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Originally published In Press as doi:10.1074/jbc.M408924200 on September 9, 2004

J. Biol. Chem., Vol. 279, Issue 46, 48404-48409, November 12, 2004
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Autophagy Gene Disruption Reveals a Non-vacuolar Cell Death Pathway in Dictyostelium*

Artemis Kosta{ddagger}§, Céline Roisin-Bouffay{ddagger}||, Marie-Françoise Luciani{ddagger}, Grant P. Otto**, Richard H. Kessin**, and Pierre Golstein{ddagger}{ddagger}{ddagger}

From the {ddagger}Centre d'Immunologie INSERM/CNRS/Université de la Mediterranée de Marseille-Luminy, Case 906, Avenue de Luminy, 13288 Marseille Cedex 9, France and the **Department of Anatomy and Cell Biology, Columbia University, New York, New York 10032

Received for publication, August 4, 2004 , and in revised form, August 11, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Types of cell death include apoptosis, necrosis, and autophagic cell death. The latter can be defined as death of cells containing autophagosomes, autophagic bodies, and/or vacuoles. Are autophagy and vacuolization causes, consequences, or side effects in cell death with autophagy? Would control of autophagy suffice to control this type of cell death? We disrupted the atg1 autophagy gene in Dictyostelium discoideum, a genetically tractable model for developmental autophagic vacuolar cell death. The procedure that induced autophagy, vacuolization, and death in wild-type cells led in atg1 mutant cells to impaired autophagy and to no vacuolization, demonstrating that atg1 is required for vacuolization. Unexpectedly, however, cell death still took place, with a non-vacuolar and centrally condensed morphology. Thus, a cell death mechanism that does not require vacuolization can operate in this cell death model showing conspicuous vacuolization. The revelation of non-vacuolar cell death in this protist by autophagy gene disruption is reminiscent of caspase inhibition revealing necrotic cell death in animal cells. Thus, hidden alternative cell death pathways may be found across kingdoms and for diverse types of cell death.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Three main forms of cell death have been described, caspase-dependent apoptosis, and caspase-independent necrosis autophagic/vacuolar cell death. The latter, also called Type II cell death, can be operationally defined as the death of cells containing autophagosomes, autophagic bodies, and/or vacuoles. Numerous instances of autophagic/vacuolar cell death have long been known (1–3) and were recently reviewed (4–9). They include certain Caenorhabditis elegans mutants with neuronal degeneration (10), developmental circumstances such as death upon insect metamorphosis of intersegmental muscle cells (11–13) of given motoneurons (7) or of salivary gland cells (14), and several pathological circumstances, especially neurological ones in human (5).

Outside the cell death field, autophagy is induced, for instance, by starvation (15–17). Autophagy helps ensure, at least for some time, the survival of cells deprived of extracellular nutrients or encumbered with aggregates of polyglutamine proteins (18). The relationship between autophagy and autophagic/vacuolar cell death is not completely clear. How to reconcile the role of autophagy in ensuring cell survival and its apparent role within a mechanism of cell death? A possibility is that autophagy leads to the disappearance of crucial cellular organelles, resulting in death. Thus, when some cells were induced to undergo cell death in the presence of caspase inhibitors and then returned to normal conditions, they survived for several days, but during this period their mitochondria disappeared. Bcl2 overexpression blocked this removal of mitochondria (19). Perhaps related, autophagic stimulation of rat hepatocytes by serum deprivation and glucagon (1 M) caused an increase of spontaneously depolarizing mitochondria, which moved into acidic vacuoles (20). Alternatively, autophagy may be irrelevant to the mechanism of death proper but instead be a consequence, perhaps aiming at clearing dead cell remains as effected by engulfment in other circumstances. In short, it is often not clear whether autophagy is a cause, a consequence, or an irrelevant side effect of autophagic mammalian cell death. A point at stake is whether control of autophagy in cells would help control some types of programmed cell death, for instance in pathological circumstances.

