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NEOPLASIA
Abolition of stress-induced protein synthesis sensitizes leukemia cells to
anthracycline-induced death
Gro Gausdal,1,2 Bjørn Tore Gjertsen,2,3 Emmet McCormack,2 Petra Van Damme,4,5 Randi Hovland,6 Camilla Krakstad,1
Øystein Bruserud,2,3 Kris Gevaert,4,5 Joël Vandekerckhove,4,5 and Stein Ove Døskeland1
1Department of Biomedicine, University of Bergen, Bergen, Norway; 2Institute of Medicine, Hematology Section and 3Department of Internal Medicine,
Haukeland University Hospital, Bergen, Norway; 4Department of Medical Protein Research, Flanders Institute for Biotechnology (VIB), Ghent, Belgium;
5Department of Biochemistry, Ghent University, Ghent, Belgium; and 6Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital,
Bergen, Norway
Anthracycline action has been thought to
involve the neosynthesis of proapoptotic
gene products and to therefore depend
on protein synthesis for optimal effect.
We found that inhibition of general, but
not rapamycin-sensitive (cap-dependent),
protein synthesis in the preapoptotic period enhanced anthracycline-induced
acute myelogenous leukemia (AML) cell
death, both in vitro and in several animal
AML models. Pre-apoptotic anthracyclineexposed AML cells had altered translational specificity, with enhanced synthesis of a subset of proteins, including
endoplasmatic reticulum chaperones. The
altered translational specificity could be
explained by perturbation (protein degradation, truncation, or dephosphorylation)
of the cap-dependent translation initiation machinery and of proteins control-
ing translation of specific mRNAs. We
propose that judiciously timed inhibition
of cap-independent translation is considered for combination therapy with anthracyclines in AML. (Blood. 2008;111:
2866-2877)
© 2008 by The American Society of Hematology
Introduction
Despite recent achievements,1,2 progress in therapy is slow for most
malignancies, including acute myelogenous leukemia (AML).3
A more thorough understanding of how the most successful current
drugs, such as the anthracyclines, induce cancer cell death can
reveal novel therapeutically valuable death mechanisms, which can
be targeted more efficiently by specifically tailored compounds
than by current drugs. It can also reveal that present drugs activate
cancer cell survival pathways in ways that can be overcome by, for
example, coadministering complementary agents.
The aim of the present study was to undertake a proteomicsbased survey4 of protein expression and modification in
anthracycline-treated AML cells to explain in molecular terms
previous observations that inhibitors of translation could protect
completely5 or partially6-9 against anthracycline-induced cell death.
Anthracycline treatment up-regulates the activity of signaling
pathways stimulating the transcription factor nuclear factor-␬B,10
and we have found that drug-resistant AML cells have up-regulated
signal transducers and activators of transcriptions 3 and 5.11
Because these factors induce genes coding mainly, although not
exclusively, for presumed survival promoting proteins,12-15 the
reported dependence on protein synthesis for death implies either
that the proapoptotic genes are induced in excess, that anthracycline treatment tips the balance between translation of mRNA
coding for proapoptotic and prosurvival proteins in favor of the
former, or can convert short-lived prosurvival proteins to prodeath
proteins by, for example, dephosphorylation16 or cleavage.17-19
We found that the first-line AML anthracycline drugs, such as
daunorubicin (DNR), modified or altered the expression of several
Submitted July 25, 2007; accepted December 16, 2007. Prepublished online as
Blood First Edition paper, January 8, 2008; DOI 10.1182/blood-2007-07-103242.
G.G. and B.T.G. contributed equally to this work.
The online version of this article contains a data supplement.
2866
proteins known to alter the translation of specific mRNAs or direct
translation from cap dependence to internal ribosome entry site
dependence. This might explain that the DNR-treated AML cells in
the late preapoptotic phase had enhanced synthesis of a subset of
proteins, including ER chaperones. The preapoptotic protein synthesis served, contrary to expectations based on previous reports, a
prosurvival function, because anthracycline-induced AML cell
death was enhanced by protein synthesis inhibitor in vitro as well
as in 3 separate intact animal (rodent) models for AML. Judiciously
timed inhibition of the stress-induced cap-independent translation
might therefore be considered in combination with anthracyclines
for AML therapy.
