1 CNRS-UMR8125, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France
2 INSERM U487, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France
3 Institut André Lwoff, INSERM U504, 16 avenue Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France
4 Institute of Biochemistry, University of Kiel, 24098 Kiel, Germany
Author for correspondence (e-mail: kroemer{at}igr.fr)
Accepted 20 April 2005
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Summary |
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Key words: Bcl-2, caspases, LAMP1, LAMP2, Lysosomes, Mitochondria
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Introduction |
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Type 2 cell death has been described in clinically relevant circumstances for example, in neurodegeneration, retinal degeneration (Shintani and Klionsky, 2004), bacterial infection (Nakagawa et al., 2004
), and tumor cells succumbing to chemotherapy in vitro (Daido et al., 2004
; Kanzawa et al., 2004
; Opipari et al., 2004
). However, functional reports indicating that silencing of Atg genes can reduce cell death are scarce. Thus, small interfering RNA (siRNA) specific for Atg genes can inhibit the autophagic cell death of L929 cells induced by the pan-caspase inhibitor Z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) (Yu et al., 2004
) as well as that of Bax/Bak/ mouse embryonic fibroblasts (MEF) treated with etoposide (Shimizu et al., 2004
). Nonetheless, there is also evidence that autophagy can exert a cytoprotective function (Alva et al., 2004
; Shintani and Klionsky, 2004
) for example, when the digestion of endogenous macromolecules compensates for the shortage of exogenous metabolites (Kuma et al., 2004
) and/or when damaged organelles including mitochondria have to be removed (Rodriguez-Enriquez et al., 2004
; Teckman et al., 2004
). Thus, in conditions of nutrient depletion, inhibition of autophagy can induce cell death, and this cell death depends on mitochondrial outer membrane permeabilization and subsequent caspase activation (Boya et al., 2005
). The pro-survival effect of autophagy was revealed by inhibiting the formation of autophagic vacuoles for example, by siRNA specific for Atg5, Atg6/Beclin, Atg10 and Atg12 or by the addition of the pharmacological autophagy inhibitor 3-methyladenine (Boya et al., 2005
). In these conditions (that is, starvation + siRNA specific for Atg5, Atg6/Beclin, Atg10 and Atg12), cells directly succumbed to apoptotic cell death, without any signs of autophagic vacuolization (Boya et al., 2005
). However, lysosomotropic agents that reduce the lysosomal pH and/or destabilize lysosomal membranes (such as hydroxychloroquine, bafilomycin A1 or monensin) (Boya et al., 2003b
) inhibited the fusion of lysosomes with autophagic vacuoles and hence produced a different morphotype. In the presence of these lysosomotropic agents, autophagic vacuoles progressively accumulated in the cytoplasm of starving cells, thus producing a morphology that initially resembled autophagic cell death. With prolonged starvation and lysosomal inhibition, such cells acquired features of apoptosis including nuclear pyknosis and karyorhexis (Boya et al., 2005
). This result suggested a possible shift from type 1 cell death to type 2 cell death, yet was based on the utilization of pharmacological agents with limited specificity.
On the basis of these premises, we looked for a genetic manipulation that might block the fusion between autophagic vacuoles and lysosomes in a more specific fashion. As shown here, targeting of LAMP2 can inhibit the fusion of autophagosomes and lysosomes required for the late stage of the autophagic process. Lysosome-associated membrane proteins-1 and -2 (LAMP1, LAMP2) are homologous C-type transmembrane proteins (37% identity in humans) specific for lysosomes. LAMP1-deficient mice are viable and fertile (Andrejewski et al., 1999). However, the knockout of LAMP2 causes embryonic lethality and the massive accumulation of autophagic vacuoles in various tissues (Eskelinen et al., 2002
; Tanaka et al., 2000
). Loss of LAMP2 is also at the etiology of Danon disease, an X-linked lysosomal glycogen storage disease affecting infants or adolescents with a clinical triad of cardiomyopathy, myopathy and mental retardation (Nishino et al., 2000
). LAMP2 loss does not lead to a major perturbation of lysosomal structure and function, except a mild accumulation of cholesterol (Eskelinen et al., 2002
; Tanaka et al., 2000
). The double knockout (DKO) of LAMP1 and LAMP2 causes a decrease in lysosomal density and a change in cholesterol traffic, which is detectable as cholesterol accumulation in late endosomal/lysosomal vesicles in MEF (Eskelinen et al., 2004
).
