Abstract

Mitochondrial (mt) DNA depletion syndromes can arise from genetic deficiencies for enzymes of dNTP metabolism, operating either inside or outside mitochondria. MNGIE is caused by the deficiency of cytosolic thymidine phosphorylase that degrades thymidine and deoxyuridine. The extracellular fluid of the patients contains 10-20 microM deoxynucleosides leading to changes in dTTP that may disturb mtDNA replication. In earlier work, we suggested that mt dTTP originates from two distinct pathways: (i) the reduction of ribonucleotides in the cytosol (in cycling cells) and (ii) intra-mt salvage of thymidine (in quiescent cells). In MNGIE and most other mtDNA depletion syndromes, quiescent cells are affected. Here, we demonstrate in quiescent fibroblasts (i) the existence of small mt dNTP pools, each usually 3-4% of the corresponding cytosolic pool; (ii) the rapid metabolic equilibrium between mt and cytosolic pools; and (iii) the intra-mt synthesis and rapid turnover of dTTP in the absence of DNA replication. Between 0.1 and 10 microM extracellular thymidine, intracellular thymidine rapidly approaches the extracellular concentration. We mimic the conditions of MNGIE by maintaining quiescent fibroblasts in 10-40 microM thymidine and/or deoxyuridine. Despite a large increase in intracellular thymidine concentration, cytosolic and mt dTTP increase at most 4-fold, maintaining their concentration for 41 days. Other dNTPs are marginally affected. Deoxyuridine does not increase the normal dNTP pools but gives rise to a small dUTP and a large dUMP pool, both turning over rapidly. We discuss these results in relation to MNGIE.

Highlights

  • Mitochondrial DNA depletion syndromes can arise from genetic deficiencies for enzymes of dNTP metabolism, operating either inside or outside mitochondria

  • DNTP Pools in Cycling and Quiescent Fibroblast Cultures—In earlier experiments, we investigated the sources of mt dNTP pools in cultures from established tumor cell lines where between 30 and 40% cells were in S-phase [5, 6]

  • Genetic mtDNA depletion syndromes challenge our understanding of the metabolism of mt dNTPs

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Summary

Mitochondrial Deoxynucleotide Pools in Quiescent Fibroblasts

Body fluids of the afflicted individuals contain large amounts (10 –20 ␮M) of thymidine and deoxyuridine [16, 17], probably resulting in an increase of the intracellular dTTP pool and possibly derangements of other dNTP pools It is not clear why derangements in the cytosol would give rise to a mt disease. The present work has two major aims: (i) to study the relation between mt and cytosolic dNTP pools in contact-inhibited fibroblasts as representatives of quiescent cells (earlier results [5, 6] were from tumor cells in exponential growth), and (ii) to provide a model for MNGIE by investigating changes induced in quiescent fibroblasts by thymidine and/or deoxyuridine.

EXPERIMENTAL PROCEDURES
RESULTS
Specific activity of thymidine kinase
Lung fibroblasts
DISCUSSION
Full Text
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