Autophagy is a lysosome-based catalytic process mainly activated
in cells under metabolic stress (Klionsky and Emr, 2000). In the
early phase, autophagy generates additional energy supplies for
starved cells, which maintains cell survival, while sustained cellular
digestion through the process of autophagic cycling eventually
results in cell death, known as autophagy-dependent cell death
(Kourtis and Tavernarakis, 2009). Recently, a large body of literature
demonstrated that there are multiple interchanges at different
molecular levels between autophagy and apoptosis pathways
(Maiuri et al., 2007).
Currently, little is known about the genetic control of necrotic
cell death. Traditionally, necrosis was considered as a passive form
of cell death resulting from physical damage or toxic insults (Walker
et al., 1988). However, new evidence that has emerged in the past
few years has shown that necrosis might not be an accidental form
of cell death, but a programmed cell death with evolutionarily design
and a distinctive biochemical cascade (Hitomi et al., 2008). In certain
experimental settings where apoptotic machinery is blocked by
pharmacological or genetic means, apoptotic stimuli induce necrotic
cell death by activating intracellular signaling pathways (Chan et
al., 2003; Zong et al., 2004; Okada et al., 2004; Degterev et al.,
2005). In an elegant model system of atg1-deficient Dictyostelium
cells in which the machinery for both apoptosis and autophagy is
destroyed, irreversible lysosomal permeabilization was identified
as the causal factor for necrotic cell death after mitochondrial
uncoupling (Giusti et al., 2009). Consistently, in a genetically
modified C. elegans model, the requirement of a lysosomal-
dependent autophagic response was confirmed for necrotic cell death
(Samara et al., 2008). These data suggested that lysosomal damage
plays a critical role in necrotic cell death in addition to autophagy
and apoptosis (Boya and Kroemer, 2008). However, it is not fully
understood which molecular switches determine cell fate among
the different types of cell death after a death inducing signal.
Glycogen synthase kinase-3 (GSK-3) is an ancient protein with
a diverse range of cellular functions (reviewed by Forde and Dale,
2007). There are two isoforms of GSK-3 in mammals, GSK-3a
and GSK-3b. Although these two isoforms are expressed
ubiquitously and share over 98% identity within their kinase
domains, they are not redundant in vivo. For example, in miceGsk3b
knockout resulted in embryonic death due to hepatocyte apoptosis,
indicating that GSK-3b loss is not compensated by GSK-3a
(Hoeflich et al., 2000). Unlike other protein kinases, GSK-3b is
constantly active and its activity is even higher under serum
starvation, indicating that GSK-3bplays a role in cell survival under
resting conditions. Currently, it is not fully clear how GSK-3b
controls cell survival under serum-free conditions.
Bax interacting factor 1 (Bif-1; also known as SH3GLB1) was
initially cloned as a binding partner of the pro-apoptotic Bax protein
(Cuddeback et al., 2001; Pierrat et al., 2001). Later, Bif-1 was shown
to promote Bax conformational change under the control of Src
kinase in apoptotic cells (Takahashi et al., 2005; Yamaguchi et al.,
2008). Most interestingly, Bif-1 was recently found to modulate
autophagy by interacting with beclin-1–VPS34 complex through
the UVRAG protein (Takahashi et al., 2007). Moreover, a recent
study suggested that Bif-1 might act as a driving force for
autophagosome formation (Etxebarria et al., 2009) (reviewed by
Takahashi et al., 2009). In this study, we discovered that Bif-1 is
GSK-3b b promotes cell survival by modulating Bif-1-
dependent autophagy and cell death
Jun Yang1,2, Yoshinori Takahashi3, Erdong Cheng1, Jihong Liu2, Paul F. Terranova4, Bin Zhao5,
J. Brantley Thrasher1, Hong-Gang Wang3and Benyi Li1,4,5,*
1Department of Urology, The University of Kansas Medical Center, Kansas City, Kansas 66160, USA
2Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
3Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
4Departments of Molecular and Integrated Physiology, and Obstetrics and Gynecology, The University of Kansas Medical Center, Kansas City,
KS 66160, USA
5Department of Neurology, The Affiliated Hospital, Guangdong Medical College, Zhanjiang 524001, China
*Author for correspondence (firstname.lastname@example.org)
Accepted 14 December 2009
Journal of Cell Science 123, 861-870
© 2010. Published by The Company of Biologists Ltd
Glycogen synthase kinase 3 beta (GSK-3b) is constantly active in cells and its activity increases after serum deprivation, indicating
that GSK-3b might play a major role in cell survival under serum starvation. In this study, we attempted to determine how GSK-3b
promotes cell survival after serum depletion. Under full culture conditions (10% FBS), GSK-3b inhibition with chemical inhibitors or
siRNAs failed to induce cell death in human prostate cancer cells. By contrast, under conditions of serum starvation, a profound necrotic
cell death was observed as evidenced by cellular morphologic features and biochemical markers. Further analysis revealed that GSK-
3b-inhibition-induced cell death was in parallel with an extensive autophagic response. Interestingly, blocking the autophagic response
switched GSK-3b-inhibition-induced necrosis to apoptotic cell death. Finally, GSK-3b inhibition resulted in a remarkable elevation of
Bif-1 protein levels, and silencing Bif-1 expression abrogated GSK-3b-inhibition-induced autophagic response and cell death. Taken
together, our study suggests that GSK-3b promotes cell survival by modulating Bif-1-dependent autophagic response and cell death.
Key words: Bif-1, GSK-3b, Necrosis, Apoptosis, Autophagy
Journal of Cell Science
required for GSK-3 inhibitor-induced autophagic response, necrosis
and apoptosis. Under serum-free conditions, suppressing GSK-3
activity induced a Bif-1-dependent autophagic response and
subsequent massive necrotic cell death. Suppressing autophagy at
either the early phase or later stage did not rescue cells but redirected
necrotic cell death to apoptosis through a mechanism involving Bif-
1-dependent Bax conformational change.
GSK-3b b suppression results in cell death under serum
Although GSK-3b is proposed to promote cell survival (Forde
and Dale, 2007), the mechanism is not fully clear. We recently
reported that inhibition of GSK-3b activity by lithium chloride
(LiCl) suppressed human cancer cell proliferation without
affecting cell survival under full culture (10% FBS) conditions
(Sun et al., 2007). To explore the functional role of GSK-3b in
cell survival after serum withdrawal, we utilized multiple GSK-
3b inhibitors, one of them is a pseudo-substrate peptide L803-
mts (Plotkin et al., 2003) and another one is a non-ATP competitive
small chemical TDZD8 (Martinez et al., 2002). Consistent with
our previous report (Sun et al., 2007), treatment of human cancer
PC-3 cells with these inhibitors in serum-containing medium did
not induce obvious cell death although cell proliferation was
significantly suppressed compared with the control (Fig. 1A).
Surprisingly, under serum-free conditions, a significant cell death
was found in both L803-mts- and TDZD8-treated cells compared
with the solvent control. In addition to L803-mts and TDZD8,
two other structurally unrelated GSK-3b inhibitors, AR-A014418
(Bhat et al., 2003) and SB216763 (Carmichael et al., 2002) also
induced a dose-dependent cell death (supplementary material Fig.
