From the Department of Medicine, University of
Minnesota Medical School, Minneapolis, Minnesota 55455 and § First Department of Internal Medicine, Osaka
Medical College, Takatsuki, Osaka 569-8686, Japan
Received for publication, August 28, 2002, and in revised form, November 15, 2002
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ABSTRACT |
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Eukaryotic translation initiation factor 4E
(eIF4E) markedly reduces cellular susceptibility to apoptosis. However,
the mechanism by which the translation apparatus operates on the
cellular apoptotic machinery remains uncertain. Here we show that
eIF4E-mediated rescue from Myc-dependent apoptosis is
accompanied by inhibition of mitochondrial cytochrome c
release. Experiments achieving gain and loss of function demonstrate
that eIF4E-mediated rescue is governed by pretranslational and
translational activation of bcl-x as well as by additional
intermediates acting directly on, or upstream of, the mitochondria.
Thus, our data trace a pathway controlling apoptotic susceptibility
that begins with the activity state of the protein synthesis machinery
and leads to interdiction of the apoptotic program at the mitochondrial checkpoint.
The integration of extracellular information by a cell into a
decision to live or die is of fundamental importance during development, wound healing, immune responses, and tumorigenesis (1, 2).
Most studies addressing the regulation of apoptosis have focused
on transcriptional control, signal transduction, and other
post-translational regulatory events. More recently, it has become
evident that apoptosis is also subject to translational control.
Cap-dependent translation involves the assembly of
initiation factors at the 5' mRNA terminus to form the trimolecular
cap binding complex, eIF4F. These factors include the cap-binding
protein, eIF4E,1 an
ATP-dependent RNA helicase, eIF4A, and an eIF4G polypeptide (eIF4GI or eIF4GII), which serves as a docking site for eIF4E and eIF4A
(3). The function of eIF4E is negatively regulated by members of the
translational repressor family, the eIF4E-binding proteins, that
sequester eIF4E in a translationally inactive complex (4). Growth
factors and other pro-survival stimuli promote phosphorylation of the
eIF4E-binding proteins. Hyperphosphorylated eIF4E-binding protein has a
decreased affinity for eIF4E, resulting in its liberation to initiate translation.
The activity of the cap-dependent translation initiation
apparatus is a major determinant of cell fate during development (5, 6)
and post-natal life (7). A constitutively active initiation apparatus
sustains morphogenesis ex vivo (5), promotes cell cycle
transit (8, 9), and leads to malignant transformation (9-12), whereas
repression of aberrant translation initiation reverses oncogenesis
(13-15). We have previously shown that apoptosis can be governed by
the activity of eIF4E. Overexpression of eIF4E rescues cells from
apoptosis (16, 17), whereas sequestration of eIF4E by overexpressed
eIF4E-binding protein 1, triggers apoptosis and markedly diminishes
tumorigenesis (18, 19).
The apoptotic program can be triggered through at least two distinct
signaling pathways with the potential for cross-talk. One pathway,
leading to activation of caspase-8, is triggered by ligation of
specific cell surface death receptors, such as Fas/CD95 or tumor
necrosis factor Despite the strong connection between translational control and
apoptosis, little is known about the underlying mechanisms. We have
previously demonstrated that over-expressed eIF4E averts Myc-dependent apoptosis, at least in part, through a cyclin
D1-dependent process (17). To provide further insight into
the mechanism of eIF4E rescue, we examined which steps in the apoptotic
cascade were blocked by eIF4E in rat embryo fibroblasts sensitized to apoptosis by constitutive expression of c-Myc. Here we show that cells
rescued by eIF4E neither release cytochrome c from their mitochondria nor do they activate any downstream steps in the apoptotic
cascade. A survey of Bcl-2 family members in rescued fibroblasts
revealed most to be in the basal state. However, Bcl-XL was
dramatically increased due to the selective recruitment of Bcl-XL mRNA to ribosomes by eIF4E as well as from
activation of pre-translational stages of Bcl-XL
production. Experiments achieving gain and loss of Bcl-XL
function indicated that factors other than Bcl-XL are
involved in eIF4E-dependent blockade of cytochrome c release. These data provide the first direct link between
translationally mediated antiapoptotic signaling and the apoptotic
machinery and underscore the pleotropic nature of survival signaling
downstream of up-regulated eIF4E.
Cell Lines--
Cell lines were derived from rat embryonic
fibroblasts (REF) as described (17). REF/Myc cells, which overexpressed
c-Myc, were provided by Dr. Weinberg (Whitehead Institute, Cambridge, MA). REF/Myc/4E cells were generated by transducing REF/Myc with retrovirus CRE-BCS encoding wild-type murine eIF4E (retrovirus was kindly provided by P. Leboulch, Massachusetts Institute of Technology, Cambridge, MA). Both REF/Myc and REF/Myc/4E cells were
grown in complete medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 4.5 g/liter glucose, 100 units/ml penicillin, 100 units/ml streptomycin, and 250 ng/ml amphotericin).
Analysis of Apoptosis--
Cells were subjected to proapoptotic
conditions as specified in the text and figure legends. Both
adherent and detached cells were collected, fixed in 70% ethanol,
washed with PBS, and stained with propidium iodide stain mixture (50 µg/ml of propidium iodide, 0.1% Triton X-100, 32 µg/ml EDTA, 2.5 µg/ml RNase in PBS) for 45 min at 37 °C. DNA content was
determined by quantitative flow cytometry using the CellQuest program.
