Bioenergetic metabolites regulate base excision repair-dependent cell death in response to DNA damage

Abstract

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance [DNA polymerase beta (Polbeta) deficiency or repair inhibition] enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polbeta triggers cell death dependent on poly(ADP-ribose) (PAR) polymerase activation yet independent of PAR-mediated apoptosis-inducing factor nuclear translocation or PAR glycohydrolase, suggesting that cytotoxicity is not from PAR or PAR catabolite signaling. Cell death is rescued by the NAD(+) metabolite beta-nicotinamide mononucleotide and is synergistic with inhibition of NAD(+) biosynthesis, showing that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polbeta-deficient cells, suggesting a linkage between DNA repair, cell survival, and cellular bioenergetics.

Figure 1. PARP activation due to BER failure

(A) Immunoblot of PAR to determine activation of PARP in the cell lines indicated before and after exposure to TMZ (1.5mM) for the time indicated. PARP1 and PCNA protein expression levels are also shown. (B) (Upper panel) TMZ induced cytotoxicity in LN428, LN428/MPG and two clones of LN428/MPG cells complemented with Flag-Pol ß-WT. After treatment (48 hrs), viable cells were determined using a modified MTT assay. Plots show the % viable cells as compared to untreated (control) cells. Means are calculated from quadruplicate values in each experiment. Results indicate the mean ± S.E. of three independent experiments. [LN428, open square; LN428/MPG, open circle; LN428/MPG/Flag-Polß-WT, clones 1 and 6, filled triangle] (Lower panel) Immunoblot of PAR to determine activation of PARP1 after exposure to TMZ (1.5mM) for the time indicated. PCNA is shown as a loading control. (C) (Upper panel) TMZ induced cytotoxicity in LN428, LN428/MPG and two clones of LN428/MPG cells complemented with Flag-Pol ß-K72A, measured as in Figure 1B. [LN428, open square; LN428/MPG, open circle; LN428/MPG/Flag-Polß-K72A, clones 5 and 16, filled triangle] (Lower panel) Immunoblot of PAR to determine activation of PARP1 after exposure to TMZ (1.5mM) for the time indicated. PCNA is shown as a loading control.

Figure 2. PARP activation mediates cellular hypersensitivity in BER defective cells

(A) TMZ induced PARP activation in LN428 and LN428/MPG cells in the presence or absence of the PARP1/PARP2 inhibitor PJ34. Cells were pre-treated with PJ34 (4 μM) or vehicle control for 30 min before exposure to TMZ (1.5 mM) plus PJ34 (2 μM) for another 30 min. PCNA was used as a loading control. (B) TMZ induced cytotoxicity (LN428 and LN428/MPG cell lines) in the presence (solid lines) or absence (dashed lines) of the PARP1/PARP2 inhibitor PJ34. Viable cells were measured 48 hours after exposure as in Figure 1B. [LN428, open and filled square; LN428/MPG, open and filled circle]

Figure 3. Absence of PAR or PAR-catabolite mediated cell death following BER failure

(A) Model depicting the nexus of BER, the synthesis of PAR and the generation of PAR catabolites in response to BER failure-induced PARP1/PARP2 hyperactivation. (B) Absence of mitochondria to nucleus translocation of AIF due to BER failure as determined by confocal microscopy. BER deficient cells (LN428/MPG) were treated with media (left panel) or 1.5 mM MMS (right panel) for 1 hour and then washed and allowed to recover in media for 5 hours prior to fixation and staining for AIF (green), actin (red) and nucleus (blue). (C) PARG KD prevented degradation of DNA damage-induced PAR. (Left Panel) Immunoblot of PAR to determine the degradation of PAR in LN428/MPG/PARG-KD cells following treatment with 1.5 mM TMZ. PCNA protein expression level was shown as a loading control. (Right Panel) Preventing generation of PAR catabolites from degradation of PAR via PARG KD enhances TMZ-induced cytotoxicity. LN428 and LN428/MPG cells with (black solid bars) or without (white empty bars) PARG KD were exposed to TMZ (1 mM) or vehicle control (DMSO) for 48 hours. Viable cells were determined as in Figure 1B and reported as percentage relative to vehicle control treated cells (% control). Results indicate the mean ± S.E. of three independent experiments. (D) HMGB1 released into the cell culture media, as demonstrated by immunoblot. LN428 and LN428/MPG cells were pre-treated with PJ34 (4 μM) or vehicle control for 30 min and then exposed to TMZ (1.5 mM) with or without PJ34 (2 μM) for 12 hours. HMGB1 was then captured using immobilized heparin and analyzed by immunoblot, as described in the Materials and Methods section.

Figure 4. BER failure induced cell death depends on NAD⁺ availability

(A) Alkylation damage promotes NAD⁺ and ATP depletion in BER defective cells. NAD⁺ content (left panel): Cells were treated with media (white bars) or 0.5 mM MMS (black bars) for 1 hour prior to collection for NAD⁺ content analysis via enzymatic assay as described in the Materials and Methods section. ATP content (right panel): Cells were treated with media (white bars), 0.5 mM MMS (grey bars) or 1.5 mM MMS (black bars) for one hour. ATP content was measured after 1-hour recovery in normal media via the luminescence ATP assay described in the Materials and Methods section. NAD⁺ levels or ATP levels shown are the average of three independent experiments and are reported as percent control of the untreated control cell line. (B) PARG-KD does not rescue alkylation damage induced NAD⁺ and ATP depletion in BER defective cells. NAD⁺ content (left panel): PARG-KD cell lines were treated with media (white bars) or 0.5 TMZ (black bars) for one hour prior to collection for NAD⁺ content analysis as described in the Materials and Methods section. ATP content (right panel): PARG-KD cells were treated with media (white bars), 0.5 mM TMZ (dotted bars), 1.0 mM TMZ (grey bars) or 1.5 mM TMZ (black bars) for one hour. ATP content was measured after 1-hour recovery in normal media via the luminescence ATP assay described in the Materials and Methods section. NAD⁺ levels or ATP levels shown are the average of three independent experiments and are reported as percent control of the untreated control cell line. (C) Bioenergetic metabolites rescue Pol ß deficient cells from DNA damage-induced cell death. LN428 and LN428/MPG cells were pre-treated with NMN, NA or vehicle control (media) for 24 hours and were then exposed to TMZ (1 mM) in the presence or absence of NMN or NA for 48 hours. Viable cells were measured as in Figure 1B and were reported as percentage relative to vehicle control treated cells (% control). Results indicate the mean ± S.E. of three independent experiments. (D) NAD⁺ biosynthesis inhibition augments BER failure-induced cell death. Cells were pretreated for 24 hours with a non-toxic 10nM dose of FK-866 (black bars) or DMSO (white bars). Cells were then exposed to media control or MMS (0.5 mM) for 1 hour. Viable cells were determined as in Figure 1B and results indicate the mean ± S.E. of three independent experiments.