The Experts below are selected from a list of 300 Experts worldwide ranked by ideXlab platform
Staffan Eriksson - One of the best experts on this subject based on the ideXlab platform.
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Down-regulation of mitochondrial thymidine Kinase 2 and Deoxyguanosine Kinase by didanosine: implication for mitochondrial toxicities of anti-HIV nucleoside analogs.
Biochemical and biophysical research communications, 2014Co-Authors: Ren Sun, Staffan Eriksson, Liya WangAbstract:Abstract Mitochondrial thymidine Kinase 2 (TK2) and Deoxyguanosine Kinase (dGK) catalyze the initial rate limiting phosphorylation of deoxynucleosides and are essential enzymes for mitochondrial function. Chemotherapy using nucleoside analogs is often associated with mitochondrial toxicities. Here we showed that incubation of U2OS cells with didanosine (ddI, 2′,3′-dideoxyinosine), a purine nucleoside analog used in the highly active antiretroviral therapy (HAART), led to selective degradation of both mitochondrial TK2 and dGK while the cytosolic deoxycytidine Kinase (dCK) and thymidine Kinase 1 (TK1) were not affected. Addition of guanosine to the ddI-treated cells prevented the degradation of mitochondrial TK2 and dGK. The levels of intracellular reactive oxygen species and protein oxidation in ddI-treated and control cells were also measured. The results suggest that down-regulation of mitochondrial TK2 and dGK may be a mechanism of mitochondrial toxicity caused by antiviral and anticancer nucleoside analogs.
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Mitochondrial Thymidine Kinase 2 but Not Deoxyguanosine Kinase Is Up-Regulated During the Stationary Growth Phase of Cultured Cells
Nucleosides nucleotides & nucleic acids, 2014Co-Authors: Ren Sun, Staffan Eriksson, Liya WangAbstract:Mitochondrial thymidine Kinase 2 (TK2) and Deoxyguanosine Kinase (dGK) catalyze the initial phosphorylation of pyrimidine and purine deoxyribonucleosides, and are essential for maintaining mitochondrial dNTP pools for mitochondrial DNA replication. Here the expression of mitochondrial TK2 and dGK in relation to cell growth phases in cultured cells was investigated. TK2 and dGK protein levels in isolated mitochondria and TK2 activity in total cell extracts from U2OS and TK1 deficient L929 cells were determined. We found that TK2 levels were negatively correlated with cell growth rates and there was an exponential increase in TK2 levels in cells entering stationary phase. The expression of dGK did not change and appeared to be constitutive.
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molecular mechanisms of mitochondrial dna depletion diseases caused by deficiencies in enzymes in purine and pyrimidine metabolism
Nucleosides Nucleotides & Nucleic Acids, 2008Co-Authors: Staffan Eriksson, Liya WangAbstract:Mitochondrial DNA depletion syndrome (MDS), a reduction of mitochondrial DNA copy number, often affects muscle or liver. Mutations in enzymes of deoxyribonucleotide metabolism give MDS, for example, the mitochondrial thymidine Kinase 2 (TK2) and Deoxyguanosine Kinase (dGK) genes. Sixteen TK2 and 22 dGK alterations are known. Their characteristics and symptoms are described. Levels of five key deoxynucleotide metabolizing enzymes in mouse tissues were measured. TK2 and dGK levels in muscles were 5- to 10-fold lower than other nonproliferating tissues and 100-fold lower compared to spleen. Each type of tissue apparently relies on de novo and salvage synthesis of DNA precursors to varying degrees.
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The pattern of deoxycytidine- and Deoxyguanosine Kinase activity in relation to messenger RNA expression in blood cells from untreated patients with B-cell chronic lymphocytic leukemia.