Immunoblot Analysis--
Cultured cells were rinsed in PBS,
trypsinized, and collected by centrifugation. Cells were resuspended in
lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl,
1 mM EDTA, 0.1% SDS, 1% Triton-X, 1% sodium
deoxycholate) supplemented with protease inhibitor mixture (0.1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml each of
leupeptin, aprotinin, and pepstatin A) and microcentrifuged at
12,000 × g for 10 min, and the supernatant was
retained. The protein concentration was measured using the Pierce BCA
protein assay kit. 20 µg of total protein was resolved by 15%
SDS/PAGE, followed by immunoblotting with the following antibodies:
anti-Bcl-XL (Trevigen), anti-Bcl-2 (Trevigen), anti-Bax
(Santa Cruz Biotechnology), anti-Bad (Santa Cruz Biotechnology),
anti-actin (Sigma), anti-tubulin (Sigma), anti-caspase-3 (Santa Cruz
Biotechnology), anti-poly(ADP-ribose) polymerase (PARP; Upstate
Biotechnology). Cytochrome c was analyzed by subjecting 50 µg of cell free extract to 12% SDS/PAGE, transferred to
nitrocellulose membranes, and incubated with mouse anti-cytochrome c antibody (BD Biosciences). Blots were incubated
with an appropriate horseradish peroxidase-coupled secondary antibody
and detected by ECL (Amersham Biosciences).
Caspase Activity Assay--
Cells were subjected to proapoptotic
conditions, and both detached and adherent cells were collected,
incubated with lysis buffer, and centrifuged for 5 min at 10,000 × g. Active caspase-3 was measured using a CleavaLite
caspase-3 activity assay kit (Chemicon). 50 µg of lysate was added to
a CleavaLite Renilla luciferase bioluminescent substrate that contains
the caspase-3 cleavage site, DEVD. After incubation for 1 h at
37 °C, fresh luciferase substrate was added, and luminescence was
read in a Lumat luminometer (EG&G Berthold). For caspase-9 activity,
the cell lysate was centrifuged at 10,000 × g for 1 min, supernatant was collected, and caspase-9 activity was determined
by cleavage of LEHD-p-nitroanilide using a caspase-9 colorimetric assay (R&D systems).
Determination of Cytochrome c Subcellular
Distribution--
Adherent and floating cells were pooled and
suspended in mitochondrial buffer (250 mM sucrose, 20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml leupeptin, 20 µg/ml aprotinin, 1 µg/ml
pepstatin A). Cells were mechanically lysed using a loose-fitting
Wheaton cell homogenizer, and centrifuged at 1000 × g
for 10 min to remove nuclei and unbroken cells. The supernatant was
centrifuged at 13,000 × g for 20 min to pellet the
mitochondria. The resulting supernatant (cytosolic fraction) was
centrifuged at 100,000 × g for 1 h. Samples were
diluted to obtain a total protein concentration of 0.4 µg/µl, and
cytochrome c concentration in each sample was determined by
enzyme-linked immunosorbent assay using an R&D systems Quantikine
murine cytochrome c immunoassay kit.
Measurement of Mitochondrial Membrane Potential--
Cells were
seeded on glass cover slips precoated with fetal calf serum and
incubated (24 h at 37 °C) in Dulbecco's modified Eagle's medium,
10% fetal calf serum. Cultures were continued with or without 5 µM lovastatin for an additional 24 h. Mitochondria were stained by exposing cells to 0.5 µg/ml rhodamine 123 dye for 45 min at 37 °C. Medium was changed to PBS immediately before analyzing
the staining pattern of the cells.
Polyribosome Preparation--
15 plates of actively
proliferating cells in 100-mm dishes were treated with cycloheximide
(100 µg/ml) for 5 min, harvested by trypsinization, and lysed as
described (50) using 60 strokes in a Dounce homogenizer. Lysate was
centrifuged at 10,000 × g for 10 min, and the nuclei
pellet was removed. Cytoplasmic extract (1.5 mg measured at
A260) was layered onto a 5-ml, 0.5-1.5
M sucrose gradient. The sucrose gradients were centrifuged
at 200,000 × g in a Beckman SW50 rotor for 90 min at
4 °C. The gradients were fractionated using an ISCO density gradient
fractionator monitoring absorbance at 254 nm. Five 1-ml fractions were
collected from each sample into tubes containing 100 µl of 10% SDS.
