MOLECULAR AND CELLULAR BIOLOGY, Feb. 2005, p. 1025–1040
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 3
Inhibition of Macroautophagy Triggers Apoptosis†
Patricia Boya,1‡§ Rosa-Ana Gonza ´lez-Polo,1‡ Noelia Casares,1Jean-Luc Perfettini,1Philippe Dessen,1
Nathanael Larochette,1Didier Me ´tivier,1Daniel Meley,2Sylvie Souquere,3Tamotsu Yoshimori,4
Ge ´rard Pierron,3Patrice Codogno,2and Guido Kroemer1*
CNRS-UMR8125, Institut Gustave Roussy,1and Laboratoire Replication de l’ADN
et Ultrastructure du Noyau, UPR-1983,3and INSERM U504,2Institut
Andre ´ Lwoff, Villejuif, France, and National Institute of Genetics,
Received 6 May 2004/Returned for modification 8 June 2004/Accepted 19 October 2004
Mammalian cells were observed to die under conditions in which nutrients were depleted and, simulta-
neously, macroautophagy was inhibited either genetically (by a small interfering RNA targeting Atg5, Atg6/
Beclin 1-1, Atg10, or Atg12) or pharmacologically (by 3-methyladenine, hydroxychloroquine, bafilomycin A1, or
monensin). Cell death occurred through apoptosis (type 1 cell death), since it was reduced by stabilization of
mitochondrial membranes (with Bcl-2 or vMIA, a cytomegalovirus-derived gene) or by caspase inhibition.
Under conditions in which the fusion between lysosomes and autophagosomes was inhibited, the formation of
autophagic vacuoles was enhanced at a preapoptotic stage, as indicated by accumulation of LC3-II protein,
ultrastructural studies, and an increase in the acidic vacuolar compartment. Cells exhibiting a morphology
reminiscent of (autophagic) type 2 cell death, however, recovered, and only cells with a disrupted mito-
chondrial transmembrane potential were beyond the point of no return and inexorably died even under
optimal culture conditions. All together, these data indicate that autophagy may be cytoprotective, at least
under conditions of nutrient depletion, and point to an important cross talk between type 1 and type 2 cell
Type 1 (apoptotic) cell death and type 2 (autophagic) cell
death are viewed as clearly distinct subroutines of cellu-
lar demise (9, 42, 47). Apoptosis, which is currently viewed
as the quantitatively most important death modality, is mor-
phologically defined by cellular and nuclear shrinkage (py-
knosis), chromatin condensation, blebbing, nuclear fragmenta-
tion (karyorrhexis), and formation of apoptotic bodies (32). At
the biochemical level, apoptosis of mammalian cells is charac-
terized by mitochondrial membrane permeabilization (MMP)
and/or massive caspase activation (1, 21, 68). Autophagic cell
death is characterized by the accumulation of autophagic vacu-
oles (AV) (see below). Although less frequent, type 2 cell
death is pathophysiologically relevant. For example, type 2 cell
death affects degenerating neurons in some pathologies (17,
39), participates in retinal degeneration (24), seals the fate of
Salmonella-infected macrophages (26), and sometimes medi-
ates chemotherapy-induced tumor killing (30, 31, 55). More-
over, an important autophagy-regulatory gene such as Beclin 1
functions as a haploinsufficient tumor suppressor gene (60, 76),
further underscoring the likely clinical importance of type 2
cell death. Nonetheless, the biochemical mechanisms account-
ing for cell killing remain largely unexplored in type 2 cell
death, and the mutual relationship between apoptotic and au-
tophagic death is currently debated (20, 27, 41–43, 48, 62, 70,
Autophagy is a regulated process of degradation and recy-
cling of cellular constituents, participating in organelle turn-
over and in the bioenergetic management of starvation (37).
During autophagy, part of the cytoplasm or entire organelles
are sequestered into double-membraned vesicles, called AV or
autophagosomes. Autophagosomes ultimately fuse with lyso-
somes, thereby generating single-membraned autophagolyso-
somes and degrading their content (73). Autophagy has been
extensively studied in Saccharomyces cerevisiae, especially at
the genetic level, leading to the discovery of autophagy-rele-
vant Apg and Aut genes, now renamed Atg genes (36, 61).
Several Atg proteins have been implicated in autophagosome
formation. The ubiquitinization of Atg5 and Atg12 by the
E1-like enzymes Atg7 and Atg10 is required to recruit other
proteins to the autophagosomal membrane and to form the
autophagic vacuole in a pathway, which was first elucidated for
yeast and then confirmed for mammalian cells (51, 53). LC3
is the mammalian equivalent of yeast Atg8. It exists in two
forms, LC3-I and its proteolytic derivative LC3-II (18 and
16 kDa, respectively), which are localized in the cytosol
(LC3-I) or in autophagosomal membranes (LC3-II). LC3-II
thus can be used to estimate the abundance of autophago-
somes before they are destroyed through fusion with lyso-
somes (29, 51). Similarly, LC3-green fluorescent protein
(GFP) fusion protein redistributes from a diffuse to a vac-
uolar pattern when AV are formed (29, 51). Finally, Beclin
1 is the mammalian orthologue of yeast Atg6 (45). Beclin 1
localizes to the trans-Golgi network, belongs to the class III
phosphatidylinositol 3-kinase complex, and participates in
autophagosome formation (33, 45). Beclin 1 is monoalleli-
* Corresponding author. Mailing address: CNRS-UMR 8125, Insti-
tut Gustave Roussy, Pavillon de Recherche 1, 39 rue Camille-Desmou-
lins, F-94805 Villejuif, France. Phone: 33-1-42 11 60 46. Fax: 33-1-42 11
60 47. E-mail: firstname.lastname@example.org.
‡ P.B. and R.-A.G.-P. contributed equally to this work.