Biochemical pharmacology, 2006Co-Authors: Kourosh Lotfi, Staffan Eriksson, Anna Fyrberg, Curt Peterson, Karin Karlsson, Gunnar Juliusson, Viggo Jønsson, Freidoun AlbertioniAbstract:Deoxycytidine Kinase (dCK) and Deoxyguanosine Kinase (dGK) catalyze the first step in the intracellular cascade of fludarabine (2-fluoroadenine-beta-D-arabinofuranoside) and cladribine (2-chlorodeoxyadenosine) phosphorylation, which leads to activation of these prodrugs, commonly used for treatment of chronic lymphocytic leukemia (CLL). Thus, resistance to nucleoside analogues may primarily be due to low levels of deoxynucleoside Kinase activity. The purpose of this study was to investigate the activity profiles of dCK and dGK and characterize the possible relationship between the levels of dCK enzymatic activities and mRNA levels in B-CLL cells from untreated patient samples in an attempt to determine the best approach for predicting sensitivity to nucleoside analogues and thereby optimizing treatment of CLL. For this purpose, dCK and dGK analyses were done in blood cells from 59 untreated symptomatic patients with CLL. The dGK activity towards 2-chlorodeoxyadenosine was significantly lower than of dCK (median 73 pmol/mg protein/min (85-121, 95% CI) versus 353 pmol/mg protein/min (331-421)). The median dCK mRNA level was 0.107 (0.096-0.120, 95% CI). There was a lack of correlation between the activities of dCK and dGK, which indicates that these proteins are regulated independently. We also found that the dCK and dGK activity measurement towards their endogenous substrates were comparable to the nucleoside analogues tested. Such variations in enzyme activities and mRNA levels may well explain differences in clinical responses to treatment. There was no correlation between the levels of dCK mRNAs and enzymatic activities using a quantitative real-time PCR procedure. Sequencing of dCK mRNA did not reveal alternate splicing or mutations in the coding region. The relation between activity and mRNA levels was studied by short interfering RNA (siRNA) method, which showed that in the siRNA treated cells the down-regulation of dCK expression, and activity followed each other. However, in control cells the mRNA levels remained stable but the protein activity markedly decreased. These data demonstrate that the dCK activity is not reflected by dCK mRNA expression that indicates a post-translational mechanism(s).
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Molecular insight into mitochondrial DNA depletion syndrome in two patients with novel mutations in the Deoxyguanosine Kinase and thymidine Kinase 2 genes
Molecular genetics and metabolism, 2005Co-Authors: Liya Wang, Anna Limongelli, Maya R. Vilà, Franco Carrara, Massimo Zeviani, Staffan ErikssonAbstract:Thymidine Kinase 2 (TK2) and Deoxyguanosine Kinase (dGK) are the two key enzymes in mitochondrial DNA (mtDNA) precursor synthesis. Deficiencies in TK2 or dGK activity, due to genetic alteration, have been shown to cause tissue-specific depletion of mtDNA. In the case of TK2 deficiency, affected individuals suffer severe myopathy and, in the case of dGK deficiency, devastating liver or multi-systemic disease. Here, we report clinical and biochemical findings from two patients with mtDNA depletion syndrome. Patient A was a compound heterozygote carrying the previously reported T77M mutation and a novel mutation (R161K) in the TK2 gene. Patient B carried a novel mutation (L250S) in the dGK gene. The clinical symptoms of patient A included muscular weakness and exercise intolerance due to a severe mitochondrial myopathy associated with a 92% reduction in mtDNA. There was minimal involvement of other organs. Patient B suffered from rapidly progressive, early onset fatal liver failure associated with profoundly decreased mtDNA levels in liver and, to a lesser extent, in skeletal muscle. Site-directed mutagenesis was used to introduce the mutations detected in patients A and B into the TK2 and dGK cDNAs, respectively. We then characterized each of these recombinant enzymes. Catalytic activities of the three mutant enzymes were reduced to about 2-4% for TK2 and 0.5% for dGK as compared to the wild-type enzymes. Altered competition between dCyd and dThd was observed for the T77M mutant. The residual activities of the two mitochondrial enzymes correlated directly with disease development.
Anna Karlsson - One of the best experts on this subject based on the ideXlab platform.
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Acute cytotoxicity of arabinofuranosyl nucleoside analogs is not dependent on mitochondrial DNA.
Experimental cell research, 2009Co-Authors: Sophie Curbo, Magnus Johansson, Jan Balzarini, Lionel D. Lewis, Anna KarlssonAbstract:Abstract The nucleoside analogs 9-β- D -arabinofuranosylguanine (araG) and 1-β- d -arabinofuranosylthymine (araT) are substrates of mitochondrial nucleoside Kinases and have previously been shown to be predominantly incorporated into mtDNA of cells, but the pharmacological importance of their accumulation in mtDNA is not known. Here, we examined the role of mtDNA in the response to araG, araT and other anti-cancer and anti-viral agents in a MOLT-4 wild-type (wt) T-lymphoblastoid cell line and its petite mutant MOLT-4 ρ0 cells (lacking mtDNA). The mRNA levels and activities of Deoxyguanosine Kinase (dGK), deoxycytidine Kinase (dCK), thymidine Kinase 1 (TK1) and thymidine Kinase 2 (TK2) were determined in the two cell lines. Compared to that in the MOLT-4 wt cells the mRNA level of the constitutively expressed TK2 was higher (p
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Depletion of mitochondrial DNA by down-regulation of Deoxyguanosine Kinase expression in non-proliferating HeLa cells.
