Activation of guanine-β-D-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints.
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.
Leanza
Luigi
L
Department of Biology, University of Padova, Padova, Italy.
Miazzi
Cristina
C
Ferraro
Paola
P
Reichard
Peter
P
Bianchi
Vera
V
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001087:Arabinonucleosides / Q000378:metabolism* / Q000494:pharmacology; D001088:Arabinonucleotides / Q000096:biosynthesis / Q000378:metabolism*; D055162:Biocatalysis; D016466:CHO Cells; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D003842:Deoxycytidine Kinase / Q000235:genetics / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000096:biosynthesis / Q000378:metabolism*; D003849:Deoxyguanosine / Q000378:metabolism* / Q000494:pharmacology; D003854:Deoxyribonucleotides / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology; D016193:G1 Phase / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D054218:Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology*; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism; D016196:S Phase / Q000187:drug effects
D001087:Arabinonucleosides; D001088:Arabinonucleotides; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; C028771:9-arabinofuranosylguanine; C022206:9-beta-D-arabinofuranosylguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; C029603:deoxyguanosine triphosphate; D004247:DNA; D011683:Purine-Nucleoside Phosphorylase; D007041:Hypoxanthine Phosphoribosyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C019165:deoxyguanosine kinase; D003842:Deoxycytidine Kinase; D020169:Caspases; D003849:Deoxyguanosine