† Supplemental material for this article may be found at http://mcb
§ Present address: Consejo Superior de Investigaciones Cientı ´ficas,
E-28040 Madrid, Spain.
cally deleted in many human patients with sporadic breast,
ovarian, and prostate cancer (45). Moreover, Beclin 1?/?
mutant mice show a high incidence of spontaneous tumors
and decreased autophagy in vitro (60, 76), suggesting that
autophagy (and perhaps autophagic cell death) may prevent
cellular transformation (13).
We previously observed that lysosomotropic agents can lead
to cytoplasmic vacuolization and cell death that involves hall-
marks of apoptosis (6, 7). We therefore explored the relation-
ship between autophagic vacuolization and subsequent cellular
demise. Unexpectedly, we found that the accumulation of AV
that is typical for the morphology of type 2 cell death can be
due to an actual inhibition of macroautophagy at the level of
the fusion between autophagosomes and lysosomes and that
this accumulation by itself is not lethal. Rather, in numerous
instances, induction of autophagic vacuolization ultimately
triggers a cell death program that is suppressed by MMP in-
hibitors or caspase antagonists. Thus, biochemical hallmarks of
type 1 cell death may be involved in the execution of morpho-
logical type 2 cell death, pointing to a major cross talk between
the two lethal subroutines.
MATERIALS AND METHODS
Cell lines and culture conditions. HeLa cells were stably transfected with the
pcDNA3.1 control vector (Neo), human Bcl-2 (Bcl-2), or the cytomegalovirus
UL37 exon 1 gene coding for the viral mitochondrial inhibitor of apoptosis
(vMIA, kindly provided by V. Goldmacher) (3, 19). Cells were cultured in
Dulbecco modified Eagle medium supplemented with 10% fetal calf serum
(FCS), 1 mM pyruvate, and 10 mM HEPES at 37°C under 5% CO2. Simian virus
40-transformed mouse embryonic fibroblasts whose genotype was either wild
type or double knockout (DKO), provided by S. Korsmeyer (69), were cultivated
in Dulbecco modified Eagle medium (Life Technologies) supplemented with
10% FCS–1? nonessential amino acids (Sigma) at 37°C under 5% CO2.
Transfection and RNA interference. Small interfering RNAs (siRNAs) were
synthesized by Proligo France SAS. For Beclin 1 (National Center for Biotech-
nology Information accession number AF077301), RNA sequences started at
positions 189 (CUCAGGAGAGGAGCCAUUU) and 1206 (GAUUGAAGAC
ACAGGAGGC) from ATG (oligoribonucleotides Beclin 100 [B110] and Beclin
168 [B168], respectively); for Atg5 (accession number BC002699), the sequence
started at position 453 (GCAACUCUGGAUGGGAUUG); for Atg10 (accession
number NM_031482), the sequence started at position 391 (GGAGUUCAUG
AGUGCUAUA); and for Atg12 (accession number NM_004707), the sequence
started at position 131 (CAGAGGAACCUGCUGGCGA). As controls, siRNA
ribonucleotides scrambled from B110 and targeting the unrelated protein emerin
(25) were used. Cells were cultured in six-well plates and transfected at 80%
confluence with Oligofectamine reagent (Invitrogen) according to the manufac-
turer’s instructions. After 3 h, 10% FCS was added, and cells were left for
another 24 to 48 h before they were trypsinized and used for experiments.
Transient transfection was performed with Lipofectamine 2000 reagent (Invitro-
gen), and cells were used 24 h after transfection. The formation of AV was
followed by means of an LC3-GFP plasmid (29). To label lysosomes, we used
the SytVII-GFP plasmid (kindly provided by N. W. Andrews) (49) and
lgp120-GFP (provided by J. Lippincott-Schwartz) (57). Mitochondria were
labeled with the commercial mitochondrion-targeted DsRed (mtDsRed)
RNA extraction and quantitative RT-PCR. mRNA preparations were ob-
tained with the RNeasy mini kit (QIAGEN) and quality controlled with an
Agilent 2100 bioanalyzer (Agilent Technologies). For quantitative reverse tran-
scription (RT)-PCR, cDNAs were synthesized from 1 ?g of total RNA with
Moloney murine leukemia virus reverse transcriptase (Roche). Then, the Taq-
Man Universal PCR was performed on an ABI PRISM 7000 sequence detection
system (Applied Biosystems), according to the manufacturer’s instructions, using
primers specific for Atg5, Atg10, and Atg12 (TaqMan assays reagents from Ap-
Reagents and cell death induction. Hydroxychloroquine (HCQ; Sanofi-
Synthelabo) was used from a stock solution at 30 ?g/ml unless otherwise spec-
ified. Bafilomycin A1 (Baf A1; 0.1 ?M; Sigma), monensin (10 ?M; Calbiochem),
3-methyladenine (3-MA; 10 mM; Fluka), staurosporine (STS; 100 nM; Sigma).
Anti-CD95 (200 nM; Immunotech) was used in combination with cycloheximide
(1 ?g/ml). For caspase inhibition, N-benzyloxycarbonyl-Val-Ala-Asp-fluorom-
ethylketone (Z-VAD-fmk; Bachem) was added at the same time as cell death
inducers at 25 ?M (8).