Experimental cell research, 2007Co-Authors: Maribel Franco, Magnus Johansson, Anna KarlssonAbstract:Purine deoxyribonucleotides required for mitochondrial DNA replication are either imported from the cytosol or derived from phosphorylation of deoxyadenosine or Deoxyguanosine catalyzed by mitochondrial Deoxyguanosine Kinase (DGUOK). DGUOK deficiency has been linked to mitochondrial DNA depletion syndromes suggesting an important role for this enzyme in dNTP supply. We have generated HeLa cell lines with 20-30% decreased levels of DGUOK mRNA by the expression of small interfering RNAs directed towards the DGUOK mRNA. The cells with decreased expression of the enzyme showed similar levels of mtDNA as control cells when grown exponentially in culture. However, mtDNA levels rapidly decreased in the cells when cell cycle arrest was induced by serum starvation. DNA incorporation of 9-beta-d-arabino-furanosylguanine (araG) was lower in the cells with decreased Deoxyguanosine Kinase expression, but the total rate of araG phosphorylation was increased in the cells. The increase in araG phosphorylation was shown to be due to increased expression of deoxycytidine Kinase. In summary, our findings show that DGUOK is required for mitochondrial DNA replication in resting cells and that small changes in expression of this enzyme may cause mitochondrial DNA depletion. Our data also suggest that alterations in the expression level of DGUOK may induce compensatory changes in the expression of other nucleoside Kinases.
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Differential incorporation of 1‐β‐D‐arabinofuranosylcytosine and 9‐β‐D‐arabinofuranosylguanine into nuclear and mitochondrial DNA
FEBS Letters, 2000Co-Authors: Chaoyong Zhu, Magnus Johansson, Anna KarlssonAbstract:The anti-leukemic nucleoside analogs 1-β-D-arabinofuranosylcytosine (araC) and 9-β-D-arabinofuranosylguanine (araG) are dependent on intracellular phosphorylation for pharmacological activity. AraC is efficiently phosphorylated by deoxycytidine Kinase (dCK). Although araG is phosphorylated by dCK in vitro, it is a preferred substrate of mitochondrial Deoxyguanosine Kinase. We have used autoradiography to show that araC was incorporated into nuclear DNA in Molt-4 and CEM T-lymphoblastoid cells as well as in Chinese hamster ovary cells. In contrast, araG was predominantly incorporated into mitochondrial DNA in the investigated cell lines, without detectable incorporation into nuclear DNA. These data suggest that the molecular targets of araG and araC may differ.
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Phosphorylation of anticancer nucleoside analogs by human mitochondrial Deoxyguanosine Kinase
Biochemical pharmacology, 1998Co-Authors: Chaoyong Zhu, Magnus Johansson, Johan Permert, Anna KarlssonAbstract:Abstract The kinetic properties of recombinant human mitochondrial Deoxyguanosine Kinase (dGK, EC 2.7.1.113) for 2′-Deoxyguanosine and the clinically important nucleoside analogs 2-chloro-2′-deoxyadenosine (CdA), 9-β -D-arabinofuranosylguanine (araG) and 2′,2′,-difluoroDeoxyguanosine (dFdG) were determined. The Michaelis–Menten kinetic parameters, comparing ATP and UTP as phosphate donors, demonstrated a marked increase in phosphorylation efficiency ( V max K m ) with UTP in comparison with ATP for both CdA and araG. The difluoro analog dFdG was an efficient substrate for recombinant dGK with an apparent K m of 16 μM with ATP as phosphate donor. We compared the kinetic properties of dGK with those of the related enzyme deoxycytidine Kinase (dCK, EC 2.7.1.74). Although the purines 2′-Deoxyguanosine (dGuo) and 2′-deoxyadenosine are substrates for both dGK and dCK, only CdA among the purine nucleoside analogs tested was an efficient substrate for both dCK and dGK. In competition with dGuo, the most efficient analog for phosphorylation by dGK was araG, as indicated by a lower K i value than for CdA and dFdG. Of the purine analogs tested as substrates for dCK, only CdA could compete with 2′-deoxycytidine (dCyd). No inhibition of dCK-mediated dCyd phosphorylation was found by either araG or dFdG. In crude cell extract of HeLa and Capan 2 cells, the major CdA phosphorylation was contributed by dCK, while most araG phosphorylation was a result of dGK activity. Our study with pure recombinant enzymes confirms that dGK is mainly responsible for araG and dFdG phosphorylation, whereas dCK is the most important enzyme for activation of CdA and 2′,2′-difluorodeoxycytidine (dFdC).