Quantification of bcl-x mRNA by Real-time PCR--
The RNA
from each fraction of the sucrose gradient was extracted using
Tri-reagent (Sigma) and quantitated. RNA (5 µg) from each fraction
was treated with DNase I (DNA-freeTM, Ambion, Austin)
according to the manufacturer's directions. cDNA was synthesized
from 2 µg of each RNA sample with a Taqman reverse transcriptase
reagent kit (Applied Biosystems) primed with oligo-dT. Rat
bcl-x DNA sequences for upper
(5'-GGAGACCCCCAGTGCCATCAAT-3') and lower
(5'-AGTGCCCCGCCAAAGGAGAAA-3') primers and rat bcl-2 DNA
sequences for upper (5'-CACCCCTGGCATCTTCTCCTTCC-3') and lower (5'-GCATCCCAGCCTCCGTTATCCT-3') primers were selected using the DNA STAR program (DNASTAR, Inc., Madison, WI), and the resulting sequences were synthesized in the University of Minnesota microchemical facility and purified by HPLC. Real time PCR was performed using a
LightCycler FastStart DNA Master SYBR Green I Kit (Roche Molecular Biochemicals). The LightCycler PCR protocol consisted of a 10-min denaturation followed by 42 cycles of 95 °C for 10 s, 68 °C
for 5 s, and 72 °C for 16 s. Reactions were set up as
recommended by the manufacturer, optimized with 4 µM
MgCl2 with each primer at 1 µM. A portion of
the cDNA reaction (9.6 µl) was used for amplification of each
gradient fraction. Quantification of mRNA was carried out by
comparison (linear interpolation) of the number of cycles to saturation
in each sample, with the number of cycles in a concurrently run
standard containing a known amount of target mRNA (5 concentrations
of the standard were used which spanned the range of values for the samples).
Northern Blot Analysis--
Total RNA was isolated using an
RNeasy Total RNA kit (Qiagen, Santa Clarita, CA), and 15 µg RNA/lane
was electrophoresed through 1% agarose, 2.2 M formaldehyde gel and
transferred to nylon filters. A synthetic 40-mer oligonucleotide
(5'-GGTGGTCATTCAGGTAGGTGGCCATCCAACTTGCAATCCG-3'), which was designed
specifically to recognize bcl-XL (2), was 3'
end-labeled with 50 µCi of [ Messenger RNA Stability--
RNA stability was quantified as
described previously (29). To assess stability of bcl-x
mRNA, cells were treated with 5 µg/ml actinomycin D (Sigma) for
the time intervals specified in the text and figure legends.
Total RNA was isolated and analyzed by Northern blot.
Generation of REF/Myc/Bcl-XL Clonal Cell
Lines--
REF/Myc cells were stably transfected with a pAPuro vector
containing an 800-bp fragment encoding wild type Bcl-XL (a
gift from Dr. E. Prochownik, Children's Hospital, Pittsburgh, PA)
using the FuGENE 6 (Roche Diagnostics) transfection technique.
Selection of transfected cells was begun after 24 h in complete
medium containing 4 µg/ml puromycin. Resistant clones were isolated
after 12-16 days.
Bcl-XL Antisense
Oligonucleotides--
Phosphorothioate antisense and nonsense DNA
oligodeoxynucleotides were synthesized and purified by HPLC (Operon
Technologies, Inc., Alameda, CA). An 18-mer antisense
oligodeoxynucleotide sequence spanning the translation start codon of
bcl-x mRNA and a control scrambled sequence were
(a) antisense 5'-CCG GTT GCT CTG AGA CAT-3' and
(b) scramble 5'-CTG AAC GGA GAG ACC CTT-3'. Cells were
seeded into chambers of 8-well glass chamber slides overnight and
shifted to Dulbecco's modified Eagle's medium containing 0.1% fetal
calf serum with or without 5 µM lovastatin, 40 µM oligodeoxynucleotides, or both. Cells were cultivated
for 72 h with one media change. Cells were fixed with ice-cold
70% ethanol and stained with acridine orange, and apoptosis was
quantified using morphological criteria, as described (16, 17).
Cytochrome c Immunostaining--
Cells were seeded at a density
of 5 × 103/cm2 in 24-well clusters onto
glass cover slips and cultured for 24 h in complete medium. Cultures were either continued in complete medium alone or containing 5 µM lovastatin for 18 or 24 h. Cells were rinsed with
PBS and fixed in PBS containing 4% paraformaldehyde. The fixed cells
were incubated in blocking buffer (PBS containing 5% normal goat
serum, 1% bovine serum albumin, and 0.3% Triton X-100) for 30 min and for an additional 2 h in PBS containing 1% bovine serum albumin, 1% normal goat serum, and 1 µg of anti-cytochrome c
antibody (Promega) per ml. Cells were washed 3 times in PBS and
incubated for 30 min in PBS containing 1% bovine serum albumin, 1%
normal goat serum, and 1 µg of fluorescein isothiocyanate-conjugated
anti-mouse antibody (Sigma) per ml. Cells were rinsed 3 times in PBS,
nuclei-stained with DAPI (0.1 mg/ml), and cover slips were mounted onto slides.
Increased Expression of eIF4E Inhibits Death Receptor-mediated and
Stressor-induced Apoptotic Pathways--
We previously reported that
ectopic expression of eIF4E rescues REF harboring constitutively
expressed c-myc (REF/Myc) from apoptosis triggered by either
serum restriction (16) or cytotoxic stress (17). To evaluate whether
eIF4E also suppresses death receptor-mediated apoptosis, we examined
its impact on REF/Myc cell viability after treatment with tumor
necrosis factor family ligands. When eIF4E was ectopically
overexpressed in REF/Myc cells (REF/Myc/4E), apoptosis was inhibited in
response to each apoptotic trigger tested (Fig.
1), indicating that eIF4E promoted
suppression of the death receptor-mediated as well as the
stress-induced apoptotic cascades. However, the magnitude of kill in
response to tumor necrosis factor- Ectopic Expression of eIF4E Blocks Release of Cytochrome c from
Mitochondria--
The cleavage of PARP into an 85-kDa daughter
fragment is a downstream step common to most forms of apoptosis. We
have traced the apoptotic pathway upstream of PARP cleavage in both
REF/Myc and REF/Myc/4E cells (Fig. 2).