Flow cytometry and cell sorting. The following fluorochromes were employed
to determine apoptosis-associated changes by cytofluorometry: 3,3?-dihexyloxa-
carbocyanine iodide [DiOC6(3), 40 nM] for mitochondrial transmembrane po-
tential (??m) quantification, propidium iodide (PI; 1 ?g/ml) and 44,6-diamino-
2-phenylindole (DAPI; 2.5 ??) for determination of cell viability (all from
Molecular Probes), and annexin V conjugated with fluorescein isothiocyanate
(Bender Medsystems) for the assessment of phosphatidylserine (PS) exposure
(10, 77). To label lysosomes, LysoTracker Red (LTR; 500 nM; Molecular Probes)
was used. Cells were trypsinized and labeled with the fluorochromes at 37°C,
followed by cytofluorometric analysis with a fluorescence-activated cell sorter
(FACS) scan (Becton Dickinson). For cell sorting, HeLa cells were treated with
60 ?g of HCQ/ml for 8 h, trypsinized, and labeled with LTR, DiOC6(3), and
DAPI for 15 min at 37°C, followed by purification with a Vantage FACS (Becton
Dickinson). After being sorted, the different populations were either fixed
(for assessment of cytochrome c relocation), relabeled, and reanalyzed for
DiOC6(3), LTR, and DAPI or cultured again in complete medium (CM) at
37°C for 16 h.
Light microscopy and immunofluorescence. Cells cultured on coverslips were
stained with Cell Tracker Green 5-chloromethylfluorescein diacetate (CMFDA;
1 ?M; Molecular Probes) and Hoechst 33342 (2 ?M; Sigma), followed by fluo-
rescence microscopic assessment with a Leica IRE2 microscope equipped with a
Leica DC300F camera. For Giemsa staining, cells were fixed in methanol and
stained with a kit from Sigma. Alternatively, cells were fixed with paraformalde-
hyde (4%, wt/vol) and picric acid (0.19%, vol/vol) for LC3-GFP and immuno-
fluorescence assays (15). Cells were stained for the detection of cytochrome c
(monoclonal antibody 6H2.B4 from Pharmingen), LAMP2 (monoclonal antibody
H4B4 from Affinity BioReagents), or activated caspase-3 (polyclonal antibody
from Cell Signaling Technology), developed by goat anti-mouse or anti-rabbit
immunoglobulin Alexa fluor conjugates (Molecular Probes) (12). Confocal
microscopy was performed with a Zeiss LSM 510 microscope equipped with
a 63? objective. To determine the percentage of colocalization, green and
merged images were loaded into Image J software and the ratio of green to
merged cells was determined with the colocalization plug-in. Scale bars indicate
Electron microscopy. Cells were fixed for 1 h at 4°C in 1.6% glutaraldehyde in
0.1 M So ¨rensen phosphate buffer (pH 7.3), washed and fixed again in aqueous
2% osmium tetroxide, and embedded in Epon. Electron microscopy was per-
FIG. 1. HCQ-mediated induction of acidic vacuoles. (A and B) Vacuolar acidic compartment- and apoptosis-associated parameters in HCQ-
treated cells. HeLa cells were exposed to HCQ (30 ?g/ml) for the indicated periods, and cells were stained with LTR, DiOC6(3), annexin
V-fluorescein isothiocyanate, or PI, followed by FACS analysis. Data shown are representative FACS profiles (A) or means of results from five
independent experiments (x ? standard errors of the mean [SEM]) (B). Bars in panel A indicate the window representing each population. CRT,
control. (C) Effect of mitochondrion-stabilizing proteins on LTR staining. HeLa cells stably transfected with Bcl-2 or vMIA were incubated for 5 h
with HCQ, followed by LTR staining and FACS analysis. Vector-only control cells (unpublished results) behaved as cells for which results are
shown in the leftmost graph of panel A. (D and E) Light microscopic evidence for HCQ-induced vacuolization. Cells treated with HCQ for the
indicated periods were stained with Giemsa (D) or Cell Tracker Green CMFDA (E). The arrow indicates the apoptotic nucleus. (F) Staining of
lysosomes with a LAMP2 antibody. HCQ-treated HeLa cells were immunofluorescence stained and counterstained with Hoechst 33324. (G) Chro-
nological hierarchy of vacuolization and MMP. Cells were stained with CMFDA, an anti-cytochrome c (Cyt c) antibody (revealed as red
fluorescence), and Hoechst 33342 (blue fluorescence), and the frequencies of cells with enhanced vacuolization, mitochondrion-released cyto-
chrome c, and apoptotic nuclei were determined (x ? SEM; n ? 4).
1026BOYA ET AL.MOL. CELL. BIOL.
formed with a Zeiss EM 902 transmission electron microscope, at 90 kV, on
ultrathin sections (80 nm thick) stained with uranyl acetate and lead citrate.
Quantification of AV was performed as described previously (65).
Western blot analysis. Cells were washed in cold phosphate-buffered saline
(PBS) at 4°C and lysed in a buffer containing 50 mM Tris HCl (pH 6.8), 10%
glycerol, 2% sodium dodecyl sulfate, 10 mM dithiothreitol, and 0.005% blue
bromophenol. Forty micrograms of protein was loaded on a 15% sodium dodecyl
sulfate–polyacrylamide gel and transferred to nitrocellulose. The membrane was
incubated for 1 h in PBS-Tween 20 (0.05%) containing 5% nonfat milk. Primary
antibodies (LC3 and Beclin 1; Santa Cruz Biotechnology) and activated
caspase-3 (Cell Signaling Technology) were revealed with the appropriate horse-
radish peroxidase-labeled secondary antibodies (Southern Biotechnologies As-
sociates) and detected by SuperSignal West Pico chemiluminescent substrate
(Pierce). Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Chemi-
con) was used to ensure equal loadings.
Analysis of protein degradation. HeLa cells were incubated with 0.2 ?Ci of
L-valine (Perkin-Elmer Life Science)/ml in CM for 24 h (54). Unincorporated
radioisotope was removed by three rinses with PBS (pH 7.4). Cells were then
incubated in nutrient-free medium (NF) plus 0.1% bovine serum albumin in
the presence of 10 mM cold valine for 18 h (prechase period). After this time,
the media were replaced by the appropriate fresh medium plus cold valine (10
mM) in the presence of 10 mM 3-MA and 60 ?g of HCQ/ml for 4 h (chase
period). Cells and radiolabeled proteins were precipitated in 10% (vol/vol)
trichloroacetic acid at 4°C. The precipitated proteins were separated from the
soluble radioactivity by centrifugation at 600 ? g for 20 min and then dis-
solved in Soluene 350. The rate of protein degradation was calculated as
the level of acid-soluble radioactivity recovered from both cells and media
Statistical analysis. Data were analyzed with JMP IN 4 software using one-way
analyses of variance. Differences between the control and treated samples were
analyzed by using individual contrasts when the factor consisted of more than two
levels. P values of ?0.05 were considered statistically significant.
