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Fig. 1. Conceptual model of macroautophagy.

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Fig. 3. Action and subversion ofautophagy during bacterial infection.Bacteria may be taken up byphagocytosis and the resultingphagosome can fuse with endosomesand then the lysosome; the bacteriaare then degraded within thephagolysosome (not shown). Somepathogens such as L. monocytogenes,escape this pathway by lysing thephagosome membrane. The bacteriamay subsequently become targets forautophagy.In addition, degradation of host cellproteins within the lateautophagosome orautophagolysosome may supply thenutrients needed for growth of thepathogen.

Fig. 2. Schematic model of autophagic regulation.

as demonstrated in yeast, inhibits autophagy. PTEN dephosphorylates 3-phosphoinositides and antagonizesthe action of class I PtdIns 3-kinase. A class III PtdIns 3-kinase complex, which includes Beclin 1/Atg6,generates PtdIns(3)P (purple circles) to control the membrane dynamics that are associated withautophagosome formation. Rapamycin inhibits mTor, while wortmannin and 3-methyladenine inhibit the classIII PtdIns 3-kinase; the effect is to induce or inhibit autophagy, respectively. Autophagy is also regulatedthrough heterotrimeric G proteins and other kinases and phosphatases that are not depicted.

Stimulation of the class IPtdIns 3-kinase at theplasma membrane throughthe insulin receptor resultsin the generation ofPtdIns(3,4)P2 andPtdIns(3,4,5)P3 (dark pinkcircles). Thesephosphoinositides allowbinding and activation ofAkt/PKB and its activatorPDK-1. Along with aminoacids, Akt/PKB activatesmTor (additionalcomponents in this pathwayare not depicted).Subsequentphosphorylation of adownstream effector,possibly analogous to Atg1or other ATG gene products

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Following growth factor withdrawal, Bax- /-Bak-/- cells activate autophagy, undergoprogressive atrophy, and ultimately succumbto cell death.

An additional consequence of growth factorlimitation is a rapid decline in the surfaceexpression of nutrient transporters including themajor glucose transporter GLUT1, the LDLreceptor, amino acid transporters and receptorsfor iron uptake

This decrease in nutrient transporter expressionhas been proposed to perturb mitochondrialphysiology resulting in the induction of apoptoticcell death.

An alternative explanation is that the decline insurface expression of nutrient transporters simplyreflects a secondary response to the decreasedmetabolic demand on the cell following thecessation of growth and the withdrawalfrom the cell cyclc.

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Cells from Bax-/- Bak-/- animals fail to undergoapoptosis in response to serum deprivation, lossof attachment, and growth factor withdrawal.Thus, Bax and Bak are essential and redundantregulators of apoptosis and extracellular signals.

(A) Two independent clones of Bax -/-Bak -/- IL-3-dependent cells (parental)stably transfected with either Bax, Bak, orempty vector (vec) were generated, andexpression levels were assessed byWestern blot. The IL-3-dependent Bax /Bak / cell line FL5.12 is shownfor comparison.

Figure 1. Bax -/- Bak -/- Cells Undergo Atrophy and Maintain ProlongedSurvival Following Withdrawal of Growth Factor

(B) Kinetics of cell death in Bax- or Bak-reconstituted cells following IL-3withdrawal. Viability was measured bypropidium iodide exclusion. Data areaverages of three experiments standarddeviation (SD).

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(D) Cell numbers of cultures that weregrown in the presence or absence of IL-3and were cultured as in (C). Data areaverages of three independentexperiments SD.

(E) Cell size of cultures that weregrown in the presence or absenceof IL-3 and were cultured as in (C).Data are averages of threeindependent experiments SD.

(C) Cell viability of Bax -/- Bak -/- cells in the presenceor absence of IL-3. Cells were washed and cultured inthe presence (open squares) or absence (closeddiamonds) of IL-3. At the indicated time points, cellswere collected and viability was assessed. Cells grownin the presence of IL-3 were passaged every 2–3 daysto restore a cell concentration of 7.5 105 cells/ml. Themedium in IL-3-deprived cultures was replaced with anidentical volume of fresh complete medium without IL-3every 10 days. Data are averages of three independentexperiments SD.

(A) Glycolytic rate of cells grown in the absence of IL-3 as measuredby the conversion of 5-3H-glucose to 3H2O at the indicated timepoints. The data presented at week 0 represent values of controlcells growing in IL-3 throughout the time course of the experiment.Data are averages of three experiments SD.

Figure 2. Metabolic Effects of IL-3 Withdrawal on Bax-/- Bak -/- Cells

(B) Western blot analysis of GLUT1 expressionin cells cultured in the absence of IL-3. TheGLUT1 expression at week 0 is representativeof GLUT1 expression of cells grown in IL-3.

time-dependent loss of GLUT1, the major glucosetransporter expressed on these cells

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(C) Mitochondrial membrane potentialas measured by TMRE stainingin cells grown without IL-3 (solidhistogram) at the indicatedtime point. Baseline TMRE wasdetermined by using cells treatedwith the uncoupler CCCP (dottedhistogram). The numbers in thetop right corner indicate the avergemean fluorescence intensityof three independent experiments. Theweek 0 time point indicatesthe mean fluorescence intensity of cellsgrowing in IL-3 and is representativeof the values obtained for such cellsover the time courseof the experiment.

Figure 2. Metabolic Effects of IL-3 Withdrawal on Bax-/- Bak -/- Cells

Coincident with the decline in glycolysis, there was adecline in mitochondrial membrane potential

(D) ATP levels in cells grown without IL-3 and expressed as arbitraryunits (AU). ATP levels for IL-3-growncells did not decline significantlyover the time course of the experiment(data not shown).Data are averages of threeindependent experiments SD.

