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. 2000 Dec 19;97(26):14376-81.
doi: 10.1073/pnas.97.26.14376.

An alternative, nonapoptotic form of programmed cell death

S Sperandio  1 , I de Belle, D E Bredesen
Affiliations

An alternative, nonapoptotic form of programmed cell death

S Sperandio et al. Proc Natl Acad Sci U S A. .

Abstract

The term apoptosis often has been used interchangeably with the term programmed cell death. Here we describe a form of programmed cell death that is distinct from apoptosis by the criteria of morphology, biochemistry, and response to apoptosis inhibitors. Morphologically, this alternative form of programmed cell death appears during development and in some cases of neurodegeneration. Despite its lack of response to caspase inhibitors and Bcl-x(L), we show that this form of cell death is driven by an alternative caspase-9 activity that is Apaf-1-independent. Characterization of this alternative form of programmed cell death should lead to new insight into cell death programs and their roles in development and degeneration.

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Figures

Figure 1
Figure 1
The IGFIR-IC induces cell death. (A) Comparison between the cell death induced by pIGFIR-IC and nonmyristylated form of the IC domain and deletion mutants. Asterisks indicate a highly significant difference from the control as determined by one-way ANOVA (F = 63.3; P < 0.0001); P < 0.0001 for pIGFIR-IC and pIGFIR-ICΔ1264–1276, Bonferroni/Dunn posthoc test. Error bars represent the SEM from three independent experiments. (B) Light microscopy, using Hoffman optics, demonstrating morphological changes induced by IGFIR-IC. IGFIR-IC expression caused a rounding of the cells and intracellular vacuoles. (C) Ultrastructural characteristics of IGFIR-induced cell death. 293T cells transfected with control empty vector pcDNA3 (a), pC9 (b), or pIGFIR-IC (c and d). Although caspase-9-transfected cells displayed characteristic features of apoptosis, including chromatin condensation, IGFIR-IC-transfected cells did not. In contrast, extensive cytoplasmic vacuolization was observed in the absence of nuclear fragmentation, cellular blebbing, or apoptotic body formation. Note that the cell shown in b displays both the chromatin condensation characteristic of apoptosis and membrane disruption characteristic of necrosis, suggesting secondary necrosis after apoptosis. (Original magnification: ×6,000.)
Figure 2
Figure 2
IGFIR-IC-induced cell death is nonapoptotic. (A) Internucleosomal DNA fragmentation of cells transfected as in Fig 1. Soluble DNA was extracted from cells 24 or 48 h after transfection. Whereas the Bax-transfected cells demonstrated internucleosomal DNA fragments, the chromatin in IGFIR-IC transfected cells remained intact at 24 and 48 h after transfection. (B) TUNEL staining of cells transfected as in A. Cells were assayed 48 h after transfection. (Upper) FITC staining of the TUNEL labeling. Bax, but not IGFIR-IC, transfected cells show TUNEL-positive staining. (Lower) The same fields of cells stained with propidium iodide (PI). Cells were permeabilized before staining. Similar results were obtained at 24 h. (C) Transcription and protein synthesis are required for IGFIR-induced cell death. Actinomycin D (Act.D) or cycloheximide (CHX) (numbers represent μg/ml) were added 8.5 h after the transfections, and the medium was replaced after 12 h with fresh medium without drugs. Floating cells were collected after 24 h and counted in the presence of trypan blue. Control samples were treated with solvent only (ethanol 0.05%). The expression of IGFIR-IC at the time of administration of the drugs was comparable to the expression at 24 h, as assessed by Western blot analysis. The error bars represent the SEM of four independent experiments. Within the samples transfected with pIGFIR-IC, the asterisks indicate a highly significant difference (P = 0.0011 for 0.25 μg/ml CHX, and P < 0.0001 for 0.5 μg/ml CHX, and 0.5 μg/ml and 1 μg/ml Act.D; Bonferroni/Dunn posthoc test) from the pIGFIR-IC transfected sample treated with solvent only (EtOH), as determined by one-way ANOVA (F = 20.727; P < 0.0001).