RESULTS AND DISCUSSION
Prelethal accumulation of AV in HCQ-treated cells. HeLa
cells exposed to the lysosomotropic agent HCQ manifested a
transiently increased level of staining with LTR (LTRhighcells)
(Fig. 1A); LTR is a fluorochrome which measures the volume
of the acidic vacuolar compartment (14, 18). This increased
LTR labeling was observed well before the ??m[as measured
with DiOC6(3), a ??m-sensitive dye] dropped, before PS res-
idues were exposed on the plasma membrane surface (quan-
tified with an annexin V-fluorescein isothiocyanate conjugate),
and before irreversible plasma membrane permeabilization
(determined with PI) occurred (Fig. 1B). Overexpression of
two unrelated MMP-inhibitory proteins (Bcl-2 and vMIA) (19)
retarded the acquisition of apoptotic parameters (??mloss, PS
exposure, and membrane permeabilization) induced by HCQ
(reference 7 and unpublished results). However, Bcl-2 and
vMIA did not affect LTR staining (Fig. 1C), indicating that the
increase in the acidic vacuolar compartment is an upstream
preapoptotic event. In addition, HCQ-treated cells manifested
cytoplasmic vacuolization, as determined by Giemsa staining
(Fig. 1D). Such cytoplasmic vacuoles were also detectable as
“holes” not staining with CMFDA (Fig. 1E) and correlated
with an increase in the volume of vesicles exhibiting a positive
immunofluorescence for the lysosomal marker LAMP2 (Fig.
1F). Vacuolization occurred well before cytochrome c was
released from mitochondria (Fig. 1G) and before nuclei
condensed (Fig. 1D to F). Double staining with LTR and
DiOC6(3) revealed that the increase in LTR staining, observed
8 h after HCQ addition, affected only the DiOC6(3)highpop-
ulation of cells (Fig. 2A), correlating with an increased disper-
sion of light (side scatter), which suggests an elevated degree of
vacuolization (Fig. 2B). FACS-sorted LTRhigh[and hence
DiOC6(3)high] cells contained double-membraned (that is,
bona fide autophagic) vacuoles filled with cytoplasmic material
yet lacked any ultrastructural sign of chromatin condensation
(Fig. 2C). This phenotype was observed in the vast majority
(?95%) of cells of the LTRhighpopulation. In contrast,
DiOC6(3)low, still-viable (DAPI-excluding) cells showed empty
(presumably apoptotic) vacuoles in addition to the autophagic
ones and underwent pyknosis and karyorrhexis (Fig. 2C and
D). Accordingly, kinetic experiments revealed that HCQ-in-
duced vacuolization, as determined by electron microscopy, led
first to the accumulation of AV and then to that of empty
vacuoles (Fig. 2E). Importantly, the FACS-purified LTRhigh
population did not progress to a loss in ??mwhen it was
recultured overnight in CM and maintained the integrity of its
plasma membrane (as determined with the vital dye DAPI),
whereas DiOC6(3)lowcells progressively died upon reculture
(Fig. 2F). Accordingly, LTRhigh[but not DiOC6(3)low] cells
retained cytochrome c in their mitochondria, even after recul-
ture (Fig. 2G). All together, these data suggest that AV accu-
mulation (and a transient phase of LTRhighstaining) precedes
the ??mloss (accompanied by a loss of LTRhighstaining),
which marks the point of no return and imminent cell death as
well as apoptotic vacuolization.
In conclusion, it appears that HCQ-induced AV accumula-
tion by itself is not sufficient to induce cell death. However,
after a latency period, HCQ does induce a type of cell death
that bears some hallmarks of apoptosis, such as ??mdissi-
pation, cytochrome c translocation, and chromatin conden-
FIG. 2. Determination of the point of no return of HCQ-induced cell death. (A) Cytofluorometric purification of subpopulations. Cells left
untreated (control) or treated with HCQ (8 h, 60 ?g/ml) were simultaneously stained with LTR, DiOC6(3), and the vital dye DAPI. The cells (gated
on DAPIlowevents with normal forward scatter) were then subjected to FACS. The gates shown in panels A indicate the FACS-sorted populations
[control, cells incorporating normal levels of LTR (LTRN), LTRhigh, and DiOC6(3)lowcells]. Note that the definition of LTRhighcells is more
restrictive than in Fig. 1A. (B) Side scatter characteristics (SSC) of cells sorted in panels A. CRT, control. (C) Representative electron microscopic
images obtained from such sorted cells as shown in panels A and B. Bars indicate 1 ?m. E, empty vesicle; N, nucleus. Arrows indicate double
membranes. (D) Quantitation of vacuolization patterns as determined by electron microscopy (50 replicates). Either the number of vesicles
(autophagic vesicles, AV, or empty vesicles) per cell was determined or the percentage of the cell volume occupied by vesicles was measured by
morphometry. CO, control. (E) Quantitation of vacuolization patterns of cells exposed to HCQ (60 ?g/ml) without FACS purification. (F and G)
Mortality of FACS-purified populations. Cells purified as described for panels A were either restained with DiOC6(3) and DAPI immediately after
FACS purification or cultured for another 16 h, followed by DiOC6(3) and DAPI staining (F). Note that only the DiOC6(3)lowpopulation tends
to lose its viability and to become DAPIhigh. Alternatively, cells were centrifuged on slides and subjected to immunofluorescent staining (0 h) or
recultured for 16 h and then labeled with a cytochrome c-specific antibody (G). The frequency of cells with a diffuse staining pattern indicative of
mitochondrial cytochrome c release is indicated in panels D (x ? SEM; n ? 3).
