The pivotal role of the mitochondrial amidoxime reducing component 2 in protecting human cells against apoptotic effects of the base analog N6-hydroxylaminopurine

Abstract

N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal.

Keywords: MOSC; N-reduction; N6-hydroxyadenosine; N6-hydroxylaminopurine; RNA interference (RNAi); apoptosis; cell metabolism; flow cytometry; mARC; nucleoside/nucleotide analogue.

FIGURE 1.

N-Reductive metabolism of HAP in HEK-293. A, N-reduction of HAP to adenine. B, effect of mARC knockdown on HAP reduction. HEK-293 cells were transfected with 10 nm mARC1 or non-targeting (NC) siRNA. The siRNA-mediated down-regulations of the proteins of interest were verified by Western blot using anti-mARC1, or anti-calnexin antibody. Calnexin levels were used as loading control. N-Reductive activities were determined on day 3 after transfection as described under “Experimental Procedures.” Results are presented as means ± S.D. (n = 3). ***, p < 0.001.

FIGURE 2.

Metabolism of HAPR in HEK-293. A, possible inosine formation pathways from HAPR in human cells. B, effect of mARC knockdown on inosine formation from HAPR. HEK-293 cells were transfected with 10 nm mARC1 or non-targeting (NC) siRNA. The siRNA-mediated down-regulations of the proteins of interest were verified by Western blot using anti-mARC1, or anti-calnexin antibody. Calnexin levels were used as loading control. N-reductive activities were determined with 4 mm HAPR on day 3 after transfection as described under “Experimental Procedures.” Results are presented as means ± S.D. (n = 3). ***, p < 0.001. C, metabolism of HAPR or adenosine with concurrent inhibition of adenosine deaminase (ADA) in HEK-293. Formation of inosine from adenosine (2 mm) or from HAPR (2 mm) and formation of adenosine from HAPR (2 mm) with simultaneous incubation with dipyridamole as inhibitor were determined as described under “Experimental Procedures.”

FIGURE 3.

HAPR or adenosine as substrates of recombinant human adenosine deaminase. The dehydroxylamination of HAPR and the deamination of adenosine catalyzed by recombinant adenosine deaminase were characterized as described under “Experimental Procedures.”

FIGURE 4.

Flow cytometric analysis of Annexin V-PE/7-AAD stained HeLa cells. Representative data showing HeLa cells with or without mARC2 knockdown that were grown in the presence of 2 mm HAP or 1% (v/v) DMSO for 48 h. Etoposide treatment (100 μm, 24 h or 48 h) was used as a positive control (A) fluorescence dotplots (B) light scatter dotplots.

FIGURE 5.

Effect of mARC knockdown on HAP-induced apoptosis in HeLa. HeLa cells were grown in the presence of various concentrations of HAP or 1% (v/v) DMSO. Etoposide treatment (100 μm) was used as a positive control. Apoptotic cell death was quantified by flow cytometric analysis of Annexin V-PE/7-AAD-stained cells as described under “Experimental Procedures.” Results are presented as means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. A, evaluation after 24 h cultivation. B, evaluation after 48 h of cultivation. C, verification of mARC knockdown in HeLa. Down-regulation of the proteins of interest were verified by Western blot when cells were plated for HAP treatment using anti-mARC1, anti-mARC2, or anti-calnexin antibody. Calnexin levels were used as loading control.

FIGURE 6.

Effect of mARC knockdown on HAP-induced PARP cleavage in HeLa. HeLa cells were grown in the presence of various concentrations of HAP or 1% (v/v) DMSO. Etoposide treatment (100 μm) was used as a positive control. Caspase-dependent apoptotic cell death was evaluated by immunoblot analysis of cleaved PARP using anti-PARP, or anti-GAPDH antibody. GAPDH levels were used as loading control. Analyzed lysates were pools of triplicates.

FIGURE 7.

Nuclear morphology in HeLa after HAP treatment. HeLa cells with or without mARC knockdown were grown in the presence of 2 mm HAP or 1% (v/v) DMSO. Changes in chromatin morphology were evaluated by Hoechst staining as described under “Experimental Procedures.” Arrows indicate examples for nuclei with prominent chromatin condensation.