To determine the GSK-3bspecificity of these inhibitors, siRNAs
for GSK-3b and GSK-3a were transfected into PC-3 cells and cell
death was monitored for up to a week under serum-free conditions.
As shown in Fig. 1B, GSK-3b siRNAs but not GSK-3a siRNAs
Journal of Cell Science 123 (6)
Fig. 1. GSK-3b b suppression leads to
cell death under serum starvation.
(A)PC-3 cells were plated in 12-well
plates overnight and then treated with
L803-mts (100M) or TDZD8 (10M)
in FBS-supplied or serum-free medium
for up to 3 days. At each time points as
indicated, live cells were counted using
the Trypan Blue exclusion assay as
described in our previous publication
(Liao et al., 2005). (B)PC-3 cells were
transfected with GSK-3 siRNAs or a
negative control siRNA mixture, as
indicated, at a final concentration of
200 nM in serum-free medium. At each
time point, as indicated, cells were
counted as above. Inset: proteins were
extracted from cells transfected with
GSK-3 siRNA or the control siRNA for
3 days, and then subjected to SDS-
PAGE and immunoblotting with anti-
GSK-3 antibodies. (C)PC-3 cells were
plated in 35 mm dishes overnight and
then treated with the solvent, L803-mts
alone (100M), 3-MA alone (10 mM)
or L803-mts plus 3-MA in serum-
supplied (10% FBS) or serum-free
medium. Photomicrographs were taken
24 hours after treatment. Original
magnification ?200. Black arrows
indicate dead cells with cytolytic
features. White arrows indicate
collapsed cells without membrane
rupture. (D)PC-3 cells were treated
with SB216763 at the indicated doses
for 3 days, and cell death was assessed
by YoPro-1–PI-staining-based flow
cytometry assay. In A, B and D data are
means ± s.e.m. from three independent
experiments. Asterisks indicate
significant difference compared with
the control (ANOVA analysis, P<0.05).
Journal of Cell Science
Bif-1 in cell death
significantly induced cell death. These data indicate that GSK-3b
activity is essential for cell survival under serum-free conditions.
To examine the characteristics of cell death induced by GSK-3b
inhibition, we first used microscopy to examine cellular
morphological change during L803-mts-induced cell death. As
shown in Fig. 1Cd, a cytolytic event, as evidenced by cytoplasmic
membrane rupture and cell lysis, was observed after L803-mts
treatment under serum-free condition. By contrast, cells treated with
L803-mts in the presence of serum or treated with the solvent in
serum-free medium did not show any sign of cytolysis (Fig. 1Ca-c).
We then characterize the cell death with a flow cytometry-based
YoPro-1–propidium iodine (PI) staining assay since the impermeant
nuclear dye YoPro-1 stains apoptotic cells accurately. The
combination of YoPro-1 with PI dye can provide a distinction
between apoptotic (YoPro-1 positive) and non-apoptotic cell death
(PI positive, YoPro-1 negative) (Idziorek et al., 1995). As shown
in Fig. 1D, GSK-3b inhibition did not increase apoptotic death
(YoPro-1 positive) but significantly increased non-apoptotic death
(PI-positive, YoPro-1-negative) in a dose-dependent manner. Also,
a clear dose-dependent transition from living (PI negative, YoPro-
1 negative) to non-apoptotic death (PI positive, YoPro-1 negative)
occurred after SB216763 treatment (supplementary material Fig.
S2). These data indicate that GSK-3b inhibition resulted in a non-
apoptotic cell death under serum starvation.
GSK-3b b inhibitor-induced cell death is necrotic under
Next, we confirmed the non-apoptotic cell death using two classical
apoptosis markers, PARP cleavage and caspase-3 processing. A
DNA damaging agent camptothecin (CPT) was included as a
positive control for apoptotic cell death. As expected, CPT treatment
induced a time-dependent caspase-3 processing and PARP cleavage
in PC-3 cells. By contrast, L803-mts treatment failed to induce either
event (Fig. 2). These data clearly indicate that GSK-3b-inhibition-
induced cell death is a non-apoptotic event.
Nuclear protein HMGB-1 is retained within the nuclear
compartment in apoptotic cells but is released during necrotic cell
death or autophagic response to cytotoxic reagents (Zong et al.,
2004; Thorburn et al., 2009). Therefore, we determined if HMGB-
1 was released during L803-mts-induced cell death. HMGB-1 levels
in conditioned medium were assessed after treatment with L803-
mts or CPT. As expected, CPT treatment did not induce HMGB-1
release (Fig. 2, bottom panel). Conversely, HMGB-1 release was
detected at 16 h after L803-mts treatment. Taken together, these
data suggest that GSK-3b inhibition induced necrotic cell death
under serum-free conditions.
GSK-3b b suppression promotes autophagy under serum
To determine whether GSK-3b inhibition triggered autophagic
response under serum starvation, we first assessed vacuole formation
(vacuolization) with transmission electronic microscopy (TEM). As
shown in Fig. 3A, L803-mts-treated PC-3 cells underwent a
remarkable vacuolization at 12 hours post-treatment under serum-
free conditions. Consistent with cell survival data, L803-mts did
not induce notable vacuolization under full culture (10% FBS)
Next, we used another indicator of autophagic response, LC3
translocation. As an important component of the autophagy
machinery, LC3 is processed from LC3-I to the membrane-bound
form LC3-II by conjugating with phosphatidylethanolamine and
subsequently accumulates in autophagosomes during the induction
of autophagy (Kabeya et al., 2000). PC-3 cells with stably
transfected GFP-LC3 were treated with or without GSK-3b
inhibitors for 8 hours under serum-free or serum-supplied
conditions. As shown in Fig. 3B,C, the defuse pattern of GFP-LC3
expression was not changed after treatment of cells with L803-mts
or TDZD8 in serum-supplied (10% FBS) medium. By contrast,
under serum-free conditions, L803-mts or TDZD8 treatment
dramatically increased the punctate foci of GFP-LC3 expression,
which was consistent with the TEM data (Fig. 3A). Then, GSK-
3b siRNAs was used to confirm the specificity of the chemical
inhibitor-induced GFP-LC3 translocation. As shown in Fig. 3D,E,
GSK-3b siRNAs remarkably increased the formation of GFP-LC3
punctate foci under serum-free conditions compared with the
control siRNAs or under the full culture (10% FBS) conditions.
To determine the correlation of the autophagic response and cell
death induced by GSK-3b suppression, we analyzed LC3
processing, PARP cleavage and HMGB-1 release by western
blotting. As shown in Fig. 3F,G, L803-mts dramatically enhanced
LC3-I processing as evidenced by increased levels of the LC3-II
fragment at 8 hours post-treatment. Meanwhile, PARP cleavage was
not detected and HMGB-1 release was observed at 16 hours after
L803-mts treatment. We also determined whether the autophagic
flux induced by GSK-3b inhibition was completed by assessing
p62 protein level, a marker for autophagy flux (Klionsky et al.,
2008). As shown in Fig. 3F, p62 protein levels dramatically
declined following L803-mts treatment. These data suggest that
GSK-3b inhibition triggered a strong autophagic flux that was
followed by a necrotic cell death.