1028 BOYA ET AL.MOL. CELL. BIOL.
1030BOYA ET AL.MOL. CELL. BIOL.
FIG. 4. Effect of HCQ and other autophagy inhibitors on the subcellular localization and biochemical status of the autophagic vacuole marker
LC3. (A and B) Immunoblot analyses of accumulating LC3-II protein in control (CM) and starved (NF, 6 h) cells treated with HCQ (A) or a range
of established autophagy inhibitors (B). (C and D) Redistribution of LC3-GFP. Twenty-four hours after transient transfection with an LC3-GFP
chimera, cells were treated for the indicated times (24 h in panels C in the presence of serum) with HCQ, Baf A1, monensin, or 3-MA; fixed; and
counterstained with Hoechst 33342. Representative cells are shown in panels C, and the frequency (x ? SEM; n ? 4) of cells with a clear vacuolar
distribution of LC3-GFP (LC3-GFPVac) or apoptotic nuclei was scored. CO, control; Mon, monensin.
FIG. 3. HCQ inhibits autophagy. (A) Effect of HCQ on protein degradation. The rate of (54) valine-labeled long-lived proteins was measured
in cells incubated in either CM or NF, alone or in the presence of the indicated autophagy inhibitors. CO, control; Baf, Baf A1; Mon, monensin.
(B) Effect of HCQ on the colocalization of mitochondria and lysosomes. Cells were transfected with mtDsRed and lysosome-targeted GFP
(SytVII-GFP). Twenty-four hours later, cells were treated with NF (2 h) in the presence or absence of HCQ, and the cells were subjected to
confocal laser microscopy. Inserts illustrate the increased size of SytVII-GFP-marked structures in HCQ-treated, nutrient-depleted cells. The
graphs (right panels) represent the fluorescence distribution determined for sections of the cell, as indicated by the orientation of the arrow.
(C) Quantitation of mitochondrial-lysosomal colocalization. Percentage overlaps were calculated with an image analyzer and plotted for control
cells (in CM) or cells starved (in NF) in the presence of the indicated autophagy inhibitors. For results with control versus 3-MA-treated cells, P was
?0.05; for results with control versus Baf A1-, monensin-, and HCQ-treated cells, P was ?0.001. (D) Inhibitory effect of HCQ on the colocalization
of LAMP2 and LC3-GFP. Cells transfected with LC3-GFP (24 h before the initiation of the experiment) were subjected to nutrient starvation (NF,
2 h) in the absence or presence of HCQ (30 ?g/ml) and immunostained for LAMP2 detection to visualize the overlap with LC3-GFP (yellow).
Representative images are shown.
VOL. 25, 2005 APOPTOSIS AND AUTOPHAGY1031
FIG. 5. Autophagy inhibition sensitizes cells to nutrient depletion-induced cell death. (A) Vacuolization induced by starvation plus autophagy
inhibition. HeLa cells were cultured in CM or NF for 24 h in the presence or absence (control [CO]) of HCQ, Baf A1, monensin, or 3-MA and
finally stained with CMFDA and Hoechst 33342 (inset). Note that HCQ, Baf A1, and monensin enhance the formation of AV (visible as holes
in the green fluorescent staining) and induce nuclear apoptosis. Arrows mark apoptotic nuclei. (B) Quantitative assessment of synergic cell death
induction. Cells cultured in CM or NF, in the presence of the indicated inhibitors or none of them (control), were stained to determine the loss
of ??m[with DiOC6(3)] and viability (with PI). Data shown are means of results of five independent experiments ? SEM. (C) Caspase-3 activation
(Casp-3a) triggered by nutrient depletion plus autophagy inhibition. Cells were stained with an antibody recognizing the 17-kDa subunit of active
1032 BOYA ET AL.MOL. CELL. BIOL.
Pharmacological inhibition of macroautophagy precipitates
starvation-induced cell death. Although HCQ induced an in-
crease in AV (Fig. 1 and 2), it inhibited the progression of the
autophagic process, based on several criteria. On the one hand,
HCQ reduced the turnover of long-lived proteins, in particular
in the absence of serum (Fig. 3A). On the other hand, HCQ
prevented the colocalization of mitochondria and lysosomes,
induced by starvation (that is, culture in serum and amino
acid-free NF). Thus, nutrient-depleted cells exhibited an in-
creased colocalization of mtDsRed- and lysosome-targeted
SytVII-GFP, and this colocalization was inhibited by HCQ
(Fig. 3B). Thus, HCQ acted in a fashion similar to that of other
known inhibitors of autophagy, namely, Baf A1 (an inhibitor of
vacuolar H?ATPase) (5, 74), monensin (which mediates the
exchange of protons for potassium or sodium) (2, 23), and
3-MA (4, 9, 63), all of which prevented the starvation-induced
colocalization of mitochondrial and lysosomal markers (Fig.
3C) and reduced the turnover of long-lived proteins (Fig. 3A).
Moreover, HCQ prevented the nutrient starvation-induced co-
localization of the lysosomal marker LAMP2 and the AV
marker LC3-GFP (Fig. 3D) and hence prevented the forma-
tion of autophagolysosomes, as has been shown previously for
Baf A1 (71).
HCQ also caused the accumulation of the AV marker
LC3-II (29, 51) both in CM and under conditions of starvation,
as determined by immunoblotting (Fig. 4A). This effect was
again similar to that induced by Baf A1 and monensin (Fig.
