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

Abstract

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.

Blood–Brain Barrier Efflux Transport of Pyrimidine Nucleosides and Nucleobases in the Rat

“…Uridine is phosphorylated in the cells by uridine kinase; this activity is the major entry step in the salvage pathway [34]; thymidine in the brain is phosphorylated by thymidine kinase 2 [35]. 3 0 -Azido-3 0 -deoxythymidine (AZT) competitively inhibits thymidine kinase 2, with a half maximal inhibitory concentration being between 7.0 ± 1.0 lM (rat heart) and 14.4 ± 2.6 lM (rat liver) [36,37]. In the presence of 50-66 lM AZT in the brain ISF, the supernatant/pellet ratio for [ 14 C]thymidine did not differ from the supernatant/ pellet ratio obtained 10 min after intracerebral injection of [ 3 H]sucrose and was significantly higher than the supernatant/pellet ratio obtained 10 min after intracerbral injection of [ 14 C]thymidine alone (without AZT, Fig.…”

A novel 11C-labeled thymidine analog, [11C]AZT, for tumor imaging by positron emission tomography

“…Although AZT is known to be phosphorylated to 5′-monophosphate by enzymes in the thymidine-phosphorylation pathway (thymidine kinase and thymidylate kinase), little AZT is converted to 5′-di- and 5′-tri-phosphate forms in humans [ 23 ], rats [ 24 ], and mice [ 25 ]. Therefore, the one major metabolite (metabolite a ) that was observed in tumor tissues (Fig.…”

“…Thymidine analog nucleoside reverse transcriptase inhibitors (NRTIs) such as zidovudine (AZT) and stavudine (d4T) suppress the replication of human immunodeficiency virus (HIV) and are now used in the treatment of acquired immunodeficiency syndrome (AIDS) [ 1 , 2 ]. Although AZT was the first anti-HIV drug to be approved worldwide, it was originally designed as an antitumor agent due to its prevention of DNA elongation by inhibiting the incorporation of thymidine into the DNA of cancer cells [ 3 , 4 ].…”

Impact of Mitochondrial Toxicity of HIV-1 Antiretroviral Drugs on Lipodystrophy and Metabolic Dysregulation

“…Thymidine kinase exists in two forms -thymidine kinase 1 (TK1), involved in cell division and expressed in mitotically active cells, and thymidine kinase 2 (TK2), which is localized to mitochondria and is highly expressed in quiescent cells [56]. This suggests that some cell types may not be as dependent on the thymidine salvage pathway, perhaps as they are able to synthesise pyrimidines de novo and thus not be as susceptible to toxicity [59]. TK1 phosphorlyates AZT more efficiently compared to TK2, while TK2 is better able to phosphorlyate other NRTIs such as didanosine and zalcitabine [57].…”