Although classical biological models of cell death have provided invaluable information, especially on mechanisms of apoptosis, alternative models might provide further information, in particular on non-apoptotic cell death mechanisms (21). Specifically, questions on autophagic/vacuolar cell death could be explored in Dictyostelium (22) or Podospora (23, 24). The Dictyostelium HMX44A cell line could be induced to undergo as a monolayer (25) autophagic vacuolar cell death (22, 26) in a caspase- and paracaspase-independent manner (27, 28). In the course of this monolayer cell death, believed to mimic Dictyostelium developmental cell death (29), cells undergo a paddle cell stage, round up through actin depolymerization while acquiring a cellulose shell, and develop a large vacuole that occupies most of the cell volume. Finally, their cytoplasmic membrane ruptures (22, 26).

Induction of Dictyostelium cell death in this monolayer system requires both starvation and addition of differentiation-inducing factor (DIF),1 a small dichlorinated molecule that is a main natural morphogen in Dictyostelium (25). Although other strains produce DIF upon starvation, HMX44A cells produce very little DIF and thus require addition of exogenous DIF for differentiation. Using this strain thus allows us to investigate the respective roles of starvation and DIF in cell death (25). Adding only DIF, in rich medium, has no detectable effect. Applying only starvation does not lead to vacuolar cell death while triggering autophagy, showing that autophagy is not sufficient to lead to cell death in this system (26). In the presence of DIF, is starvation required because autophagy is required, or is starvation required for a reason other than autophagy? Is autophagy necessary at all in this autophagic vacuolar cell death?

The occurrence and the course of autophagy are governed by a number of genes studied mostly in yeast but conserved throughout the eukaryotes (17, 30–33), in particular in Dictyostelium (32, 34). These genes are now referred to as atg for autophagy-related genes (33). In yeast, the protein kinase Atg1 is believed to play a role in early events of autophagosome formation (see Ref. 35 and references therein).

We inactivated by homologous recombination the atg1 gene in Dictyostelium strain HMX44A cells and studied the impact of this inactivation on the cell death process. We found that autophagy was decreased and vacuolization was suppressed, but unexpectedly cell death still took place, with a different, "centrally condensed" morphotype. This demonstrated the existence of a cell death mechanism in Dictyostelium that did not require vacuolization. Also, the autophagy gene inactivation leading to condensed cell death described in this report is curiously similar to the previously reported inhibition of apoptosis leading to necrotic cell death (36–39). These results have therapeutic, phylogenetic, and mechanistic implications.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells, Cell Culture, and Microscopy—For cell death induction, unless stated otherwise Dictyostelium vegetative cells in growth phase were washed once and resuspended in HL5 medium at a concentration of 2 x 105 cells/ml. One ml of this cell suspension was distributed in each well of Lab-Tek culture chambers (155380; Nalge Nunc, Naperville, IL). After overnight incubation, HL5 was discarded, and each well received 1 ml of phosphate-buffered saline, pH 6.7 (Soerensen buffer, SB) containing 3 mM cAMP. After 8 h of incubation at 22 °C in SB and cAMP, cells were washed once in SB and incubated at 22 °C in either SB or SB in the presence of the differentiation factor DIF-1 (D-3450; Interchim, Montluçon, France) at a final concentration of 10-7 M. Further details on Dictyostelium strains, culture, microscopy and image processing, double labeling with phalloidin and CMXRos, simple labeling with calcofluor, fluorescein diacetate, or propidium iodide, electron microscopy methods, and evaluation of cell regrowth have been previously described (22). Cells were stained with 10 µg/ml acridine orange (A-6014; Sigma) or with 20 µg/ml Bodipy 493/503 (D-3922; Molecular Probes) for 30 min, washed, and left in SB for 30 min and then observed using a fluorescein filter, or with 40 nM Dioc6(3) (D273; Molecular Probes) for 15 min, washed, and then observed using a fluorescein filter, or with 2.5 µg/ml Nile Red (N-3013; Sigma) in buffer (50 mM trizma, pH 7.5, (T4628; Sigma), containing 20 mg/ml of polyvinylpyrrolidone, WT 40,000, (PVP-40; Sigma)) for 5 min, and observed using a fluorescein filter without removing the staining buffer, or with 10 µM Lysosensor Yellow/Blue (DND-160; Molecular Probes) for 3 min, washed in SB, and examined using a DAPI filter for a very short time because of marked cell destruction under fluorescent light.