Figure 2. Metabolic Effects of IL-3 Withdrawal on Bax-/- Bak -/- Cells

Cellular ATP levels also fell, but the decline in glucose transporterexpression was greater than that expected based on the ATP decline,suggesting that cells were utilizing alternative substrates to maintain theirbioenergetics.

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The continued decline in cell size of the G0/G1 arrestedcells following growth factor withdrawal suggestedthe possibility that cells were utilizing macroautophagyto catabolize intracellular substrates to maintaintheir survival.

(A) Electron microscopy of cellsgrown in the absence of IL-3 for48 hr (a–c) showing thepresence of autophagosomes.Arrowheads depictrepresentative autophagosomesquantitated in (d). Scale bar, 100nm. (d) Quantitation of thenumber of autophagosomes percross-sectioned cell cultured inthe presence or absence of IL-3for 48 hr. Error bar representsSD. Statistical significancedetermined by Student’s t test.

Figure 3. Growth Factor Withdrawal Induced Autophagosome Formation IsRequired for Survival

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(B) Immunofluoresence with anti-LC3*antibody on cells grown in the presence (a)or absence (b) of IL-3 for 48 hr.

(*) antibody specific for themammalian homolog of theyeast Atg8 protein,microtubule-associatedprotein-1 light chain-3 (LC3).

(C) Immunoblot analysis of LC3-I processing into LC3-IIin cells transfected with control or two independentshRNA constructs against ATG5 (hp-2 and hp-7)followed by culture in the presence or absence of IL-3for 48 hr. Actin was used as a loading control.

(D) Time course of cell viability following IL-3withdrawal in cells with inactivation of ATG5. Dataare averages of three experiments SD.Western blot analysis of ATG5 protein expressionin cells transfected with vector control, hp-2, or hp-7 shRNA is shown as a representativeexperiment. Actin was used as loading control.

(E) Time course of cell viability following IL-3withdrawal in cells transfected with FITCtagged-siRNA for ATG7 (Yu et al., 2004) or acontrol siRNA. Cells which had incorporatedthe siRNA for ATG7 or control were purified byFACS sorting based on FITC-positive cells,and viability was assessed at the indicatedtime points. Data are averages of threeexperiments SD.

Inhibition of Autophagy Leads to Cell Death

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(A) Electron microscopy of cells grown in the presence (a) orabsence (b) of IL-3 for 6 weeks. Scale bar, 8.5 m. Magnificationimage of a cell grown in the presence (c) or absence (d) of IL-3showing autophagosomes (arrows). Scale bar, 2.3 m.

Figure 4. Persistent Autophagy in Long-Term Growth Factor-Withdrawn Cells

IL-3 -IL-3

Higher magnification of cells grown in the absence of IL-3 (e and f). Arrowheads depictautophagosomes in cells containing recognizable cellular material (e) or a late autophagosomefusing with a lysosome (f). Arrowheads depict representative autophagosomes quantitated in (B).

Long termdeprivation(6 weeks)

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(A) Viability of cells grownin the presence (top panel)or absence (bottom panel)of IL-3 for 6 weeks treatedwith 5 mM 3-MA (closedsquares) or 10 M CQ(open triangles). PBS wasused as a vehicle control(closed diamonds).

Figure 5. Cell Death Following Inhibition of Autophagy Is Reversed by Methylpyruvate

3MA (3-Methyladenine) and CQ (chloroquine) = inhibitors of automacrophagy

IL-3

-IL-3

(B) Immunofluorescence staining of LC3 in cells grown in the presence (a) or absence (b) ofIL-3 for 6 weeks. Cells grown in the presence or absence of IL-3 were treated for 18 hr with 5mM 3-MA (c and d) or 10 M CQ (e and f) followed by LC3 staining. PBS was used as avehicle control.(C) DNA fragmentation assay was performed on Bax / Bak / cells grown in the presence orabsence of IL-3 for 6 weeks and treated for 36 hr with 5 mM 3-MA, 10 M CQ, or PBS as avehicle control. IL-3-dependent Bax -/- Bak -/- FL5.12 cells grown in the absence of IL-3 for36 hr were used as a positive control for DNA laddering.

NoDNAladder

DNA ladder

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A cell-permeable form of pyruvate, methylpyr- uvate (MP), wasadded to the cultures at the time 3-MA or CQ treatment. Onceinternalized, this substrate can be oxidized in the tricarboxylicacid cycle to produce NADH to fuel electron transport and ATPproduction.

Figure 7. IL-3 Restimulates Glycolysis and Growth/Proliferation in Growth Factor-Deprived Cells

Despite the loss of cell surface nutrient transporters,the absence of an observable Golgi/ER, and aprofound decline in total protein content, the cellscultured in the absence of IL-3 had higher levels ofsurface IL-3 receptor than cells grown in the presenceof IL-3.

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(E) Cell size recovery following IL-3 readdition is dependent onthe duration of deprivation. Histogram of mean cell size (fL) incells restimulated with IL-3 for the indicated number of daysfollowing 2 (left panel) or 6 (right panel) weeks of growth factorwithdrawal.

2 weeks withdrawal 6 weeks withdrawal

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Based on the results, macroautophagy appears to be an evolutionarilyconserved survival strategy. Macroautophagy can support growthfactor-independent cell survival of hematopoietic cells for severalweeks.

Macroautophagy Is a Conserved but Self-LimitedSurvival Mechanism

Thus, it appears eukaryotic cells share a common survival pathwaythat promotes cell-autonomous survival in the face of starvationand/or neglect. Animal cells may have evolved an apoptoticresponse in part to limit this form of cell-autonomous survival.Nevertheless, as previously demonstrated in unicellular organisms,macroautophagy is a self limited survival strategy and ultimately willresult in cell death if not reversed.