4B). HCQ, Baf A1, and monensin also led to the redistribution
of an LC3-GFP fusion construct into punctate cytoplasmic
structures (Fig. 4C), indicating an increase in AV. Kinetic
experiments revealed that the LC3-GFP redistribution to vacu-
oles occurred in cells well before nuclear apoptosis increased
above background levels (Fig. 4D). However, all autophagy
inhibitors, including 3-MA, did induce nuclear apoptosis when
they were added to starved cells (Fig. 5A). While starvation
alone did not cause major cell death (as defined by a loss in
??mor staining with PI), the combination of starvation and
autophagy inhibition (with HCQ, Baf A1, monensin, or 3-MA)
had a synergistic lethal effect (Fig. 5B). Similar results were
obtained when PS exposure was measured (unpublished re-
sults). Moreover, we found that caspase-3 was synergistically
activated by nutrient depletion and autophagy inhibition (Fig.
5C). In strict contrast, autophagy inhibition did not increase
the lethal actions of classical apoptosis inducers such as STS
(Fig. 5D) and the CD95 agonistic antibody 7C11 (unpub-
lished results). As a result, it appears that pharmacological
autophagy inhibition can sensitize cells to starvation-induced
Autophagic vacuolization-associated cell death is reduced
by mitochondrial stabilization and caspase inhibition. To de-
termine by which mechanism starvation coupled to autophagy
inhibition killed cells, we introduced Bcl-2 and vMIA, two
MMP inhibitors, into HeLa cells or used the pan-caspase in-
hibitor Z-VAD-fmk as an apoptosis suppressor. Bcl-2 and
vMIA reduced the losses in ??mand viability induced by
starvation plus autophagy inhibitors (Fig. 6A and B). Caspase
inhibition with Z-VAD-fmk (25 ?M) reduced the loss of via-
bility induced by autophagy suppression yet had no significant
effect on the loss in ??m(Fig. 6A and B), suggesting that, as
in other paradigms of apoptosis (21, 22, 38), caspase activation
occurred downstream of MMP. Of note, neither MMP nor the
caspase inhibitors (Bcl-2, vMIA, and Z-VAD-fmk) affected the
formation of AV, as determined by assessing the autophago-
somal redistribution of LC3-GFP induced by HCQ (Fig. 6C),
Baf A1, or monensin (see Fig. S1 in the supplemental material)
in the presence or absence of serum. To further substantiate
the role of MMP in the death process, we used mouse embry-
onic fibroblasts that lacked both the proapoptotic multidomain
Bcl-2 family proteins Bax and Bak (DKO) cells. While DKO
cells failed to die, wild-type control cells readily succumbed to
starvation plus treatment with HCQ, Baf A1, monensin, or
3-MA (Fig. 7A and B). However, the absence of Bax and Bak
did not inhibit vacuolization induced by HCQ, monensin (Fig.
7C), or Baf A1 (see Fig. S2 in the supplemental material) in the
absence or presence of serum. All together, these data indicate
that two biochemical processes that define apoptosis (MMP
and caspase activation) are required for the demise of starved,
Knockdown of essential components of the autophagic ma-
chinery sensitizes cells to starvation-induced death. Pharma-
cological inhibition of autophagy might be affected by nonwar-
ranted side effects of drugs endowed with limited selectivity.
Therefore, we used siRNA to knock down Atg genes impli-
cated in the autophagic pathway. First, we targeted the essen-
tial Beclin 1/Atg6 gene (45). Two different siRNA constructs
designed to down-regulate the expression of Beclin 1 (Fig. 8A)
reduced the colocalization of mitochondria and lysosomes in-
duced by starvation compared to the level of colocalization
produced by a scrambled siRNA control (Fig. 8B). This ma-
nipulation also enhanced the losses in ??mand viability in-
duced by starvation yet had no effect on cell death induction by
the universal apoptosis inducer STS (Fig. 8C). Addition of the
broad-spectrum caspase inhibitor Z-VAD-fmk (Fig. 8C) or
transfection with Bcl-2 or vMIA (Fig. 8D) had a selective effect
on viability (with inhibitory effects of Z-VAD-fmk, Bcl-2, and
vMIA) and ??mdissipation (with inhibition by Bcl-2 and
vMIA yet no effect by Z-VAD-fmk). Since Beclin 1 has been
shown to interact with Bcl-2 (45), it might have been possible
that the Beclin 1 effects involved direct cross talk with mito-
chondria. We therefore used siRNA constructs designed to
target other Atg genes (Atg5, Atg10, and Atg12) (Fig. 9A),
which did prevent mitochondrial-lysosomal colocalization (Fig.
9B). siRNA of these Atg gene products enhanced starvation-
induced cell death (but not STS-mediated killing). Cooperative
killing of Atg5, Atg10, or Atg12 by starvation plus treatment
with siRNA was inhibited by Z-VAD-fmk (which, however,
caspase-3, and the frequency of positive cells was scored after culture in the presence or absence of nutrient and autophagy inhibitors (as described
for panel B, at 24 h). Ho, Hoechst 33342; Baf, Baf A1; Mon, monensin. (D) Autophagy inhibition does not sensitize cells to STS-induced cell death.
Cells were cultured with 100 nM STS in the presence of the indicated inhibitors, and cell death-related parameters were measured as described
for panels B. Results are mean values ? SEM of three to five independent determinations.