Methods
Reagents and cells
Cycloheximide (CHX), rapamycin, puromycin, and emetine were from
Sigma-Aldrich (St Louis, MO). Calyculin A was from Calbiochem (San
Diego, CA). DNR was from Sigma-Aldrich (cell experiments) or sanofiaventis (Bridgewater, NJ; animal experiments). Idarubicin (IDA) was from
Sigma-Aldrich (cell experiments) or Pfizer (New York, NY; animal
experiments).
HL60 cells were from GCMCC (Braunschweig, Germany; http://
www.dsmz.de), and NB4 cells were from Dr M. Lanotte (Hôpital St Louis,
Paris, France). They were cultured in RPMI 1640 medium (Invitrogen,
Carlsbad, CA) with 10% fetal bovine serum (FBS) and L-glutamine
(2 mmol/L), and kept in logarithmic growth until studied at approximately
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2008 by The American Society of Hematology
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
0.5 ⫻ 106 cells/mL. The IPC-81 rat promyelocytic leukemia cell line was
cultured as described previously.20 BN rat acute myelocytic leukemia
(BNML) cells were a gift from Dr P. O. Iversen (University of Oslo, Oslo,
Norway) and serially passaged in vivo. Blasts from AML patients were
isolated and prepared as described previously.21 Detailed information about
patient material is given in Table S2 (available on the Blood website; see the
Supplemental Materials link at the top of the online article).
Assessment of apoptosis
Cells were fixed and screened for apoptosis using differential interference
contrast microscopy to visualize surface budding, and fluorescence microscopy for chromatin condensation.18 The chromatin pattern in cells incubated with the autofluorescent anthracyclines was identical to that observed
with the DNA-specific dyes bisbenzimide (Hoechst 33342) and 4,6diamidino-2-phenylindole. Cell viability was also scored by the WST1
assay (Millipore Bioscience Research Reagents, Temecula, CA), and flow
cytometry. For examination of ultrastructure, HL60 cells were fixed and
further processed as described previously.22
Metabolic labeling of cells
Metabolic labeling of phosphoproteins with 32Pi was performed as described previously.23
For protein labeling, the cells were transferred to methionine-free
medium supplemented with dialyzed FBS and [35S]methionine
(0.02 mCi/mL; SJ1515; GE Healthcare, Chalfont St Giles, United Kingdom). For pulse-chase, labeled cells were washed in medium with
unlabeled methionine, left for 0.5 hours, and then exposed to experimental
agents. For determination of protein synthesis, cells labeled with [35S]methionine were washed and precipitated in 7% trichloroacetic acid (TCA). The
pellets were washed in 5% TCA, and then with water-saturated ether before
being dissolved in 5 mmol/L Tris buffer, pH 8, containing 8.4 M urea, 2%
3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), and
100 mmol/L dithioerythritol. Aliquots were diluted in sodium dodecyl
sulfate (SDS) loading buffer for SDS-polyacrylamide gel electrophoresis
(PAGE) analysis,22 or the incorporated radioactivity determined by liquid
scintillation counting.24
Isolation of RNA and its use as template for in vitro
protein synthesis
HL60 cells (107 cells) were pelleted, the pellet was lysed in 1 mL of TRIzol
(Invitrogen), and RNA was isolated.25 In vitro protein synthesis was
performed in rabbit reticulocyte lysate (Promega, Madison, WI) as described previously26 with 0.8 mCi/mL [35S]methionine and 0.2 mg/mL
RNA. Aliquots were precipitated with 7% TCA and processed for 2dimensional gel analysis.18
Gel electrophoresis, immunoblotting, evaluation of spot
intensity, and picking for MS analysis
One-dimensional SDS-PAGE, two-dimensional PAGE (2DE), and immunoblotting were performed essentially as described previously.18 Antibody
against pICln was from Dr K. Strange (Vanderbilt University, Nashville,
TN), caspase 3 and actin were from Santa Cruz Biotechnology (Santa Cruz,
CA), and p70S6K, phospho-p70S6K, 4E-BP1, phospho-4E-BP1, eEF2K,
eEF2, mTOR, and phospho-mTOR were from Cell Signaling Technology
(Danvers, MA). Some gels were silver-stained, dried, and exposed to
autoradiography films. For mass spectrometry (MS) analysis, gels were
stained with Sypro Ruby (Bio-Rad Laboratories, Hercules, CA), and spots
were picked manually under illumination in a Fuji LAS3000 (FujiFilm,
Tokyo, Japan). [35S]Methionine-labeled spots were excised from dried,
unfixed gels using filmless autoradiographic analysis printout as overlay.
