Human thymidine kinase 2: molecular cloning and characterisation of the enzyme activity with antiviral and cytostatic nucleoside substrates

Wang, Liya; Munch‐Petersen, Birgitte; Sjöberg, Anita Herrström; Hellman, Ulf; Bergman, Tomas; Jörnvall, Hans; Eriksson, Staffan

doi:10.1016/s0014-5793(98)01711-6

Cited by 144 publications

(173 citation statements)

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“…Both tissues display negative cooperativity in the phosphorylation of thymidine and AZT. This is consistent with previous observations in purified thymidine kinase 2 [25].…”

Section: Discussionsupporting

confidence: 94%

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3′-Azido-3′-deoxythymidine (AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: A possible mechanism of AZT hepatotoxicity

Lynx

Bentley

McKee

2006

Biochemical Pharmacology

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Abstract3′-Azido-3′-deoxythymidine (AZT) is a staple of highly active antiretroviral therapy (HAART). Prior to HAART, long-term use of high-dosage AZT caused myopathy, cardiomyopathy, and hepatotoxicity, associated with mitochondrial DNA depletion. As a component of HARRT, AZT causes cytopenias and lipodystrophy. AZT-5′-triphosphate (AZTTP) is a known inhibitor of the mitochondrial polymerase γ and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart mitochondria, AZT phosphorylation beyond AZT-5′-monophosphate (AZTMP) was not detected. Rather, AZT was shown to be a more potent inhibitor of thymidine phosphorylation (50% inhibitory concentration (IC 50 ) of 7.0 ± 1.0 μM) than AZTTP is of polymerase γ (IC 50 of >100 μM), suggesting that depletion of mitochondrial stores of TTP may limit replication. This work has investigated whether an identical mechanism might account for the hepatotoxicity seen with long-term use of AZT. Isolated rat liver mitochondria were incubated with labeled thymidine or AZT, and the rate and extent of phosphorylation were determined by HPLC analysis of acid-soluble extracts of the incubated mitochondria. The results showed that in the phosphorylation of thymidine to TMP, liver mitochondria exhibit a higher V max and K m than heart mitochondria, but otherwise heart and liver mitochondria display similar kinetics. AZT is phosphorylated to AZTMP, but no further phosphorylated forms were detected. In addition, AZT inhibited the production of TTP, with an IC 50 of 14.4 ± 2.6 μM AZT. This is higher, but comparable to, the results seen in isolated rat heart mitochondria. KeywordsAZT; Liver mitochondria; Thymidine; Mitochondrial toxicity; Thymidine kinase 2; Reverse transcriptase inhibitors Abbreviations AIDS, acquired immunodeficiency syndrome; AZT, 3′-azido-3′-deoxythymidine; AZTDP, 3′-azido-3′-deoxythymidine-5′-diphosphate; AZTMP, 3′-azido-3′-deoxythymidine-5′-monophosphate; AZTTP, 3′-azido-3′-deoxythymidine-5′-triphosphate; HAART, highly active antiretroviral therapy; HIV, human immunodeficiency virus; IC 50 , 50% inhibitory concentration; S.E.M., standard error of mean

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“…Both tissues display negative cooperativity in the phosphorylation of thymidine and AZT. This is consistent with previous observations in purified thymidine kinase 2 [25].…”

Section: Discussionsupporting

confidence: 94%

“…3B, inset) is 0.67. Both the biphasic Eadie-Hofstee plot and the Hill coefficient of less than 1 are highly suggestive of negative cooperativity, which has been previously observed with purified thymidine kinase 2 [25].…”

Section: Kinetics Of Thymidine and Azt Phosphorylation In Isolated Rasupporting

confidence: 71%

3′-Azido-3′-deoxythymidine (AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: A possible mechanism of AZT hepatotoxicity

Lynx

Bentley

McKee

2006

Biochemical Pharmacology

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“…[133][134][135][136] Cytoplasmic dGK may also contribute to the supply of purine deoxynucleotides for nuclear DNA replication and repair. 132 In this way, nucleoside ana- logues phosphorylated by dGK may exert their cytotoxic effects by interference with both mitochondrial and nuclear DNA synthesis.…”