atg1 Gene Inactivation by Homologous Recombination—The homologous recombination constructs for the deletion allele atg1–2 included 5' and 3' arms obtained separately by PCR from genomic DNA. Restriction sites were generated at the ends of the PCR products by inclusion of appropriate sequences in the primers. For the atg1–2 deletion (302 bp), the 5' arm (1082 bp) was from nt -673 to nt +409, and the 3' arm (963 bp) from nt +712 to nt +1675 of the atg1 gene. The 5' and 3' arms were used as templates within the same PCR reactions to obtain a 2045-bp sequence. This sequence was ligated into pGEM-T Easy (Promega). The blasticidin resistance cassette was removed from pBSR519 with BamHI and cloned into the BamHI site of the above sequence. The resulting deletion construct and the previously obtained insertion construct (40) were used for electroporation. Transformants were cloned the next day and selected with blasticidin (10 µg/ml) in HL5 medium for 10 days. Transformants were screened by PCR for homologous recombination of the insertion or deletion constructs with the endogenous atg1 locus. For Northern blot analysis, cells were deposited on nitrocellulose filters under starvation conditions for development, and the filters were harvested after 8 h for RNA extraction. Total RNA (5 µg) of each cell type was size-fractionated on a 1.2% agarose gel, transferred to nitrocellulose, and hybridized with a random primer-labeled DNA probe.

Transfections for Complementation or Dominant Negative—The CFP-Atg1 expression vector was described previously (40). For the Atg1 expression vector, the atg1 insert was extracted from pDXA-CFP-Atg1 with SacI and ligated into a SacI-digested pDneo2 vector. Expression of both CFP-Atg1 and Atg1 was driven by an actin 15 promoter. 20 µg of construct DNA was electroporated in HMX44A or HMX44A.atg1–1 cells. Selection was with neomycin (20–40 µg/ml).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Insertional Disruption of the atg1 Gene in Dictyostelium HMX44A Cells Abolished Vacuolization, but Not Cell Death— When HMX44A cells were incubated in starvation medium, but in the absence of exogenous DIF, they flattened and did not die (Fig. 1A). If DIF was added, they underwent a programmed sequence of cell death events (22, 26), including differentiation into paddle cells (Fig. 1B, 16 h), rounding, vacuolization (Fig. 1B, 40–64 h), and membrane rupture (22, 26).



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  		FIG. 1.
The impact of atg1 inactivation on Dictyostelium cell death. Wild-type Dictyostelium HMX44A (A, B) and HMX44A.atg1–1 (C, D) cells were incubated for 8 h in cAMP in SB and then washed and further incubated in SB alone (A, C) or in SB and DIF (B, D). Within each of the four conditions, smaller panels show phase contrast microscopy of cells after 16, 40, or 64 h of incubation after the post-cAMP wash. Note in the presence of DIF the shift in cell death type from vacuolar for wild-type cells (B) to non-vacuolar with central condensation for atg1–1 cells (D).

 
We disrupted the atg1 autophagy gene in HMX44A cells using a previously described insertion construct (40). The corresponding mutant cells are designated throughout as HMX44A.atg1–1 or atg1–1. Upon starvation without addition of exogenous DIF, HMX44A.atg1–1 cells adopted a round shape (Fig. 1D and Fig. 2) and showed the expected (40) impaired autophagy compared with HMX44A wild-type cells. The latter showed yellow-green dots when stained with Lysosensor Yellow Blue and bright orange dots when stained with acridine orange (Fig. 2, A and B), which are believed to represent acidic vacuoles (41, 42). Also, upon staining with Bodipy 493/503 or with Nile Red, bright yellow dots could be seen (Fig. 2, C and D), which are believed to label lipidic bodies appearing upon stress (43, 44). All of these stained small acidic vacuoles or lipidic bodies were less numerous in HMX44A.atg1–1 cells (Fig. 2, E–H). Also, most of the electron microscopy sections of starved cells showed at least one large autophagosome for HMX44A cells and none for HMX44A.atg1–1 cells (Fig. 2, I and J). Although these data show altered autophagy, it is difficult to assess whether in these cells autophagy is only partially or fully impaired.



