Transient expression of Drosophila melanogaster deoxynucleoside kinase gene enhances cytotoxicity of nucleoside analogs.

The Drosophila melanogaster deoxynucleoside kinase gene was introduced into HeLa cells with cationic lipids to allow its transient expression, and cytotoxic effects of several nucleoside analogs in the transfected cells were examined. Of the analogs tested, cytotoxicities of 1-beta-D-arabinofuranosylcytosine (araC), 5-fluorodeoxyuridine (FUdR), and 1-(2-deoxy-2-methylene-beta-D-erythro-pentofuranosyl)cytosine (DMDC) were increased by the deoxynucleoside kinase gene. These results suggest that the combination of the transient expression of the Drosophila deoxynucleoside kinase gene and these nucleoside analogs is a candidate for the suicide gene therapy.

Inhibition of murine retrovirus-induced immunodeficiency disease by dideoxycytidine and dideoxycytidine 5'-triphosphate.

The antiretroviral activity of many nucleoside analogues depends not only on their ability to inhibit the virus reverse transcriptase but also on the specific cellular pools of natural deoxynucleosides and on the level of the enzymes responsible for their phosphorylation. In an attempt to overcome these limitations, we have tested the efficacy of the oral administration of 2',3'-dideoxycytidine (DDC) and the administration of its phosphorylated derivative, 2',3'-dideoxycytidine 5'-triphosphate (DDCTP) encapsulated into autologous red blood cells in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS). The results obtained showed that both single treatments are quite effective in preventing the typical signs of MAIDS. Combined treatment with both oral DDC and encapsulated DDCTP yields an additive response in some, but not all the parameters investigated. Furthermore, animals receiving the simultaneous administration of DDC and DDCTP show a reduction of animal body weight, a persistent high concentration of IgM, and a high titer of anti-LP-BM5 gag immunoglobulins. Thus, the administration of the same drug in different molecular forms and/or with different delivery systems should be carefully evaluated in preclinical animal models because of the unpredictability of the effects of these treatments from the conclusion drawn by studies on single treatment.

Effects of dNTPs on the sister-chromatid exchange (SCE) frequency in human lymphocytes.

The effects of deoxynucloside triphosphates (dNTPs) on the frequency of sister-chromatid exchange (SCE) was studied in human peripheral lymphocytes. Treatment with each dCTP and dTTP did not change the SCE frequency; however, dGTP caused a significant dose-dependent increase in SCE frequency, whereas dATP caused a significantly decreased in SCE frequency. The SCE-increasing effect of the treatment with dGTP could be totally reversed by treatment with equal concentrations of dATP or dCTP. Treatment with a mixture of four equal concentrations of dATP, dGTP, dCTP, dTTP did not alter the SCE frequency. These results suggest that the dNTP pool imbalance was a SCE-effecting factor in human lymphocytes, and that dGTP may be mainly responsible for this effect.

[The mutagenic activity of N4-(4-aminobutoxy)-2'-deoxycytidine-5'-triphosphate]. / O metagennoi aktivnosti N4-(4-aminobutoksi)-2'-dezoksitsitidin-5'- trifosfata.

We have shown that deoxycytidine-5'-triphosphate modified by O-(4-aminobutyl)hydroxylamine in the pyrimidine ring, is effectively incorporated into DNA synthesizing in vitro, replacing deoxythymidine-5'-triphosphate or deoxycytidine-5'-triphosphate and inducing A-->G and G-->A transitions, respectively. UV spectroscopy and NMR spectroscopy have shown that the modified cytidine-5'-triphosphate is identical to N4-(4-aminobutoxy)-2'-deoxycytidine-5'-triphosphate. When the modified deoxycytidine-5'-triphosphate was inserted into DNA in vitro by DNA polymerase I of E. coli Klenow fragment, retardation sites correlating with poly-A sites (when the modified triphosphate replaced deoxythymidine-5'-triphosphate) or with poly-G sites (when it replaced deoxycytidine-5'-triphosphate) were revealed. Our data show high mutagenic effect of the modified deoxycytidine-5'-triphosphate inserted into DNA, allowing us to recommend this compound for localized static mutagenesis.

Induction of mutation in vitro in phage phi X174 am3 by N4-aminodeoxycytidine triphosphate.

When phi X174 am3-phage-infected E. coli is treated with N4-aminocytidine, reversion of the phage to the wild type is efficiently induced. The mechanism of this reversion is considered to consist of metabolic conversion of N4-aminocytidine into its deoxynucleoside 5'-triphosphate followed by incorporation of the nucleotide into the replicating phage DNA, thereby causing AT-to-GC transition at the am3 locus. The second half of this mechanism has now been experimentally proved, using an in vitro mutagenesis system. Thus, by nick-translation, N4-aminodeoxycytidine 5'-triphosphate was incorporated into the replicative form of phi X174 am3 DNA, and the DNA was used to transfect CA++-treated E. coli HF4714 (sup+). The reversion frequency of the phage produced was up to one-order of magnitude greater than that of the control in which the nick-translation had been done without the addition of N4-aminodeoxycytidine triphosphate. This nucleotide analog may be useful as a reagent for in vitro site-directed mutagenesis.

Relationship between antiviral activity and host toxicity: comparison of the incorporation efficiencies of 2',3'-dideoxy-5-fluoro-3'-thiacytidine-triphosphate analogs by human immunodeficiency virus type 1 reverse transcriptase and human mitochondrial DNA polymerase.

Emtricitabine [(-)FTC; (-)-beta-L-2'-3'-dideoxy-5-fluoro-3'-thiacytidine] is an oxathiolane nucleoside analog recently approved by the Food and Drug Administration for the treatment of human immunodeficiency virus (HIV). Structurally, (-)FTC closely resembles lamivudine [(-)3TC] except that the former is 5-fluorinated on the cytosine ring. In HIV-1 reverse transcriptase (RT) enzymatic assays, the triphosphate of (-)FTC [(-)FTC-TP] was incorporated into both DNA-DNA and DNA-RNA primer-templates nearly 3- and 10-fold more efficiently than (-)3TC-TP. Animal studies and clinical trial studies have demonstrated a favorable safety profile for (-)FTC. However, a detailed study of the incorporation of (-)FTC-TP by human mitochondrial DNA polymerase gamma, a host enzyme associated with nucleoside toxicity, is required for complete understanding of the molecular mechanisms of inhibition and toxicity. We studied the incorporation of (-)FTC-TP and its enantiomer (+)FTC-TP into a DNA-DNA primer-template by recombinant human mitochondrial DNA polymerase in a pre-steady-state kinetic analysis. (-)FTC-TP was incorporated 2.9 x 10(5)-, 1.1 x 10(5)-, 1.6 x 10(3)-, 7.9 x 10(3)-, and 100-fold less efficiently than dCTP, ddCTP, (+)3TC-TP, (+)FTC-TP, and (-)3TC-TP, respectively. The rate of removal of (-)FTC-MP from the corresponding chain-terminated 24-mer DNA by polymerase gamma's 3'-->5' exonuclease activity was equal to the removal of (+)FTC-MP, 2-fold slower than the removal of (-)3TC-MP and (+)3TC-MP, and 4.6-fold slower than the excision of dCMP. These results demonstrate that there are clear differences between HIV-1 RT and polymerase gamma in terms of preferences for substrate structure.