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J. Biol. Chem., Vol. 281, Issue 32, 22720-22728, August 11, 2006

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Mitochondrial DNA Depletion and Thymidine Phosphate Pool Dynamics in a Cellular Model of Mitochondrial Neurogastrointestinal Encephalomyopathy^*

Giovanna Pontarin, Paola Ferraro, Maria L. Valentino, Michio Hirano, Peter Reichard, and Vera Bianchi¹

From the Department of Biology, University of Padova, I-35131 Padova, Italy and the Department of Neurology, Columbia University Medical Center, New York, New York 10032

Mitochondrial (mt) neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease associated with depletion, deletions, and point mutations of mtDNA. Patients lack a functional thymidine phosphorylase and their plasma contains high concentrations of thymidine and deoxyuridine; elevation of the corresponding triphosphates probably impairs normal mtDNA replication and repair. To study metabolic events leading to MNGIE we used as model systems skin and lung fibroblasts cultured in the presence of thymidine and/or deoxyuridine at concentrations close to those in the plasma of the patients, a more than 100-fold excess relative to controls. The two deoxynucleosides increased the mt and cytosolic dTTP pools of skin fibroblasts almost 2-fold in cycling cells and 8-fold in quiescent cells. During up to a two-month incubation of quiescent fibroblasts with thymidine (but not with deoxyuridine), mtDNA decreased to 50% without showing deletions or point mutations. When we removed thymidine, but maintained the quiescent state, mtDNA recovered rapidly. With thymidine in the medium, the dTTP pool of quiescent cells turned over rapidly at a rate depending on the concentration of thymidine, due to increased degradation and resynthesis of dTMP in a substrate (=futile) cycle between thymidine kinase and 5'-deoxyribonucleotidase. The cycle limited the expansion of the dTTP pool at the expense of ATP hydrolysis. We propose that the substrate cycle represents a regulatory mechanism to protect cells from harmful increases of dTTP. Thus MNGIE patients may increase their consumption of ATP to counteract an unlimited expansion of the dTTP pool caused by circulating thymidine.

Received for publication, May 10, 2006 , and in revised form, June 13, 2006.

^* This work was supported by grants from AIRC, Italian Association for Cancer Research, Italian Telethon Grant GGP05001, and the Italian Ministry of Education and Research (Prin Project 2005) (to V. B.), an Italian Telethon Fellowship grant (to M. L. V.), and National Institutes of Health Grant P01NS11766, the Muscular Dystrophy Association, and the Marriott Mitochondrial Disorder Clinical Research Fund (MMDCRF) (to M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biology, University of Padova, I-35131 Padova, Italy. Tel.: 39-049-8276282; Fax: 39-049-8276280; E-mail: vbianchi{at}bio.unipd.it.

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