Cytotoxicity of 3′-azido-3′-deoxythymidine correlates with 3′-azidothymidine-5′-monophosphate (AZTMP) levels, whereas antihuman immunodeficiency virus (HIV) activity correlates with 3′-azidothymidine-5′-triphosphate (AZTTP) levels in cultured CEM T-lymphoblastoid cells

Yvonne Törnevik, Buddy Ullman, Jan Balzarini, Britta Wahren, Staffan Eriksson

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Abstract

Activation of the anti-human immunodeficiency virus (HIV) compound 3′-azido-3′-deoxythymidine (AZT) is dependent on its 5′-phosphorylation by cellular nucleoside and nucleotide kinases. Azidothymidine 5′-triphosphate (AZTTP) is considered to be the metabolite responsible for both the anti-HIV effect of AZT, via inhibition of reverse transcriptase, and cytoxicity by interference with cellular DNA polymerases. During the characterization of AZT metabolism in cultured human T-lymphoblastoid CEM cells, a spontaneously occurring variant cell line, CEM/Ag-1, was found that showed approximately 10-fold resistance to AZT growth inhibition as compared to wild type (wt) cells (EC50 = 2 mM as compared to 350 μM for wt cells). CEM/Ag-1 cells had a 3-fold reduced capacity to accumulate azidothymidine monophosphate (AZTMP) compared to wt cells whereas similar levels of AZTTP were found in both cell lines. The intracellular half-life of AZTMP was approximately 70 min in both wt and CEM/Ag-1 cells. A 3-fold lower specific activity of cytoplasmic thymidine kinase was observed in CEM/Ag-1 extracts as compared to wt. The reduced thymidine kinase activity was not correlated to a decreased level of thymidine kinase mRNA. Syncytium formation of CEM/Ag-1 cells infected with HIV-2 as well as HIV-1 antigen production was inhibited at the same concentrations of AZT (approx. 0.01 μM) as were HIV-1 and HIV-2 infected wt cells. Thus, minor decreases in cellular thymidine kinase levels may markedly affect the cytoxicity of AZT but have no major effect on the antiviral activity of AZT. Our results strongly suggest that AZTMP is responsible for a major part of the growth inhibitor effects, while AZTTP mainly mediates the antiviral activity of AZT.

Original languageEnglish (US)
Pages (from-to)829-837
Number of pages9
JournalBiochemical Pharmacology
Volume49
Issue number6
DOIs
StatePublished - Mar 15 1995

Fingerprint

Zidovudine
Cytotoxicity
Viruses
HIV
Thymidine Kinase
HIV-2
Antiviral Agents
nucleoside phosphotransferase
Cells
HIV-1
Growth Inhibitors
Phosphorylation
RNA-Directed DNA Polymerase
DNA-Directed DNA Polymerase
Metabolites
triphosphoric acid
Cell Line
Nucleosides
Metabolism
Giant Cells

Keywords

  • antiviral chemotherapy
  • cytotoxicity
  • deoxynucleoside kinases
  • metabolism
  • nucleoside analogs

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

Cite this

@article{817cf63ad8634a18b6fa2ec74dd9f340,
title = "Cytotoxicity of 3′-azido-3′-deoxythymidine correlates with 3′-azidothymidine-5′-monophosphate (AZTMP) levels, whereas antihuman immunodeficiency virus (HIV) activity correlates with 3′-azidothymidine-5′-triphosphate (AZTTP) levels in cultured CEM T-lymphoblastoid cells",
abstract = "Activation of the anti-human immunodeficiency virus (HIV) compound 3′-azido-3′-deoxythymidine (AZT) is dependent on its 5′-phosphorylation by cellular nucleoside and nucleotide kinases. Azidothymidine 5′-triphosphate (AZTTP) is considered to be the metabolite responsible for both the anti-HIV effect of AZT, via inhibition of reverse transcriptase, and cytoxicity by interference with cellular DNA polymerases. During the characterization of AZT metabolism in cultured human T-lymphoblastoid CEM cells, a spontaneously occurring variant cell line, CEM/Ag-1, was found that showed approximately 10-fold resistance to AZT growth inhibition as compared to wild type (wt) cells (EC50 = 2 mM as compared to 350 μM for wt cells). CEM/Ag-1 cells had a 3-fold reduced capacity to accumulate azidothymidine monophosphate (AZTMP) compared to wt cells whereas similar levels of AZTTP were found in both cell lines. The intracellular half-life of AZTMP was approximately 70 min in both wt and CEM/Ag-1 cells. A 3-fold lower specific activity of cytoplasmic thymidine kinase was observed in CEM/Ag-1 extracts as compared to wt. The reduced thymidine kinase activity was not correlated to a decreased level of thymidine kinase mRNA. Syncytium formation of CEM/Ag-1 cells infected with HIV-2 as well as HIV-1 antigen production was inhibited at the same concentrations of AZT (approx. 0.01 μM) as were HIV-1 and HIV-2 infected wt cells. Thus, minor decreases in cellular thymidine kinase levels may markedly affect the cytoxicity of AZT but have no major effect on the antiviral activity of AZT. Our results strongly suggest that AZTMP is responsible for a major part of the growth inhibitor effects, while AZTTP mainly mediates the antiviral activity of AZT.",
keywords = "antiviral chemotherapy, cytotoxicity, deoxynucleoside kinases, metabolism, nucleoside analogs",
author = "Yvonne T{\"o}rnevik and Buddy Ullman and Jan Balzarini and Britta Wahren and Staffan Eriksson",
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T1 - Cytotoxicity of 3′-azido-3′-deoxythymidine correlates with 3′-azidothymidine-5′-monophosphate (AZTMP) levels, whereas antihuman immunodeficiency virus (HIV) activity correlates with 3′-azidothymidine-5′-triphosphate (AZTTP) levels in cultured CEM T-lymphoblastoid cells