Here, we show that targeting of LAMP2 expression by RNA interference inhibits the fusion of lysosomes and autophagic vacuoles, thereby increasing the number of autophagic vacuoles in conditions of prolonged nutrient starvation. In these conditions, cells progressively adopt characteristics of type 1 and type 2 cell death. This argues against the formal, sharp distinction between the two death modalities.
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Materials and Methods |
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Transfection and RNA interference
siRNAs for human lamp1 and lamp2 (National Center for Biotechnology Information, accession numbers NM_005561 and NM_002294) were synthesized by Proligo France, or Eurogentec Belgium. For lamp1, the siRNA sequence of the + strand started at position 311 (AGAAAUGCAACACGUUA, LAMP1A) or at position 904 (GGAAUCCAGUUGAAUACAA, LAMP1B). For lamp2 the siRNA sequences were chosen to start at position 64 (GCUGUGCGGUCUUAUGCAU, LAMP2-64) and at position 69 (GCGGUCUUAUGCAUUGGAA, LAMP2-69). As a control, we targeted the nuclear envelope protein Emerin, whose knockdown does not induce any known phenotype (Harborth et al., 2001). The concentrations of LAMP siRNA was 100 nM in single siRNA experiments, as well as in double siRNA experiments, whereas that of Emerin siRNA was 200 nM. Cells were cultured and transfected with siRNAs at 80% confluence with Oligofectamine reagent (InVitrogen). Transient transfections with cDNAs was performed with Lipofectamine 2000TM (Invitrogen) to label mitochondria with mt-dsRed plasmid (Clontech), lysosomes with SytVII-GFP (synaptotagmin VII-green fluorescent protein) plasmid (kindly provided by N. W. Andrews, Yale University, New Haven, CT) (Martinez et al., 2000
) and autophagic vacuoles with LC3-GFP plasmid (Kabeya et al., 2000
).
Flow cytometry
To determine apoptosis-associated changes by cytofluorometry we used 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3), 40 nM) for the mitochondrial transmembrane potential (m) quantification, and propidium iodide (PI, 1 µg/ml) for determination of cell viability (all of them from Molecular Probes) (Boya et al., 2003c
; Castedo et al., 2002a
). After different experimental conditions, cells were trypsinized and incubated with the fluorochromes for 15 minutes at 37°C, followed by cytofluorometric analysis with a FACS Scan (Becton Dickinson).
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Western blot analysis
Cells were washed by cold PBS at 4°C and lysed in a buffer containing 50 mM Tris HCl pH 6.8, glycerol 10%, 2% SDS, 10 mM DTT and 0.005% bromophenol blue. Forty micrograms of protein were loaded on a 10% SDS-PAGE and transferred to nitrocellulose. The membrane was incubated for 1 hour in PBS-Tween 20 (0.05%) containing 5% nonfat milk. Primary antibodies (anti-LAMP2 mAb H4B4 and LAMP1 mouse IgG2b) (from ABR Affinity BioReagents and BD Transduction Laboratories, respectively) were incubated for 15 hours at 4°C and detected with the appropriated horseradish peroxidase-labeled secondary antibodies (Southern Biotechnologies Associates) and revealed by SuperSignal West Pico chemoluminiscent substrate (Pierce). Anti-GAPDH (Chemicon) was used to control equal loading.
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Statistical analysis
All the data are representative of at least three independent experiments (with comparable results). Statistical significance of the data was evaluated after the calculation of one way analysis of variance (ANOVA). Values of P<0.05 were considered statistically significant.
