deoxyguanosine-triphosphate and 9-arabinofuranosylguanine

Forodesine and nelarabine (the pro-drug of ara-G) are 2 nucleoside analogues with promising anti-leukemic activity. To better understand which pediatric patients might benefit from forodesine or nelarabine (ara-G) therapy, we investigated the in vitro sensitivity to these drugs in 96 diagnostic pediatric leukemia patient samples and the mRNA expression levels of different enzymes involved in nucleoside metabolism. Forodesine and ara-G cytotoxicities were higher in T-cell acute lymphoblastic leukemia (T-ALL) samples than in B-cell precursor (BCP)-ALL and acute myeloid leukemia (AML) samples. Resistance to forodesine did not preclude ara-G sensitivity and vice versa, indicating that both drugs rely on different resistance mechanisms. Differences in sensitivity could be partly explained by significantly higher accumulation of intracellular dGTP in forodesine-sensitive samples compared with resistant samples, and higher mRNA levels of dGK but not dCK. The mRNA levels of the transporters ENT1 and ENT2 were higher in ara-G-sensitive than -resistant samples. We conclude that especially T-ALL, but also BCP-ALL, pediatric patients may benefit from forodesine or nelarabine (ara-G) treatment.

Topics: Antineoplastic Agents; Arabinonucleosides; Cell Line, Tumor; Child; Deoxycytidine Kinase; Deoxyguanine Nucleotides; Drug Resistance, Neoplasm; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Gene Expression; Humans; In Vitro Techniques; Leukemia, Myeloid, Acute; Leukemia, Prolymphocytic, B-Cell; Phosphotransferases (Alcohol Group Acceptor); Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Prodrugs; Purine Nucleosides; Purines; Pyrimidinones; RNA, Messenger; RNA, Neoplasm

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.

Topics: Animals; Apoptosis; Arabinonucleosides; Arabinonucleotides; Biocatalysis; Caspases; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; CHO Cells; Cricetinae; Cricetulus; Cytosol; Deoxycytidine Kinase; Deoxyguanine Nucleotides; Deoxyguanosine; Deoxyribonucleotides; DNA; DNA Replication; Fibroblasts; G1 Phase; Guanosine Triphosphate; Humans; Hypoxanthine Phosphoribosyltransferase; Kinetics; Mitochondria; Phosphotransferases (Alcohol Group Acceptor); Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Purine-Nucleoside Phosphorylase; S Phase

Deoxyguanosine is selectively cytotoxic to leukemic cells from patients with T-acute lymphoblastic leukemia (T-ALL), whereas all other leukemic cell types were significantly less sensitive. Arabinosylguanine, a deoxyguanosine analog resistant to cleavage by purine nucleoside phosphorylase, is a more potent inhibitor of DNA synthesis in T-leukemic cells than deoxyguanosine and retains a selective cytotoxic activity for T-leukemic cells. Deoxyguanosine and arabinosylguanine are phosphorylated to deoxyGTP and arabinosylGTP, respectively, by T cells but not by other cell types. The phosphorylation and the cytotoxicity of arabinosylguanine are prevented by deoxycytidine. The selectivity of arabinosylguanine for malignant T cells, the exquisite sensitivity of these cells to the drug, and the failure of PNP to cleave the nucleoside indicate its potential in the treatment of T-ALL.

Topics: Arabinonucleosides; Cells, Cultured; Deoxyguanine Nucleotides; Deoxyguanosine; Guanine; Humans; Leukemia, Lymphoid; T-Lymphocytes