Zidovudine Induces Downregulation of Mitochondrial Deoxynucleoside Kinases: Implications for Mitochondrial Toxicity of Antiviral Nucleoside Analogs

Abstract

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of deoxynucleosides in the synthesis of the DNA precursors required for mitochondrial DNA (mtDNA) replication and are essential for mitochondrial function. Antiviral nucleosides are known to cause toxic mitochondrial side effects. Here, we examined the effects of 3=-azido-2=,3=-dideoxythymidine (AZT) (zidovudine) on mitochondrial TK2 and dGK levels and found that AZT treatment led to downregulation of mitochondrial TK2 and dGK in U2OS cells, whereas cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) levels were not affected. The AZT effects on mitochondrial TK2 and dGK were similar to those of oxidants (e.g., hydrogen peroxide); therefore, we examined the oxidative effects of AZT. We found a modest increase in cellular reactive oxygen species (ROS) levels in the AZT-treated cells. The addition of uridine to AZT-treated cells reduced ROS levels and protein oxidation and prevented the degradation of mitochondrial TK2 and dGK. In organello studies indicated that the degradation of mitochondrial TK2 and dGK is a mitochondrial event. These results suggest that downregulation of mitochondrial TK2 and dGK may lead to decreased mitochondrial DNA precursor pools and eventually mtDNA depletion, which has significant implications for the regulation of mitochondrial nucleotide biosynthesis and for antiviral therapy using nucleoside analogs. N ucleoside analogs are widely used as antiviral and anticancer agents, and today Ͼ50% of the FDA-approved antiviral and anticancer drugs are nucleoside or nucleobase analogs. These analogs are administered as prodrugs that need to be activated by cellular enzymes to exert their therapeutic potential. Deoxynucleoside kinases catalyze the initial phosphorylation of nucleoside analogs; once phosphorylated, these nucleotides are trapped inside the cell and are further metabolized to their active forms, which interact with the final targets. There are four deoxynucleoside kinases in mammalian cells, i.e., cytosolic thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK) and mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) (1, 2). Recent studies using TK1-and dCK-knockout mouse models indicate that cytosolic TK1 and dCK regulate hematopoiesis by linking salvage deoxynucleotide synthesis and replication stress (3, 4). The mitochondrial enzymes TK2 and dGK are vital for mitochondrial function, because deficiency in either causes severe mitochondrial DNA (mtDNA) depletion syndrome (MDS) in human patients and in TK2-knockout mouse models (5-8). TK2 has also been shown to play an important role in providing dTTP for nuclear DNA repair and thus genomic stability in quiescent cells (9).The nucleoside analogs used in antiviral and anticancer therapy are often associated with mitochondrial toxicity. Mitochondrial myopathy, cardiomyopathy, and lipodystrophy associated with anti-HIV treatment using zidovudine (AZT) or 2=,3=-dideoxyinosine (ddI) (didanosine) wer...

The mitochondrial deoxyguanosine kinase is required for cancer cell stemness in lung adenocarcinoma

“…Next, we sought to determine whether the over‐reliance of lung CSC on DGUOK could be exploited to inhibit tumor growth and to prevent tumor initiation. There is currently no specific DGUOK inhibitor available; however, several FDA‐approved anti‐HIV reverse transcriptase inhibitors such as didanosine (DDI) and zidovudine have been shown to inhibit DGUOK and mtDNA homeostasis in cell culture and mouse models (Sun et al , ,c). Interestingly, there is anecdotal evidence that DDI treatment might be responsible for the long‐term survival in a HIV‐positive small‐cell lung cancer patient (Kato et al , ).…”

“…Although there is currently no specific DGUOK inhibitor available, several reports indicated that FDA‐approved anti‐HIV reverse transcriptase inhibitors such as DDI and zidovudine could also inhibit DGUOK and TK2 (Sun et al , ,c). Indeed, it was reported that HIV‐infected patients receiving DDI treatment displayed 47% decrease in mtDNA copies in liver biopsies (Walker et al , ).…”

Beyond the polymerase-γ theory: Production of ROS as a mode of NRTI-induced mitochondrial toxicity

“…However, plasma concentrations of 50μM have been observed for AZT in the clinic [ 32 ]. In vitro experiments commonly use concentrations between 10–200μM [ 11 , 74 , 75 ]. Drugs can be exposed to nematodes on solid media, by either mixing them in with the bacterial lawn or adding them directly to the agar plates [ 76 , 77 ].…”

Effects of ARVs and HIV on mitochondrial function and immune activation in monocyte derived macrophages in vitro

“…Compared to the individuals who were administered a placebo, AZT patients had significantly more cases of anemia and neutropenia as well as more AZT patients needed red-cell transfusions compared to the placebo group 40 . It has also been reported that AZT decreases cell viability in vitro 41 and induces mitochondrial toxicity at physiological concentrations [42][43][44] . As years passed, ART improved, with decreases in toxicity shown in NRTIs TFV, emtricitabine (FTC), and lamivudine (3TC) compared to other commonly prescribed ART such as AZT, didanosine (ddI), ritonavir (RTV), lopinavir (LPV), and stavudine (d4T) 45,46 .…”

“…ROS have also been shown to be important signaling molecules, critical to inflammatory and antiviral immune responses, especially in TLR4 signaling and immune activation 256 . Superoxides, the main type of mitochondrial ROS, are produced from the reduction of oxygen by one electron, which leak out of the ETC 43 . Superoxides are produced mainly through ETC complexes I and III, and are used mainly in signaling pathways or to combat invading pathogens, but also have toxic abilities if they accumulate 119,120 .…”