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. 2000 Aug 21;192(4):571-80.
doi: 10.1084/jem.192.4.571.

Two distinct pathways leading to nuclear apoptosis

S A Susin  1 , E Daugas, L Ravagnan, K Samejima, N Zamzami, M Loeffler, P Costantini, K F Ferri, T Irinopoulou, M C Prévost, G Brothers, T W Mak, J Penninger, W C Earnshaw, G Kroemer
Affiliations

Two distinct pathways leading to nuclear apoptosis

S A Susin et al. J Exp Med. .

Abstract

Apaf-1(-/-) or caspase-3(-/-) cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1(-/-) or caspase-3(-/-) cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralization of AIF and CAD is required to suppress the nuclear DNA loss caused by cytoplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletion alone suffices to suppress the nuclear DNA loss contained in extracts from apoptotic Apaf-1(-/-) or caspase-3(-/-) cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptosis. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation. The other pathway, which is caspase-independent, involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin condensation.

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Figures

Figure 1
Figure 1
Mitochondrial and nuclear features of apoptosis in Apaf-1−/− and caspase-3−/− cells. MEFs from control mice (wild-type), Apaf-1−/−, or caspase-3−/− knockout mice were treated for 24 h with 2 μM STS (A and B), 100 μM etoposide, 150 μM cisplatin, 50 μM arsenite (B) and/or 50 μM Z-VAD.fmk (right panels only), followed by determination of the fall of the ΔΨm (measured as a reduction of the red fluorescence emitted by the ΔΨm-sensitive dye JC-1), mitochondrio-nuclear, and mitochondrio-cytosolic translocation of AIF or Cyt-c respectively (determined by immunostaining), proteolytic activation of caspase-3 (detected with an antibody specific for active caspase-3), or nuclear morphology (detected with Sytox-green). Individual cells demonstrated in A are shown after 8 h (wild-type) or 24 h (Apaf-1−/−, caspase-3−/−) of treatment with STS and are representative for the dominant phenotype. The chromatin condensation of wild-type cells cultured with STS was regarded as stage II of nuclear apoptosis, whereas Apaf-1−/− or caspase-3−/− cells manifest a stage I pattern of chromatin condensation. Kinetic analyses of apoptotic parameters induced by STS (▵), etoposide (□), cisplatin (▪), arsenite (○) and/or Z-VAD.fmk (right panels) are shown in B. This experiment has been repeated three times, yielding comparable results. Electron microscopy (C), large scale DNA fragmentation (D), and oligonucleosomal DNA fragmentation (E) of cells treated as in A are also shown.
Figure 1
Figure 1
Mitochondrial and nuclear features of apoptosis in Apaf-1−/− and caspase-3−/− cells. MEFs from control mice (wild-type), Apaf-1−/−, or caspase-3−/− knockout mice were treated for 24 h with 2 μM STS (A and B), 100 μM etoposide, 150 μM cisplatin, 50 μM arsenite (B) and/or 50 μM Z-VAD.fmk (right panels only), followed by determination of the fall of the ΔΨm (measured as a reduction of the red fluorescence emitted by the ΔΨm-sensitive dye JC-1), mitochondrio-nuclear, and mitochondrio-cytosolic translocation of AIF or Cyt-c respectively (determined by immunostaining), proteolytic activation of caspase-3 (detected with an antibody specific for active caspase-3), or nuclear morphology (detected with Sytox-green). Individual cells demonstrated in A are shown after 8 h (wild-type) or 24 h (Apaf-1−/−, caspase-3−/−) of treatment with STS and are representative for the dominant phenotype. The chromatin condensation of wild-type cells cultured with STS was regarded as stage II of nuclear apoptosis, whereas Apaf-1−/− or caspase-3−/− cells manifest a stage I pattern of chromatin condensation. Kinetic analyses of apoptotic parameters induced by STS (▵), etoposide (□), cisplatin (▪), arsenite (○) and/or Z-VAD.fmk (right panels) are shown in B. This experiment has been repeated three times, yielding comparable results. Electron microscopy (C), large scale DNA fragmentation (D), and oligonucleosomal DNA fragmentation (E) of cells treated as in A are also shown.
Figure 1
Figure 1