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Results |
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Upon close inspection, we found that a subpopulation of LAMP2-depleted cells exhibited chromatin condensation, as detectable with the nuclear stain Hoechst 33342 (Fig. 1D,E). Cytofluorometric quantification revealed that a significant fraction of starved LAMP2 knockdown cells lost the capacity to retain the dye DiOC6(3) and hence dissipated the mitochondrial transmembrane potential (m) (Fig. 2A,B). Among this
mlow population, a fraction of cells incorporated the vital dye propidium iodide (PI) and thus lost the barrier function of their plasma membrane (Fig. 2A). The frequency of dead (PI+) cells increased with concurrent depletion of LAMP2 and nutrients (Fig. 2C). Again, these effects (enhanced nuclear condensation,
m loss and cell death in response to starvation) were less pronounced for LAMP1 depletion than for LAMP2 knockdown (Fig. 2). Electron microscopic examination of cells dying after LAMP2 depletion and nutrient depletion revealed typical signs of nuclear apoptosis (pyknosis and karyorhexis), as well as a pronounced vacuolization of the cytoplasm (Fig. 2D).
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LAMP2 depletion blocks the colocalization of autophagosomal and lysosomal markers
LAMP2 may be involved in lysosomal biogenesis and/or the fusion between autophagosomes and lysosomes required for the final catabolism of autophagic material (Eskelinen et al., 2002; Eskelinen et al., 2004
; Levine and Klionsky, 2004
; Shintani and Klionsky, 2004
; Tanaka et al., 2000
). To discriminate between these possibilities, we labeled cells with an acidophilic, lysosomotropic agent, Lysotracker® Red (LTR), and simultaneously tracked autophagic vacuoles with LC3-GFP. LAMP2 (or LAMP1) knockdown had no negative effect on the intensity of LTR staining, neither in normal nor in starving conditions (Fig. 4A and data not shown). However, LAMP2-specific siRNA, alone or in combination with LAMP1-specific siRNA (but not LAMP1-specific siRNA alone), reduced the colocalization of LC3-GFP and LTR induced by starvation (Fig. 4A,B). This suggests that LAMP2 (but not LAMP1) is required for the formation of autophagolysosomes. Mitochondria are known to undergo progressive autophagic depletion in conditions of nutrient or growth factor depletion (Chang et al., 2003
; Rodriguez-Enriquez et al., 2004
; Xue et al., 2001
). We therefore measured the colocalization of a mitochondrial marker (mt-DsRed) and that of a lysosomal marker (synaptotagmin VII fused to GFP, SytVII-GFP) (Martinez et al., 2000
) in starving cells in which either of the two LAMP proteins were knocked down. After nutrient depletion, a fraction of mitochondria colocalized with SytVII-GFP in control cells treated with the control siRNA specific for emerin. LAMP2-specific siRNA reduced the colocalization of both organellar markers (Fig. 5A,B), again suggesting that LAMP2 is involved in the fusion of lysosomes with autophagic vacuoles. Very similar results were obtained in LAMP1/2 DKO fibroblasts from murine origin (Fig. 6A,B), underscoring that LAMP proteins have similar roles in different species and cell types. Data indicating a failure in the colocalization of autophagic vacuoles and lysosomes or mitochondrial and lysosomes induced by LAMP2 deficiency were obtained at different time points after starvation, namely 4 hours after nutrient depletion (shown in Figs 4, 5, 6), as well as after 16 hours (not shown), when a significant fraction of cells were dead or dying (Fig. 2).
