Progressive loss of mitochondrial DNA in thymidine kinase 2-deficient mice.

Deficient enzymatic activity of the mitochondrial deoxyribonucleoside kinases deoxyguanosine kinase (DGUOK) or thymidine kinase 2 (TK2) cause mitochondrial DNA (mtDNA)-depletion syndromes in humans. Here we report the generation of a Tk2-deficient mouse strain and show that the mice develop essentially normally for the first week but from then on exhibit growth retardation and die within 2-4 weeks of life. Several organs including skeletal muscle, heart, liver and spleen showed progressive loss of mtDNA without increased mtDNA mutations or structural alterations. There were no major histological changes in skeletal muscle, but heart muscle showed disorganized and damaged muscle fibers. Electron microscopy showed mitochondria with distorted cristae. The Tk2-deficient mice exhibited pronounced hypothermia and showed loss of hypodermal fat and abnormal brown adipose tissue. We conclude that Tk2 has a major role in supplying deoxyribonucleotides for mtDNA replication and that other pathways of deoxyribonucleotide synthesis cannot compensate for loss of this enzyme.

Origin of the U87MG glioma cell line: Good news and bad news.

Human tumor-derived cell lines are indispensable tools for basic and translational oncology. They have an infinite life span and are easy to handle and scalable, and results can be obtained with high reproducibility. However, a tumor-derived cell line may not be authentic to the tumor of origin. Two major questions emerge: Have the identity of the donor and the actual tumor origin of the cell line been accurately determined? To what extent does the cell line reflect the phenotype of the tumor type of origin? The importance of these questions is greatest in translational research. We have examined these questions using genetic profiling and transcriptome analysis in human glioma cell lines. We find that the DNA profile of the widely used glioma cell line U87MG is different from that of the original cells and that it is likely to be a bona fide human glioblastoma cell line of unknown origin.

A novel variant in plakophilin-2 gene detected in a family with arrhythmogenic right ventricular cardiomyopathy.

AIMS: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by fibrofatty replacement of muscular fibers predominantly in the right ventricle and with ventricular arrhythmias as the main clinical manifestation. Mutations in several components of the desmosome genes have been identified and mutations of the plakophilin-2 (PKP-2) gene are a common cause of ARVC. The aim of this study is to investigate the correlation between genotype and phenotype in a family with a novel PKP-2 variant. METHODS AND RESULTS: This study describes the clinical findings and genetic analysis in a family with ARVC. A part of the family has been followed clinically long term for up to 27 years. Two not previously reported PKP-2 variants (L506P and T526A) have been identified in this family. Even though all members of this family share the novel variant L506P, the clinical features, i.e., their phenotypes are different. The L506P variant is located in exon 7 and affects a highly conserved residue. The same amino acid, leucine, is present in all species evaluated, indicating a functional importance and the variant is predicted to be damaging. The novel L506P variant in the PKP-2 gene is thus a possible pathogenic alteration in the described family with ARVC. In contrast, the T526A variant is weakly conserved and predicted to be tolerated. CONCLUSION: While many of the reported ARVC mutations are truncating mutations, the possibly damaging variant found in this family, is a missense alteration affecting a highly conserved residue 506 located in exon 7.

Retained sensitivity to cytotoxic pyrimidine nucleoside analogs in thymidine kinase 2 deficient human fibroblasts.

Thymidine kinase 2 (TK2) is a mitochondrial deoxyribonucleoside kinase that phosphorylates several nucleoside analogs used in anti-viral and anti-cancer therapy. A fibroblast cell line with decreased TK2 activity was investigated in order to obtain insights in the effects of TK2 deficiency on nucleotide metabolism. The role of TK2 for the sensitivity against cytotoxic nucleoside analogs was also investigated. The TK2 deficient cells retained their sensitivity against all pyrimidine nucleoside analogs tested. This study suggests that nucleoside analog phosphorylation mediated by TK2 may be less important, compared to other deoxyribonucleoside kinases, for the cytotoxic effects of these compounds.

Increased mitochondrial DNA copy-number in CEM cells resistant to delayed toxicity of 2',3'-dideoxycytidine.

The nucleoside analog 2',3'-dideoxycytidine (ddC) has been used for treatment of human immunodeficiency virus (HIV) infections. ddC causes delayed toxicity when cells are exposed to the drug at low concentration for prolonged periods of time. The delayed toxicity is due to inhibition of mitochondrial DNA (mtDNA) replication, which results in mtDNA depletion and mitochondrial dysfunction. In the present study we have cultured CEM T-lymphoblast cells in the presence of low concentrations of ddC to generate two cell lines resistant to the delayed toxicity of the drug. Both cell lines were resistant to mtDNA depletion by ddC. The mechanism of ddC resistance was investigated and we showed that the resistant cells had decreased mRNA expression of the nucleoside kinases deoxycytidine kinase and thymidine kinase 2. We also studied the mitochondrial DNA in the cells and showed that the ddC-resistant cells had structurally intact mtDNA but 1.5-2-fold increased mtDNA copy-number as well as increased levels of the mitochondrial transcription factor A (Tfam). Our study suggests that cells may increase their level of mtDNA to counteract mtDNA depletion induced by ddC, while keeping pronounced antiviral activity of the drug.

Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is sequence-related to three human deoxyribonucleoside kinases and to herpes simplex virus type-1 thymidine kinase. Dm-dNK phosphorylates both purine and pyrimidine deoxyribonucleosides and nucleoside analogues although it has a preference for pyrimidine nucleosides. We performed site-directed mutagenesis on residues that, based on structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of purine nucleoside substrates to create an improved enzyme to be used in suicide gene therapy. A Q81N mutation showed a relative increase in deoxyguanosine phosphorylation compared with the wild-type enzyme although the efficiency of deoxythymidine phosphorylation was 10-fold lower for the mutant. In addition to residue Q81 the function of amino acids N28, I29 and F114 was investigated by different substitutions. All of the mutated enzymes showed decreased efficiency of thymidine phosphorylation in comparison with the wild-type enzyme supporting their importance for substrate binding and/or catalysis as proposed by the recently solved structure of Dm-dNK.