Mitochondrial and nuclear features of apoptosis in Apaf-1−/− and caspase-3−/− cells. MEFs from control mice (wild-type), Apaf-1−/−, or caspase-3−/− knockout mice were treated for 24 h with 2 μM STS (A and B), 100 μM etoposide, 150 μM cisplatin, 50 μM arsenite (B) and/or 50 μM Z-VAD.fmk (right panels only), followed by determination of the fall of the ΔΨm (measured as a reduction of the red fluorescence emitted by the ΔΨm-sensitive dye JC-1), mitochondrio-nuclear, and mitochondrio-cytosolic translocation of AIF or Cyt-c respectively (determined by immunostaining), proteolytic activation of caspase-3 (detected with an antibody specific for active caspase-3), or nuclear morphology (detected with Sytox-green). Individual cells demonstrated in A are shown after 8 h (wild-type) or 24 h (Apaf-1−/−, caspase-3−/−) of treatment with STS and are representative for the dominant phenotype. The chromatin condensation of wild-type cells cultured with STS was regarded as stage II of nuclear apoptosis, whereas Apaf-1−/− or caspase-3−/− cells manifest a stage I pattern of chromatin condensation. Kinetic analyses of apoptotic parameters induced by STS (▵), etoposide (□), cisplatin (▪), arsenite (○) and/or Z-VAD.fmk (right panels) are shown in B. This experiment has been repeated three times, yielding comparable results. Electron microscopy (C), large scale DNA fragmentation (D), and oligonucleosomal DNA fragmentation (E) of cells treated as in A are also shown.
Figure 2
Figure 2
Microinjection of apoptosis-regulatory proteins into Apaf-1−/− and caspase-3−/− cells. MEFs from control mice (wild-type), Apaf-1−/−, or caspase-3−/− knockout mice were microinjected with the indicated protein (5 μM AIF, 5μM AIFΔ1-351, 25 μM Cyt-c 1U/μl caspase-3, 250 nM ICAD/CAD protein, or 250 nM activated CAD), followed by staining with Hoechst 33342 (blue fluorescence) and the ΔΨm-sensitive dye CMXRos (red fluorescence). Representative phenotypes obtained 2 h after injection are shown in A. Mean values ± SD of three independent experiments are shown in B. Note that two stages (I and II) of nuclear chromatin condensation can be distinguished. Stage I resembles the nuclear apoptosis of Apaf-1 −/− cells (Fig. 1A and Fig. c), whereas stage II corresponds to that observed in apoptotic wild-type cells stimulated with STS (Fig. 1A and Fig. c).
Figure 3
Figure 3
Apoptotic nuclear features induced by AIF and CAD in a cell-free system. HeLa nuclei were exposed to 200 nM AIF (120 min), 250 nM activated CAD (recombinant CAD/ICAD protein digested with caspase-3 inhibited or not with Ac-DEVD.fmk; 15 or 120 min), followed by staining with Hoechst 33342 (A) or electron microscopy (B). (C–E) In addition, nuclei were left untreated 1 or incubated for 120 min with: 200 nM AIF 2; AIF and 30 μM of PCMPS 3; AIF and 500 nM ICAD 4; caspase-3 5; 250 nM CAD–ICAD 6; 250 nM activated CAD 7; CAD–ICAD treated with 50 μM of Ac-DEVD.fmk before addition of caspase-3 8; CAD–ICAD treated with 50 μM of Ac-DEVD.fmk after activation of CAD by caspase-3 9; active CAD and ICAD 10; and active CAD and PCMPS. This was followed by staining with propidium iodide and flow cytometric determination of the frequency of hypoploid nuclei (C; X ± SD of four experiments), determination of large scale DNA fragmentation (D) and assessment of oligonucleosomal DNA fragmentation (E). (F) The kinetics of large scale DNA fragmentation induced by CAD (100 nM) was determined at different time points: nuclei untreated 1; incubated for 5 min 2; incubated for 15 min 3; incubated for 30 min 4; or incubated for 60 min 5. (G) Oligonucleosomal DNA fragmentation was assessed after exposure of control nuclei 1 to AIF alone (200 nM; 120 min; 2), CAD alone (250 nM; 120 min; 3), or after sequential exposure of nuclei, first to AIF, then to CAD 4.
Figure 4
Figure 4
Inhibition of AIF and CAD reveals two parallel pathways of nuclear apoptosis. (A) HeLa nuclei were left untreated (control nuclei) or incubated for 2 h with cytosolic extracts obtained from untreated cells (Control) or cytosols (CS) from STS-treated wild-type MEFs, followed by staining with propidium iodide and flow cytometric quantitation of DNA content. Extracts were subjected to immunodepletion of AIF, sham immunodepletion, and/or addition of recombinant ICAD protein (500 nM). (B) Comparison of cytosols obtained from wild-type, Apaf-1−/−, and caspase-3−/− MEFs stimulated with three different apoptosis inducers (STS, etoposide, and cisplatin). Cytosols were evaluated for their capacity to induce nuclear DNA loss in purified HeLa nuclei (quantitated as in A) after depletion of AIF and/or addition of ICAD, as indicated. Cytosols from untreated control cells induced ≥10% of nuclear apoptosis. Results are means of triplicates (X ± SD) and are representative of three independent determinations.
Figure 5
Figure 5
Scenario for the different phases of nuclear Apoptosis. AIF causes large scale chromatin fragmentation and a peripheral chromatin condensation (stage I). CAD causes a more advanced oligonucleosomal DNA fragmentation and DNA condensation (stage II).
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