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  		FIG. 2.
atg1 inactivation in Dictyostelium HMX44A cells affects autophagy. HMX44A (A–D) or HMX44A.atg1–1 (E–H) cells were incubated first with cAMP in SB and then for 17–22 h in SB alone. They were then stained with Lysosensor Yellow Blue (A, E), acridine orange (B, F), Bodipy 493/503 (C, G), or Nile Red (D, H). atg1–1 cells were consistently less labeled than their wild-type counterparts. Electron micrographs of starved cells showed most frequently the presence of large autophagosomes in HMX44A cells (I) and their absence in HMX44A.atg1–1 cells (J). The insert in panel I shows details of a large autophagosome, including its double membrane. Horizontal scale bars represent 1 µm for panels I and J and 0.1 µm for the insert.

 
In the presence of DIF and in contrast to wild-type cells, the atg1–1 cells did not show any vacuolization (Fig. 1D) even after 96 h in DIF (not shown). We infer from these results that atg1 gene disruption suppresses vacuolization. We do not know whether this is through impaired autophagy. Although not vacuolizing, atg1–1 cells subjected to both starvation and DIF nevertheless died. Not only were they round, but they also showed central condensation (Fig. 1D), giving them a characteristic "fried egg" appearance. With time, these cells tended to swell, and finally their external membrane ruptured (Fig. 1D and not shown).

To further study the relationships between atg1, vacuolization, and cell death, we created an atg1 deletion mutant in HMX44A cells that we call atg1–2 (Fig. 3A). This new mutant showed the same cell death mutant phenotype as did the original insertion mutant, with no vacuolization and with central condensation of organelles (Fig. 3C). The insertion is in the 9th of the 11 subdomains of the atg1 catalytic protein kinase domain as defined in the homologous yeast atg1 (45). The deletion removes three such subdomains, including two conserved residues whose mutation abolishes the protein kinase activity (45). The same non-vacuolar cell death pattern was also observed (not shown) in monolayer tests of the previously obtained atg1–1 insertion mutant in strain DH1 (40). Non-vacuolar cell death was thus observed in two strain backgrounds and with two distinct atg1 inactivation constructs. In addition, transformation of HMX44A cells with the previously described CFP-Atg1 expression vector (40), but not with an Atg1 expression vector, led to a very similar phenotype of non-vacuolar cell death (Fig. 3C), most probably through a dominant negative effect of CFP-Atg1 in this case. These multiple independent instances of atg1 alteration abolishing vacuolization, but not cell death, strongly indicated an atg1 requirement for vacuolization. Furthermore, transformation with an Atg1 expression vector of the atg1–1 insertion mutant led to mutant complementation, namely recovery of the usual vacuolar pattern of Dictyostelium cell death (Fig. 3C). The identity of, in particular, the dominant negative and the complemented cells was counterchecked by PCR (Fig. 3D) and resistance to blasticidin and/or neomycin (not shown). Complementation formally demonstrated that atg1 is required for vacuolization.



