Transfection and RNA interference.

Small interfering RNAs (siRNAs) were synthesized by Proligo France SAS. For Beclin 1 (National Center for Biotechnology Information accession number AF077301), RNA sequences started at positions 189 (CUCAGGAGAGGAGCCAUUU) and 1206 (GAUUGAAGACACAGGAGGC) 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 (GGAGUUCAUGAGUGCUAUA); 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 manufacturer'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 (Invitrogen), 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) plasmid (Clontech).

RNA extraction and quantitative RT-PCR.

mRNA preparations were obtained with the RNeasy mini kit (QIAGEN) and quality controlled with an Agilent 2100 bioanalyzer (Agilent Technologies). For quantitative reverse transcription (RT)-PCR, cDNAs were synthesized from 1 μg of total RNA with Moloney murine leukemia virus reverse transcriptase (Roche). Then, the TaqMan 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 Applied Biosystems).

Reagents and cell death induction.

Hydroxychloroquine (HCQ; Sanofi-Synthelabo) was used from a stock solution at 30 μg/ml unless otherwise specified. Bafilomycin A1 (Baf A1; 0.1 μM; Sigma), monensin (10 μM; 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 μg/ml). For caspase inhibition, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk; Bachem) was added at the same time as cell death inducers at 25 μM (8).

Flow cytometry and cell sorting.

The following fluorochromes were employed to determine apoptosis-associated changes by cytofluorometry: 3,3′-dihexyloxacarbocyanine iodide [DiOC₆(3), 40 nM] for mitochondrial transmembrane potential (ΔΨ_m) quantification, propidium iodide (PI; 1 μg/ml) and 44,6-diamino-2-phenylindole (DAPI; 2.5 μΜ) 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 μg 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 μM; Molecular Probes) and Hoechst 33342 (2 μM; Sigma), followed by fluorescence 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 paraformaldehyde (4%, wt/vol) and picric acid (0.19%, vol/vol) for LC3-GFP and immunofluorescence 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 10 μm.

Electron microscopy.

Cells were fixed for 1 h at 4°C in 1.6% glutaraldehyde in 0.1 M Sörensen phosphate buffer (pH 7.3), washed and fixed again in aqueous 2% osmium tetroxide, and embedded in Epon. Electron microscopy was performed 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 horseradish peroxidase-labeled secondary antibodies (Southern Biotechnologies Associates) and detected by SuperSignal West Pico chemiluminescent substrate (Pierce). Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Chemicon) was used to ensure equal loadings.

Analysis of protein degradation.

HeLa cells were incubated with 0.2 μCi 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 μg 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 dissolved in Soluene 350. The rate of protein degradation was calculated as the level of acid-soluble radioactivity recovered from both cells and media (58).

Pharmacological inhibition of macroautophagy precipitates starvation-induced cell death.

Although HCQ induced an increase in AV (Fig. ​(Fig.11 and ​and2),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. ​(Fig.3A).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 increased colocalization of mtDsRed- and lysosome-targeted SytVII-GFP, and this colocalization was inhibited by HCQ (Fig. ​(Fig.3B).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. ​(Fig.3C)3C) and reduced the turnover of long-lived proteins (Fig. ​(Fig.3A).3A). Moreover, HCQ prevented the nutrient starvation-induced colocalization of the lysosomal marker LAMP2 and the AV marker LC3-GFP (Fig. ​(Fig.3D)3D) and hence prevented the formation of autophagolysosomes, as has been shown previously for Baf A1 (71).

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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 μg/ml) and immunostained for LAMP2 detection to visualize the overlap with LC3-GFP (yellow). Representative images are shown.

