Human ALKBH7 is required for alkylation and oxidation-induced programmed necrosis

Abstract

Programmed necrosis has emerged as a crucial modulator of cell death in response to several forms of cellular stress. In one form of programmed necrotic cell death, induced by cytotoxic alkylating agents, hyperactivation of poly-ADP-ribose polymerase (PARP) leads to cellular NAD and ATP depletion, mitochondrial dysfunction, reactive oxygen species formation, and ensuing cell death. Here, we show that the protein encoded by the human AlkB homolog 7 (ALKBH7) gene plays a pivotal role in DNA-damaging agent-induced programmed necrosis by triggering the collapse of mitochondrial membrane potential and large-scale loss of mitochondrial function that lead to energy depletion and cellular demise. Depletion of ALKBH7 suppresses necrotic cell death induced by numerous alkylating and oxidizing agents while having no effect on apoptotic cell death. Like wild-type cells, ALKBH7-depleted cells undergo PARP hyperactivation and NAD depletion after severe DNA damage but, unlike wild-type cells, exhibit rapid recovery of intracellular NAD and ATP levels. Consistent with the recovery of cellular bioenergetics, ALKBH7-depleted cells maintain their mitochondrial membrane potential, plasma membrane integrity, and viability. Our results uncover a novel role for a mammalian AlkB homolog in programmed necrosis, presenting a new target for therapeutic intervention in cancer cells that are resistant to apoptotic cell death.

Keywords: ALKBH7; AlkB; DNA damage; death; necrosis.

Figure 1.

ALKBH7 is targeted to mitochondria. (A) ALKBH7 contains a MTS at the N terminus, as predicted through primary amino acid sequence analysis and subcellular localization prediction. (B) Transiently expressed ALKBH7-GFP colocalizes with mitochondria in HeLa human cervical carcinoma cells. (C) ALKBH7 fractionates into a higher-molecular-weight species in the cytoplasm and a lower-molecular-weight form in purified mitochondrial fractions, indicative of mitochondrial import and processing of ALKBH7.

Figure 2.

Depletion of ALKBH7 in 293T human embryonic kidney cells confers resistance to alkylating and oxidizing agents. (A) Immunoblot analysis of ALKBH7 levels in 293T cells expressing a nonsilencing shRNA (control-sh) or either of two shRNAs targeting different regions of the ALKBH7 mRNA transcript (ALKBH7-sh1 and ALKBH7-sh2). The amount of ALKBH7 remaining in each cell type (%ALKBH7) is expressed relative to the control-sh cell line after normalization to the levels of GAPDH. (B) Representative growth of control-sh versus ALKBH7-depleted 293T cell lines after treatment with the indicated amount of MMS. (C) Viability of control-sh and ALKBH7-depleted 293T cell lines after treatment with the indicated concentration of MMS, as measured by trypan blue dye exclusion. (D) Immunoblot analysis and post-MMS viability of 293T cells transfected with nonsilencing siRNA (scramble) or pooled siRNAs targeting a non-ALKBH protein-coding transcript (PHGDH) or ALKBH7 mRNA. (E) Confirmation of ALKBH7 expression in 293T cell lines transfected with plasmids expressing the indicated ALKBH7 protein and viability of the transfected cells after treatment with MMS. (F) Viability of control-sh and ALKBH7-depleted cell lines after exposure to the indicated alkylating or oxidizing agent. Cells were treated with MMS, MNNG, H₂O₂, and t-buOOH at the indicated concentration for 1 h in serum-free medium, while mechlorethamine and temozolomide were left in culture medium until analysis. Error bars represent the standard deviation of at least three independent experiments (C,D,F) or three technical replicates (E). (*) P < 0.05 (***); (**) P < 0.01; P < 0.001.

Figure 3.