Figure 3
Figure 3
IGFIR-IC-induced cell death is blocked by a catalytic mutant of pro-caspase-9. (A and B) 293T cells were transfected with 2 μg of DNA consisting of either pcDNA3, 0.5 μg pIGFR-IC + 1.5 μg pcDNA3, or 0.5 μg pIGFR-IC + 1.5 μg dominant negative caspase-9 (pC9DN, in A) or dominant negative caspase-7 (pC7DN, in B) expression constructs. Forty-eight hours after transfection, dead cells were counted. Asterisks denote a highly significant difference from the control as determined by one-way ANOVA. In A, F = 25.2, P = 0.0002 with P = 0.0001 for pIGFIR-IC (Bonferroni/Dunn posthoc test). In B, F = 118.487, P < 0.0001, with P < 0.0001 for pIGFIR-IC and pIGFIR-IC + pC7DN (Bonferroni/Dunn posthoc test). Error bars represent the SEM from three independent experiments. (C) Protein extracts from cells cotransfected with pIGFR-IC wild type or mutants, as indicated, and FLAG-tagged C9DN expression constructs were subjected to immunoprecipitation (IP) using an anti-IGFIR polyclonal antibody. Immunoprecipitates then were resolved by 15% SDS/PAGE, and Western blotting was performed by using a mAb against the FLAG epitope to detect caspase-9. WCE = whole-cell extract.
Figure 4
Figure 4
Caspase-9 induces both apoptosis and nonapoptotic cell death. (A) 293T cells were transfected with the indicated pcDNA3-based caspase zymogen expression constructs. The general caspase inhibitor BAF was added at 50 μM final concentration. Cell death was assayed 48 h after transfection. Although BAF was able to inhibit caspase-7-induced cell death completely, it only inhibited caspase-9-induced cell death partially (30%). Asterisks indicate a highly significant difference from the control as determined by one-way ANOVA analysis (F = 34.624, P < 0.0001), P < 0.0001 for pC9, P = 0.0002 for pC7, and P = 0.0001 for pC9 + BAF (Bonferroni/Dunn posthoc test). Error bars represent the SEM from three independent experiments. (B) Light microscopy of 293T cells transfected with control vector (pcDNA3), pro-caspase-9 alone (pC9), or in the presence of 50 μM BAF (pC9 + BAF). Cells were photographed 24 h after transfection. Expression of pro-caspase-9 resulted in the induction of a typical apoptotic phenotype, including cell shrinkage and the formation of apoptotic bodies (Middle). BAF suppressed the apoptotic phenotype, but cells displayed a vacuolated morphology similar to that observed with IGFIR-IC-induced cell death (Bottom, arrowheads). (C) Internucleosomal DNA fragmentation of 293T cells transfected with the indicated pro-caspase expression constructs alone or in the presence of 50 μM BAF. Internucleosomal DNA fragmentation was completely inhibited by BAF. (D) TUNEL staining of 293T cells 48 h after transfection with the indicated constructs. Cells were stained as described in Fig. 2B. (E) Electron micrograph of 293T cells transfected with an expression construct for pro-caspase-9 in the presence of zVAD.fmk (40 μM). Morphology similar to IGFIR-IC-induced cell death was observed, including cytoplasmic vacuolation and the absence of chromatin condensation, nuclear fragmentation, and apoptotic body formation. (Original magnification: ×6,000.)
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Comment in

  • More than one way to go.
    Wyllie AH, Golstein P. Wyllie AH, et al. Proc Natl Acad Sci U S A. 2001 Jan 2;98(1):11-3. doi: 10.1073/pnas.98.1.11. Proc Natl Acad Sci U S A. 2001. PMID: 11136242 Free PMC article. No abstract available.

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