AU - Törnevik, Yvonne

AU - Ullman, Buddy

AU - Balzarini, Jan

AU - Wahren, Britta

AU - Eriksson, Staffan

PY - 1995/3/15

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N2 - Activation of the anti-human immunodeficiency virus (HIV) compound 3′-azido-3′-deoxythymidine (AZT) is dependent on its 5′-phosphorylation by cellular nucleoside and nucleotide kinases. Azidothymidine 5′-triphosphate (AZTTP) is considered to be the metabolite responsible for both the anti-HIV effect of AZT, via inhibition of reverse transcriptase, and cytoxicity by interference with cellular DNA polymerases. During the characterization of AZT metabolism in cultured human T-lymphoblastoid CEM cells, a spontaneously occurring variant cell line, CEM/Ag-1, was found that showed approximately 10-fold resistance to AZT growth inhibition as compared to wild type (wt) cells (EC50 = 2 mM as compared to 350 μM for wt cells). CEM/Ag-1 cells had a 3-fold reduced capacity to accumulate azidothymidine monophosphate (AZTMP) compared to wt cells whereas similar levels of AZTTP were found in both cell lines. The intracellular half-life of AZTMP was approximately 70 min in both wt and CEM/Ag-1 cells. A 3-fold lower specific activity of cytoplasmic thymidine kinase was observed in CEM/Ag-1 extracts as compared to wt. The reduced thymidine kinase activity was not correlated to a decreased level of thymidine kinase mRNA. Syncytium formation of CEM/Ag-1 cells infected with HIV-2 as well as HIV-1 antigen production was inhibited at the same concentrations of AZT (approx. 0.01 μM) as were HIV-1 and HIV-2 infected wt cells. Thus, minor decreases in cellular thymidine kinase levels may markedly affect the cytoxicity of AZT but have no major effect on the antiviral activity of AZT. Our results strongly suggest that AZTMP is responsible for a major part of the growth inhibitor effects, while AZTTP mainly mediates the antiviral activity of AZT.

AB - Activation of the anti-human immunodeficiency virus (HIV) compound 3′-azido-3′-deoxythymidine (AZT) is dependent on its 5′-phosphorylation by cellular nucleoside and nucleotide kinases. Azidothymidine 5′-triphosphate (AZTTP) is considered to be the metabolite responsible for both the anti-HIV effect of AZT, via inhibition of reverse transcriptase, and cytoxicity by interference with cellular DNA polymerases. During the characterization of AZT metabolism in cultured human T-lymphoblastoid CEM cells, a spontaneously occurring variant cell line, CEM/Ag-1, was found that showed approximately 10-fold resistance to AZT growth inhibition as compared to wild type (wt) cells (EC50 = 2 mM as compared to 350 μM for wt cells). CEM/Ag-1 cells had a 3-fold reduced capacity to accumulate azidothymidine monophosphate (AZTMP) compared to wt cells whereas similar levels of AZTTP were found in both cell lines. The intracellular half-life of AZTMP was approximately 70 min in both wt and CEM/Ag-1 cells. A 3-fold lower specific activity of cytoplasmic thymidine kinase was observed in CEM/Ag-1 extracts as compared to wt. The reduced thymidine kinase activity was not correlated to a decreased level of thymidine kinase mRNA. Syncytium formation of CEM/Ag-1 cells infected with HIV-2 as well as HIV-1 antigen production was inhibited at the same concentrations of AZT (approx. 0.01 μM) as were HIV-1 and HIV-2 infected wt cells. Thus, minor decreases in cellular thymidine kinase levels may markedly affect the cytoxicity of AZT but have no major effect on the antiviral activity of AZT. Our results strongly suggest that AZTMP is responsible for a major part of the growth inhibitor effects, while AZTTP mainly mediates the antiviral activity of AZT.

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