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Cell death-induced accumulation of autophagic vacuoles involves hallmarks of apoptosis
The accumulation of autophagic vacuoles induced by starvation and LAMP2 depletion was followed by caspase-3 activation, as shown for HeLa cells (Fig. 7A,B) and MEF (Fig. 3E, see above). We therefore wondered whether inhibition of caspases with Z-VAD-fmk would prevent cell death in this model. Z-VAD-fmk partially reduced the mortality (as assessed with the vital dye PI) of cells depleted from LAMPs and nutrients (Fig. 7D). However, Z-VAD-fmk did not stabilize the m, which was lost in an increasing fraction of such cells (Fig. 7C), presumably as a sign of metabolic insufficiency (Zong et al., 2004
) or of incipient apoptosis (Zamzami et al., 1995
). Next, we addressed the question as to whether the preservation of mitochondrial function by overexpressed Bcl-2 and the mitochondrion-targeted Cytomegalovirus protein vMIA would prevent apoptosis induced by the accumulation of autophagic vacuoles. Transfection-enforced overexpression of either of these two proteins stabilized the
m of cells depleted from nutrients and from LAMP proteins (Fig. 8A). Moreover, Bcl-2 and vMIA prevented cell death in these circumstances (Fig. 8B). Thus, two different strategies of apoptosis inhibition, caspase inhibition and mitochondrial stabilization, both preserved the cellular viability yet differed in their capacity to maintain the
m. Of note, apoptosis inhibition did not affect the accumulation of autophagic vacuoles (as measured with LC3-GFP) (Fig. 8C), supporting the contention that autophagic vacuolization occurs upstream and independently of the apoptotic process.
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Discussion |
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It should be noted that a shift from an initial type 2 to a final type 1 morphology has been observed in another model system. In Drosophila, developmental cell death in the salivary gland occurs in a stepwise fashion, with a caspase-independent, reversible type 2 morphology that drifts to a caspase-dependent, irreversible type 1 pattern (Lee and Baehrecke, 2001; Martin and Baehrecke, 2004
). Thus, at least in some paradigms of type 2 cell death, cells may ultimately succumb to biochemical processes such as caspase activation, which are typically associated with apoptosis. However, it remains to be determined whether type 2 cell death shifts to type 1 cell death in other examples of cell death, for instance in human pathologies.
Another important aspect emerging from this study is that lesions affecting distinct organellar systems can trigger cell death through a final mitochondrial pathway (Ferri and Kroemer, 2001; Green and Kroemer, 2004
). vMIA is a predominantly mitochondrial protein that suppresses outer membrane permeabilization through its capacity to interact with Bax and to convert Bax into an apoptosis inhibitor (Arnoult et al., 2004
; Poncet et al., 2004
). Similar to Bcl-2, vMIA can inhibit cell death induced by damage that primarily affects nuclei (Andreau et al., 2004
; Castedo et al., 2004
), the endoplasmic reticulum (Boya et al., 2002
), lysosomes (Boya et al., 2003a
; Boya et al., 2003c
) and, as shown here, the autophagic compartment. This underscores the importance of mitochondrial events in sealing irreversible cell fate, even when the primary lethal lesion affects other organelles.
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Acknowledgments |
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Footnotes |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, J. M. (2003). Ways of dying: multiple pathways to apoptosis. Genes Dev. 17, 2481-2495.
Alva, A. S., Gultekin, S. H. and Baehrecke, E. H. (2004). Autophagy in human tumors: cell survival or death? Cell Death Differ. 11, 1046-1048.[CrossRef][Medline]
Andreau, K., Castedo, M., Perfettini, J.-L., Roumier, T., Pichart, E., Souquere, S., Larochette, N., Pierron, G. and Kroemer, G. (2004). Pre-apoptotic chromatin condensation upstream of the mitochondrial checkpoint. J. Biol. Chem 279, 55937-55945.
Andrejewski, N., Punnonen, E. L., Guhde, G., Tanaka, Y., Lullmann-Rauch, R., Hartmann, D., von Figura, K. and Saftig, P. (1999). Normal lysosomal morphology and function in LAMP-1-deficient mice. J. Biol. Chem. 274, 12692-12701.
Arnoult, D., Bartle, L. M., Skaletskaya, A., Poncet, D., Zamzami, N., Park, P. U., Sharpe, J., Youle, R. J. and Goldmacher, V. S. (2004). Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria. Proc. Natl. Acad. Sci. USA 101, 7988-7989.