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Enhanced Cytotoxicity of Nucleoside Analogs by Overexpression of Mitochondrial Deoxyguanosine Kinase in Cancer Cell Lines
The Journal of biological chemistry, 1998Co-Authors: Chaoyong Zhu, Magnus Johansson, Johan Permert, Anna KarlssonAbstract:Abstract The cytotoxic anti-cancer purine nucleoside analogs 2-chloro-2′-deoxyadenosine (CdA), 9-β-d-arabinofuranosylguanine (araG), and 2′,2′-difluoroDeoxyguanosine (dFdG) are phosphorylated by human mitochondrial Deoxyguanosine Kinase (dGK) in vitro. We overexpressed dGK as a fusion protein to the green fluorescent protein in the human pancreatic cancer cell lines PanC-1 and MIA PaCa-2 to determine the importance of dGK-mediated nucleoside analog phosphorylation. The transfected cells showed mitochondrial fluorescence patterns, and the mitochondrial locations of endogenous and overexpressed dGK were verified by Western blot analysis of cell extracts with polyclonal anti-dGK antibodies. The increase of dGK activity in the overexpressing cells was ∼4-fold. These cell lines exhibited increased sensitivity to CdA, araG, and dFdG as compared with the untransfected parent cell lines. This is, to our knowledge, the first demonstration of a correlation between the activity of a mitochondrial deoxyribonucleoside Kinase and the cytotoxicity of nucleoside analogs. Our data imply that the dGK activity is rate-limiting for the efficacy of nucleoside analogs in the cell lines investigated.
Liya Wang - One of the best experts on this subject based on the ideXlab platform.
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MPV17 loss of function affects the purine branch of mitochondrial salvage pathway.
2016Co-Authors: Ilaria Dalla Rosa, Yolanda Cámara, Romina Durigon, Chloe F. Moss, Sara Vidoni, Gokhan Akman, Lilian Hunt, Mark A. Johnson, Sarah Grocott, Liya WangAbstract:Representative immunoblot thymidine Kinase 2 (TK2) in (A) control and MPV17-mutant fibroblasts in dividing and quiescent cells, and (B) in the liver of wild-type (WT) and knockout (KO) mice. (C) Steady state levels of adenylate Kinase 2 and 3 (AK2 and AK3) and Deoxyguanosine Kinase (Dguok) in the liver of wild-type (WT) and knockout (KO) mice. The arrow indicates the Dguok isoform downregulated in KO mouse liver. The samples were from 2 month-old mice unless indicated. (D) AK2, AK3 and DGUOK steady state levels in control and MPV17 mutant fibroblasts in proliferating and quiescent cells. Vinculin, GAPDH, and Tom20 were used as loading control.
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Down-regulation of mitochondrial thymidine Kinase 2 and Deoxyguanosine Kinase by didanosine: implication for mitochondrial toxicities of anti-HIV nucleoside analogs.
Biochemical and biophysical research communications, 2014Co-Authors: Ren Sun, Staffan Eriksson, Liya WangAbstract:Abstract Mitochondrial thymidine Kinase 2 (TK2) and Deoxyguanosine Kinase (dGK) catalyze the initial rate limiting phosphorylation of deoxynucleosides and are essential enzymes for mitochondrial function. Chemotherapy using nucleoside analogs is often associated with mitochondrial toxicities. Here we showed that incubation of U2OS cells with didanosine (ddI, 2′,3′-dideoxyinosine), a purine nucleoside analog used in the highly active antiretroviral therapy (HAART), led to selective degradation of both mitochondrial TK2 and dGK while the cytosolic deoxycytidine Kinase (dCK) and thymidine Kinase 1 (TK1) were not affected. Addition of guanosine to the ddI-treated cells prevented the degradation of mitochondrial TK2 and dGK. The levels of intracellular reactive oxygen species and protein oxidation in ddI-treated and control cells were also measured. The results suggest that down-regulation of mitochondrial TK2 and dGK may be a mechanism of mitochondrial toxicity caused by antiviral and anticancer nucleoside analogs.