Blocking autophagy switches GSK-3b b inhibitor-induced
necrosis to apoptosis
Since we observed GSK-3b-inhibition-induced autophagic response
followed by necrotic cell death, we then asked if blocking autophagy
would reduce GSK-3b-inhibition-induced cell death. To test this
hypothesis, we treated cells with L803-mts plus the autophagy
inhibitor 3-MA (Selgen et al., 1982) with or without serum
supplement. Under serum-supplied (10% FBS) conditions, treatment
with 3-MA alone had no obvious effect on cell survival (Fig. 1Ce,f),
Fig. 2. GSK-3b b inhibitor induces necrotic cell death. PC-3 cells were plated
in six-well plates and then treated with camptothecin (CPT; 3.0M) or L803-
mts (100M) in serum-free medium. Equal amount of cellular proteins were
used in western blotting to detect caspase processing and PARP cleavage as
indicated on the left. Actin was used as a protein loading control. To analyze
HMGB-1 release, cell culture media were collected and the supernatants were
concentrated using a 3.0-kDa cutoff column. Equal amount of proteins from
the concentrated supernatants were subjected to SDS-PAGE followed by
immunoblotting with HMGB-1 antibody. The experiment was repeated twice.
Journal of Cell Science
similar to the results when L803-mts was used alone (Fig. 1Cc).
Unexpectedly, under serum-free conditions, a Trypan-Blue-based
cell counting assay revealed that cell survival was similarly
attenuated by 3-MA plus L803-mts treatment compared with L803-
mts alone (Fig. 4A). In addition, a similar pattern of lactate
dehydrogenase (LDH) release, the marker of cytotoxic effect that
increases in both necrosis and apoptosis (Kroemer et al., 2009),
was observed after treatment with either L803-mts alone or L803-
mts plus 3-MA (Fig. 4B). However, microscopic examination
revealed that cellular collapse but not plasma membrane rupture or
cytolysis was induced after L803-mts plus 3-MA treatment
(Fig. 1Ch) under serum-free conditions, indicating that 3-MA
addition switched L803-mts-induced necrosis to a different type of
Then, we went on to determine if the cell death induced by L803-
mts plus 3-MA is apoptotic. In addition to 3-MA, another autophagy
blocker chloroquine (CQ), was included to block the autophagy
flux. Cell death was examined with the YoPro-1–PI assay. As
expected, 3-MA and CQ alone did not induce obvious cell death
compared with the control (Fig. 4Cb and c vs a), but treatment with
L803-mts alone resulted in a significant increase of PI-positive cells
in the population, a sign of necrotic cell death (Fig. 4Cd).
Journal of Cell Science 123 (6)
Fig. 3. GSK-3b b suppression induces strong
autophagic response. (A)PC-3 cells were treated
with L803-mts (100M) or the solvent as control
for 6-12 hours under FBS-supplied or serum-free
conditions. Transmission electron microscopy
was used to evaluate the vacuolization. Note that
massive vacuolization was observed in cells after
L803-mts treatment under serum-free conditions.
(B,C)PC-3 cells stably transfected with GFP-LC3
(PC3/GFP-LC3) were treated with the solvent or
L803-mts (100M), TDZD8 (10M) for 12
hours under serum-supplied (10% FBS) or serum-
free conditions. Photomicrographs were taken
under a fluorescent microscope at a magnification
of ?200. Quantitative data of the average GFP-
positive loci per cell are summarized in C.
(D,E)PC3/GFP-LC3 cells were transfected with
GSK-3b siRNAs (siGSK-3b, 200 nM) or the
control siRNA (200 nM) for 48 hours. Cells were
then cultured in serum-supplied (10% FBS) or
serum-free conditions, as indicated, for 12 hours.
Quantitative data of the average GFP-positive loci
per cell are summarized in E. (F,G)PC-3 cells
were plated in six-well pates and treated with
L803-mts (100M) under serum-free conditions.
Cells were harvested at the indicated time points.
Conditioned culture media were collected for
HMGB-1 analysis as described earlier. Equal
amount of cellular proteins were used in western
blotting analysis for LC3 processing, p62
degradation and PARP cleavage. Actin served as a
loading control. Quantitative data of band
densities are summarized in G. Data are from
three experiments and error bars indicate s.e.m.
Asterisks indicate a significant difference
compared with the control (ANOVA, P<0.01).
Journal of Cell Science
Bif-1 in cell death
Interestingly, addition of 3-MA or CQ to L803-mts treatment
resulted in a dramatic increase in YoPro-1-positive, PI-positive cell
population (Fig. 4Ce,f), suggesting that blocking autophagy flux
redirected necrosis to apoptotic cell death.
Next, we used a fluorescent dye JC-1, an indicator of
mitochondrial damage due to apoptosis (Smiley et al., 1991), to
ascertain the shift of apoptotic cell death. As shown in Fig. 4D,
JC-1 staining of mitochondria (red color, evidence of functional
Fig. 4. Blocking autophagy flux
redirects GSK-3b b suppression-induced
necrosis to apoptosis. (A)PC-3 cells
were plated in 12-well plates and then
treated with 3-MA (10 mM), L803-mts
(100M) or 3-MA plus L803-mts in
serum-free conditions for 24 hours. Live
cells were counted after Trypan Blue
staining, as described earlier. (B)PC-3
cells were plated in 24-well plates and
then treated with L803-mts (100M) with
or without 3-MA (10 mM) under serum-
free conditions. Cell culture media were
collected at the indicated time points and
subjected to LDH assay as described in
the text. Data in A and B are means from
four independent experiments and error
bars indicate s.e.m. (C)PC-3 cells were
plated in six-well plates and then treated
with L803-mts in serum-free medium in
the presence of 3-MA (10 mM), CQ
(5M) or the solvent for 48 hours. Cells
were harvested and stained with YoPro-1
and PI dyes. Representative graphics of
cell profiles from the flow cytometry
analysis are shown. Note the pattern shift
of cell distribution from necrosis (d) to
apoptotic death (e and f). (D)PC-3 cells
were treated with L803-mts (100M),
AR-A011418 (3M), LiCl (10 mM),
SB216763 (30M) in the presence of
3-MA (10 mM) or the solvent for 24
hours in serum-free medium. Cells were
then stained with JC-1 from a
mitochondrial membrane potential assay
kit, as described in our previous
publication (Liao et al., 2005). Original
magnification ?200. (E)PC-3 cells were
plated in six-well plates overnight and
then treated with L803-mts (100M) in
serum-free medium for different time
periods, as indicated, in the presence or
absence of 3-MA (10 mM). Cells were
harvested and equal amounts of cellular
proteins were used for immunoblotting
with antibodies as indicated. HMGB-1
analysis was described earlier. The
molecular masses of the bands are
indicated on the right side of the blots.