Belzacq, A. S., El Hamel, C., Vieira, H. L. A., Cohen, I., Haouzi, D., Metivier, D., Marchetti, P., Goldmacher, V., Brenner, C. and Kroemer, G. (2001). The adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437. Oncogene 20, 7579-7587.[CrossRef][Medline]
Boya, P., Cohen, I., Zamzami, N., Vieira, H. L. A. and Kroemer, G. (2002). Endoplasmic reticulum stress-induced cell death requires mitochondrial membrane permeabilization. Cell Death Differ. 9, 465-467.[CrossRef][Medline]
Boya, P., Andreau, K., Poncet, D., Zamzami, N., Perfettini, J.-L., Metivier, D., Ojcius, D. M., Jaattela, M. and Kroemer, G. (2003a). Lysosomal membrane permeabilization induces cell death in a mitochondrion-dependent fashion. J. Exp. Med. 197, 1323-1334.
Boya, P., Gonzalez-Polo, R.-A., Poncet, D., Andreau, K., Roumier, T., Perfettini, J.-L. and Kroemer, G. (2003b). Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine. Oncogene 22, 3927-3936.[CrossRef][Medline]
Boya, P., Morales, M. C., Gonzalez-Polo, R.-A., Andreau, K., Gourdier, I., Perfettini, J.-L., Larochette, N., Deniaud, A., Baran-Marszak, F., Fagard, R. et al. (2003c). The chemopreventive agent 4-hydroxyphenylretinamide induces apoptosis through a mitochondrial pathway regulated by proteins from the Bcl-2 family. Oncogene 22, 6220-6230.[CrossRef][Medline]
Boya, P., Gonzalez-Polo, R.-A., Casares, N., Perfettini, J.-L., Dessen, P., Larochette, N., Metivier, D., Meley, D., Souquere, S., Pierron, G. et al. (2005). Inhibition of macroautophagy triggers apoptosis. Mol. Cell. Biol. 25, 1025-1040.
Bursch, W. (2001). The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ. 8, 569-581.[CrossRef][Medline]
Castedo, M., Ferri, K. F., Blanco, J., Roumier, T., Larochette, N., Barretina, J., Amendola, A., Nardacci, R., Metivier, D., Este, J. A. et al. (2001). Human immunodeficiency virus 1 envelope glycoprotein complex-induced apoptosis involves mammalian target of rapamycin/FKBP12-rapamycin-associated protein-mediated p53 phosphorylation. J. Exp. Med. 194, 1097-1110.
Castedo, M., Ferri, K., Roumier, T., Metivier, D., Zamzami, N. and Kroemer, G. (2002a). Quantitation of mitochondrial alterations associated with apoptosis. J. Immunol. Methods 265, 39-47.[CrossRef][Medline]
Castedo, M., Roumier, T., Blanco, J., Ferri, K. F., Barretina, J., Andreau, K., Perfettini, J.-L., Armendola, A., Nardacci, R., LeDuc, P. et al. (2002b). Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the human immunodeficiency virus-1 envelope. EMBO J. 21, 4070-4080.
Castedo, M., Perfettini, J.-L., Roumier, T., Valent, A., Raslova, H., Yakushijin, K., Horne, D. A., Feunteun, J., Lenoir, G., Vainchenker, W. et al. (2004). Mitotic catastrophe. A special case of apoptosis preventing aneuploidy. Oncogene 23, 4362-4370.[CrossRef][Medline]
Chang, L. K., Schmidt, R. E. and Johnson, E. M. J. (2003). Alternating metabolic pathways in NGF-deprived sympathetic neurons affect caspase-independent death. J. Cell Biol. 162, 245-256.
Daido, S., Kanzawa, T., Yamamoto, A., Takeuchi, H., Kondo, Y. and Kondo, S. (2004). Pivotal role of the cell death factor BNIP3 in ceramide-induced autophagic cell death in malignant glioma cells. Cancer Res. 64, 4286-4293.
Edinger, A. L. and Thompson, C. B. (2004). Death by design: apoptosis, necrosis and autophagy. Curr. Opin. Cell Biol. 16, 663-669.[CrossRef][Medline]
Eskelinen, E. L., Illert, A. L., Tanaka, Y., Schwarzmann, G., Blanz, J., Von Figura, K. and Saftig, P. (2002). Role of LAMP-2 in lysosome biogenesis and autophagy. Mol. Biol. Cell 13, 3355-3368.
