd, ddCyd and ddAdo). All ddeNs except ddeUrd are potent and/or selective inhibitors of human immunodeficiency virus (HIV) in vitro, ddeCyd being the most potent (MIC50, 0.30 microM). The inhibitory effect of ddeCyd on ATH8 cell proliferation and HIV-induced cytopathogenicity is comparable to that of ddCyd. ddeThd is a more potent anti-HIV agent than ddThd (MIC50, 3.4 microM and 84 microM, respectively), but also more cytostatic (ID50, 172 microM and greater than 2000 microM, respectively). However, its in vitro chemotherapeutic index is higher than that of 3'-azido-2',3'-dideoxythymidine, a drug which has recently proven effective in the treatment of acquired immunodeficiency syndrome. ddeAdo has a weaker anti-HIV and a stronger cytostatic effect than ddAdo. Neither ddeUrd nor ddUrd shows significant anti-retroviral activity at 500 microM. In contrast to their anti-retroviral activity, both ddNs and ddeNs lack any appreciable inhibitory activity against a series of nononcogenic RNA and DNA viruses, pointing to their selectivity as anti-retroviral agents. All ddeNs show a progressive loss of anti-retroviral effect upon prolonged incubation with virus-infected cells. This phenomenon is most likely due to the chemical instability of these compounds, and not to a preferential enzymatic phosphorolytic cleavage of the ddeNs. Evidence is presented that ddeCyd and ddCyd, and ddeThd and ddThd are phosphorylated by cellular dCyd kinase and dThd kinase, respectively. However, the Ki values as alternate substrate inhibitors for their respective kinases are high (greater than 500 microM), indicating poor substrate activity and, thus, poor anabolism in ATH8 cells.

On the mechanism of deoxyribonucleoside toxicity in human T-lymphoblastoid cells. Reversal of growth inhibition by addition of cytidine.

High levels of deoxyadenosine and deoxyguanosine in patients with inherited deficiency of either adenosine deaminase or purine-nucleoside phosphorylase, respectively, are considered to be responsible for the associated immunological disorder. The mechanism involves phosphorylation to the corresponding deoxyribonucleoside triphosphates which subsequently inhibit the CDP-reducing activity of ribonucleotide reductase. Addition of deoxycytidine protects cells from the cytotoxic effects of deoxyadenosine and deoxyguanosine by competition for phosphorylation and by replenishing dCTP, the apparent limiting DNA precursor. Addition of cytidine, but not uridine, led to a reversal of deoxyguanosine and thymidine growth inhibition, comparable to that obtained with deoxycytidine. Analysis of the intracellular nucleotide pools showed that increased levels of cytidine ribonucleotides were sufficient to overcome the inhibitory effects of dGTP and dTTP on CDP reduction, thereby circumventing a depletion of the dCTP pool. A partial reversal of deoxyadenosine toxicity was also obtained with addition of cytidine. In this case little change in the dCTP level was observed, but a decreased dGTP pool appeared to be correlated with growth inhibition. High cytidine ribonucleotide levels partially prevented this effect. The present results may encourage the use of cytidine in combination with deoxycytidine as a pharmacological regime in treatment of immunodeficiency disease associated with increased deoxyribonucleotide levels.

Mutator phenotype in a mutant of S49 mouse T-lymphoma cells with abnormal sensitivity to thymidine.

We have selected and characterized a thymidine-sensitive S49 mutant line, MC-3-3. MC-3-3 cells are 35-fold more sensitive to the cytotoxic effects of thymidine and 15-fold more sensitive to the cytotoxic effects of 5-bromodeoxyuridine than wild type S49 cells. In contrast, the MC-3-3 mutant line does not exhibit increased sensitivity to the cytotoxic action of 5-fluorodeoxyuridine. The MC-3-3 mutant line possesses levels of thymidylate synthetase and thymidine kinase activity which are equivalent to the levels in wild type S49 cells, but the ribonucleotide reductase activity in MC-3-3 cells, using CDP as a substrate, is only 10-30% of that in wild type cells. Using ADP as a substrate, the ribonucleotide reductase activity in permeabilized MC-3-3 cells is slightly higher than that in wild type S49 cells. The deoxyribonucleotide pools in exponentially growing MC-3-3 cells are approximately 40-50% of those in wild type S49 cells. By hybrid analysis, we determined that the thymidine sensitivity of the MC-3-3 cells is recessive. The MC-3-3 mutant line displays a rate of spontaneous mutation which is 15-30-fold higher than that of wild type S49 cells. The MC-3-3 mutant cells are also 5-10-fold more sensitive than wild type cells to the cytotoxic effects of tunicamycin and compactin. These results suggest that the MC-3-3 mutant line possesses a mutation in the dTTP binding site in ribonucleotide reductase; abnormal regulation of this enzyme results in an increase in the rate of spontaneous mutation.