(F,G)PC-3 cells were transfected with
Atg5 siRNAs (siAtg5; 200 nM) or the
negative control siRNA (siControl;
200 nM) for 48 hours, and then left
untreated (F) or treated with L803-mts
(100M) with or without 3-MA (10 mM)
in serum-free medium for 18 hours. Equal
amounts of proteins from cell lysates
were subjected to SDS-PAGE and
immunoblotting with the antibodies
indicated. Data in C-G are the results
from two independent experiments each.
Journal of Cell Science
mitochondria) was not changed after treatment with GSK-3b
inhibitors alone. Conversely, treatment with GSK-3binhibitors plus
3-MA resulted in green JC-1 staining, an indication of severe
damage of mitochondrial membrane potential and apoptotic cell
Thirdly, we examined this pattern change of cell death with two
classical apoptotic hallmarks, caspase-3 processing and PARP
cleavage. As shown in Fig. 4E, 3-MA addition to L803-mts
treatment dramatically reduced L803-mts-induced LC3 processing,
p62 degradation and HMGB-1 release but increased PARP cleavage
compared with L803-mts single treatment.
Finally, a gene silencing approach was used to confirm the effect
of pharmacological blockage of autophagy flux on the switch from
necrosis to apoptosis. Atg5, a critical component of the autophagy
machinery (Kuma et al., 2004), was silenced with a pooled siRNA
mixture. As shown in Fig. 4F, compared with the control siRNAs,
transfection with Atg5 siRNAs largely reduced Atg5 protein levels.
As expected, Atg5 siRNA dramatically suppressed serum starvation-
induced LC3 processing (Fig. 4F lane 3 vs lane 2), confirming the
blockade of autophagy. Consistently, L803-mts treatment-induced
LC3 processing and p62 degradation were largely attenuated in Atg5
siRNA-transfected cells (Fig. 4G, lane 4 vs lane 2), similar to 3-
MA treatment (Fig. 4G, lane 3). Meanwhile, PARP cleavage was
detected in Atg5-silenced cells but not in the control siRNA-
transfected cells after L803-mts treatment. Taken together, these
data demonstrated that blocking autophagy flux switches GSK-3b-
inhibition-induced necrosis to apoptotic cell death.
Bif-1 is required for GSK-3b b inhibitor-induced cell death
under serum starvation
As described so far, GSK-3binhibition induced a strong autophagic
response and subsequent necrotic cell death that was redirected to
apoptosis when autophagy was blocked. To determine the
mechanisms involved in GSK-3b-inhibition-induced autophagy flux
and cell death, we assessed the protein levels of several autophagy-
and apoptosis-related genes in response to L803-mts treatment with
or without 3-MA. As shown in Fig. 5A, a remarkable increase of
Bif-1 protein levels was found after L803-mts treatment with or
without 3-MA addition. No dramatic change was observed for Bcl-
2, Bax, beclin-1 and VPS34 proteins. Under serum-free conditions,
the increase of Bif-1 protein is in a L803-mts dose- and time-
dependent manner (Fig. 5A,C). By contrast, under culture conditions
with 10% FBS, Bif-1 protein levels remained unchanged after GSK-
3b inhibition (Fig. 5D), which is consistent with the cytotoxicity
data. These results indicate that Bif-1 is involved in GSK-3b-
inhibition-induced cell death.
Recent reports have shown that Bif-1 participates in both the
autophagic pathway (Takahashi et al., 2007) and apoptotic cell death
(Takahashi et al., 2005). Thus, we reasoned that Bif-1 might act as
a molecular switch between GSK-3b-inhibition-induced apoptosis and
necrosis. To test this hypothesis, we established a stable subline of
PC-3 cells in which Bif-1 expression was silenced (PC-3/shBif-1).
An empty-vector-transfected cell subline (PC-3/Puro) was established
as a control. These cells were treated with L803-mts with or without
3-MA. Similar to the parental PC-3 cells, in PC-3/Puro cells, L803-
mts treatment significantly reduced cell survival (Fig. 6A), enhanced
LC3 processing and p62 degradation, as well as caused HMGB-1
release (Fig. 6B). Addition of 3-MA did not attenuate L803-mts-
induced cell death (Fig. 6A) but resulted in PARP cleavage and
abolished HMGB-1 release (Fig. 6B). In PC-3/shBif-1 cells, in a sharp
contrast, L803-mts treatment did not induced cell death, and all of
LC-3 processing, p62 degradation, HMGB-1 release and PARP
cleavage were abolished, indicating that Bif-1 is required for GSK-
3b-inhibition-induced autophagy flux and cell death.
Journal of Cell Science 123 (6)
Fig. 5. GSK-3b b suppression causes Bif-1 protein accumulation. (A,B)PC-3 cells were left untreated or treated with L803-mts at indicated doses (M) in the
presence or absence of 3-MA (10 mM) for 18 hours in serum-free medium. After harvesting, equal amounts of cellular proteins were subjected to SDS-PAGE and
immunoblotting with the indicated antibodies. The relative band densities were normalized against the anti-actin blot and summarized in B. Data are from two
separate experiments and error bars indicate the s.e.m. Asterisks indicate significant difference compared with the control (ANOVA, P<0.05). (C)PC-3 cells were
treated with the solvent or L803-mts (100M) in serum-free medium. Cells were harvested at the indicated time points and Bif-1 protein levels were assessed by
western blotting. (D)PC-3 cells were treated with the solvent, L803-mts (100M) or LiCl (10 mM) in serum-supplied (FBS, 10%) medium. Cells were harvested
at the indicated time points and western blotting was conducted with the primary antibodies as indicated. The anti-actin blot served as a protein loading control.
Journal of Cell Science
Bif-1 in cell death
Because Bif-1 is required for apoptotic cell death induced by
L803-mts plus 3-MA, we then determined whether Bax was
activated during apoptotic cell death. In PC-3/Puro cells, the level
of active Bax protein, determined with the conformational specific
Bax antibody 6A7 (Hsu et al., 1997), remained largely unchanged
after L803-mts treatment, compared with the control. However,
addition of 3-MA to the L803-mts treatment dramatically increased
the levels of the active Bax form (Fig. 6B, lane 3 vs 2). Most
interestingly, Bax activation was abrogated in PC-3/shBif-1 cells
after L803-mts treatment with or without addition of 3-MA (Fig.
6B, lanes 4 and 5), which was in agreement with the cell survival
data as shown in Fig. 6A. Taken together, these results clearly
indicate that Bif-1 is required for GSK-3b-inhibition-induced
autophagic response and cell death, and blocking autophagy flux
redirects necrosis to apoptosis, which is associated with Bif-1-
dependent Bax conformational change and/or activation.
It has been shown that in response to metabolic stress, Bif-1
interacts with a beclin-1–VPS34 complex, leading to VPS34
activation and autophagy induction (Takahashi et al., 2007). Thus,
we determined whether GSK-3b inhibition increases Bif-1
interaction with the beclin-1–VPS34 complex. As shown in Fig.