Treatment with lovastatin led to the appearance of the 85-kDa fragment
of PARP in REF/Myc cells but not in REF/Myc/4E cells (Fig.
2A). Similarly, the proform of caspase-3 (32 kDa) was
cleaved into its active 17-kDa form in REF/Myc but not in REF/Myc/4E
(Fig. 2B). Consistent with this observation, REF/Myc cells
manifested a progressive increase in caspase-3 activation, whereas
caspase-3 activity remained low in REF/Myc/4E cells throughout the
entire interval of observation (Fig. 2C). Caspase-9 followed
the same pattern. Activation was noted as early as 4 h in REF/Myc
cells, reaching levels more than 3-fold that of control by 16 h
(Fig. 2D). In REF/Myc/4E cells caspase-9 remained in the
inactive state.
Cytochrome c normally resides between the inner and outer
mitochondrial membranes and is not usually found in the cytoplasm (30).
Because cytochrome c is required for activation of caspase-9 (31), we examined the subcellular distribution of cytochrome c after exposure of cells to lovastatin. At base line,
cytochrome c was detectable at low levels in the cytoplasm
of REF/Myc cells, a value that increased more than 3-fold after 20 h of lovastatin treatment (Fig.
3A). In contrast, cytochrome
c remained near basal levels in the cytoplasm of REF/Myc/4E
cells after lovastatin treatment, a result corroborated by immunoblot
analysis (Fig. 3B).
Mitochondrial inner transmembrane potential collapse frequently
precedes cytochrome c release and caspase activation (32). To examine whether this was the case in our system, we treated cells
with lovastatin for 24 h and measured the uptake of rhodamine 123, a cationic fluorophore that enters mitochondria in direct proportion to
membrane potential (33). REF/Myc and REF/Myc/4E cells took up the dye
similarly after lovastatin treatment, suggesting that the suppression
of cytochrome c release in REF/Myc/4E cells was not due to
eIF4E-induced alterations in mitochondrial membrane depolarization
(data not shown).
Overexpressed eIF4E Selectively Stimulates Expression of
Bcl-XL--
Deregulated c-Myc mediates apoptosis in some
transformed cell lines by selectively decreasing the mRNA and
protein levels of the death antagonists Bcl-2 and Bcl-XL
(34, 35). We therefore examined whether eIF4E had any effect on this
interplay between c-Myc and the Bcl-2 family proteins. Immunoblot
analysis demonstrated that overexpression of eIF4E resulted in a 7-fold
increase of Bcl-XL protein in REF/Myc cells without
significantly affecting the expression levels of Bcl-2, Bax, or Bad
(Fig. 4).
Overexpression of eIF4E Increases Recruitment of Bcl-XL
mRNA to Ribosomes--
To examine whether overexpression of eIF4E
increased cellular levels of Bcl-XL protein by direct
translational activation, total RNA from REF/Myc and REF/Myc/4E was
stratified by sucrose gradient centrifugation to separate
translationally active transcripts (more bound ribosomes resulting in
more rapid transit through the gradient) from less translationally
active transcripts. The resulting fractions were subjected to
quantitative PCR analysis for the bcl-XL
transcript, comparing it to the bcl-2 transcript as a
control. Examination of the absorbance pattern revealed a bias toward
heavier polyribosomes, with an increased proportion of RNA in fractions
4 and 5 in cells ectopically expressing eIF4E (Fig.
5A). Real time PCR
quantification indicated that the transcript for bcl-2 was
distributed similarly in REF/Myc and REF/Myc/4E cells, with fraction 2 (the least translationally active) and fraction 5 (the most
translationally active) containing similar amounts of transcript (Fig.
5B). In marked contrast, there was a significant increase in
the quantity of bcl-XL mRNA appearing in
heavy polyribosomes in REF/Myc/4E cells (Fig. 5C). The
average number of bound ribosomes per bcl-XL transcript in
REF/Myc/4E cells was 3.7 compared with 1.5 in REF/Myc cells, indicating
a 2.5-fold increase in the rate of bcl-XL mRNA
translation initiation (36, 37). Thus, one mechanism for the increase
in Bcl-XL protein was direct translational activation of
its mRNA.
Overexpressed eIF4E Increases Cellular Levels of Bcl-XL
mRNA--
A growing number of studies show that eIF4E directly or
indirectly participates in a variety of pre-translational stages of protein synthesis including production and processing of mRNA and
its nuclear-cytoplasmic transport (38). To gain insight into the
influence of eIF4E on pretranslational events in the synthesis of
Bcl-XL, we measured the abundance and stability of its
mRNA. Quantitative PCR analysis demonstrated a dramatic effect of
eIF4E on steady state levels of bcl-XL mRNA
but not on the levels of bcl-2 mRNA (Fig.
6A). The level of
bcl-XL mRNA was increased nearly 30-fold in
REF/Myc/4E cells, a result supported by Northern blot analysis (Fig.
6B). The kinetics of bcl-XL mRNA
degradation were only marginally altered by ectopic eIF4E (half-life of
3 h for REF/Myc and 2.5 h for REF/Myc/4E; Fig.