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  		FIG. 3.
Cell death, vacuolization, and atg1 disruption, deletion, and complementation. A, structure of the Dictyostelium atg1 gene. Nt 1 corresponds to the first nucleotide of the codon for the start methionine of the 2329-nt Dictyostelium atg1 gene (accession number AY191011 [GenBank] ). The insertion and deletion are as indicated under"Experimental Procedures."B, Northern blot of 8-h-starved HMX44A (lane 1), HMX44A.atg1–1 insertion (lane 2), and HMX44A.atg1–2 deletion (lane 3) cells. Upper panel, a filter probed with a 443-bp EcoRI/HincII fragment corresponding to the 5' end of the atg1 gene. Lower panel, a gel photograph of ethidium bromide-stained RNA before transfer. Lanes 1–3 were loaded with 5 µg of total RNA. The approximate length of atg1 transcripts, indicated by arrows, are 2.4 kb for the wild-type, 1.2 kb for the insertion mutant, and 1.7 kb for the deletion mutant. The residual transcripts in cells bearing the insertion or deletion result from transcription of the 5'-most atg1 sequences and of part of the inserted blacticidin-resistance cassette. C, cell death phenotype of cells of HMX44A origin after 16–17 h of incubation in starvation buffer and DIF. Wild-type, untransfected. Insertion, HMX44A.atg1–1. Deletion, HMX44A. atg1–2. Dom. neg., dominant negative, HMX44A transfected with CFP-Atg1 (the same mutant phenotype was obtained in each of several clones of two independent transformations; in contrast, transformation with atg1 did not modify the wild-type phenotype, not shown). Complem., complementation, mutant HMX44A.atg1–1 cells transfected with Atg1, yielding back the wild-type phenotype. D, PCR confirmation of the identity of three HMX44A (1–3) and three HMX44A.atg1–1 (4–6) cell populations, untransformed (lanes 1 and 4) or transformed with an Atg1 (lanes 2 and 5) or a CFP-Atg1 expression vector (lanes 3 and 6). The primer pairs were in atg1 on each side of the blasticidin resistance cassette (upper panel) or in atg1 and in this cassette (lower panel). All groups showed the expected pattern, taking into account that when two bands are expected, the shorter one prevails. The dominant negative (lane 3) and the atg1–1 mutant (lane 4), which have the same non-vacuolar cell death phenotype, show distinct PCR patterns. The wild-type cells (lane 1) and the complemented mutant (lane 5), which have the same vacuolar cell death phenotype, also show distinct PCR patterns.

 
Importantly, in all these experiments DIF addition induced death of both wild-type and atg1 mutant starved cells. The state of atg1 affected the morphology (with vacuoles or with central condensation), not the occurrence, of this DIF-induced cell death.

General Features of Non-vacuolar atg1–1 Mutant Cell Death—A closer examination of dying atg1–1 cells by differential interference contrast microscopy (Fig. 4A) and electron microscopy (Fig. 4B) showed that central condensation reflected clustering of cell organelles, including the nucleus, in the central region of cells. The nucleus did not show obvious condensation or fragmentation at these stages (Fig. 4, B and D). Staining with the mitochondrial stains CmxRos or Dioc6(3) further highlighted the concentration of organelles toward the center of cells (Fig. 4C). Perinuclear clustering of mitochondria has been reported in several instances of cell signaling often leading to death and/or involving alterations of the cytoskeleton (46–48). In many of these cells, peripheral staining with phalloidin was observed concentric to the central CmxRos-stained condensation, suggesting the presence of peripheral F-actin (Fig. 4C).



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  		FIG. 4.
Some characteristics of non-vacuolar HMX44A.atg1–1 cell death. A, images of the beginning of central condensation by phase contrast (left) or differential interference contrast (right). B, electron micrograph showing central nucleus, perinuclear clustering of mitochondria, and organelle-free periphery. Scale bar, 1 µm. C, top, phase contrast (left) and staining of mitochondria with CMXRos (red) and of F-actin with phalloidin (green) (right). Bottom, phase contrast (left) and staining of mitochondria with Dioc6(3) (right). D, phase contrast (upper left) and staining with fluorescein diacetate (upper right), propidium iodide (bottom left) or calcofluor (bottom right). After 16–17 h (A and B) and after 23 h (C and D) in SB and DIF. Filled arrow, nucleus. Open arrow, mitochondria.

 
Starvation conditions alone led to rupture of cytoplasmic membranes in a small proportion of cells (Fig. 1C). This occurred significantly earlier and at higher frequency in the presence of DIF (Fig. 1D), as confirmed by time lapse video microscopy (not shown). Although many cells with central condensation were initially viable as measured by fluorescein diacetate labeling, with time an increasing proportion of these cells showed nuclear staining by propidium iodide (Fig. 4D), indicative of membrane rupture. Some dying cells were calcofluor-positive (Fig. 4D), showing that atg1–1 cells could synthesize cellulose, but less efficiently than wild-type cells (22).