Biochemical basis for the enhanced toxicity of deoxyribonucleosides toward malignant human T cell lines.

Human malignant T cell lines have high levels of deoxyribonucleoside phosphorylating activity and low levels of deoxyribonucleotide dephosphorylating activity. When incubated with deoxyadenosine or thymidine, the malignant T cell lines rapidly accumulate toxic concentrations of dATP and dTTP, respectively. This unusual pattern of deoxyribonucleotide metabolism renders the malignant T cells especially vulnerable to the toxic effects of deoxyribonucleosides and related analogues.

Purinogenic immunodeficiency diseases: selective toxicity of deoxyribonucleosides for T cells.

Deoxyadenosine at low concentrations and in the presence of an inhibitor of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) is markedly toxic to lymphoblast cell lines of T cell origin but does not impair growth of B cell lines. Deoxyguanosine is also more toxic for T lymphoblasts. In the presence of deoxyadenosine or deoxyguanosine, elevation of the corresponding deoxyribonucleoside triphosphate (dATP or dGTP) occurs in T cell, but not in B cell, lines. The addition of deoxycytidine or dipyridamole results in lower dATP and dGTP levels and prevents deoxyribonucleoside toxicity. These findings provide a molecular basis for the immunodeficiency observed in individuals with several inborn errors of purine metabolism.

Deoxyribonucleoside toxicity in adenosine deaminase and purine nucleoside phosphorylase deficiency: implications for the development of new immunosuppressive agents.

The immunodeficient state associated with adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency may result from the selective phosphorylation by thymus-derived lymphocytes of the ADA substrate deoxyadenosine and the PNP substrate deoxyguanosine, leading to the intracellular trapping of toxic deoxyribonucleoside triphosphates. Agents such as deoxycytidine might be able to favourably modify the immunodeficient state by inhibiting deoxyribonucleoside phosphorylation. Deficiencies of other nucleotide catabolic enzymes, if selectively expressed by lymphocytes, might also lead to immunodeficiency via nucleoside trapping in lymphoid tissues. Purine deoxyribonucleoside analogues, either alone or in combination with ADA inhibitors, may have value as lymphospecific antimetabolites.

Lymphospecific toxicity in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency: possible role of nucleoside kinase(s).

Inherited deficiencies of the enzymes adenosine deaminase (adenosine aminohydrolase; EC 3.5.4.4) and purine nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase; EC 2.4.2.1) preferentially interfere with lymphocyte development while sparing most other organ systems. Previous experiments have shown that through the action of specific kinases, nucleosides can be "trapped" intracellularly in the form of 5'-phosphates. We therefore measured the ability of newborn human tissues to phosphorylate adenosine and deoxyadenosine, the substrate of adenosine deaminase, and also inosine, deoxyinosine, guanosine, and deoxyguanosine, the substrates of purine nucleoside phosphorylase. Substantial activities of adenosine kinase were found in all tissues studied, while guanosine and inosine kinases were detected in none. However, the ability to phosphorylate deoxyadenosine, deoxyinosine, and deoxyguanosine was largely confined to lymphocytes. Adenosine deaminase, but not purine nucleoside phosphorylase, showed a similar lymphoid predominance. Other experiments showed that deoxyadenosine, deoxyinosine, and deoxyguanosine were toxic to human lymphoid cells. The toxicity of deoxyadenosine was reversed by the addition of deoxycytidine, but not uridine, to the culture medium. Based upon these and other experiments, we propose that in adenosine deaminase and purine nucleoside phosphorylase deficiency, toxic deoxyribonucleosides produced by many tissues are selectively trapped in lymphocytes by phosphorylating enzyme(s).