6D, L803-mts treatment largely increased beclin-1 interaction with
both Bif-1 and VPS34 (lane 2). However, addition of 3-MA to the
L803-mts treatment reduced Bif-1 interaction with beclin-1 but
enhanced beclin-1–VPS34 interaction (lane 4). These data suggest
that increased interaction between Bif-1 and the beclin-1–VPS34
complex may be involved in the GSK-3b-inhibition-induced
autophagic response. Addition of 3-MA prevented the interaction
between Bif-1 and beclin-1, leading to Bif-1-dependent Bax
activation and apoptotic cell death. However, the significance of
increased interaction of VPS34 with beclin-1 after 3-MA addition
needs further investigation.
Fig. 6. Bif-1 is required for GSK-3b b-suppression-induced
autophagic response and cell death. (A)PC-3/Puro and PC-
3/shBif-1 cells were treated with L803-mts (100M) with or
without 3-MA (10 mM) in serum-free medium for 2 days.
Live cells were counted after Trypan Blue staining, as
described previously. Inset: exponentially grown PC-3/Puro
and PC-3/shBif-1 cells were harvested for western blotting
with Bif-1 antibodies. The membrane was re-probed with
anti-actin antibody as a loading control. (B,C)PC-3/Puro and
PC-3/shBif-1 cells were treated with the solvent or L803-mts
(100M) plus 3-MA (10 mM) for 16 hours. Cell extracts
were subjected to immunoblotting with the antibodies as
indicated. To detect Bax conformational change, cellular
extracts were prepared in Chaps buffer and then subject to
immunoprecipitation with Bax 6A7 antibodies, followed by
immunoblotting with regular Bax antibody. Relative band
densities were normalized against the anti-actin blot and
summarized in C. (D,E)PC-3 cells were treated with L803-
mts (100M) plus or minus 3-MA (10 mM) in serum-free
medium for 18 hours and cellular proteins were subjected to
anti-beclin-1 immunoprecipitation and the eluted immuno-
complexes were analyzed by western blotting with Bif-1 and
VPS34 antibodies, as indicated. Data are from two
independent experiments. Relative band densities were
shown in E. Asterisks indicate significant difference
compared with the control (ANOVA, P<0.05).
Journal of Cell Science
In this study, we examined the functional role of GSK-3b in cell
survival under serum-free conditions and the mechanisms involved
in GSK-3b-inhibition-induced cell death. We demonstrated that
inhibition of GSK-3bactivity results in a strong autophagic response
and subsequent necrotic cell death, suggesting a critical role of GSK-
3b in fine-tuning autophagic response and in promoting cell
survival after serum deprivation. Most importantly, we identified
that Bif-1 plays a key role in the autophagic response and necrotic
cell death after GSK-3b inhibition. Blockage of autophagy flux
using pharmacological reagents and genetic means redirected Bif-
1-dependent necrosis to apoptosis, which was associated with a
conformational change in Bax. Therefore, we propose that Bif-1 is
a part of the cell death determinant that is modulated by GSK-3b
In recent years, autophagy has been the subject of considerable
attention because of its important role in cell fate determination and
human diseases (Klionsky and Emr, 2000). It has been shown that
autophagy suppression had different effects, either triggering or
preventing apoptotic cell death, depending on the death stimulus
(Boya et al., 2005; Takacs-Vellai et al., 2005; Wang et al., 2008).
However, under certain experimental settings, blunting the apoptotic
pathway leads to increased autophagic response and necrotic cell
death (Raymond et al., 2003; Cao et al., 2006; Ullman et al., 2008),
and simultaneous blocking of both autophagy and apoptosis induces
necrotic cell death (Degenhardt et al., 2006). Therefore, there may
be interactions among different cell death routes, of which the
autophagy-related mechanism might be the one to activate for cell
death fate (Mora and Regnier-Vigouroux, 2009). However, it is not
entirely clear, at the molecular level, which mechanism determines
the cell death route following a given death stimulus.
In this study, we demonstrated that Bif-1, the molecule associated
with both autophagy and apoptosis, was required for both necrotic
and apoptotic cell death induced by GSK-3binhibition. We showed
that Bif-1 was upregulated at the protein level and was required for
the autophagic response and cell death (either necrotic or apoptotic
death). Based on these data, we propose that under serum-free
conditions or possibly other metabolic stresses, the autophagic
response is activated in cells seeking survival. As a survival factor,
GSK-3b acts as a modulator to fine-tune Bif-1 protein levels and
the associated autophagy process in order to avoid excessive self-
digestion and cell death. Suppression of GSK-3bactivity diminishes
the tight modulation of Bif-1 levels, resulting in Bif-1 accumulation
and profound autophagic response, which eventually leads to
necrotic cell death. If the autophagic response is blocked, then Bif-
1 turns on Bax-dependent apoptotic machinery (Fig. 7).
Although necrosis has been recently considered as one type of
programmed cell death (Zong and Thompson, 2006; Golstein and
Kroemer, 2007), there is a controversy regarding the interplay
among different death pathways. Frequently, necrosis inducers, such
as ischemia or nutrient stress, also stimulate autophagic responses
as a survival mechanism in the early stage towards necrosis,
however, blocking autophagy suppresses necrosis, indicating that
autophagy is critical for necrosis (Samara et al., 2008). Conversely,
in apoptosis-deficient mammalian cells, blocking autophagy induces
necrotic death under conditions of nutrient limitation (Degenhardt
et al., 2006). In this study, we found that blocking autophagy flux
redirects GSK-3b-inhibition-induced necrosis to apoptosis under
serum-free conditions. Therefore, the interchange between different
death routes may be based on the death stimulus and the genetic
background of cellular models.
Currently, it is largely unknown how necrotic cell death and its
interplay with other types of cell death mechanisms are regulated,
although lysosomal biogenesis and mitochondrial dysfunction were
found to be critical for induction of necrosis in D. discoideum or
C. elegans (Artal-Sanz et al., 2006; Giusti et al., 2009). In
mammalian cells, the death domain kinase receptor interacting
protein 1 and 3 (RIP1 and 3) and BH3-only Bcl-2 modifying factor
(Bmf) were found to regulate the canonical signal-induced
necroptosis, a type of necrotic cell death when apoptosis is blocked
(Holler et al., 2000; Hitomi et al., 2008; Cho et al., 2009; He et al.,
2009; Zhang et al., 2009). In this study, we found that a strong
autophagic response proceeded necrotic cell death after GSK-3b
inhibition. Most importantly, our data revealed that Bif-1, a binding
partner of the pro-apoptotic Bax protein, plays a key role in the
autophagic response and cell death. It has been demonstrated that
Bif-1 forms a complex with beclin-1 through UVRAG and knockout
or knockdown of Bif-1 reduces the number of autophagosomes
induced by nutrient starvation (Takahashi et al., 2007). However,
precisely how Bif-1 regulates autophagy remains to be investigated.