Section: Deoxyguanosine Kinasementioning

confidence: 99%

Nucleoside analogues: mechanisms of drug resistance and reversal strategies

2001

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“…1): (i) deoxynucleotide transporters in the membrane introduce nucleotides from the cytosol (1,2), and (ii) thymidine kinase 2 (TK2) in the mt matrix phosphorylates thymidine to dTMP (3)(4)(5)(6), which is further phosphorylated by nucleotide kinases to dTTP. Preliminary evidence for a third pathway via an intra-mt ribonucleotide reductase (7) has not been followed up, nor could we confirm it.…”

mentioning

confidence: 99%

Origins of mitochondrial thymidine triphosphate: Dynamic relations to cytosolic pools

Pontarin

Gallinaro

Ferraro

et al. 2003

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

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Nuclear and mitochondrial (mt) DNA replication occur within two physically separated compartments and on different time scales. Both require a balanced supply of dNTPs. During S phase, dNTPs for nuclear DNA are synthesized de novo from ribonucleotides and by salvage of thymidine in the cytosol. Mitochondria contain specific kinases for salvage of deoxyribonucleosides that may provide a compartmentalized synthesis of dNTPs. Here we investigate the source of intra-mt thymidine phosphates and their relationship to cytosolic pools by isotope-flow experiments with [ 3 H]thymidine in cultured human and mouse cells by using a rapid method for the clean separation of mt and cytosolic dNTPs. In the absence of the cytosolic thymidine kinase, the cells (i) phosphorylate labeled thymidine exclusively by the intra-mt kinase, (ii) export thymidine phosphates rapidly to the cytosol, and (iii) use the labeled dTTP for nuclear DNA synthesis. The specific radioactivity of dTTP is highly diluted, suggesting that cytosolic de novo synthesis is the major source of mt dTTP. In the presence of cytosolic thymidine kinase dilution is 100-fold less, and mitochondria contain dTTP with high specific radioactivity. The rapid mixing of the cytosolic and mt pools was not expected from earlier data. We propose that in proliferating cells dNTPs for mtDNA come largely from import of cytosolic nucleotides, whereas intra-mt salvage of deoxyribonucleosides provides dNTPs in resting cells. Our results are relevant for an understanding of certain genetic mitochondrial diseases.M itochondria contain two separate potential pathways to provide dTTP for mitochondrial (mt) DNA replication ( Fig. 1): (i) deoxynucleotide transporters in the membrane introduce nucleotides from the cytosol (1, 2), and (ii) thymidine kinase 2 (TK2) in the mt matrix phosphorylates thymidine to dTMP (3-6), which is further phosphorylated by nucleotide kinases to dTTP. Preliminary evidence for a third pathway via an intra-mt ribonucleotide reductase (7) has not been followed up, nor could we confirm it. We do not further consider it here.The first pathway in Fig. 1 relies mainly on de novo synthesis of deoxyribonucleoside diphosphates by ribonucleotide reductase in the cytosol (8) and to a minor extent on the activity of the cytosolic thymidine kinase 1 (TK1) (9). Both enzymes are active only during the S phase of the cell cycle (10, 11). The first pathway is therefore absent from terminally differentiated cells. The second pathway in Fig. 1 uses thymidine imported from the extracellular milieu and is active also outside S phase because TK2 is not cell-cycle regulated. Regulation of this pathway may occur by an intra-mt substrate cycle involving TK2 and 5Ј(3Ј)-deoxyribonucleotidase 2 (dNT-2) (12), similar to the cytosolic substrate cycle between TK1 and deoxyribonucleotidase 1 (13). Several genetic diseases affecting mtDNA replication arise from malfunction of enzymes in either mt pathway (14, 15). Also, the genetic loss of the cytosolic thymidine phosphorylase results in mutatio...

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Human thymidine kinase 2: molecular cloning and characterisation of the enzyme activity with antiviral and cytostatic nucleoside substrates

Cited by 144 publications

References 19 publications

3′-Azido-3′-deoxythymidine (AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: A possible mechanism of AZT hepatotoxicity

3′-Azido-3′-deoxythymidine (AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: A possible mechanism of AZT hepatotoxicity

Nucleoside analogues: mechanisms of drug resistance and reversal strategies

Origins of mitochondrial thymidine triphosphate: Dynamic relations to cytosolic pools

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