Role of poly(ADP-ribose) polymerase in rapid intracellular acidification induced by alkylating DNA damage

Abstract

In response to high levels of DNA damage, catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) triggers necrotic death because of rapid consumption of its substrate beta-nicotinamide adenine dinucleotide and consequent depletion of ATP. We examined whether there are other consequences of PARP activation that could contribute to cell death. Here, we show that PARP activation reaction in vitro becomes acidic with release of protons during hydrolysis of beta-nicotinamide adenine dinucleotide. In the cellular context, we show that Molt 3 cells respond to DNA damage by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with a dose-dependent acidification within 30 min. Whereas acidification by 0.15 pH units induced by 10 microM MNNG is reversed within 1 h, 100 , microinduced acidification by 0.5-0.6 pH units is persistent up to 7 h. Acidification is a general DNA damage response because H(2)O(2) exposure also acidifies Molt 3 cells, and MNNG causes acidification in Jurkat, U937, or HL-60 leukemia cells and in PARP(+/+) fibroblasts. Acidification is significantly decreased in the presence of PARP inhibitors or in PARP(-/-) fibroblasts, suggesting a major role for PARP activation in acidification. Inhibition of proton export through ATP-dependent Na(+)/H(+) exchanger is another major cause of acidification. Using the pH clamp method to either suppress or introduce changes in cellular pH, we show that brief acidification by 0.5-0.6 pH units may be a negative regulator of apoptosis while permitting necrotic death of cells with extensively damaged DNA.

Figure 1

Acidification during PARP activation reaction in vitro. (A) pH changes during PARP activation reaction. Purified PARP was activated for 20 min in the unbuffered complete assay (■), in which either 100 μM DHQ (▴) or 100 mM Tris, pH 7.4 (×) were added or PARP was excluded (○). Data (mean ± SD) was derived from three experiments, each carried out in duplicate. (B) PARP immunoblot. Samples from reactions in A at 0 and 20 min were immunoblotted for PARP. (C) Polymer immunoblot. Samples similar to B were immunoblotted with anti-polymer 10H. Blots in B and C represent one of three experiments with identical results.

Figure 2

MNNG-induced acidification response. (A) Intracellular acidification in MNNG-treated Molt 3 cells. Changes in pH were monitored in BCECF-loaded control (○) or 100 μM MNNG-treated cells (□). Results (mean ± SD) were obtained from five experiments, each in triplicate. (B) Dose-dependent acidification response to MNNG. Molt 3 cells were treated as above with 10–100 μM MNNG for 30 min, and pH was measured. Results (mean ± SD) were obtained from two experiments, each in triplicate. (C) Acidification response to MNNG and H₂O₂. Molt 3 cells were treated for 1 h with 100 μM MNNG (○) or 300 μM H₂O₂ (□) as above, and pH changes were measured. Results (mean ± SD) were obtained from five experiments, each in triplicate. (D) MNNG-induced acidification in other cells. Jurkat, U937, and HL-60 cells were loaded with BCECF and treated with 100 μM MNNG for 30 min before analysis of pH. Results (mean ± SD) were obtained from two experiments, each in triplicate.

Figure 3

MNNG-induced PARP activation and acidification in Molt 3 cells. (A) Polymer immunoblot. Molt 3 cells were treated with 10 or 100 μM MNNG for a given time and immunoblotted with anti-polymer LP96–10. The blot represents one of the four experiments with identical results. (B) NAD and ATP depletion. Samples of Molt 3 cells, treated with 10 or 100 μM MNNG as above, were analyzed for NAD (○) or ATP (□). Results (mean ± SD) were obtained from four experiments, each in triplicate. (C) Time course of acidification. Molt 3 cells were treated with 10 (◊) or 100 (■) μM MNNG, and changes in pH were monitored by BCECF method up to 7 h. Results (mean ± SD) were obtained from four experiments, each in triplicate.

Figure 4

Role of PARP in acidification response. (A) Suppression of PARP activation with DHQ. Molt 3 cells were exposed to 100 μM MNNG after 5-min pretreatment with 100 μM DHQ and immunoblotted for polymer with LP96–10. This blot represents one of the four experiments with identical results. (B) Suppression of acidification with PARP inhibitor. BCECF-loaded Molt 3 cells were exposed to 100 μM MNNG or 300 μM H₂O₂ with or without 5-min pretreatment with 100 μM DHQ, and changes in pH were measured at 30 min. Results (mean ± SD) were obtained from four experiments, each in triplicate. (C) MNNG-induced polymer synthesis in PARP^+/+ and PARP^−/− fibroblasts. Cells with two PARP genotypes were treated with 300 μM MNNG and immunoblotted with anti-polymer LP96–10. This blot represents one of the three experiments with identical results. (D) MNNG-induced acidification in PARP^+/+ and PARP^−/− fibroblasts. The pH changes in BCECF-loaded cells were measured at 30 min after exposure to 300 μM MNNG. Results (mean ± SD) were obtained from two experiments, each in triplicate.

Figure 5

Role of PARP and NHE in MNNG-induced acidification in Molt 3 cells. (A) pH recovery after acid loading with NH₄Cl. BCECF-loaded cells were acidified with an NH₄Cl pulse, and pH recovery was monitored in the absence (◊) or presence (□) of 10 μM of NHE inhibitor EIPA. Results (mean ± SD) were obtained from two experiments, each in quadruplicate. (B) Effect of inhibitors of PARP and NHE on MNNG-induced acidification. BCECF-loaded cells were treated with 100 μM MNNG for 30 min and incubated for 30 more min without any inhibitor (◊, M) or with 10 μM EIPA (□) or 100 μM DHQ (▵) before measurement of pH. Results (mean ± SD) were obtained from three experiments, each in triplicate. (C) Acidification effect of second MNNG treatment. BCECF-loaded Molt 3 cells were treated with 100 μM MNNG for 30 min before a second treatment for 30 min with 100 μM MNNG alone (◊, M) or with 10 μM EIPA (□) or 100 μM DHQ (▵). Results (mean ± SD) were obtained from three experiments, each in triplicate. (D) Lack of acidification by NHE inhibition per se. The pH changes in BCECF-loaded Molt 3 cells were monitored in the absence (◊) or presence (□) of 10 μM EIPA. Results (mean ± SD) were obtained from six experiments, each in triplicate.

Figure 6

Impact of acidification on mode of cell death. (A) PARP activation in pH-clamped cells. Molt 3 cells were treated for 60 min with 10 μM MNNG without pH clamp, as in Fig. 3A, or with pH 6.8 clamp (lanes 1–5). Another set of cells was treated with 100 μM MNNG without pH clamp, as in Fig. 3A, or with pH 7.4 clamp (lanes 6–10). Samples were immunoblotted with antipolymer LP96–10. (B) Flow cytometry analysis of mode of cell death. Cells treated for 1 h with 10 or 100 μM MNNG with or without pH clamp were allowed to recover for 10 h, stained with annexin V-FITC and propidium iodide, and analyzed by flow cytometry. The viable cells were identified by low signals for both the dyes, whereas apoptotic cells were detected by exclusion of propidium iodide and staining with annexin V. In contrast, necrotic cells were detected by high uptake of both the dyes. (C) Caspase 3-immunoblot analysis of cell death. Cells treated as described in B were immunoblotted for caspase 3. All lanes marked C represent DMSO-treated controls, and etoposide-treated HL-60 cells were used as positive apoptosis control in both C and D (lane 9). (D) PARP immunoblot analysis of cell death. Cells treated as described in B were also immunoblotted for PARP. All data represent one of three experiments with identical results.