Effects of 8-aminoguanosine on the toxicity of guanosine and deoxyguanosine for malignant and normal lymphoid cells (original) (raw)
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The Journal of Immunology
The effect of deoxyguanosine on mitogen- and antigen-induced proliferation of peripheral blood lymphocytes from healthy donors was studied. Deoxyguanosine was found to inhibit the proliferative response to mitogens and antigens. Concentrations of deoxyguanosine causing 50% inhibition of the proliferation proved to be dependent on the activity of catabolic enzymes, such as purine nucleoside phosphorylase (PNP), in sera used in the culture media. The inhibitory effect of deoxyguanosine on phytohemagglutinin (PHA)-induced cell proliferation was prevented by deoxycytidine as well as by hypoxanthine. These findings were analyzed further by determination of intracellular (deoxy)-nucleotide levels. Stimulation of lymphocytes by PHA in the presence of deoxyguanosine leads to intracellular accumulation of dGTP. The presence of hypoxanthine in addition to deoxyguanosine abolished the inhibitory effect but did not prevent dGTP accumulation. On the other hand, the addition of deoxycytidine in c...
The expression of deoxyguanosine toxicity in T lymphocytes at different stages of maturation
The Journal of Immunology
Different human T cell populations were assayed for susceptibility of DNA synthesis to inhibition by deoxyguanosine. T lymphocytes from the thymus were most sensitive to inhibition of proliferation by deoxyguanosine (90% inhibition at 10 microM deoxyguanosine). This exquisite sensitivity of thymocytes appeared related to an enhanced ability of these cells for uptake and phosphorylation of deoxyguanosine to deoxyGTP and by their reduced ability to degrade accumulated deoxyGTP. Compared to more mature T lymphocytes and B cells, thymocytes contained the highest level of the salvage enzyme deoxynucleoside kinase and the lowest level of the nucleotide degrading enzyme, 5'-nucleotidase. The present study suggests that the levels of these 2 enzymes can serve as differentiation markers, identifying T cells at various stages of maturation, and that the loss of sensitivity to deoxyguanosine toxicity may be a stepwise process. Further, a deficiency in purine nucleoside phosphorylase may pr...
Different pathways for deoxyguanosine toxicity in T-lymphocytes of various developmental stages
The basis of the selective cellular immunodeficiency which occurs in patients with purine nucleoside phosphorylase (PNP) deficiency still is not completely understood. We studied the mechanism of deoxyguanosine (dGuo) toxicity in proliferating lymphoid T-cells of different maturation stage, i.e. in T-cells of adult peripheral blood and cord blood and in CD3 + and CD3 subfractions of thymocytes. The mitogeninduced proliferation of T-cells from peripheral blood and cord blood and of CD3 + thymocytes, as well as the spontaneous proliferation of CD3 thymocytes, are inhibited by dGuo. CD3 ~ and CD3 thymocytes are significantly more sensitive to dGuo than T-cells from peripheral blood or cord blood. Among the thymocyte subfractions CD3 thymocytes appeared to be extremely sensitive. In all cell types studied, inhibition of proliferation is accompanied by intracellular increases in both guanosine triphosphate (GTP) and deoxyguanosine triphosphate (dGTP) concentrations. By use of the PNP inhibitor 8-aminoguanosine, or the metabolites hypoxanthine or deoxycytidine, the metabolism of dGuo could be selectively directed to the formation of GTP or to dGTP. Based on the pattern of rescue from dGuo intoxication under these different metabolic conditions we conclude that in CD3 thymocytes dGuo toxicity is mediated by dGTP. In all other cell types studied GTP mediates dGuo intoxication. Altogether the results show that during the maturation from immature thymocytes to mature peripheral blood T-cells a shift occurs in the pattern of dGuo toxicity since dGuo toxicity in the former is primarily caused via the dCyd kinase pathway, and in the latter mainly the degradation route is involved. Since in PNP deficiency mature T-cells do occur in the peripheral blood, we must conclude that some cells escape the stage of T-cell maturation in the thymus which is extremely sensitive to dGuo. Furthermore, the results imply that as far as T-cell development in the normal thymus is concerned, survival and death of cells might be regulated by local (deoxy) nucleoside availability.