Non-vacuolar cell death was quantified through 1) an increase in percentage of propidium iodide-positive cells (Table I), 2) a decrease in numbers of plaque-forming cells on bacterial lawns (not shown), 3) a decrease in cell numbers upon regrowth as cell populations in rich medium (Table II), and 4) a decrease in the numbers of clone-forming cells in limiting dilution tests in rich medium (not shown). Of note, in each of these tests using either cell membrane rupture or loss of clonogenicity as cell death criteria, DIF induced cell death in both starved wild-type cells with vacuolization and atg1–1 cells without vacuolization, and the proportions of dead cells were similar. Also, membrane permeabilization was similarly delayed compared with growth inhibition in both the vacuolar death of HMX44A cells (22, 26) and the non-vacuolar death of atg1 mutant cells (compare Tables I and II). The main difference between death of wild-type or atg1–1 cells was not the proportions or kinetics of cell death but vacuolar or condensed morphology of the dying cells.


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  		TABLE I
atg1–1 mutant cell death measured by membrane permeabilization

 


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  		TABLE II
atg1–1 mutant cell death measured by growth inhibition

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The main finding in this report was that atg1 autophagy gene inactivation in an autophagic vacuolar developmental cell death model suppressed vacuolization, but not death. When subjected to starvation and DIF, wild-type HMX44A cells vacuolized and died, whereas mutant HMX44A.atg1 cells instead underwent a non-vacuolar type of cell death showing central condensation with organelle clustering. From a mechanistic point of view, the cellulose synthase and atg1 mutations have been found to prune cell death of apparently non-causal traits such as acquisition of a cellulose shell (22) and of a vacuole (this report). These two most conspicuous signs of Dictyostelium cell death thus turn out not to be required for cell death.

For DIF induction of Dictyostelium cell death, starvation is required. Starvation induces autophagy. Is autophagy sufficient for DIF-induced vacuolar cell death? Indeed, electron microscopy studies indicated that in developing Dictyostelium vacuoles may be related to autophagy (49–53). However, the presence of large vacuoles cannot be just because of an autophagic process secondary to incubation in starvation medium (as observed in some mutant strains of yeast cells) (16), because HMX44 cells starved without DIF do not show such large vacuoles and do not die. Thus, starvation is not sufficient for induction of DIF-induced vacuolar cell death. Is autophagy required for DIF-induced vacuolar cell death? We have demonstrated here that in Dictyostelium the atg1 autophagy gene is required for autophagy and for vacuolization in cell death. It follows that the autophagy machinery is most probably necessary for vacuolization in cell death. However, it does not follow that the autophagy machinery is required for cell death itself (see below for discussion on one or two mechanisms of cell death). Both starvation and DIF are required for induction of cell death, in particular when atg1 is inactivated and there is little or no autophagy. Thus, some step that is starvation-triggered but distinct from autophagy may be required together with DIF for induction of cell death. We do not know what this step is.

Are there one or two mechanisms for Dictyostelium cell death? The simplest interpretation of the results presented in this report is that Dictyostelium developmental cell death follows only one non-vacuolar causal mechanism. In this "one mechanism" interpretation, autophagy is a consequence of starvation and may be required for vacuolization, but the latter is merely a side effect with regard to cell death, a decoration of a single underlying mechanism of cell death. This would be cell death with, not by, autophagy and vacuolization. Alternatively, two distinct mechanisms of cell death might exist in Dictyostelium, one of which requires an intact atg1 gene and vacuolization and a second one that does not. The difference in phenomenology would reflect a difference in mechanisms. In the atg1-dependent mechanism, cell death might be vacuole-executed as discussed for some instances of plant cell death (54). atg1 alteration would reveal the other, non-vacuolar mechanism. Both interpretations imply the existence of a non-vacuolar mechanism of cell death, the unknown molecular basis of which may be amenable to analysis utilizing the genetic tractability of Dictyostelium.

