Activation of guanine-β-D-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Deoxyguanosine (GdR) and its analog guanine-ß-d-arabinofuranoside (araG) demonstrate specific toxicity towards T lymphocytes.
• This toxicity is linked to purine nucleoside phosphorylase (PNP) activity and requires intracellular phosphorylation to triphosphate forms, implicating DNA replication.
• Understanding the mechanisms of these nucleoside analogs is crucial for developing targeted therapies.

Purpose of the Study:

• To elucidate the mechanism of toxicity of guanine-ß-d-arabinofuranoside (araG) and deoxyguanosine (GdR) in T lymphocytes.
• To investigate the role of intracellular phosphorylation and subcellular localization in nucleoside analog-induced cytotoxicity.
• To determine the impact of these nucleosides on DNA synthesis, cell cycle progression, and apoptosis.

Main Methods:

• Isotope-labeled araG and flow cytometry were used to track araG phosphate accumulation and turnover in cultured cells.
• Kinetic analysis of cytosolic and mitochondrial kinases and deoxynucleotidases was performed.
• Effects on dNTP pools, ribonucleotide reductase activity, DNA synthesis, cell cycle, and apoptosis were assessed.

Main Results:

• araG phosphates rapidly accumulate and turn over, with distribution between cytosol and mitochondria influenced by kinase kinetics, leading to higher mitochondrial araGTP.
• Deoxyguanosine triphosphate (dGTP) imbalances disrupt ribonucleotide reduction, inhibit DNA synthesis, and cause G1-S cell cycle arrest.
• araGTP does not affect ribonucleotide reduction, but araG incorporation into DNA halts DNA synthesis, arresting cells in S-phase and inducing apoptosis via caspases.

Conclusions:

• Both GdR and araG induce T lymphocyte apoptosis through mechanisms involving DNA replication interference and cell cycle arrest.
• Subcellular localization of phosphorylation significantly impacts araGTP levels and subsequent cytotoxicity.
• While high araG concentrations primarily affect nuclear DNA synthesis, potential effects on mitochondrial DNA at lower concentrations warrant further investigation.