Scandinavian Journal of Immunology, 1988
Expression of Deoxyadenosine and Deoxyguanosine Toxicity al Different Stages of Lymphocyte Activation. Scand. J. Immunol. 28, 87-93. 1988 We have previously shown that deoxyguanosine (dGuo) is toxic to normal T and B lymphocytes, an effect mediated by intracellutar accumulation of guaninc ribonuclcolides. In order lo define the cellular processes that are sensitive to guanosine Iriphosphute (CiTP) we have performed studies in which the effects of dGuo on normal T cells are compared wilh those of deoxyadensoine (dAdo) on adcnosine dcaminase (ADA)-deficii;nt T cells. Kinetic studies show that dAdo exerts ils toxic effects on processes thai precede the onsel of DNA synthesis, like intcrlcukin 2 receptor expression,whereas dGuo added as lale as 24-4H h after initiation of the eullure still inhibits mitogen-induced prolilertUion. It can thus be concluded thai dGuo toxicity as medialpd through guanine ribonuclcotides is manifested relatively late during the process of T-cell activalion. whereas dAdo acts early in T-cell activalion by a mechanism ihat cannoi be explained by inhibition of ribonucleotide reductasc.
Metabolism and Disposition of O6-Benzyl-2'-deoxyguanosine in Sprague-Dawley Rats
Chemical research …, 1994
06-Benzyl-2'-deoxyguanosine is a potential antitumor drug modulator that is intended to reduce or eliminate 06-alkylguanine-DNA alkyltransferase activity in tumors prior to treatment with genotoxic chemotherapeutic alkylating agents. The rationale for using this compound instead of the more active 06-benzylguanine and its substituted benzyl derivatives a t the benzyl ring is its greater solubility in aqueous media and potential pharmacologic advantage. Metabolism and disposition of 06-benzyl-2'-deoxyguanosine was determined in adult male Sprague-Dawley rats following an ip injection of 100 mg/kg. Under these conditions, the compound was partially metabolized to yield a glucuronic acid conjugate, which was secreted exclusively in the bile. Removal of the 2'-deoxyribose or the benzyl group to yield 06benzylguanine and 2'-deoxyguanosine, respectively, occurred to a lesser extent. Metabolism accounted for the clearance of at least 58% of the total dose and took place primarily in the liver. Direct excretion of unchanged drug, mainly in urine, accounted for the remainder of the dose. Analysis of venous blood showed the presence of 06-benzyl-2'-deoxyguanosine and 06-benzylguanine a t concentrations which are considered to be effective in depleting alkyltransferase activity. Levels of the nucleoside reached a maximum of 45 pM a t 2 h, while those of 06-benzylguanine peaked to 20 pM a t 4 h and remained a t that level for a t least 4 more hours. Transport of 06-benzyl-2'-deoxyguanosine in C6 glioma cells increased linearly with the extracellular concentration of the drug up to 600 pM. Intracellular levels of the drug reached 1.2 pmol per pM of extracellular compound per lo6 cells as soon as 30 s after exposure and remained as high for a t least 1 h. Such levels indicate that entrapment of the nucleoside inside cells by either phosphorylation or other means is probably not an important feature for this drug. The extensive glucuronidation of 06-benzyl-2'-deoxyguanosine may result in the inactivation of the drug as a n alkyltransferase inhibitor, thus protecting the intestinal epithelium from being sensitized to alkylating agents. However, hydrolysis of the conjugate by bacterial /3-glucuronidases could restore the inhibitory effect of the drug in the colon, which could have pharmacologic implications in the treatment of colon cancers.
Intranuclear Distribution of 8-hydroxy-2 9 -deoxyguanosine: An Immunocytochemical Study
2000
SUMMARY We used immunofluorescence staining (monoclonal antibody N45.1) with cyto- logical imprinting to study changes in the intranuclear distribution of 8-hydroxy-2 9 -deoxy- guanosine in renal cells of male Wistar rats after oxidative stress by ferric nitrilotriacetate. In the control proximal tubule cells, small spherical signals were uniformly distributed through- out the nuclei. Under oxidative stress, immunofluorescence intensity was increased,
Experimental Cell Research, 2010
The deoxyguanosine (GdR) analog guanine-ß-D-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.
Biological effects of incorporation of O6-methyldeoxyguanosine into Chinese hamster V79 cells
Mutation research, 1983
Analysis of the biological effects of specific DNA alkylations by simple alkylating agents is complicated by the variety of sites involved. It is, therefore, of value to be able to incorporate into cellular DNA nucleosides alkylated in a single position, e.g., O6-methyldeoxyguanosine. Such cellular incorporation is particularly difficult to achieve because this nucleoside is rapidly demethylated by adenosine deaminase. We have attempted to achieve such incorporation into the DNA of V79 cells by using coformycin, an inhibitor of adenosine deaminase, and by forcing the cells to depend on exogenous purines by the use of medium containing aminopterin. The DNA of V79 cells exposed to O6-methyl-[8-3H]deoxyguanosine (2.4 microM, sp. act. 14500 Ci/mole) showed an incorporation level of 4 X 10(-8) nucleotides. When 1000-fold higher concentrations were employed (3-15 mM, sp. act. 1.6 Ci/mole), significant cytotoxicity and inhibition of DNA synthesis was observed. However, because it was not e...