VOL. 25, 2005APOPTOSIS AND AUTOPHAGY1033
1034BOYA ET AL.MOL. CELL. BIOL.
had no effect on the disruption of the ??m) (Fig. 9C). Again,
both Bcl-2 and the mitochondrion-targeted viral inhibitor vMIA
prevented death acceleration by the knockdown of diverse
Atg genes (Fig. 9D). Thus, siRNA experiments confirmed
the cytoprotective potential of autophagy under conditions
of starvation and extended the notion that impaired auto-
phagy can kill nutrient-deprived cells by an apoptotic mech-
Concluding remarks. In the present paper, we demonstrate
that the death-associated accumulation of AV may be the
result of inhibited rather than exacerbated autophagy. Inhibi-
tion of the fusion of lysosomes with autophagosomes by HCQ,
Baf A1, or monensin results in the accumulation of AV, which
was detectable at several levels (LC3 proteolysis in Fig. 4A,
LC3-GFP redistribution in Fig. 4C, extension of the acidic
compartment in Fig. 1 and 3, accumulation of two-membraned
vesicles in Fig. 2, cytoplasmic vacuolization in Fig. 1D and E,
increase in the volume of LAMP2-positive vesicles in Fig. 1F,
and increase in the volume of Syt VII-GFP-labeled vesicles in
Fig. 3B). HCQ (Fig. 3D), Baf A1 (71), and monensin (data not
shown) apparently inhibited the formation of autophagolyso-
somes through the fusion of AV and lysosomes, thereby re-
ducing the turnover of AV. This reduction in turnover is linked
to enhanced cell death, in particular under conditions of nu-
trient depletion (Fig. 5). Although autophagic vacuolization by
itself is not lethal (Fig. 2), it primes cells for death accompa-
nied by a loss in the ??m(which marks the point of no return)
(Fig. 2), relocation of cytochrome c from mitochondria to the
cytosol (Fig. 2G), caspase activation (Fig. 5C), PS exposure
(Fig. 1A and B), and terminal plasma membrane permeabili-
zation (Fig. 1A and B and 5B). Similarly, inhibition of the early
stages of autophagy, using 3-MA (Fig. 3 to 7) or more specif-
ically siRNA for the knockdown of Atg5, Atg6/Beclin 1, Atg10,
and Atg12 (Fig. 8 to 9), sensitized cells to starvation-induced
cell death, although without any signs of autophagic vacuoliza-
tion. Irrespective of the stage at which autophagy was in-
hibited, disabled autophagy accelerated starvation-induced
death via a common final pathway involving biochemical
features of apoptosis. Thus, mitochondrial stabilization (by
overexpressed Bcl-2 or vMIA in Fig. 6A and B, 8C and D,
and 9D or by knockout of Bax and Bak in Fig. 7) or caspase
inhibition (Fig. 6A to C, 8C, and 9C) succeeded in delaying
cell death induced by combined starvation and autophagy
How is it possible that apoptosis is initiated by suppressed
autophagy? In a variety of systems, disequilibria in defined or-
ganellar systems leads to local caspase activation (e.g., caspase-
12 in the endoplasmic reticulum and caspase-2 in the nucleus)
(16, 40, 52). However, caspase activation is probably not an
initiating event in this system, because Z-VAD-fmk does not
affect MMP (Fig. 6A, 8C, and 9D), which thus is likely to occur
upstream and independently from caspases. It remains an on-
going conundrum how MMP occurs in starvation-induced cell
death under conditions of autophagy inhibition. Although Bax
and Bak are clearly involved in this process (Fig. 8), it is un-
clear which upstream processes may account for Bax- or Bak-
mediated MMP. On theoretical grounds, MMP may result
from bioenergetic failure (59), perhaps as a result of nutrient
depletion, combined with the impossibility of recruiting endog-
enous nutrients by autophagy-dependent catabolic reactions.
Furthermore, autophagy is the process that leads to the re-
moval of damaged mitochondria, provided that MMP occurs
at a level below the apoptotic threshold (14). Thus, it is con-
ceivable that failure to remove individual mitochondria that
undergo MMP primes for apoptosis. In addition, or alter-
natively, the inhibition of autophagy might alter the equi-
librium state between pro- and antiapoptotic mediators with
different half-lives, much as this has been shown for protea-
some inhibition, a manipulation that induces apoptosis via
multiple (and perhaps inextricable) pathways (11, 28, 44, 50,
Irrespective of the detailed molecular pathways linking dis-
abled autophagy to mitochondrial apoptosis, the results con-
tained in this work add to the growing suspicion that type 1 and
type 2 cell death may be somehow interwoven. Thus, in Dro-
sophila development, the involution of the salivary gland—a
paradigm of type 2 cell death—is actually linked to the expres-
sion of numerous proapoptotic genes (20, 41) and requires
activation of caspases (48). In mammals, Beclin 1/Atg6 has
been shown to interact with Bcl-2 (46), and down-regulation
of Bcl-2 by the antisense approach can actually trigger type
2 cell death (62). A BH3-only protein interacting with Bcl-
2, HSpin1, has also been found to induce type 2 cell death
(72). DAP kinase, a well-known tumor suppressor gene (35),
stimulates autophagy as well as apoptosis-related membrane
blebbing (27). Finally, a series of bona fide apoptosis induc-
ers can trigger type 2 cell death in some cell types (31, 55,
Two important notions emerge from our findings. First, au-
tophagy may preserve cellular homeostasis while suppressing
the latent apoptotic program, meaning that under conditions
of nutrient deprivation, autophagy can actually suppress cell
death, presumably by providing endogenous metabolites when
exogenous nutrients are missing. While it is not a new finding
that autophagy can rescue cells under conditions of starvation
(34, 37, 66), it is novel that autophagy actually prevents the
apoptotic default pathway to be activated. This finding may be
incorporated into a more general hypothesis suggesting the
existence of a double switch between the two principal lethal
FIG. 6. Effect of apoptosis inhibitors on starvation-induced cell death occurring in autophagy-inhibited cells. (A and B) Effect of the inhibitors
on cell death markers. HeLa cells stably transfected with vector only (Neo), left untreated (Neo Co), or treated with the pan-caspase inhibitor
Z-VAD-fmk human or cells transfected with Bcl-2 or cytomegalovirus-derived vMIA were cultured under the indicated conditions (not starved in
CM and starved in NF) in the absence (control [CO]) or presence of the indicated autophagy inhibitors for 24 h. Then, the percentages of
cells (x ? SEM; n ? 4) with low-level DiOC6(3) incorporation (A) and PI-permeable plasma membranes (B) were determined by cyto-
fluorometry. Mon, monensin. (C) Apoptosis inhibition does not affect the accumulation of autophagosomes. Cells (Neo-, Bcl-2-, or vMIA-
transfected cells as described for panels B and C) were transfected with the LC3-GFP chimera and then treated with HCQ (24 h) in the presence
(CM) or absence (NF) of serum, and in the presence of Z-VAD-fmk where indicated, followed by counterstaining with Hoechst 33342 (Ho) and
VOL. 25, 2005APOPTOSIS AND AUTOPHAGY1035
1036BOYA ET AL.MOL. CELL. BIOL.