It is possible that the SH3 domain of Bif-1 binds to and suppresses
UVRAG-mediated autophagosome maturation, whereas the N-BAR
domain of Bif-1 is involved in the generation of autophagosomal
membranes independently of its interaction with the beclin-1
Currently, it is not clear how, after GSK-3b inhibition, blockade
of the autophagic response, either in the early stage by 3-MA or in
the late stage by CQ, redirects Bif-1 to activate Bax. One plausible
mechanism is that blocking autophagy reduces Bif-1 interaction with
the beclin-1–VPS34 complex (Fig. 6D), which subsequently leads
to increased Bif-1-dependent Bax conformational change and/or
activation for apoptosis induction (Fig. 7). Notably, a recent
publication showed that Bif-1 was not detected in a beclin-1-
Journal of Cell Science 123 (6)
Fig. 7. Proposed mechanism of GSK-3b b modulation of Bif-1-dependent
autophagy and cell death. Blue: under serum starvation conditions, GSK-3b
maintains a limited autophagic response for cell survival by modulating the
level of Bif-1 protein. Red: once GSK-3b activity is suppressed, Bif-1 protein
is increased, resulting in a massive autophagy response and necrotic cell death.
Green: if autophagy is blocked, the cell death route is redirected to apoptotic
cell death through a mechanism associated with Bif-1-dependent Bax
Journal of Cell Science
Bif-1 in cell death
containing complex isolated from mouse brain or liver tissues
(Zhong et al., 2009). Since we and others (Takahashi et al., 2007)
reproducibly found that Bif-1 interacts with the beclin-1–VPS34
complex after GSK-3b inhibition or during nutrient starvation, it
is hypothesized that Bif-1 interaction with the beclin-1–VPS34
complex is not steady but transient or dynamic in response to
extracellular stimuli or metabolic stress.
In summary, our study revealed that GSK-3b promotes cell
survival by modulating Bif-1-dependent autophagic response after
serum starvation. Inhibition of GSK-3b activity diminishes the
GSK-3bcontrol over Bif-1-dependent autophagic response, leading
to an enhanced autophagic response and eventually necrotic cell
death. In addition, Bif-1 is possibly involved in a necrosis-apoptosis
switch if autophagy is inhibited. Further investigation is under way
to elucidate the mechanism involved in GSK-3b-mediated
regulation of Bif-1 expression under serum-free conditions.
Materials and Methods
Antibodies and chemicals
Antibodies for PARP, caspase-3, VPS34, beclin-1, Bax, Bcl-2 and Atg5 were from
Cell Signaling Inc (Danvers, MA). Anti-Bif-1 antibody was from Imgenex Co. (San
Diego, CA). Anti-HMGB-1 and anti-LC3 antibodies were from GeneTex (Irvine,
CA). Anti-Bax clone 6A7 and p62/SQSTM1 clone D-3 antibodies were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). LiCl and 3-MA were from Sigma-
Aldrich (St Louis, MO). TDZD8, AR-A014418 and Chloroquine (CQ) were purchased
from Calbiochem (San Diego, CA). SB216763 was obtained from Biomol (Plymouth
Meeting, PA). L803-mts [N-myristol-GKEAPPAPPQS(P)P] was synthesized by
Genemed Synthesis (San Antonio, TX) as previously described (Plotkin et al., 2003),
and dissolved in DMSO at more than 1000-fold the final concentration used in cell
Cell culture and transfection
PC-3 cells were cultured in RPMI 1640 medium supplemented with 10% FBS (fetal
bovine serum), 100 g/ml streptomycin and 100 IU/ml penicillin. All the siRNAs
were obtained from Santa Cruz Biotechnology and transfected into cells at the
indicated concentration using the OligoFectamine reagent (Invitrogen, Carlsbad, CA).
GFP-LC3 and Bif-1 shRNA expression constructs were described previously
(Takahashi et al., 2007). Cells were transfected with plasmid DNA using LipoFectamin
(Invitrogen). To establish a stable cell line expressing GFP-LC3 or Bif-1 shRNA,
cells were co-transfected with the expression vector and pBabe-puro vector. After
24 hours transfection, single colonies were selected in the puromycin-containing
medium as described in our previous publication (Liao et al., 2005).
Cytotoxicity assays, JC-1 staining and flow cytometry
Cell viability was assessed with Trypan Blue exclusion assay. Mitochondrial
membrane potential was determined using JC-1 staining kit and the protocol obtained
from the manufacturer (Invitrogen) was followed as described previously (Liao et
al., 2005). Cell death was determined using a FACScalibur flow cytometer (BD
Biosciences) using a YoPro-1 Apoptosis Assay Kit (Invitrogen) according to the
Immunoprecipitation and immunoblotting analysis
Cells were harvested, rinsed with PBS, and lysed on ice in RIPA buffer (Sigma-
Aldrich). Equal amount of proteins from each lysate was loaded onto SDS-PAGE
gels and immunoblotted (IB) with antibodies as indicated in the figures. For detection
of Bax conformational change, total cell lysates were prepared in Chaps-containing
cell extract buffer (Cell Signaling Technology), and subjected to anti-Bax 6A7
immunoprecipitation (IP) followed by immunoblotting (IB) analysis with regular Bax
antibody. For beclin-1 IP, cell lysates were prepared with RIPA buffer. Anti-beclin-
1 (Cell Signaling Technology) antibodies were used in the IP assay overnight and
thereafter with protein A and/or G plus agarose (Santa Cruz Biotechnology) for 2
hours. Antibodies-bound beads were collected by centrifugation and washed five times
with lysis buffer. Immunoprecipitates were eluted in sample buffer and subjected to
western blot analysis. Band densities on the immunoblots were scanned and the relative
band densities were normalized against anti-actin blots. The band densities from the
control was set as a value of 1.
HMGB-1 and LDH release assay
For the HMGB-1 release assay, cell culture medium was collected and spun at 1000
g for 10 minutes and the supernatant was then concentrated through Millipore
centrifugal filter devices YM-3. Equal amount of the concentrated supernatants were
subjected to western blotting with anti-HMGB-1 antibody. For the LDH release assay,
culture medium was collected and LDH activity was assessed using a LDH
cytotoxicity assay kit (Cayman Chemical, Ann Arbor, MI) according to the
Phase-contrast, fluorescence and electron microscopy
For phase-contrast microscopy, PC-3 cells were plated in 35 mm dishes and then
were treated with the solvent L803-mts at 100 M or L803-mts (100 M) plus 3-
MA at 10 mM in serum-free medium. For fluorescence microscopy, cells were grown
on eight-well chamber slides. After fluorescent dye loading, slides were mounted
with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame,
CA). Photomicrographs were taken with a QImaging Retiga 4000R digital camera
driven by QCapture Pro 5.1 image capture software under a Olympus 1X71 inverted
microscope. For electron microscopy, cells were fixed in 2.5% glutaraldehyde
overnight at 4°C and post-fixed in 0.5% osmium tetroxide for 30 minutes. Specimens
were stained with 2% uranyl acetate in 50% ethanol. The cells were then dehydrated
in a graded ethanol series, infiltrated with plastic, embedded in Epon-812 and
polymerized at 60°C for 2 days. Sections were cut at thickness of 80 nm and stained
with lead citrate, and then photographed on JEOL 100CX II transmission electron
Images of western blots and immunostaining are from a representative experiment.