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Mitochondrial Thymidine Kinase 2 but Not Deoxyguanosine Kinase Is Up-Regulated During the Stationary Growth Phase of Cultured Cells
Nucleosides nucleotides & nucleic acids, 2014Co-Authors: Ren Sun, Staffan Eriksson, Liya WangAbstract:Mitochondrial thymidine Kinase 2 (TK2) and Deoxyguanosine Kinase (dGK) catalyze the initial phosphorylation of pyrimidine and purine deoxyribonucleosides, and are essential for maintaining mitochondrial dNTP pools for mitochondrial DNA replication. Here the expression of mitochondrial TK2 and dGK in relation to cell growth phases in cultured cells was investigated. TK2 and dGK protein levels in isolated mitochondria and TK2 activity in total cell extracts from U2OS and TK1 deficient L929 cells were determined. We found that TK2 levels were negatively correlated with cell growth rates and there was an exponential increase in TK2 levels in cells entering stationary phase. The expression of dGK did not change and appeared to be constitutive.
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molecular mechanisms of mitochondrial dna depletion diseases caused by deficiencies in enzymes in purine and pyrimidine metabolism
Nucleosides Nucleotides & Nucleic Acids, 2008Co-Authors: Staffan Eriksson, Liya WangAbstract:Mitochondrial DNA depletion syndrome (MDS), a reduction of mitochondrial DNA copy number, often affects muscle or liver. Mutations in enzymes of deoxyribonucleotide metabolism give MDS, for example, the mitochondrial thymidine Kinase 2 (TK2) and Deoxyguanosine Kinase (dGK) genes. Sixteen TK2 and 22 dGK alterations are known. Their characteristics and symptoms are described. Levels of five key deoxynucleotide metabolizing enzymes in mouse tissues were measured. TK2 and dGK levels in muscles were 5- to 10-fold lower than other nonproliferating tissues and 100-fold lower compared to spleen. Each type of tissue apparently relies on de novo and salvage synthesis of DNA precursors to varying degrees.
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Molecular insight into mitochondrial DNA depletion syndrome in two patients with novel mutations in the Deoxyguanosine Kinase and thymidine Kinase 2 genes
Molecular genetics and metabolism, 2005Co-Authors: Liya Wang, Anna Limongelli, Maya R. Vilà, Franco Carrara, Massimo Zeviani, Staffan ErikssonAbstract:Thymidine Kinase 2 (TK2) and Deoxyguanosine Kinase (dGK) are the two key enzymes in mitochondrial DNA (mtDNA) precursor synthesis. Deficiencies in TK2 or dGK activity, due to genetic alteration, have been shown to cause tissue-specific depletion of mtDNA. In the case of TK2 deficiency, affected individuals suffer severe myopathy and, in the case of dGK deficiency, devastating liver or multi-systemic disease. Here, we report clinical and biochemical findings from two patients with mtDNA depletion syndrome. Patient A was a compound heterozygote carrying the previously reported T77M mutation and a novel mutation (R161K) in the TK2 gene. Patient B carried a novel mutation (L250S) in the dGK gene. The clinical symptoms of patient A included muscular weakness and exercise intolerance due to a severe mitochondrial myopathy associated with a 92% reduction in mtDNA. There was minimal involvement of other organs. Patient B suffered from rapidly progressive, early onset fatal liver failure associated with profoundly decreased mtDNA levels in liver and, to a lesser extent, in skeletal muscle. Site-directed mutagenesis was used to introduce the mutations detected in patients A and B into the TK2 and dGK cDNAs, respectively. We then characterized each of these recombinant enzymes. Catalytic activities of the three mutant enzymes were reduced to about 2-4% for TK2 and 0.5% for dGK as compared to the wild-type enzymes. Altered competition between dCyd and dThd was observed for the T77M mutant. The residual activities of the two mitochondrial enzymes correlated directly with disease development.
Magnus Johansson - One of the best experts on this subject based on the ideXlab platform.
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Acute cytotoxicity of arabinofuranosyl nucleoside analogs is not dependent on mitochondrial DNA.
