Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis

Abstract

Apoptosis and necrosis are considered conceptually and morphologically distinct forms of cell death. Here, we report that demise of human T cells caused by two classic apoptotic triggers (staurosporin and CD95 stimulation) changed from apoptosis to necrosis, when cells were preemptied of adenosine triphosphate (ATP). Nuclear condensation and DNA fragmentation did not occur in cells predepleted of ATP and treated with either of the two inducers, although the kinetics of cell death were unchanged. Selective and graded repletion of the extramitochondrial ATP/pool with glucose prevented necrosis and restored the ability of the cells to undergo apoptosis. Pulsed ATP/depletion/repletion experiments also showed that ATP generation either by glycolysis or by mitochondria was required for the active execution of the final phase of apoptosis, which involves nuclear condensation and DNA degradation.

Figure 1

ATP-depletion and the shape of cell death: apoptosis or necrosis. Cells, in a glucose-free medium supplemented with 2 mM pyruvate, were incubated with solvent (control) (squares), 1.2 μM STS (triangles), or 100 ng/ml αCD95 (circles). Pretreatment with 2.5 μM oligomycin (olig) is shown by the open symbols; (a) intracellular ATP concentration was measured at the time indicated and expressed as percentage of untreated controls (1.6 nmol/mg protein; 4.5 nmol/10⁶ cells). ATP concentrations in cells treated with oligomycin plus STS or αCD95 did not differ from those of cells treated with oligomycin alone (open squares); (b and c) the percentage of apoptotic and necrotic cells was determined after staining of cultures with H-33342 plus SYTOX; in d, Jurkat cell were incubated with 1.2 μM STS (left), STS plus oligomycin (middle), or STS plus oligomycin plus 5 mM glucose (right) for 4 h, before staining with SYTO-13 (green) and EH-1 (red). Data are means ± SD of triplicate determinations and representative of at least four experiments.

Figure 1

ATP-depletion and the shape of cell death: apoptosis or necrosis. Cells, in a glucose-free medium supplemented with 2 mM pyruvate, were incubated with solvent (control) (squares), 1.2 μM STS (triangles), or 100 ng/ml αCD95 (circles). Pretreatment with 2.5 μM oligomycin (olig) is shown by the open symbols; (a) intracellular ATP concentration was measured at the time indicated and expressed as percentage of untreated controls (1.6 nmol/mg protein; 4.5 nmol/10⁶ cells). ATP concentrations in cells treated with oligomycin plus STS or αCD95 did not differ from those of cells treated with oligomycin alone (open squares); (b and c) the percentage of apoptotic and necrotic cells was determined after staining of cultures with H-33342 plus SYTOX; in d, Jurkat cell were incubated with 1.2 μM STS (left), STS plus oligomycin (middle), or STS plus oligomycin plus 5 mM glucose (right) for 4 h, before staining with SYTO-13 (green) and EH-1 (red). Data are means ± SD of triplicate determinations and representative of at least four experiments.

Figure 2

Lack of apoptotic DNA cleavage in ATP-depleted cells. Jurkat cells were incubated with αCD95 or STS in the presence or absence of oligomycin (olig). (a) After a 90 min incubation, high molecular weight DNA fragmentation was determined by FIGE; (b) after 3 h, low molecular weight oligonucleosomal fragments were determined by CAGE. At the selected time points, the two types of DNA cleavage had reached the maximum as determined by pilot experiments.

Figure 2

Lack of apoptotic DNA cleavage in ATP-depleted cells. Jurkat cells were incubated with αCD95 or STS in the presence or absence of oligomycin (olig). (a) After a 90 min incubation, high molecular weight DNA fragmentation was determined by FIGE; (b) after 3 h, low molecular weight oligonucleosomal fragments were determined by CAGE. At the selected time points, the two types of DNA cleavage had reached the maximum as determined by pilot experiments.

Figure 3

Changes in the mode of cell death after clamping the intracellular ATP concentration at defined levels or at different times. (a) Jurkat cells in glucose-free medium containing pyruvate (2 mM) were exposed to 2.5 μM oligomycin plus the indicated concentrations of glucose. Intracellular ATP concentrations were determined at the times indicated; (b and c) Jurkat cells were preincubated for 45 min in medium containing 2.5 μM oligomycin plus the concentration of glucose indicated. STS (b) or αCD95 (c) were then added and the mode of cell death was determined after further 3.5 h; (d ) intracellular ATP levels were manipulated during incubation of Jurkat cells with either STS or αCD95. Left, cells were incubated in pyruvate-supplemented medium without glucose and challenged (at t = 0) with 100 ng/ml αCD95 or 1.2 μM STS. At the times indicated oligomycin was added to deplete ATP, and the mode of cell death was determined 4 h after the challenge. Right, cells were first depleted of ATP by preincubation with oligomycin (at t = −1 h). At t = 0, STS or αCD95 were added. At the times indicated, intracellular ATP was replenished by adding 10 mM glucose to the incubation medium. The dashed bold line indicates the cellular ATP concentration (percentage of untreated control cultures in standard pyruvate medium), which was reached 15 min after each glucose supplementation. Data are means ± SD of triplicate determinations.