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. ​(Fig.4A).4A). This effect was again similar to that induced by Baf A1 and monensin (Fig. ​(Fig.4B).4B). HCQ, Baf A1, and monensin also led to the redistribution of an LC3-GFP fusion construct into punctate cytoplasmic structures (Fig. ​(Fig.4C),4C), indicating an increase in AV. Kinetic experiments revealed that the LC3-GFP redistribution to vacuoles occurred in cells well before nuclear apoptosis increased above background levels (Fig. ​(Fig.4D).4D). However, all autophagy inhibitors, including 3-MA, did induce nuclear apoptosis when they were added to starved cells (Fig. ​(Fig.5A).5A). While starvation alone did not cause major cell death (as defined by a loss in ΔΨ_m or staining with PI), the combination of starvation and autophagy inhibition (with HCQ, Baf A1, monensin, or 3-MA) had a synergistic lethal effect (Fig. ​(Fig.5B).5B). Similar results were obtained when PS exposure was measured (unpublished results). Moreover, we found that caspase-3 was synergistically activated by nutrient depletion and autophagy inhibition (Fig. ​(Fig.5C).5C). In strict contrast, autophagy inhibition did not increase the lethal actions of classical apoptosis inducers such as STS (Fig. ​(Fig.5D)5D) and the CD95 agonistic antibody 7C11 (unpublished results). As a result, it appears that pharmacological autophagy inhibition can sensitize cells to starvation-induced cell death.

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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-GFP^Vac) or apoptotic nuclei was scored. CO, control; Mon, monensin.

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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 ΔΨ_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 activecaspase-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.

Autophagic vacuolization-associated cell death is reduced by mitochondrial stabilization and caspase inhibition.

To determine 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 inhibitor Z-VAD-fmk as an apoptosis suppressor. Bcl-2 and vMIA reduced the losses in ΔΨ_m and viability induced by starvation plus autophagy inhibitors (Fig. 6A and B). Caspase inhibition with Z-VAD-fmk (25 μM) reduced the loss of viability induced by autophagy suppression yet had no significant effect on the loss in ΔΨ_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 autophagosomal redistribution of LC3-GFP induced by HCQ (Fig. ​(Fig.6C),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 embryonic 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. ​(Fig.7C),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, autophagy-inhibited cells.

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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 cytofluorometry. 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 fluorescent microscopy.

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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.

Knockdown of essential components of the autophagic machinery sensitizes cells to starvation-induced death.

Pharmacological inhibition of autophagy might be affected by nonwarranted side effects of drugs endowed with limited selectivity. Therefore, we used siRNA to knock down Atg genes implicated in the autophagic pathway. First, we targeted the essential Beclin 1/Atg6 gene (45). Two different siRNA constructs designed to down-regulate the expression of Beclin 1 (Fig. ​(Fig.8A)8A) reduced the colocalization of mitochondria and lysosomes induced by starvation compared to the level of colocalization produced by a scrambled siRNA control (Fig. ​(Fig.8B).8B). This manipulation also enhanced the losses in ΔΨ_m and viability induced by starvation yet had no effect on cell death induction by the universal apoptosis inducer STS (Fig. ​(Fig.8C).8C). Addition of the broad-spectrum caspase inhibitor Z-VAD-fmk (Fig. ​(Fig.8C)8C) or transfection with Bcl-2 or vMIA (Fig. ​(Fig.8D)8D) had a selective effect on viability (with inhibitory effects of Z-VAD-fmk, Bcl-2, and vMIA) and ΔΨ_m dissipation (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 mitochondria. We therefore used siRNA constructs designed to target other Atg genes (Atg5, Atg10, and Atg12) (Fig. ​(Fig.9A),9A), which did prevent mitochondrial-lysosomal colocalization (Fig. ​(Fig.9B).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, had no effect on the disruption of the ΔΨ_m) (Fig. ​(Fig.9C).9C). Again, both Bcl-2 and the mitochondrion-targeted viral inhibitor vMIA prevented death acceleration by the knockdown of diverse Atg genes (Fig. ​(Fig.9D).9D). Thus, siRNA experiments confirmed the cytoprotective potential of autophagy under conditions of starvation and extended the notion that impaired autophagy can kill nutrient-deprived cells by an apoptotic mechanism.

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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. ​Fig.3.3. (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.

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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. ​Fig.3B.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.

We thank Jennifer Lippincott-Schwartz (Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md.), Victor Goldmacher (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 microscopy.

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, respectively.