Eskelinen, E. L., Schmidt, C. K., Neu, S., Willenborg, M., Fuertes, G., Salvador, N., Tanaka, Y., Lullmann-Rauch, R., Hartmann, D., Heeren, J. et al. (2004). Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/LAMP-2 double-deficient fibroblasts. Mol Biol Cell. 15, 3132-3145.
Ferri, K. F. and Kroemer, G. K. (2001). Organelle-specific initiation of cell death pathways. Nat. Cell Biol. 3, E255-E263.[CrossRef][Medline]
Goldmacher, V. S., Bartle, L. M., Skletskaya, S., Dionne, C. A., Kedersha, N. L., Vater, C. A., Han, J. W., Lutz, R. J., Watanabe, S., McFarland, E. D. C. et al. (1999). A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2. Proc. Natl. Acad. Sci. USA 96, 12536-12541.
Green, D. R. and Kroemer, G. (2004). The pathophysiology of mitochondrial cell death. Science 305, 626-629.
Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T. and Weber, K. (2001). Identification of essential genes in cultured mammalian cells using small interfering RNAs. J. Cell Sci. 114, 4557-4565.[Medline]
Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y. and Yoshimori, T. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720-5728.
Kanzawa, T., Germano, I. M., Komata, T., Iton, H., Kondo, Y. and Kondo, S. (2004). Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ. 11, 448-457.[CrossRef][Medline]
Kihara, A., Kabeya, Y., Ohsumi, Y. and Yoshimori, T. (2001). Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep. 2, 330-335.
Klionsky, D. J., Cregg, J. M., Dunn, W. A. J., Emr, S. D., Sakai, Y., Sandoval, I. V., Sibirny, A., Subramani, S., Thumm, M., Veenhuis, M. et al. (2003). A unified nomenclature for yeast autophagy-related genes. Dev. Cell 5, 539-545.[CrossRef][Medline]
Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H., Yoshimori, T., Ohsumi, Y., Tokuhisa, T. and Mizushima, N. (2004). The role of autophagy during the early neonatal starvation period. Nature 432, 1032-1036.[CrossRef][Medline]
Lee, C. Y. and Baehrecke, E. H. (2001). Steroid regulation of autophagic programmed cell death during development. Development 128, 1443-1455.
Leist, M. and Jaattela, M. (2001). Four deaths and a funeral: from caspases to alternative mechanisms. Nat. Rev. Mol. Cell Biol. 2, 589-598.[CrossRef][Medline]
Levine, B. and Klionsky, D. J. (2004). Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev. Cell 6, 463-477.[CrossRef][Medline]
Lockshin, R. A. and Zakeri, Z. (2001). Programmed cell death and apoptosis: origins of the theory. Nat. Rev. Mol. Cell Biol. 2, 545-550.[CrossRef][Medline]
Martin, D. N. and Baehrecke, E. H. (2004). Caspases function in autophagic programmed cell death in Drosophila. Development 131, 275-284.
Martinez, I., Chakrabarti, S., Hellevik, T., Morehead, J., Fowler, K. and Andrews, N. W. (2000). Synaptotagmin VII regulates Ca(2+)-dependent exocytosis of lysosomes in fibroblasts. J. Cell Biol. 148, 1141-1149.
Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi, Y., Kabeya, Y., Suzuki, K., Tokuhisa, T., Ohsumi, Y. and Yoshimori, T. (2001). Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J. Cell Biol. 152, 657-668.
Nakagawa, I., Amano, A., Mizushima, N., Yamamoto, A., Yamaguchi, H., Kamimoto, T., Nara, A., Funao, J., Nakata, M., Tsuda, K. et al. (2004). Autophagy defends cells against invading group A Streptococcus. Science 306, 1037-1040.
Nemoto, T., Tanida, I., Tanida-Miyake, E., Yokota, M., Ohsumi, M., Ueno, T. and Kominami, E. (2003). The mouse APG10 homologue, an authentic E2-like enzyme for Apg12p-Apg5p conjugation system, facilitates MAP-LC3 processing. J. Biol. Chem. 278, 39517-39526.