The mean and standard error of the mean (s.e.m.) from cell counting, autophagosome,
GFP-loci counts, band densities of immunoblots and flow cytometry experiments are
shown. The significance of the differences between treatment and control was analyzed
using SPSS software (SPSS, Chicago, IL).
We thank Barbara Fegley for her excellent assistance in electronic
microscopy. We also thank Joyce Slusser, Director of KUMC Flow
Cytometry Core Facility that is funded by an NIH COBRE program
from the National Center for Research Resources (P20 RR016443), for
her technical support. We are also grateful to the Imaging Core Facility
of the Center for Reproductive Sciences at KUMC for image processing.
This study was partially supported by the KU William L. Valk
Endowment, grants from Kansas Mason’s Foundation, Department of
Defense PCRP program (W81XWH-04-1-0214 and W81XWH-07-
1-0021) to B.L., and NIH (CA82197 and CA129682) to H.-G.W.
Deposited in PMC for release after 12 months.
Supplementary material available online at
Artal-Sanz, M., Samara, C., Syntichaki, P. and Tavernarakis, N. (2006). Lysosomal
biogenesis and function is critical for necrotic cell death in Caenorhabditis elegans. J.
Cell Biol. 173, 231-239.
Bhat, R., Xue, Y., Berg, S., Hellberg, S., Ormö, M., Nilsson, Y., Radesäter, A. C., Jerning,
E., Markgren, P. O., Borgegård, T. et al. (2003). Structural insights and biological
effects of glycogen synthase kinase 3-specific inhibitor AR-A014418. J. Biol. Chem.
Boya, P. and Kroemer, G. (2008). Lysosomal membrane permeabilization in cell death.
Oncogene 27, 6434-6451.
Boya, P., González-Polo, R. A., Casares, N., Perfettini, J. L., Dessen, P., Larochette,
N., Métivier, D., Meley, D., Souquere, S., Yoshimori, T. et al. (2005). Inhibition of
macroautophagy triggers apoptosis. Mol. Cell. Biol. 25, 1025-1040.
Cao, C., Subhawong, T., Albert, J. M., Kim, K. W., Geng, L., Sekhar, K. R., Gi, Y. J.
and Lu, B. (2006). Inhibition of mammalian target of rapamycin or apoptotic pathway
induces autophagy and radiosensitizes PTEN null prostate cancer cells. Cancer Res. 66,
Carmichael, J., Sugars, K. L., Bao, Y. P. and Rubinsztein, D. C. (2002). Glycogen
synthase kinase-3beta inhibitors prevent cellular polyglutamine toxicity caused by the
Huntington’s disease mutation. J. Biol. Chem. 277, 33791-33798.
Chan, F. K., Shisler, J., Bixby, J. G., Felices, M., Zheng, L., Appel, M., Orenstein, J.,
Moss, B. and Lenardo, M. J. (2003). A role for tumor necrosis factor receptor-2 and
receptor-interacting protein in programmed necrosis and antiviral responses. J. Biol.
Chem. 278, 51613-51621.
Cho, Y. S., Challa, S., Moquin, D., Genga, R., Ray, T. D., Guildford, M. and Chan, F.
K. (2009). Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates
programmed necrosis and virus-induced inflammation. Cell 137, 1112-1123.
Cuddeback, S. M., Yamaguchi, H., Komatsu, K., Miyashita, T., Yamada, M., Wu, C.,
Singh, S., Wang, H. G. (2001). Molecular cloning and characterization of Bif-1. A
novel Src homology 3 domain-containing protein that associates with Bax. J. Biol. Chem.
Degenhardt, K., Mathew, R., Beaudoin, B., Bray, K., Anderson, D., Chen, G.,
Mukherjee, C., Shi, Y., Gélinas, C., Fan, Y. et al. (2006). Autophagy promotes tumor
cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell 10,
Degterev, A., Huang, Z., Boyce, M., Li, Y., Jagtap, P., Mizushima, N., Cuny, G. D.,
Mitchison, T. J., Moskowitz, M. A. and Yuan, J. (2005). Chemical inhibitor of
Journal of Cell Science
870 Download full-text
nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat. Chem.
Biol. 1, 112-119.
Etxebarria, A., Terrones, O., Yamaguchi, H., Landajuela, A., Landeta, O., Antonsson,
B., Wang, H. G. and Basañez, G. (2009). Endophilin B1/Bif-1 stimulates BAX
activation independently from its capacity to produce large scale membrane
morphological rearrangements. J. Biol. Chem. 284, 4200-4212.
Forde, J. E. and Dale, T. C.(2007). Glycogen synthase kinase 3, a key regulator of cellular
fate. Cell Mol. Life Sci. 64, 1930-1944.
Giusti, C., Luciani, M. F., Klein, G., Aubry, L., Tresse, E., Kosta, A. and Golstein, P.
(2009). Necrotic cell death: From reversible mitochondrial uncoupling to irreversible
lysosomal permeabilization. Exp. Cell Res. 315, 26-38.
Golstein, P. and Kroemer, G.(2007). Cell death by necrosis: towards a molecular definition.
Trends Biochem. Sci. 32, 37-43.
He, S., Wang, L., Miao, L., Wang, T., Du, F., Zhao, L. and Wang, X. (2009). Receptor
interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell
Hitomi, J,., Christofferson, D. E., Ng, A., Yao, J., Degterev, A., Xavier, R. J. and Yuan,
J.(2008). Identification of a molecular signaling network that regulates a cellular necrotic
cell death pathway. Cell 135, 1311-1323.
Hoeflich, K. P., Luo, J., Rubie, E. A., Tsao, M. S., Jin, O. and Woodgett, J. R. (2000).
Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB
activation. Nature 406, 86-90.
Holler, N., Zaru, R., Micheau, O., Thome, M., Attinger, A., Valitutti, S., Bodmer, J.
L., Schneider, P., Seed, B. and Tschopp, J. (2000). Fas triggers an alternative, caspase-
8-independent cell death pathway using the kinase RIP as effector molecule. Nat.
Immunol. 1, 489-495.
Hsu, Y. T. and Youle, R. J. (1997). Nonionic detergents induce dimerization among
members of the Bcl-2 family. J. Biol. Chem. 272, 13829-13834.
Idziorek, T., Estaquier, J., De Bels, F. and Ameisen, J. C. (1995). YOPRO-1 permits
cytofluorometric analysis of programmed cell death (apoptosis) without interfering with
cell viability. J. Immunol. Methods 185, 249-258.
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-
Klionsky, D. J. and Emr, S. D. (2000). Autophagy as a regulated pathway of cellular
degradation. Science 290, 1717-1721.
Klionsky, D. J., Abeliovich, H., Agostinis, P., Agrawal, D. K., Alievm. G., Askew, D.
S., Baba, M., Baehrecke, E. H., Bahr, B. A., Ballabio, A. et al. (2008). Guidelines
for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.
Autophagy 4, 151-175.
Kourtis, N. and Tavernarakis, N. (2009). Autophagy and cell death in model organisms.
Cell Death Differ. 16, 21-30.