Experimental cell research, 2009Co-Authors: Sophie Curbo, Magnus Johansson, Jan Balzarini, Lionel D. Lewis, Anna KarlssonAbstract:Abstract The nucleoside analogs 9-β- D -arabinofuranosylguanine (araG) and 1-β- d -arabinofuranosylthymine (araT) are substrates of mitochondrial nucleoside Kinases and have previously been shown to be predominantly incorporated into mtDNA of cells, but the pharmacological importance of their accumulation in mtDNA is not known. Here, we examined the role of mtDNA in the response to araG, araT and other anti-cancer and anti-viral agents in a MOLT-4 wild-type (wt) T-lymphoblastoid cell line and its petite mutant MOLT-4 ρ0 cells (lacking mtDNA). The mRNA levels and activities of Deoxyguanosine Kinase (dGK), deoxycytidine Kinase (dCK), thymidine Kinase 1 (TK1) and thymidine Kinase 2 (TK2) were determined in the two cell lines. Compared to that in the MOLT-4 wt cells the mRNA level of the constitutively expressed TK2 was higher (p
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Depletion of mitochondrial DNA by down-regulation of Deoxyguanosine Kinase expression in non-proliferating HeLa cells.
Experimental cell research, 2007Co-Authors: Maribel Franco, Magnus Johansson, Anna KarlssonAbstract:Purine deoxyribonucleotides required for mitochondrial DNA replication are either imported from the cytosol or derived from phosphorylation of deoxyadenosine or Deoxyguanosine catalyzed by mitochondrial Deoxyguanosine Kinase (DGUOK). DGUOK deficiency has been linked to mitochondrial DNA depletion syndromes suggesting an important role for this enzyme in dNTP supply. We have generated HeLa cell lines with 20-30% decreased levels of DGUOK mRNA by the expression of small interfering RNAs directed towards the DGUOK mRNA. The cells with decreased expression of the enzyme showed similar levels of mtDNA as control cells when grown exponentially in culture. However, mtDNA levels rapidly decreased in the cells when cell cycle arrest was induced by serum starvation. DNA incorporation of 9-beta-d-arabino-furanosylguanine (araG) was lower in the cells with decreased Deoxyguanosine Kinase expression, but the total rate of araG phosphorylation was increased in the cells. The increase in araG phosphorylation was shown to be due to increased expression of deoxycytidine Kinase. In summary, our findings show that DGUOK is required for mitochondrial DNA replication in resting cells and that small changes in expression of this enzyme may cause mitochondrial DNA depletion. Our data also suggest that alterations in the expression level of DGUOK may induce compensatory changes in the expression of other nucleoside Kinases.
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Differential incorporation of 1‐β‐D‐arabinofuranosylcytosine and 9‐β‐D‐arabinofuranosylguanine into nuclear and mitochondrial DNA
FEBS Letters, 2000Co-Authors: Chaoyong Zhu, Magnus Johansson, Anna KarlssonAbstract:The anti-leukemic nucleoside analogs 1-β-D-arabinofuranosylcytosine (araC) and 9-β-D-arabinofuranosylguanine (araG) are dependent on intracellular phosphorylation for pharmacological activity. AraC is efficiently phosphorylated by deoxycytidine Kinase (dCK). Although araG is phosphorylated by dCK in vitro, it is a preferred substrate of mitochondrial Deoxyguanosine Kinase. We have used autoradiography to show that araC was incorporated into nuclear DNA in Molt-4 and CEM T-lymphoblastoid cells as well as in Chinese hamster ovary cells. In contrast, araG was predominantly incorporated into mitochondrial DNA in the investigated cell lines, without detectable incorporation into nuclear DNA. These data suggest that the molecular targets of araG and araC may differ.