ALKBH7 depletion inhibits necrotic cell death but not apoptosis. (A) Phase-contrast images of control-sh and ALKBH7-depleted 293T cells that were left untreated or treated with the indicated concentration of MMS. Images were taken 24 h after initiation of treatment. (B) Scatter plots of 7AAD positivity for plasma membrane permeabilization (necrosis) and annexin V staining (apoptosis), as measured simultaneously through flow cytometry in single cells at the indicated time points post-MMS treatment. (C) Caspase 3/7 activation in 293T cell lines after treatment with the indicated agents. Bars represent the standard deviation of three independent experiments. (D) Immunoblot analysis of cleaved PARP in control-sh and ALKBH7-depleted cell lines after exposure to the indicated agent or transient expression of the proapoptotic protein BAX. (E–G) Viability of control-sh and ALKBH7-depleted cell lines after exposure to the indicated agent or after transient expression of the apoptosis-inducing protein BAX. Cells were treated with MMS (1.5 mM) or H₂O₂ (1 mM) for 1 h, while STS (1 μM) or etoposide (100 μM) were left in the culture medium until analysis. UV irradiation was delivered at 80 μJ/m². In some experiments, the caspase inhibitor z-vad-fmk (100 μM) or the RIP kinase inhibitor nec-1 (50 μM) was added to the medium after transfection or treatment. Bars represent the standard deviation of at least three independent experiments.

Figure 4.

ALKBH7 prevents the recovery of NAD⁺ and ATP levels after MMS treatment that is necessary for cellular survival after exposure to MMS (1.5 mM for 1 h). (A,B) Measurement of NAD⁺ or ATP levels in the indicated 293T human embryonic kidney cell lines after MMS treatment. (C) Enzymes and metabolites of the NAD⁺ salvage pathway. (NAMPT) Nicotinamide phosphoribosyltransferase; (NMNAT) nicotinamide mononucleotide adenylyltransferase; (PP_i) inorganic pyrophosphate. (D) Viability of control-sh or ALKBH7-depleted cell lines after treatment with MMS, followed by the addition of either mock buffer, NAD⁺ (10 mM), or NMN (10 mM). (E) Viability of control-sh or ALKBH7-depleted cell lines after treatment with MMS in the absence or presence of either of the PARP inhibitors: 3AB (2 mM) or DPQ (20 μM). (F) Viability of control-sh or ALKBH7-depleted cell lines after treatment with MMS, followed by the absence or presence of the NAMPT inhibitor FK-866 (10 nM). Bars for all graphs represent the mean of at least three independent experiments.

Figure 5.

ALKBH7 depletion prevents the MPT, loss of mitochondria membrane potential, and formation of ROS that accompany MMS-induced cell death. For all experiments, cell lines were treated with 1.5 mM MMS for 1 h. (A) Measurement of MPT in control-sh or ALKBH7-depleted cell lines after exposure to MMS. Cells were loaded with calcein-FM, followed by CoCl₂ quenching. (B) Representative flow cytometry plot of untreated and MMS-treated cell lines stained with TMRE to monitor mitochondria membrane potential (Δψm). (C) Percentage of cells with low TMRE staining, indicative of reduced mitochondrial Δψm after treatment with MMS. Bars represent the mean of three independent experiments. (D) Scatter plots of JC-1 staining for mitochondria membrane potential as measured through JC-1 staining. (E) Quantification of cells with depolarized mitochondria, as measured by the loss of JC-1 red fluorescence, which is indicative of mitochondria membrane depolarization. (F) Quantification of ROS formation. After treatment with MMS, the indicated cell lines were loaded with the mitochondrial superoxide indicator MitoSox Red (2 μM), which increases in fluorescence intensity after oxidation. The fold increase in fluorescence signal relative to untreated cells represents the mean of three independent experiments. (G) Viability of control-sh or ALKBH7-depleted cell lines after treatment with MMS in the absence or presence of the ETC inhibitors antimycin (5 μM) or rotenone (25 μM). Bars for all graphs represent the mean of at least three independent experiments. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

Figure 6.

Programmed necrosis inhibits the accumulation of cells with genomic DNA damage. (A) SCEs per chromosome of the indicated cell lines with or without treatment with MNNG (20 μM). Scatter plots represent the aggregate of two independent experiments. (B) MNNG-induced frequency of SCE in the indicated cell lines.

Figure 7.

Model for alkylating and oxidizing agent-induced necrotic cell death modulated by ALKBH7. See the Discussion for details.