Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase.

Schatzmayr

Mem. Inst. Oswaldo Cruz

2003

mentioning

confidence: 99%

Human immunodeficiency virus type 1: drug resistance in treated and untreated Brazilian children

Schatzmayr

Mem. Inst. Oswaldo Cruz

2003

“…Antiviral AZT efficacy depends on its initial transformation to AZT monophosphate (AZT-MP) by cellular thymidine kinase (TK), followed by further phosphorylation steps to AZT diphosphate and AZT triphosphate (AZT-TP), catalyzed by thymidylate kinase and pyrimidine nucleoside diphosphate kinase, respectively. The last product inhibits HIV-1 replication by interrupting the viral DNA chain elongation [7]. Low levels of TK activity could therefore induce a decrease in the amount of AZT-MP available for AZT-TP synthesis, thus reducing the therapeutic efficacy of AZT.…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

AZT‐induced hypermethylation of human thymidine kinase gene in the absence of total DNA hypermethylation

et al. 1996

Genome-wide DNA hypermethylation induced by 3'-azido-3'-deoxythymidine (AZT) has been suggested to be involved in the development of AZT resistance. We used a CD4 T-lymphoblastoid CEM line and its AZT-resistant MT500 variant with reduced thymidine kinase activity. Evaluation of total DNA methylation, after AZT treatment, failed to show an increase in the 5-methylcytosine level in both parental and AZTresistant cells. The effect was instead observed at a more specific gene level, on the three HpaII sites present in exon 1 of the human thymidine kinase gene. These results suggest that AZT treatment can induce site-specific hypermethylation, even in the absence of a more general DNA hypermethylating effect.Key words: 3'-Azido-3'-deoxythymidine; Drug resistance; DNA methylation; Thymidine kinase gene; HpaII/PCR assay der conditions severely affecting cell viability. Moreover, only scanty information is available about the methylation status of specific sites of the TK gene after exposure to AZT. This paper reports data concerning the effects of AZT on human CD4 T-lymphoblastoid CEM cell line and on its derived AZT-resistant cell variant, where a reduced TK activity has been previously reported [I0]. In particular, we investigated the effects of AZT on the total DNA methylation as well as on specific methylation sites present in the TK gene. A hypermethylating effect was found, in the absence of both overall DNA hypermethylation and of any great loss of cell viability, at the analyzed TK sites, after AZT treatment of CEM cells and of its derived AZT-resistant cell variant. This phenomenon, which was more marked in the resistant cells, could be the molecular basis for the transcriptional inactivation of the TK gene.

Biochemical Pharmacology

“…Inhibition of the salvage pathway by AZT is thus more devastating in U-937 cells and results in slowed growth or death of cells. Other investigators have demonstrated an initial decrease in TTP pools in a variety of cultured cells upon treatment with AZT [11][12][13]. Fridland, et.…”

Section: Discussionmentioning

confidence: 99%

“…Earlier work by Furman et. al., [11], has shown AZTMP is a competitive inhibitor of human thymidylate kinase (Ki = 8.6 μM). However, the highest concentrations of AZTMP were observed in H9c2 cells in which TMP was not observed.…”

Section: Effect Of Azt On Thymidine Phosphorylationmentioning

confidence: 99%

Effect of AZT on thymidine phosphorylation in cultured H9c2, U-937, and Raji cell lines

Lynx¹,

Kang²,

McKee³

2008

Abstract3′-azido-3′-deoxythymidine (AZT) has been shown to be a potent inhibitor of thymidine kinase 2 in work from this laboratory. Inhibition results in decreased salvage of thymidine to TTP, which may lead to depletion of the TTP pool and result in the mitochondrial dysfunction and mt-DNA depletion observed with AZT toxicity. The effect of AZT on thymidine phosphorylation in growing cells expressing thymidine kinase 1 has not been shown. Three cell lines were used in these experiments: H9c2, derived from rat cardiomyoblasts; U-937, derived from human monocytes; and Raji, derived from human lymphoblasts. AZT inhibited growth in a concentration dependent manner in U-937 cells, but not the other cell lines. The phosphorylation of [3H]-thymidine or [3H]-AZT was determined during log growth. All cell lines salvaged and phosphorylated thymidine to TTP, with TTP the major product. The U-937 cells had a much more active salvage pathway than the other cells. All cell lines phosphorylated AZT to the triphosphate, but the major product was AZTMP. The AZT inhibition of growth of the U-937 cells did not correlate with levels of phosphorylated AZT. In contrast, pro-drug AZT was shown to inhibit thymidine phosphorylation in all lines with 50% inhibition concentrations (IC50) ranging from 4.4-21.9 μM. Since the U-937 cells expressed higher activity of the salvage pathway than the other cell lines, the U-937 cells may rely more heavily on the salvage pathway for TTP synthesis, accounting for AZT inhibition of growth.