Single Stranded Polydeoxyribonucleotides Compete with the Incorporation of [3H] Thymidine or [3H]dAMP into DNA.

Abstract

Defibrotide, the sodium salt of a single-stranded polydeoxyribonucleotide is prepared by a controlled depolymerisation of deoxyribonucleic acid (DNA) which is obtained from mammalian organs. In previous studies it was shown to mediate immunosuppressive effects (Ferraresso et al. 1993)1.In the current survey we investigated whether randomly chemically synthesized single-stranded polydeoxyribonucleotides of different length and composition could provide similar effects. For this purpose, purified T-cells were stimulated in the presence of dNTP's or single-stranded polydeoxyribonucleotides with irradiated, allogeneic PBMC's, PHA or anti-CD3/CD28 Dynabeads. Cellular proliferation was assessed by incorporation of tritium-labelled thymidine ([3H]thymidine), respectively [3H]dAMP or by staining with CFSE (carboxyfluorescein succinimidyl ester). After 72h or 120h of incubation, the incorporation of [3H]thymidine, or [3H]dAMP as well as the CFSE distribution was assessed. Cell viability was measured by trypan blue exclusion. T-cell activation was measured after 72h by quantifying the number of CD3+ T-cells expressing the activation markers CD25 and CD69. Cellular uptake of fluorochrome-labelled oligos was detected by fluorescence microscopy using an AxioCam HR and visualized by AxioVison. Each experiment was performed at least three times. Polydeoxyribonucleotides of different length, composition or concentrations (up to 5mM) did not cause cytotoxic effects to lymphocytes. But the incorporation of [3H]thymidine or [3H]dAMP was competed by polydeoxyribonucleotides. These effects were found to be dependent on length, concentration and base-composition of the nucleic acids. The proliferative capacity of the T-cells, as assessed by CFSE-staining, seemed to be unaffected. In context with data obtained by fluorescence microscopy, we hypothesise that there might be a cellular uptake of the polydeoxyribonucleotides followed by a subsequent cytoplasmatic degradation and a intracellular reutilisation via the salvage pathway for synthesis of nucleotides. The standard approach to detect cellular proliferation by incorporation of tritium-labelled nucleotides or derivatives is not useful to assess changes in cellular metabolism or proliferation in context with polydeoxyribonucleotides. Moreover, and that might be even more important, treatment approaches using nucleoside analogues like fludarabine, cytarabine e.g. in context with polydeoxyribonucleotides might be critical and diminish the efficacy of these drugs. Further experiments are necessary to elucidate the precise mechanism of these effects.