Nishino, I., Fu, J., Tanji, K., Yamada, T., Shimojo, S., Koori, T., Mora, M., Riggs, J. E., Oh, S. J., Koga, Y. et al. (2000). Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature 406, 906-910.[CrossRef][Medline]
Opipari, A. W. J., Tan, L., Boitano, A. E., Sorenson, D. R., Aurora, A. and Liu, J. R. (2004). Resveratrol-induced autophagocytosis in ovarian cancer cells. Cancer Res. 64, 696-703.
Poncet, D., Larochette, N., Pauleau, A. L., Boya, P., Jalil, A. A., Cartron, P. F., Vallette, F., Schnebelen, C., Bartle, L. M., Skaletskaya, A. et al. (2004). An anti-apoptotic viral protein that recruits Bax to mitochondria. J. Biol. Chem. 279, 22605-22614.
Qu, X., Yu, J., Bhagat, G., Furuya, N., Hibshoosh, H., Troxel, A., Rosen, J., Eskelinen, E. L., Mizushima, N., Ohsumi, Y. et al. (2003). Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J. Clin. Invest. 112, 1809-1820.
Rodriguez-Enriquez, S., He, L. and Lemasters, J. J. (2004). Role of mitochondrial permeability transition pores in mitochondrial autophagy. Int. J. Biochem. Cell Biol. 36, 2463-2472.[CrossRef][Medline]
Shimizu, S., Kanaseki, T., Mizushima, N., Mizuta, T., Arakawa-Kobayashi, S., Thompson, C. B., Korsmeyer, S. J. and Tsujimoto, Y. (2004). A role of Bcl-2 family of proteins in non-apoptotic programmed cell death dependent on autophagy genes. Nat. Cell Biol. 6, 1221-1228.[CrossRef][Medline]
Shintani, T. and Klionsky, D. J. (2004). Autophagy in health and disease: a double-edged sword. Science 306, 990-995.
Tanaka, Y., Guhde, G., Suter, A., Eskelinen, E. L., Hartmann, D., Lullmann-Rauch, R., Janssen, P. M., Blanz, J., von Figura, K. and Saftig, P. (2000). Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature 406, 902-906.[CrossRef][Medline]
Teckman, J. H., An, J. K., Blomenkamp, K., Schmidt, B. and Perlmutter, D. (2004). Mitochondrial autophagy and injury in the liver in alpha 1-antitrypsin deficiency. Am. J. Physiol. Gastrointest. Liver Physiol. 386, G851-G862.[CrossRef]
Vieira, H. L., Belzacq, A.-S., Haouzi, D., Bernassola, F., Cohen, I., Jacotot, E., Ferri, K. F., Hamel, E. H., Bartle, L. M., Melino, G. et al. (2001). The adenine nucleotide translocator: a target of nitric oxide, peroxynitrite and 4-hydroxynonenal. Oncogene 20, 4305-4316.[CrossRef][Medline]
Wang, X. (2002). The expanding role of mitochondria in apoptosis. Genes Dev. 15, 2922-2933.
Xue, L., Fletcher, G. C. and Tolkovsky, A. M. (2001). Mitochondria are selectively eliminated from eukaryotic cells after blockade of caspases during apoptosis. Curr. Biol. 6, 361-365.
Yu, L., Alva, A., Su, H., Dutt, P., Freundt, E., Welsh, S., Baehrecke, E. H. and Lenardo, M. J. (2004). Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science 304, 1500-1502.
Yue, Z., Jin, S., Yang, C., Levine, A. J. and Heintz, N. (2003). Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc. Natl. Acad. Sci. USA 100, 15077-15082.
Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssière, J.-L., Petit, P. X. and Kroemer, G. (1995). Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181, 1661-1672.
Zong, W. X., Ditsworth, D., Bauer, D. E., Wang, Z. Q. and Thompson, C. B. (2004). Alkylating DNA damage stimulates a regulated form of necrotic cell death. Genes Dev. 18, 1272-1282.
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