6C). These data indicate that in addition to direct
translational activation, ectopic eIF4E stimulates synthesis of
Bcl-XL by increasing the abundance of its transcript.
Decreasing Cellular Levels of Bcl-XL Reduces
eIF4E-mediated Rescue from Apoptosis--
To assess whether increased
expression of Bcl-XL is required for the antiapoptotic
function of eIF4E, REF/Myc/4E cells were treated with an antisense
oligodeoxynucleotide (ASO) directed against human
bcl-XL mRNA sequences in the predicted
translation initiation region. A scrambled oligonucleotide served as a
control. Incubation of cells with the ASO for 72 h significantly
reduced the level of Bcl-XL protein, whereas the scrambled
oligonucleotide had no effect (Fig.
7A). Continuation of these
cultures for an additional 24 h in the presence of lovastatin led
to a significant increase in the apoptotic frequency of ASO compared
with the scrambled oligonucleotide control (Fig. 7B).
However, despite reduction of Bcl-XL in REF/Myc/4E by ASO
below levels found in REF/Myc, the degree of apoptosis was only half
(62.5% in REF/Myc versus 30% in ASO-treated REF/Myc/4E).
These data indicate that increased expression of Bcl-XL is
necessary for the full-scale antiapoptotic function of ectopic eIF4E
and that Bcl-XL is not the sole determinant of
eIF4E-mediated rescue from apoptosis.
Ectopic Expression of Bcl-XL Does Not Fully Reproduce
eIF4E Rescue--
To determine whether increased expression of
Bcl-XL could substitute for eIF4E in the rescue of REF/Myc
cells from apoptosis, we generated clonal lines of REF/Myc cells stably
expressing a range of Bcl-XL protein. Twelve
puromycin-resistant clones derived from REF/Myc cells transfected with
a pAPuro/bcl-x vector were screened for levels of
Bcl-XL expression. Two clonal cell lines were selected for
further investigation (Fig.
8A), one line with a
Bcl-XL level that closely matched the level of
Bcl-XL expression in REF/Myc/4E cells (clone 7) and a
second line in which the level of Bcl-XL was increased more
than 5-fold (clone 13).
To validate the function of our construct, we first examined the
kinetics of cytochrome c release in the clonal lines by
immunoblot and immunolocalization. Ectopic expression of eIF4E
completely suppressed cytochrome c release in the basal
state and after lovastatin treatment (Fig. 8B). In the basal
state, 15% of the cytochrome c in REF/Myc cells was in the
cytoplasm, whereas after ectopic expression of Bcl-XL,
virtually no cytoplasmic cytochrome c was detected. After
treatment with lovastatin, levels of cytochrome c in the
cytoplasm of REF/Myc cells were stable for 12 h, gradually increasing to more than 40% of total cellular cytochrome c
by 24 h (Fig. 8B). For the Bcl-XL clonal
lines, there was a steady translocation of cytochrome c from
the mitochondria to the cytoplasm beginning at 8 h and increasing
up to 24 h. At all time points examined, the magnitude of
cytochrome c release in cells overexpressing Bcl-XL was significantly less than that observed in
REF/Myc, with a rank order of potency reflecting the expression level
of Bcl-XL (Fig. 8B).
Morphological analysis showed a perinuclear granular
pattern of cytochrome c in REF/Myc,
REF/Myc/Bcl-XL 7, REF/Myc/Bcl-XL 13, and
REF/Myc/4E cells in the basal state, consistent with a mitochondrial
subcellular localization (Fig. 8C). After lovastatin treatment for 18 h, cytochrome c acquired a diffuse
distribution (consistent with cytoplasmic localization) in REF/Myc and
REF/Myc/Bcl-XL 7, and after 24 h a diffuse pattern was
evident in REF/Myc/Bcl-XL 13. In contrast, the cytochrome
c signal remained punctate in REF/Myc/4E cells for the 24-h
observation interval. Consistent with these results, ectopic expression
of Bcl-XL significantly attenuated apoptosis in REF/Myc
cells (Fig. 8D). However, even the highest expressing clonal
line manifested an apoptotic frequency of 30%, more than 2-fold
that observed in REF/Myc/4E. These data indicate a qualitative
difference between rescue by eIF4E and rescue by
Bcl-XL.
To begin deciphering the rules governing translational control of
cell death, in this report we examined which components of the
apoptotic machinery were inhibited when apoptosis was suppressed by
ectopic overexpression of eIF4E using Myc-dependent
apoptosis in fibroblasts as a model. Here we show that eIF4E rescues
cells by blocking release of cytochrome c from the
mitochondria. Rescue by eIF4E was mediated in part by its ability to
increase cellular levels of Bcl-XL, a key apoptotic
antagonist. The eIF4E-induced increase in Bcl-XL was
robust, occurring through at least two separate mechanisms, 1) direct
translational activation and 2) increase in the abundance of the
bcl-xL transcript. However, gain and loss
of function experiments indicated that Bcl-XL did not fully
account for the potent antiapoptotic activity of eIF4E. These
observations indicate that the set point for cellular susceptibility to
apoptosis can be governed by translational control.
The rate of protein synthesis is intimately connected with the process
of programmed cell death. An ordered shut down of protein synthesis is
one of the earliest events during apoptosis, and suppression of global
translation can enhance apoptosis (37-40). Although maintenance of
global translation tends to antagonize apoptosis,
cap-dependent and IRES-driven modes of translation can impact cell death differently. One mediator of IRES-regulated translation, death-associated protein 5 (DAP5/p97/NAT1), for example, promotes cell death (39, 40). In contrast, eIF4E, the principal activator of cap-dependent translation, rescues cells from
apoptosis (16-18).