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Phosphorylation of anticancer nucleoside analogs by human mitochondrial Deoxyguanosine Kinase
Biochemical pharmacology, 1998Co-Authors: Chaoyong Zhu, Magnus Johansson, Johan Permert, Anna KarlssonAbstract:Abstract The kinetic properties of recombinant human mitochondrial Deoxyguanosine Kinase (dGK, EC 2.7.1.113) for 2′-Deoxyguanosine and the clinically important nucleoside analogs 2-chloro-2′-deoxyadenosine (CdA), 9-β -D-arabinofuranosylguanine (araG) and 2′,2′,-difluoroDeoxyguanosine (dFdG) were determined. The Michaelis–Menten kinetic parameters, comparing ATP and UTP as phosphate donors, demonstrated a marked increase in phosphorylation efficiency ( V max K m ) with UTP in comparison with ATP for both CdA and araG. The difluoro analog dFdG was an efficient substrate for recombinant dGK with an apparent K m of 16 μM with ATP as phosphate donor. We compared the kinetic properties of dGK with those of the related enzyme deoxycytidine Kinase (dCK, EC 2.7.1.74). Although the purines 2′-Deoxyguanosine (dGuo) and 2′-deoxyadenosine are substrates for both dGK and dCK, only CdA among the purine nucleoside analogs tested was an efficient substrate for both dCK and dGK. In competition with dGuo, the most efficient analog for phosphorylation by dGK was araG, as indicated by a lower K i value than for CdA and dFdG. Of the purine analogs tested as substrates for dCK, only CdA could compete with 2′-deoxycytidine (dCyd). No inhibition of dCK-mediated dCyd phosphorylation was found by either araG or dFdG. In crude cell extract of HeLa and Capan 2 cells, the major CdA phosphorylation was contributed by dCK, while most araG phosphorylation was a result of dGK activity. Our study with pure recombinant enzymes confirms that dGK is mainly responsible for araG and dFdG phosphorylation, whereas dCK is the most important enzyme for activation of CdA and 2′,2′-difluorodeoxycytidine (dFdC).
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Enhanced Cytotoxicity of Nucleoside Analogs by Overexpression of Mitochondrial Deoxyguanosine Kinase in Cancer Cell Lines
The Journal of biological chemistry, 1998Co-Authors: Chaoyong Zhu, Magnus Johansson, Johan Permert, Anna KarlssonAbstract:Abstract The cytotoxic anti-cancer purine nucleoside analogs 2-chloro-2′-deoxyadenosine (CdA), 9-β-d-arabinofuranosylguanine (araG), and 2′,2′-difluoroDeoxyguanosine (dFdG) are phosphorylated by human mitochondrial Deoxyguanosine Kinase (dGK) in vitro. We overexpressed dGK as a fusion protein to the green fluorescent protein in the human pancreatic cancer cell lines PanC-1 and MIA PaCa-2 to determine the importance of dGK-mediated nucleoside analog phosphorylation. The transfected cells showed mitochondrial fluorescence patterns, and the mitochondrial locations of endogenous and overexpressed dGK were verified by Western blot analysis of cell extracts with polyclonal anti-dGK antibodies. The increase of dGK activity in the overexpressing cells was ∼4-fold. These cell lines exhibited increased sensitivity to CdA, araG, and dFdG as compared with the untransfected parent cell lines. This is, to our knowledge, the first demonstration of a correlation between the activity of a mitochondrial deoxyribonucleoside Kinase and the cytotoxicity of nucleoside analogs. Our data imply that the dGK activity is rate-limiting for the efficacy of nucleoside analogs in the cell lines investigated.
David Dimmock - One of the best experts on this subject based on the ideXlab platform.
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Potentially diagnostic electron paramagnetic resonance spectra elucidate the underlying mechanism of mitochondrial dysfunction in the Deoxyguanosine Kinase deficient rat model of a genetic mitochondrial DNA depletion syndrome.
Free radical biology & medicine, 2016Co-Authors: Brian Bennett, Daniel Helbling, Hui Meng, Jason A. Jarzembowski, Aron M. Geurts, Marisa W. Friederich, Johan L.k. Van Hove, Michael W. Lawlor, David DimmockAbstract:A novel rat model for a well-characterized human mitochondrial disease, mitochondrial DNA depletion syndrome with associated Deoxyguanosine Kinase (DGUOK) deficiency, is described. The rat model recapitulates the pathologic and biochemical signatures of the human disease. The application of electron paramagnetic (spin) resonance (EPR) spectroscopy to the identification and characterization of respiratory chain abnormalities in the mitochondria from freshly frozen tissue of the mitochondrial disease model rat is introduced. EPR is shown to be a sensitive technique for detecting mitochondrial functional abnormalities in situ and, here, is particularly useful in characterizing the redox state changes and oxidative stress that can result from depressed expression and/or diminished specific activity of the distinct respiratory chain complexes. As EPR requires no sample preparation or non-physiological reagents, it provides information on the status of the mitochondrion as it was in the functioning state. On its own, this information is of use in identifying respiratory chain dysfunction; in conjunction with other techniques, the information from EPR shows how the respiratory chain is affected at the molecular level by the dysfunction. It is proposed that EPR has a role in mechanistic pathophysiological studies of mitochondrial disease and could be used to study the impact of new treatment modalities or as an additional diagnostic tool.