Kroemer, G., Galluzzi, L., Vandenabeele, P., Abrams, J., Alnemri, E. S., Baehrecke,
E. H., Blagosklonny, M. V., El-Deiry, W. S., Golstein, P., Green, D. R. et al. (2009).
Classification of cell death: recommendations of the Nomenclature Committee on Cell
Death 2009. Cell Death Differ. 16, 3-11.
Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H., Yoshimori, T., Ohsumi,
Y., Tokuhisa, T., Mizushima, N.(2004). The role of autophagy during the early neonatal
starvation period. Nature 432, 1032-1036.
Liao, X., Tang, S., Thrasher, J. B., Griebling, T. L. and Li, B. (2005). Small-interfering
RNA-induced androgen receptor silencing leads to apoptotic cell death in prostate cancer.
Mol. Cancer Ther. 4, 505-515.
Maiuri, M. C., Zalckvar, E., Kimchi, A. and Kroemer, G. (2007). Self-eating and self-
killing: crosstalk between autophagy and apoptosis. Nat. Rev. Mol. Cell. Biol. 8, 741-
Martinez, A., Alonso, M., Castro, A., Pérez, C. and Moreno, F. J. (2002). First non-
ATP competitive glycogen synthase kinase 3 beta (GSK-3beta) inhibitors:
thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer’s disease.
J. Med. Chem. 45, 1292-1299.
Mora, R. and Régnier-Vigouroux, A. (2009). Autophagy-driven cell fate decision maker:
activated microglia induce specific death of glioma cells by a blockade of basal autophagic
flux and secondary apoptosis/necrosis. Autophagy 5, 419-421.
Okada, M., Adachi, S., Imai, T., Watanabe, K., Toyokuni, S. Y., Ueno, M., Zervos, A.
S., Kroemer, G. and Nakahata, T. (2004). A novel mechanism for imatinib mesylate-
induced cell death of BCR-ABL-positive human leukemic cells: caspase-independent,
necrosis-like programmed cell death mediated by serine protease activity. Blood 103,
Pierrat, B., Simonen, M., Cueto, M., Mestan, J., Ferrigno, P. and Heim, J. (2001).
SH3GLB, a new endophilin-related protein family featuring an SH3 domain. Genomics
Plotkin, B., Kaidanovich, O., Talior, I. and Eldar-Finkelman, H.(2003). Insulin mimetic
action of synthetic phosphorylated peptide inhibitors of glycogen synthase kinase-3. J.
Pharmacol. Exp. Ther. 305, 974-980.
Raymond, M. A., Mollica, L., Vigneault, N., Désormeaux, A., Chan, J. S., Filep, J. G.
and Hébert, M. J. (2003). Blockade of the apoptotic machinery by cyclosporin A
redirects cell death toward necrosis in arterial endothelial cells: regulation by reactive
oxygen species and cathepsin D. FASEB J. 17, 515-517.
Samara, C., Syntichaki, P. and Tavernarakis, N. (2008). Autophagy is required for
necrotic cell death in Caenorhabditis elegans. Cell Death Differ. 15, 105-112.
Seglen, P. O. and Gordon, P. B. (1982). 3-Methyladenine: specific inhibitor of
autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc. Natl. Acad.
Sci. USA 79, 1889-1892.
Smiley, S. T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T. W., Steele,
G. D., Jr and Chen, L. B. (1991). Intracellular heterogeneity in mitochondrial
membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc.
Natl. Acad. Sci. USA 88, 3671-3675.
Sun, A., Shanmugam, I., Song, J., Terranova, P. F., Thrasher, J. B. and Li, B. (2007).
Lithium suppresses cell proliferation by interrupting E2F-DNA interaction and
subsequently reducing S-phase gene expression in prostate cancer. Prostate 67, 976-
Takacs-Vellai, K., Vellai, T., Puoti, A., Passannante, M., Wicky, C., Streit, A., Kovacs,
A. L. and Müller, F. (2005). Inactivation of the autophagy gene bec-1 triggers apoptotic
cell death in C. elegans. Curr. Biol. 15, 1513-1517.
Takahashi, Y., Karbowski, M., Yamaguchi, H., Kazi, A., Wu, J., Sebti, S. M., Youle,
R. J. and Wang, H. G.(2005). Loss of Bif-1 suppresses Bax/Bak conformational change
and mitochondrial apoptosis. Mol. Cell. Biol. 25, 9369-9382.
Takahashi, Y., Coppola, D., Matsushita, N., Cualing, H. D., Sun, M., Sato, Y., Liang,
C., Jung, J. U., Cheng, J. Q., Mul, J. J. et al. (2007). Bif-1 interacts with Beclin 1
through UVRAG and regulates autophagy and tumorigenesis. Nat. Cell Biol. 9, 1142-
Takahashi, Y., Meyerkord, C. L. and Wang, H. G. (2009). Bif-1/Endophilin B1: a
candidate for crescent driving force in autophagy. Cell Death Differ. 16, 947-955.
Thorburn, J., Horita, H., Redzic, J., Hansen, K., Frankel, A. E. and Thorburn, A.
(2009). Autophagy regulates selective HMGB-1 release in tumor cells that are destined
to die. Cell Death Differ. 16, 175-183.
Ullman, E., Fan, Y., Stawowczyk, M., Chen, H. M., Yue, Z. and Zong, W. X. (2008).
Autophagy promotes necrosis in apoptosis-deficient cells in response to ER stress. Cell
Death Differ. 15, 422-425.
Walker, N. I., Harmon, B. V., Gobé, G. C. and Kerr, J. F. (1988). Patterns of cell death.
Methods Achiev. Exp. Pathol. 13, 18-54.
Wang, Y., Singh, R., Massey, A. C., Kane, S. S., Kaushik, S., Grant, T., Xiang, Y.,
Cuervo, A. M. and Czaja, M. J. (2008). Loss of macroautophagy promotes or
prevents fibroblast apoptosis depending on the death stimulus. J. Biol. Chem. 283,
Yamaguchi, H., Woods, N. T., Dorsey, J. F., Takahashi, Y., Gjertsen, N. R., Yeatman,
T., Wu, J. and Wang, H. G. (2008). SRC directly phosphorylates Bif-1 and prevents
its interaction with Bax and the initiation of anoikis. J. Biol. Chem. 283, 19112-19118.
Zhang, D. W., Shao, J., Lin, J., Zhang, N., Lu, B. J., Lin, S. C., Dong, M. Q. and Han,
J. (2009). RIP3, an energy metabolism regulator that switches TNF-induced cell death
from apoptosis to necrosis. Science 325, 332-336.
Zhong, Y., Wang, Q. J., Li, X., Yan, Y., Backer, J. M., Chait, B. T., Heintz, N. and
Yue, Z. (2009). Distinct regulation of autophagic activity by Atg14L and Rubicon
associated with Beclin 1-phosphatidylinositol-3-kinase complex. Nat. Cell Biol.11, 468-
Zong, W. X. and Thompson, C. B. (2006). Necrotic death as a cell fate. Genes Dev. 20,
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.
Journal of Cell Science 123 (6)
Journal of Cell Science