Our finding that eIF4E modulates the mitochondrial checkpoint for
apoptosis provides a glimpse into the mechanism by which the
translational apparatus links survival signaling to the apoptotic machinery. We show that Bcl-XL is regulated by direct
control its translation, a finding in accord with a recent publication reporting that fibroblast growth factor-2 translationally activates both Bcl-XL and Bcl-2 through a Ras/MEK (mitogen-activated
protein kinase/extracellular signal-regulated kinase
kinase)-dependent signaling pathway (41). In this regard,
eIF4E is known to be activated in a MEK-dependent manner
(42), thus defining a putative survival pathway from growth factor
receptors through Ras activation of the translational apparatus to the
Bcl-XL/Bcl-2-regulated apoptotic checkpoint.
What is the mechanism by which eIF4E selectively increases translation
of the Bcl-XL transcript? It is generally agreed that activated eIF4E preferentially stimulates translation of those mRNA
with a high degree of complexity in their 5'-untranslated region
(5'-UTR), such as those with upstream open reading frames or a high
GC-content (43, 44). One documented example of translational control of
Bcl-2 family proteins through a 5'-UTR is the bcl-2 mRNA, which contains a 35-bp upstream open reading frame (45). The
5'-UTR of the bcl-XL message is relatively long
and highly GC-rich (46), suggesting that it too may be a good candidate for eIF4E-mediated regulation, which we found to be the case.
Pretranslational stages of Bcl-XL production were also
activated by overexpressed eIF4E, suggesting that transcriptional
regulation, posttranscriptional maturation, and nuclear export of its
mRNA may all be targets of translational control. Transcriptional
control of bcl-x is an established mechanism for regulating
cellular levels of Bcl-XL (47, 48). A number of
transcription factors such as Stat5 (49, 50), NF Bcl-XL is not the sole mediator of
eIF4E-dependent antiapoptotic signaling upstream of
mitochondria, since gain of Bcl-XL function could not
reproduce the robust effects of eIF4E on apoptosis nor could loss of
Bcl-XL function completely abrogate eIF4E mediated rescue,
thus supporting the available literature that translational control of
apoptosis is mediated by a set of antiapoptotic effectors (24, 54, 55).
Because the vast majority of mRNAs encoding growth factors, their
cognate receptors and signal transduction pathways have long 5'-UTRs or
contain upstream open reading frames, it is likely that a plethora of
antiapoptotic pathways emanate from activated eIF4E. In this
connection, we have previously documented that one downstream effector
of eIF4E, cyclin D1, is required for eIF4E-mediated rescue from
Myc-dependent apoptosis (17). Future investigations will be
required to establish whether cyclin D1 and Bcl-XL lie on
the same or different survival pathways downstream of eIF4E.
Our data help to clarify how c-Myc and eIF4E cooperate in
tumorigenesis. Deregulated c-Myc triggers uncontrolled cell cycle progression and apoptosis, processes that have opposite effects on
oncogenesis (56). In this regard, the oncogenic potential of c-Myc,
which is masked by its own pro-apoptotic potency, requires an
additional event that antagonizes apoptosis (57). Overexpressed eIF4E
effectively cooperates with c-Myc in malignant conversion of rodent
fibroblasts (10, 58). Our previous reports (16, 17) and present
findings suggest that the impact of eIF4E on Myc-dependent
oncogenesis is determined to a significant degree by its ability to
suppress the mitochondrial events essential for Myc-activated apoptosis.
Thus, our data trace a novel pathway that controls cell susceptibility
to apoptosis. The pathway originates at the protein synthesis machinery
and leads to pretranslational and translational modification of the
apoptotic program at the mitochondrial checkpoint. Our findings
highlight the need for developing new discovery tools that accurately
identify those transcripts that are translationally activated by eIF4E
to mitigate a proapoptotic stress. In preliminary studies, combining
polyribosome preparations to stratify mRNA by the number of bound
ribosomes with gene expression microarray has begun to show promise.
Independent of the identity of the entire set of translationally
activated messages mediating eIF4E rescue, our data begin to explain
how the translation initiation apparatus functions to suppress
apoptosis and promote oncogenesis, suggesting a plausible mechanism to
explain the dramatic oncogenic synergy between eIF4E and a variety of
pre-neoplastic alterations that promote both uncontrolled cell cycle
progression and cell death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
receptor (20). The second pathway, initiated by
various stressors such as cytotoxic drugs and radiation, is transduced
through a caspase-2-mediated series of steps into mitochondrial release
of cytochrome c (21). Subsequent formation of the
apoptosome, a complex containing cytochrome c, adapter
protein Apaf-1, and procaspase-9 leads to activation of caspase-9 (22).