Figure 3

Changes in the mode of cell death after clamping the intracellular ATP concentration at defined levels or at different times. (a) Jurkat cells in glucose-free medium containing pyruvate (2 mM) were exposed to 2.5 μM oligomycin plus the indicated concentrations of glucose. Intracellular ATP concentrations were determined at the times indicated; (b and c) Jurkat cells were preincubated for 45 min in medium containing 2.5 μM oligomycin plus the concentration of glucose indicated. STS (b) or αCD95 (c) were then added and the mode of cell death was determined after further 3.5 h; (d ) intracellular ATP levels were manipulated during incubation of Jurkat cells with either STS or αCD95. Left, cells were incubated in pyruvate-supplemented medium without glucose and challenged (at t = 0) with 100 ng/ml αCD95 or 1.2 μM STS. At the times indicated oligomycin was added to deplete ATP, and the mode of cell death was determined 4 h after the challenge. Right, cells were first depleted of ATP by preincubation with oligomycin (at t = −1 h). At t = 0, STS or αCD95 were added. At the times indicated, intracellular ATP was replenished by adding 10 mM glucose to the incubation medium. The dashed bold line indicates the cellular ATP concentration (percentage of untreated control cultures in standard pyruvate medium), which was reached 15 min after each glucose supplementation. Data are means ± SD of triplicate determinations.

Figure 4

Activation of caspases in apoptotic or necrotic cell death. (a and b) Cells were preincubated for 45 min with oligomycin and/or 75 μM zVAD-fmk as indicated and then challenged with αCD95. After 4 h, (a) the mode of cell death (closed bars, apoptotic; open bars, necrotic) or (b) DNA fragmentation were determined by dual fluorescent staining or ELISA, respectively; (c) cells were incubated with αCD95 or STS for 200 min in the presence or absence of 2.5 μM oligomycin (olig). Lamin B cleavage was determined by Western blot analysis. The original lamin band and the proteolytic fragment, typically found in apoptotic cells are indicated by arrowheads; (d ) Lamin disintegration was also studied by immunostaining. Cells untreated or treated with oligomycin were then challenged by STS. Lamins were stained with an anti-lamin antibody, whereas nuclei were counterstained with H-33342. Confocal images show that breaks/dissolution of the lamin structure (middle-left) as well as chromatin condensation (middle-right) did not occur in cells undergoing necrosis (i.e., treated with oligomycin) (bottom).

Figure 4

Activation of caspases in apoptotic or necrotic cell death. (a and b) Cells were preincubated for 45 min with oligomycin and/or 75 μM zVAD-fmk as indicated and then challenged with αCD95. After 4 h, (a) the mode of cell death (closed bars, apoptotic; open bars, necrotic) or (b) DNA fragmentation were determined by dual fluorescent staining or ELISA, respectively; (c) cells were incubated with αCD95 or STS for 200 min in the presence or absence of 2.5 μM oligomycin (olig). Lamin B cleavage was determined by Western blot analysis. The original lamin band and the proteolytic fragment, typically found in apoptotic cells are indicated by arrowheads; (d ) Lamin disintegration was also studied by immunostaining. Cells untreated or treated with oligomycin were then challenged by STS. Lamins were stained with an anti-lamin antibody, whereas nuclei were counterstained with H-33342. Confocal images show that breaks/dissolution of the lamin structure (middle-left) as well as chromatin condensation (middle-right) did not occur in cells undergoing necrosis (i.e., treated with oligomycin) (bottom).

Figure 4

Activation of caspases in apoptotic or necrotic cell death. (a and b) Cells were preincubated for 45 min with oligomycin and/or 75 μM zVAD-fmk as indicated and then challenged with αCD95. After 4 h, (a) the mode of cell death (closed bars, apoptotic; open bars, necrotic) or (b) DNA fragmentation were determined by dual fluorescent staining or ELISA, respectively; (c) cells were incubated with αCD95 or STS for 200 min in the presence or absence of 2.5 μM oligomycin (olig). Lamin B cleavage was determined by Western blot analysis. The original lamin band and the proteolytic fragment, typically found in apoptotic cells are indicated by arrowheads; (d ) Lamin disintegration was also studied by immunostaining. Cells untreated or treated with oligomycin were then challenged by STS. Lamins were stained with an anti-lamin antibody, whereas nuclei were counterstained with H-33342. Confocal images show that breaks/dissolution of the lamin structure (middle-left) as well as chromatin condensation (middle-right) did not occur in cells undergoing necrosis (i.e., treated with oligomycin) (bottom).