Altogether, in this system under starvation conditions in the absence of DIF, autophagy is a survival mechanism, because then wild-type cells survive longer than atg1 cells. When DIF is added, autophagy may be required only for vacuolization or for vacuolization and a mechanism of cell death. The latter would imply that autophagy in this system can be required for both survival and cell death.

At least in this example of cell death with autophagy and vacuolization, atg1 alteration inhibited vacuolization, but not cell death. As recently reported, in some animal cells caspase inhibition can trigger autophagic cell death (55); perhaps reciprocally, autophagy inhibition can trigger apoptosis.2 In previous studies, caspase inhibition inhibited apoptosis, but not cell death, in some animal cells in both developmental (38) and experimental death receptor-mediated (36, 37, 39) circumstances. Upon induction of cell death in these cases, caspase inhibition prevented apoptosis but led to caspase-independent necrosis. Thus, distinct cell death mechanisms such as caspase-dependent apoptosis and caspase-independent necrosis, or atg1-dependent vacuolar and atg1-independent non-vacuolar cell death (in the "two-mechanisms" interpretation) may coexist in the same cells. The present results thus extend the possibility of coexistence of cell death mechanisms within a given cell (36–39) to another kingdom and to other mechanisms. Therefore, this situation may not be rare and should be taken into account systematically and not only in the case of caspase-dependent apoptosis when attempting to trigger or block cell death for therapeutic purposes.

From an evolutionary point of view, the recurrence across different kingdoms of a hierarchical coexistence of different cell death mechanisms within the same cells is of note. Clearly, an alternative cell death mechanism can act as a back-up death-ensuring pathway in case of inhibition of the other pathway by, for example, microorganism-originating inhibitors, which represents a selective advantage. Also, on a very speculative note, the one- or two-mechanism interpretations above (vacuolization as a side effect and vacuolization as a cell death mechanism in its own right) may be sequential in evolution rather than mutually exclusive, a line of reasoning that may extend to caspase-dependent and -independent cell death. This suggests a sequence of events for the successive emergence in evolution of diverse cell death mechanisms. One or more primordial cell death mechanism(s) might have emerged earliest and might persist as underlying caspase-independent and/or atg1-independent cell death. Elements of autophagy or apoptosis would have emerged next, initially as additions to the primordial mechanism(s). Both autophagy and apoptosis help dispose of the dead cell; thus these additions could be conceived as selectively advantageous improvements of the cell death process. Next, autophagy/vacuolization or apoptosis may acquire autonomy and exist in given cells in parallel with a primordial mechanism. Eventually they may acquire exclusivity and remain the only mechanism at play. This would explain, for instance, why caspase inhibition in some instances completely blocks cell death (56), whereas in other instances it reveals a "still present" underlying necrotic mechanism. A similar situation may apply to autophagic vacuolar cell death. In the case of Dictyostelium cell death, although we do not know yet whether autophagy/vacuolization is an addition or an autonomous mechanism, clearly an underlying non-vacuolar mechanism exists.


   FOOTNOTES
 
* This work was supported by institutional grants from INSERM and CNRS and specific grants from Association pour la Recherche contre le Cancer, Ministere de la Recherche et de la Technologie, and the European Community 5th Framework Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by an INSERM poste vert. Back

Both authors contributed equally to this work. Back

|| Supported by the European Community. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Centre d'Immunologie INSERM/CNRS/Université de la Mediterranée, Case 906, Campus de Luminy, Ave. de Luminy, 13288 Marseille Cedex 9, France. Tel.: 33-0-4-91-26-94-68; Fax: 33-0-4-91-26-94-30; E-mail: golstein{at}ciml.univ-mrs.fr.

1 The abbreviations used are: DIF, differentiation-inducing factor; atg1-, bearing a disruption of the autophagy gene 1; SB, Soerensen buffer; nt, nucleotide. Back

2 G. Kroemer, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Jean-Paul Chauvin and Chantal De Chastellier for help with electron microscopy and Jonathan Ewbank for help with the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Schweichel, J.-U., and Merker, H.-J. (1973) Teratology 7, 253-266[CrossRef][Medline] [Order article via Infotrieve]
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