When activated, caspases-8 and -9 activate effectors caspases-3, -6, and/or 7, which in turn cleave critical cellular targets, resulting in
death (23). Proteins of the Bcl-2 family tightly regulate mitochondrial
release of cytochrome c. Proapoptotic proteins, such as Bid,
Bax, Bad, and Bak, form pores in the outer mitochondrial membrane,
whereas the anti-apoptotic proteins, Bcl-2 and Bcl-XL,
inhibit pore formation (24, 25). In most cell types, these two pathways
converge, and receptor-induced activation of caspase-8 also results in
mitochondrial release of death promoters with subsequent activation of
the apoptosome-dependent caspase cascade (26). Thus, the
mitochondria integrate a variety of cell death signals, and the ability
of Bcl-2/Bcl-XL to interdict apoptosis is one hallmark
of mitochondrial involvement in the apoptotic pathway (27, 28).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]dATP (3000 Ci/mmol)
using terminal deoxynucleotidyltransferase (Roche Molecular
Biochemicals). A glyceraldehyde-3-phosphate dehydrogenase cDNA
probe (Clontech, Palo Alto, CA) was random-primed
with 50 µCi of [
-32P]dCTP (3000 Ci/mmol).
Radiolabeled probes were purified using Sephadex G-25 spin columns
(Roche Molecular Biochemicals). Hybridization was performed in 50%
(v/v) formamide, 5× sodium chloride/sodium citrate (SSC), 1% SDS, 1×
Denhardt's, 10% dextran sulfate, and 250 µg/ml yeast RNA (Sigma)
with either the 3' end-labeled bcl-XL oligonucleotide probe or the random primer-labeled
glyceraldehyde-3-phosphate dehydrogenase cDNA probe at 42 °C
overnight. Blots were washed twice in 0.1× SSC, 0.1% SDS at 50 °C
for 30 min and exposed to x-ray film.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
or Fas ligand was modest and not
amenable to further study. In contrast, more than 64% of cells
underwent apoptotic death in response to lovastatin, a value reduced to
nearly the basal frequency by ectopic expression of eIF4E. Based on
this result, we restricted our subsequent studies of eIF4E rescue to lovastatin-induced apoptosis.
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Fig. 1.
eIF4E rescues cells from
Myc-dependent apoptosis. REF/Myc and REF/Myc/4E cells
were cultured in complete medium with or without 5 µM
lovastatin for 24 h or in Dulbecco's modified Eagle's medium
plus 1% fetal calf serum (FCS) alone, with 100 ng/ml tumor
necrosis factor- (TNF-
), or with 100 ng/ml Fas ligand
(Fas-L) for 24 h. Apoptosis was quantified by flow
cytometry. The data presented are the mean (±S.E.) of four independent
experiments.
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Fig. 2.
eIF4E blocks PARP cleavage and suppresses
caspase-3 and caspase-9 activation. A and B,
immunoblot analysis of PARP and caspase-3. REF/Myc and REF/Myc/4E cells
were cultured in complete medium with or without 5 µM
lovastatin for 24 h. Cleavage of PARP (A) and activated
caspase-3 (B) were detected by immunoblot analysis with
tubulin shown as a loading control. C and D,
analysis of caspase-3 and caspase-9 enzymatic activity. REF/Myc or
REF/Myc/4E cells were cultured for the time interval indicated in the
presence of 5 µM lovastatin. Caspase-3 like activity
was detected using CleavaLiteTM caspase-3 activity assay kit
(C), and caspase-9 like activity was detected with a
colorimetric assay (D); results shown are representative of
three independent experiments.
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Fig. 3.
eIF4E prevents release of cytochrome
c from the mitochondria to the cytosol.
A, immunoassay of cytoplasmic cytochrome c.
REF/Myc or REF/Myc/4E cells were cultured in complete medium with 5 µM lovastatin for the time intervals indicated. Release
of cytochrome c from mitochondria was determined by
quantifying cytochrome c concentration in the cytoplasm by
enzyme-linked immunosorbent assay. Data shown are representative of
three independent experiments. B, immunoblot analysis of
cytochrome c subcellular distribution. REF/Myc or REF/Myc/4E
cells were cultured in complete medium with or without 5 µM lovastatin for 24 h. Cytochrome c was
detected in cell extracts (cytosolic fraction and pellet) by immunoblot
analysis (with actin shown as a loading control).
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Fig. 4.
eIF4E increases steady state levels of
Bcl-XL. REF/Myc or REF/Myc/4E cells were cultured in
complete medium with or without 5 µM lovastatin for
24 h. Immunoblot analysis was carried out for Bcl-2 family
members, pro-apoptotic, Bax and Bad (upper panel), and
anti-apoptotic, Bcl-2 (middle panel) and Bcl-XL
(lower panel), with tubulin used as a loading control. Shown
below each blot is densitometric quantification expressed in arbitrary
units. O.D., optical density.
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[in a new window]
Fig. 5.
eIF4E increases recruitment of
bcl-XL transcripts to ribosomes.
A, polyribosome analysis. Polyribosomes from REF/Myc or
REF/Myc/4E cells were resolved into discrete fractions by sucrose
gradient ultracentrifugation. Shown is the optical density (O.D.
(254 nm)) as a function of sedimentation velocity with the heavier
polyribosomes corresponding to the higher numbered fractions. The
position of 40, 60, and 80 S ribosomal fractions (designated with
arrows) were off scale. B and C,
translational activity of bcl-2 and
bcl-XL mRNA. RNA from each gradient fraction
was reverse-transcribed and quantified by real time PCR. A standard
curve was generated using five concentrations of a cell extract
containing a known quantity of target mRNA that spanned the range
of mRNA content in the samples to be analyzed. Quantification of
bcl-2 and bcl-XL mRNA in REF/Myc
and REF/Myc/4E cells was carried out by linear interpolation. Shown is
the polysome distribution of bcl-2 (B) and
bcl-XL (C) mRNA in REF/Myc and
REF/Myc/4E as a function of position in the gradient.