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Recessive Deoxyguanosine Kinase deficiency causes juvenile onset mitochondrial myopathy.
Molecular Genetics and Metabolism, 2012Co-Authors: Adam H. Buchaklian, Daniel Helbling, Stephanie M. Ware, David DimmockAbstract:Deoxyguanosine Kinase (DGUOK) (MIM#601465) deficiency was originally described as the cause of an infantile onset hepatocerebral mitochondrial disease [1]. The classic features of this disorder include significant hepatic failure with nystagmus and hypotonia. Mitochondrial DNA studies reveal significant mitochondrial DNA depletion in the affected tissues. Subsequently it has been shown that the same mutations in this gene may present with isolated acute liver failure without cerebral involvement. In this paper we studied the mitochondrial DNA depletion in cells from a patient presenting with mitochondrial myopathy caused by a novel mutation in DGUOK. Subsequently we developed the method to diagnose this condition using MyoD induced fibroblasts to study the muscle specific phenotype. In addition, supplementation of MyoD induced fibroblasts with dAMP and dGMP resulted in a restoration of mtDNA quantity.
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Abnormal neurological features predict poor survival and should preclude liver transplantation in patients with Deoxyguanosine Kinase deficiency
Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation S, 2008Co-Authors: David Dimmock, Annette Feigenbaum, Lee-jun C. Wong, Peter Freisinger, J. Kay Dunn, Anthony Rupar, Rita Horvath, Bénédicte Mousson De Camaret, Fernando ScagliaAbstract:Deoxyguanosine Kinase (DGUOK) deficiency is the commonest type of mitochondrial DNA depletion associated with a hepatocerebral phenotype. In this article, we evaluate predictors of survival and therapeutic options in patients with DGUOK deficiency. A systematic search of MEDLINE, LILAC, and SCIELO was carried out to identify peer-reviewed clinical trials, randomized controlled trials, meta-analyses, and other studies with clinical pertinence. DGUOK deficiency was searched with the terms dGK, DGUOK, mitochondrial DNA depletion, mtDNA, and hepatocerebral. Bibliographies of identified articles were reviewed for additional references. Thirteen identified studies met the inclusion criteria and were used in this study. The analysis revealed that DGUOK deficiency is associated with a variable clinical phenotype. Long-term survival is best predicted by the absence of profound hypotonia, significant psychomotor retardation, or nystagmus. In the presence of these features, there is increased mortality, and liver transplantation does not confer increased survival. In summary, liver transplantation appears to be futile in the presence of specific neurological signs or symptoms in patients affected with DGUOK deficiency. Conversely, in the absence of these neurological features, liver transplantation may be considered a potential treatment.
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clinical and molecular features of mitochondrial dna depletion due to mutations in Deoxyguanosine Kinase
Human Mutation, 2008Co-Authors: David Dimmock, Eric S. Schmitt, L. Tang, Qing Zhang, Carlo Dionisivici, Rosalba Carrozzo, Joseph T C Shieh, Cavatina K Truong, Mara Sifryplatt, Simona LucioliAbstract:Published mutations in Deoxyguanosine Kinase (DGUOK) cause mitochondrial DNA depletion and a clinical phenotype that consists of neonatal liver failure, nystagmus and hypotonia. In this series, we have identified 15 different mutations in the DGUOK gene from 9 kindreds. Among them, 12 have not previously been reported. Nonsense, splice site, or frame-shift mutations that produce truncated proteins predominate over missense mutations. All patients who harbor null mutations had early onset liver failure and significant neurological disease. These patients have all died before 2-years of age. Conversely, two patients carrying missense mutations had isolated liver disease and are alive in their 4th year of life without liver transplant. Five subjects were detected by newborn screening, with elevated tyrosine or phenylalanine. Consequently, this disease should be considered if elevated tyrosine is identified by newborn screening. Mitochondrial DNA content was below 10% of controls in liver in all but one case and modestly reduced in blood cells. With this paper a total of 39 different mutations in DGUOK have been identified. The most frequent mutation, c.763_c.766dupGATT, occurs in 8 unrelated kindreds. 70% of mutations occur in only one kindred, suggesting full sequencing of this gene is required for diagnosis. The presentation of one case with apparent viral hepatitis, without neurological disease, suggests that this disease should be considered in patients with infantile liver failure regardless of the presence of neurological features or apparent infectious etiology.