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.
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.
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 wildtype enzyme supporting their importance for substrate binding and/or catalysis as proposed by the recently solved structure of Dm-dNK.Keywords: gene therapy; nucleoside analog; suicide gene.The deoxyribonucleoside kinase of the fruit fly Drosophila melanogaster (Dm-dNK) is a multisubstrate enzyme that phosphorylates pyrimidine and purine deoxyribonucleosides as well as several anticancer and antiviral nucleoside analogues [1][2][3]. Dm-dNK is sequence-related to the human deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and thymidine kinase (TK2), as well as to the herpes simplex virus type-1 thymidine kinase (HSV-1 TK) [2]. Although the human and viral deoxyribonucleoside kinases can phosphorylate multiple deoxyribonucleosides, Dm-dNK is the only enzyme in the nucleoside kinase enzyme family that has the ability to phosphorylate all naturally occurring deoxyribonucleosides required for DNA replication. In addition to its broad substrate specificity, Dm-dNK also exhibits higher catalytic rates for nucleoside and nucleoside analogue phosphorylation compared with other nucleoside kinases.Nucleoside kinases are presently being investigated for possible use as suicide genes in combined gene/chemotherapy of cancer [4]. The most commonly studied nucleoside kinase suicide gene is the HSV-1 TK gene used in combination with the guanosine nucleoside analogue ganciclovir (GCV) [4][5][6]. The suicide nucleoside kinase is rate-limiting in the pharmacological activation of the cytotoxic nucleoside analogues, and mutants of HSV-1 TK with improved biochemical properties for nucleoside analogue phosphorylation are more efficient suicide genes [7][8][9]. The broad substrate specificity of Dm-dNK and its high catalytic rate makes it an interesting candidate gene for suicide gene therapy. We have recently evaluated the possible use of Dm-dNK as a suicide gene and shown that over-expression of DmdNK enhances the sensitivity of cancer cells to several cytotoxic nucleoside analogues [10].Alt...
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