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Fig. 6.
eIF4E increases pretranslational steps in
Bcl-XL production. A, steady state levels
of bcl-XL mRNA. Total (pre-gradient) RNA
from REF/Myc or REF/Myc/4E cells was quantified by real time PCR
(left, shown as the REF/Myc/4E:REF/Myc ratio of
bcl-2 and bcl-XL mRNA) or
subjected to Northern blot analysis (right,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), shown as a
loading control). B, stability of
bcl-XL mRNA. REF/Myc or REF/Myc/4E cells
were treated with 5 µg/ml actinomycin D for the time interval
indicated. Total RNA was isolated and analyzed by Northern blot.
C, shown is RNA stability expressed as the optical
density (O.D.) ratio of Bcl-XL and
glyceraldehyde-3-phosphate dehydrogenase.
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Fig. 7.
Decreasing Bcl-XL to base-line
levels does not fully restore the apoptotic response in REF/Myc/4E
cells. A, analysis of Bcl-XL protein levels
after treatment with bcl-XL antisense
oligodeoxynucleotides. REF/Myc/4E cells were treated with either 40 µM bcl-XL ASO or a scramble
control (SCR) for 72 h. ASO was directed against human
bcl-XL mRNA sequences in the predicted
translation initiation region to inhibit synthesis of
Bcl-XL protein, which was detected by immunoblotting.
Expression of tubulin is shown as a loading control. B,
apoptosis. REF/Myc/4E cells were treated as in A and
cultured for an additional 24 h in the presence or absence of 5 µM lovastatin. The proportion of cells with hypodiploid
DNA content was quantitated by flow cytometry.
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Fig. 8.
Increasing steady state levels of
Bcl-XL attenuates but does not eliminate apoptosis in
REF/Myc cells. A, steady state levels of
Bcl-XL in clonal REF/Myc cell lines stably transfected with
Bcl-XL. REF/Myc cells were stably transfected with
Bcl-XL, and a series of clonal lines were developed. Shown
is the immunoblot analysis of Bcl-XL protein in parental
cells, clone 7 (chosen to closely match the Bcl-XL level in
REF/Myc/4E) and clone 13 (chosen to have a level of Bcl-XL
in great excess to that in REF/Myc/4E). B and C,
subcellular distribution of cytochrome c. REF/Myc,
REF/Myc/Bcl-XL 7, and REF/Myc/Bcl-XL 13 cells
were cultured in complete medium with 5 µM lovastatin for
the time intervals indicated. Shown are the relative amounts of
cytosolic cytochrome c determined by immunoblot
(B) and the cellular distribution of cytochrome c
by immunofluoresence (nuclei stained with 4,6-diamidino-2-phenylindole;
representative micrographs from three independent experiments are
shown). The scale bar equals 20 µM
(C). D, apoptosis. Clonal lines of REF/Myc cells
ectopically expressing Bcl-XL or empty puromycin
vector were cultured for 24 h in the presence or absence of 5 µM lovastatin. Apoptosis is shown for each cell
line, quantified by flow cytometry.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B (51), PAX3 (52),
and Ets2 (53) may all be involved in regulating bcl-x gene
expression. Although we speculate that eIF4E might increase the level
of bcl-XL mRNA by activating translation of
these factors or their regulators, detailed studies of the 5'- and
3'-UTR of the transcript will be needed to clarify the mechanism. Our
attempts to separately quantify the impact of pre-translational and
translational activation of Bcl-XL production by eIF4E
using actinomycin D were unsuccessful due to the short half-life of its
mRNA.2 It should be
pointed out, however, that at least two transcription factors, c-Fos
and c-Myc, are known to be targets for translation control (54). Future
studies will undoubtedly expand the list.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Janet Dubinsky (Department of Neuroscience, University of Minnesota) for helping to detect the mitochondrial transmembrane potential using rhodamine 123 dye. We also thank Drs. Nahum Sonenberg, Ronald Jemmerson, and Timothy Behrens for invaluable help and discussions and Dr. Edward Prochownik (Children's Hospital, Pittsburgh, PA) for the pAPuro/bcl-X vector.
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FOOTNOTES |
---|
* This work was supported by Department of Defense Grant BC98414 (to V. A. P.), National Institutes of Health Grant 2P50-HL50152 (to P. B. B.), and National Institutes of Health Grant HL 07741-07 (to S. L. and D. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 612-624-0999; Fax: 612-625-2174; E-mail: bitte001@umn.edu.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M208821200
2 S. Li, T. Takasu, D. M. Perlman, M. S. Peterson, D. Burrichter, S. Avdulov, P. B. Bitterman, and V. A. Polunovsky, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: eIF4E, eukaryotic translation initiation factor 4E; REF, rat embryonic fibroblasts; PBS, phosphate-buffered saline; PARP, anti-poly(ADP-ribose) polymerase; HPLC, high performance liquid chromatography; ASO, antisense oligodeoxynucleotide; UTR, untranslated region; IRES, internal ribosomal entry site; DAPI, 4,6-diamidino-2-phenylindole.
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