2'-deoxyguanosine toxicity for B and mature T lymphoid cell lines is mediated by guanine ribonucleotide accumulation.

Abstract

Abstract. Inherited deficiency of the enzyme purine nucleoside phosphorylase (PNP) results in selective and severe T lymphocyte depletion which is mediated by its substrate, 2'-deoxyguanosine. This observation provides a rationale for the use of PNP inhibitors as selective T cell immunosuppressive agents. We have studied the relative effects of the PNP inhibitor 8-aminoguanosine on the metabolism and growth of lymphoid cell lines of T and B cell origin. We have found that 2'-deoxyguanosine toxicity for T lymphoblasts is markedly potentiated by 8-aminoguanosine and is mediated by the accumulation of deoxyguanosine triphosphate. In contrast, the growth of T4' mature T cell lines and B lymphoblast cell lines is inhibited by somewhat higher concentrations of 2'-deoxyguanosine (ID50 20 and 18 ,uM, respectively) in the presence of 8-aminoguanosine without an increase in deoxyguanosine triphosphate levels. Cytotoxicity correlates instead with a three-to fivefold increase in guanosine triphosphate (GTP) levels after 24 h. Accumulation of GTP and growth inhibition also result from exposure to guanosine, but not to guanine at equimolar concentrations. B lymphoblasts which are deficient in the purine, salvage enzyme hypoxanthine guanine phosphoribosyltransferase are completely resistant to 2'-deoxyguanosine or guanosine concentrations up to 800 ,uM and do not demonstrate an increase in GTP levels. Growth inhibition and GTP accumulation are prevented by hypoxanthine or adenine, but not by 2'-deoxycytidine.

Deoxyadenosine triphosphate as a mediator of deoxyguanosine toxicity in cultured T lymphoblasts.

“…GTP levels doubled over 24 h (Table II) tivity of T lymphoblasts to 2'-deoxyguanosine is associated with the capacity of the T phenotype for accumulation of deoxyribonucleotides. B lymphoblasts (JP), as previously reported (29), failed to elevate their dGTP or dATP pools significantly. Effectofcoincubation with2-deoxycytidine.…”

“…Catabolism of 2'-deoxyguanosine and salvage to guanine ribonucleotides is an important cause of its toxicity to B lymphoblasts and mature T cell lines, but not for HGPRTase-deficient B cell lines or PNP-inhibited T lymphoblasts (29,39). In our experiments with T lymphoblasts GTP was not increased by 2'-deoxyguanosine concentrations that caused GI-phase arrest, and we have found that HGPRTase-deficient T lymphoblasts similarly block in GI.…”

“…After lyophilization, cell extracts were taken up in 500 Ml of 10 mM Tris buffer, pH 7.85, and spun at 40,000 g at 4°C for 10 min and the supernatants were stored at -20°C. dNTP pools were measured by a modification ofthe DNA polymerase assay (17,33,34 Ribonucleotide triphosphates (NTPs) were quantified by high-performance liquid chromatography as described (29). NTPs and dNTPs were eluted from a Partisil-1O SAX anion exchange column (Whatman Laboratory Products, Inc., Clifton, NJ) using a linear ammonium phosphate gradient (0.30-0.45 M, pH 3.3-3.6 over 35 min) at a flow rate of 2 ml/min.…”

“…At micromolar concentrations of 2'-deoxyguanosine in the presence of PNP inhibitor, dGTP pool elevation has been documented in T lymphoblasts and mitogen-stimulated lymphocytes, and has been correlated with inhibition of proliferation (28). In contrast, GTP has been shown to be the major toxic metabolite for B lymphoblasts and mature T cell lines (29).…”

The mononucleotide-dependent, nonantisense mechanism of action of phosphodiester and phosphorothioate oligonucleotides depends upon the activity of an ecto-5′-nucleotidase

“…[5][6][7][8] It was postulated that this effect is caused by intracellular enzymatic phosphorylation of deoxyguanosine to its monophosphate, diphosphate, and, finally, triphosphate (dGTP), which acts as an allosteric inhibitor of ribonucleoside reductase. [16][17][18] Therefore, the cytotoxic effects of mononucleotides on different cell lines may occur not only as the result of their dephosphorylation, but also as a consequence of their effective transport across cell membranes. Some nucleoside carriers responsible for cellular uptake of nucleosides can be inhibited by NBTI, dilazep, or dipyridamole.…”