Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

Journal Article · Fri Apr 08 04:00:00 EDT 2011 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/J.BBRC.2011.03.018· OSTI ID:22204869

Villarroya, Joan ^[1]; Lara, Mari-Carmen ^[1]; Dorado, Beatriz ^[2]; Garrido, Marta ^[3]; Garcia-Arumi, Elena ^[1]; Meseguer, Anna ^[1]; Hirano, Michio ^[2]; Vila, Maya R. ^[1]

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1. Institut de Recerca, Hospital Universitari de la Vall d'Hebron, Barcelona (Spain)
2. Department of Neurology, Columbia University Medical Center, New York, NY (United States)
3. Unitat de Biologia Cel.lular i Molecular, IMIM-Hospital del Mar, Barcelona (Spain)

Highlights: {yields} We impaired TK2 expression in Ost TK1{sup -} cells via siRNA-mediated interference (TK2{sup -}). {yields} TK2 impairment caused severe mitochondrial DNA (mtDNA) depletion in quiescent cells. {yields} Despite mtDNA depletion, TK2{sup -} cells show high cytochrome oxidase activity. {yields} Depletion of mtDNA occurs without imbalance in the mitochondrial dNTP pool. {yields} Nuclear-encoded ENT1, DNA-pol {gamma}, TFAM and TP gene expression is lowered in TK2{sup -} cells. -- Abstract: The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed the first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1{sup -} cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase {gamma}, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity despite profound depletion in mtDNA levels.

• OSTI ID: 22204869
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 2 Vol. 407; ISSN BBRCA9; ISSN 0006-291X
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
CYTOCHROME OXIDASE
DISEASES
DNA
GENE MUTATIONS
HOMEOSTASIS
IN VITRO
MESSENGER-RNA
MITOCHONDRIA
THYMIDINE
TRANSCRIPTION FACTORS