Secondary loss of deoxyguanosine kinase activity in purine nucleoside phosphorylase deficient mice

Snyder, Floyd F.; Jenuth, Jack P.; Dilay, Jocelyn E.; Fung, Ernest; Lightfoot, Therese; Mably, Ellen R.

doi:10.1016/0925-4439(94)90103-1

Cited by 26 publications

(22 citation statements)

References 26 publications

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“…However, it is not known if these cells retain dGK activity, and it is possible that the cells have lost both dCK and dGK activity. Indirect evidence for such an event is that a mouse model of purine nucleoside phosphorylase deficiency that accumulates high levels of deoxyguanosine shows signs of deficient dGK activity (22). The production of specific anti-dGK antibodies reported in this paper should facilitate future studies on the correlation of dCK and dGK expression and activity in purine nucleoside analog-resistant cells.…”

Section: Expression Of Dgk-gfp In Pancreatic Adenocarcinomamentioning

confidence: 96%

Enhanced Cytotoxicity of Nucleoside Analogs by Overexpression of Mitochondrial Deoxyguanosine Kinase in Cancer Cell Lines

Zhu¹,

Johansson²,

Permert³

et al. 1998

Journal of Biological Chemistry

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The cytotoxic anti-cancer purine nucleoside analogs 2-chloro-2-deoxyadenosine (CdA), 9-␤-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 ϳ4-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.

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Section: Expression Of Dgk-gfp In Pancreatic Adenocarcinomamentioning

confidence: 96%

Enhanced Cytotoxicity of Nucleoside Analogs by Overexpression of Mitochondrial Deoxyguanosine Kinase in Cancer Cell Lines

Zhu¹,

Johansson²,

Permert³

et al. 1998

Journal of Biological Chemistry

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“…A report on secondary loss of dGK activity without loss of dCK activity in purine nucleoside phosphorylase-deficient mice (27) suggests that dGK nucleoside phosphorylation can mediate cytotoxic effects. These findings may be important in studies of the purine nucleoside phosphorylase resistant nucleoside analog araG, a substrate of both dCK and dGK (6,9,12,28 (28,29).…”

Section: Discussionmentioning

confidence: 99%

Cloning and expression of human deoxyguanosine kinase cDNA.

Johansson

Karlsson

1996

Proc. Natl. Acad. Sci. U.S.A.

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A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base. The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level. The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113). The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa. Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA. dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues. Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK. To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases. Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases.

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“…Increased salvage of purine deoxynucleosides, leading to accumulation of toxic levels of dGTP in lymphocytes, is the assumed mechanism of the disease [10]. Snyder et al [11] have developed a mutant mouse strain lacking PNP activity and they observed that in all tissues from the mutant mice there was also a reduction of the dGK activity. This latter deficiency most likely protected the PNP mutants from the toxicity of dGuo, demonstrating a central role for dGK in the pathogenesis of PNP deficiency.…”

Section: Introductionmentioning

confidence: 99%

Cloning and expression of human mitochondrial deoxyguanosine kinase cDNA

1996

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Mammalian mitochondrial deoxyguanosine kinase (dGK) is responsible for phosphorylation of purine deoxyribonucleosides in the mitochondrial matrix. Using a RT-PCRgenerated probe, based on amino acid sequence information from proteolytic fragments of purified bovine dGK, we have cloned a cDNA from a human brain cDNA library that encodes a 30 kDa protein. The deduced amino acid sequence of this protein included the sequence of all six peptides isolated and sequenced from purified dGK. Expression and purification of recombinant protein from induced Escherichia coli extracts revealed that it catalyses efficient phosphorylation of dGuo, arabinosyl guanine, dAdo, 2-chloro-2'-deoxyadenosine and dlno similar to purified dGK. Northern blot analysis demonstrated one dominant positive mRNA of 1.35 kb and it was found in several tissues at similar levels. The coding sequence of dGK showed 46% identity to the coding sequence of cytosolic deoxycytidine kinase, and conserved sequence motifs among the known deoxynucleoside kinase were identified.

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Secondary loss of deoxyguanosine kinase activity in purine nucleoside phosphorylase deficient mice

Cited by 26 publications

References 26 publications

Enhanced Cytotoxicity of Nucleoside Analogs by Overexpression of Mitochondrial Deoxyguanosine Kinase in Cancer Cell Lines

Enhanced Cytotoxicity of Nucleoside Analogs by Overexpression of Mitochondrial Deoxyguanosine Kinase in Cancer Cell Lines

Cloning and expression of human deoxyguanosine kinase cDNA.

Cloning and expression of human mitochondrial deoxyguanosine kinase cDNA

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