FIG. 8. siRNA of Beclin 1 sensitizes cells to starvation-induced cell death. (A) siRNA-induced downregulation of Beclin 1, determined by
immunoblotting at the indicated times after treatment with scrambled control (SC) siRNA or two different Beclin 1-1-targeted double-stranded
oligoribonucleotides. (B) Effect of Beclin 1-specific siRNA on the colocalization of mitochondria (DsRed labeled) or lysosomes (GFP labeled)
after culture in NF. The degree of colocalization (x ? SEM; n ? 12) was determined as described for Fig. 3. (C and D) Beclin 1 knockdown
sensitizes cells to nutrient depletion-induced cell death. HeLa cells that were either wild type (C) or transfected with the neomycin resistance gene,
vMIA, or Bcl-2 (D) were exposed to the indicated siRNA and then cultured for 24 h in nutrient-rich (CM) or nutrient-deficient (NF) medium,
in the presence (C) or absence (C and D) of Z-VAD-fmk (Z-VAD) or STS (15 h) followed by DiOC6(3) or PI staining (x ? SEM; n ? 4).
?, P was ?0.05 versus values with CM; §, P ? 0.05 versus values with NF.
FIG. 7. DKO of Bax and Bak protects against starvation-induced cell death exacerbated by autophagy inhibition. Wild-type (WT) mouse
embryonic fibroblasts or Bax and Bak DKO (Bax?/?Bak?/?) fibroblasts were cultured in CM or NF and incubated with 3-MA, Baf A1, monensin
(Mon), or HCQ for 24 h and then stained with a low concentration of DiOC6(3) (A) or a high concentration of PI (B), followed by cytofluorometric
analysis (x ? SEM; n ? 3). Alternatively, cells were stained with CMFDA and Hoechst 333234 and observed under fluorescence microscopy.
Representative images for monensin- and HCQ-treated cells (in CM or NF) are shown. CO, control.
VOL. 25, 2005 APOPTOSIS AND AUTOPHAGY 1037
signaling pathways. On the one hand, inhibition of apoptosis
(induced by growth factor withdrawal) can lead to a chronic
degenerative autophagic cell death (17, 70). On the other
hand, prevention of autophagy can precipitate death, as shown
here. Second, cells that exhibit morphological hallmarks of
type 2 cell death with a massive AV accumulation, due to a
reduced formation of autophagolysosomes, are not yet com-
mitted to cell death when mitochondria are intact. Paradoxi-
cally, such cells ultimately die from apoptosis, if we define
apoptosis in biochemical terms as a process that can be inhib-
ited by Bcl-2, vMIA, the DKO of Bax and Bak, or caspase
inhibitors. Thus, the presumed mechanistic contraposition of
the type 1 and type 2 forms of cell death deserves close scrutiny
and critical reevaluation.
FIG. 9. Targeting of Atg genes enhances the lethality of nutrient depletion. (A) Reduction of mRNA levels by Atg-specific siRNA. After
treatment with the indicated siRNA, the level of mRNA was determined by quantitative RT-PCR after 24 h. Results (x ? SEM; n ? 3) are
expressed as percentages, with 100% being considered the mRNA level of untreated control cells. (B) Effect of Atg-specific siRNA on the
colocalization of mitochondria and lysosomes determined as described for Fig. 3B. (C and D) Apoptosis inhibition reduces cell death induced by
starvation plus Atg depletion. Cells with the indicated genotype (wild type in panel C; Neo, Bcl-2, or vMIA in panel D) were treated with diverse
siRNA constructs (24 h) and then starved for nutrients and/or treated with Z-VAD-fmk (Z-VAD) or STS. Data result from cytofluorometric
analyses of DiOC6(3)- or PI-stained cells (x ? SEM; n ? 4). ?, P was ?0.05 versus values with CM; §, P was ?0.05 versus values with NF.
1038 BOYA ET AL.MOL. CELL. BIOL.
We thank Jennifer Lippincott-Schwartz (Cell Biology and Metabo-
lism Branch, National Institute of Child Health and Human Develop-
ment, National Institutes of Health, Bethesda, Md.), Victor Goldma-
cher (ImmunoGen, Inc., Cambridge, Mass.), Stanley J. Korsmeyer
(Harvard Medical School, Boston, Mass.), and Norma W. Andrews
(Section of Microbial Pathogenesis and Department of Cell Biology,
Yale University School of Medicine, New Haven, Conn.) for reagents;
Yann Lecluse (IGR, Villejuif, France) for cell sorting; Ana-Maria
Cuervo (Albert Einstein College of Medicine, Bronx, N.Y.) for helpful
discussion; Patrick Fitze (Laboratory of Ecology, University Pierre and
Marie Curie, Paris, France) for statistical advice; and Abdelali Jalil
(Institut Gustave Roussy, Villejuif, France) for confocal micros-
This work has been supported by a special grant from the LNC as
well as grants from the ANRS and the European Commission (QLK3-
CT-2002-01956 to G.K.). P.B. received a fellowship from the European
Commission (MCFI-2000-00943), as did R.-A.G.-P. (FP6-2002-5000698);
N.C. and J.-L.P. received grants from the FRM and the ANRS, re-
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