Gel spots were quantified based on isotope content using a Fuji Bas5000
PhosphoImager (GE Healthcare) or by densitometry (silver-stained gels),
using mainly MultiGauge v2.3 (FujiFilm). To obtain the relative intensity, the
intensity of each spot was divided by the sum of the total spot intensity in the gel.
The theoretical molecular weight and isoelectric point (pI) of identified proteins
TRANSLATIONAL CONTROL BY ANTHRACYCLINES
2867
was calculated using the ProtParam tool (http://au.expasy.org/tools/
protparam.html).
COFRADIC, liquid chromatography-tandem mass
spectrometry, and MALDI-TOF-MS analysis
For each analysis, 100 mL of NB4 cells (0.4 ⫻ 106 cells/mL) were washed
and lysed in 1.5 mL of 20 mmol/L potassium phosphate, pH 6.8, with
1 mmol/L EDTA, 10 mmol/L CHAPS, 50 mmol/L NaF, 0.3 mmol/L
NaVO3, and Complete mini protease inhibitor (Roche Molecular Biochemicals, Indianapolis, IN). Extract was centrifuged (10 000g, 20 minutes, 4°C)
and kept at ⫺80°C until analysis.
The combined fractional diagonal chromatography (COFRADIC)–
based isolation and identification of the protein N-terminal peptides was
performed as described previously.4,27 In brief, the technique allows the
specific sorting of the trypsin-cleaved N-terminal parts of the proteins
present in the complete lysate. The relative intensities of 16O- and
18O-tagged peptides allows relative changes of protein expression between
2 different states to be measured. When a new protein N terminus is
generated, as the result of cleavage, this site is easily isolated and identified.
Thus, information of the exact cleavage site is obtained. Matrix-assisted
laser desorption ionization/time-of-flight (MALDI-TOF)-MS analysis, including the acquirement of postsource decay (PSD) spectra, was performed
as described previously.18,28,29
AML animal models and estimation of the antileukemic efficacy
of anthracyclines and CHX in vivo
NOD/SCID/B2mnull mice and Rowett (rnu/rnu) rats were bred and maintained under defined flora conditions in a high-efficiency particulate
arrester-filtered atmosphere. Male BN/Rij rats were from TNO (Rijswijk,
the Netherlands). All experiments were approved by the Norwegian Animal
Research Authority and conducted according to the European Convention
for the Protection of Vertebrates Used for Scientific Purposes.
Male NOD/SCID/B2mnull mice 6 to 8 weeks old were irradiated (BCC
Dynaray CH4.4 megavolt irradiation source) with 2.5 Gy (100 cGy/min)
24 hours before transplantation of 10 ⫻ 106 NB4 cells (day 0) via the tail
vein. rnu/rnu rats (60-80 g) were similarly injected with 20 ⫻ 106 IPC-81
cells, whereas BN/Rij rats (170-230 g) received 5 ⫻ 106 BNML cells from
the spleen of a terminal stage leukemic animal.
IDA (1 mg/mL in water) was given by mouth to male rnu/rnu rats once
daily (3 mg/kg) on days 14 to 16 after transplantation, whereas BN/Rij rats
received 1.5 mg/kg on days 3 to 5. DNR (1 mg/mL in saline for rat and
0.1 mg/mL for mouse) was injected intravenously into BN/Rij rats
(1.5 mg/kg per day) on days 3 to 5 and to NOD/SCID/B2mnull mice
(0.5 mg/kg) on days 10 to 12. CHX (1 mg/mL in saline for rat and 5 mg/mL
for mice) was injected intraperitoneally 1 hour after the administration of
anthracycline, at 1.5, 0.8, and 5 mg/kg for rnu/rnu rats, BN/Rij rats, and
NOD/SCID mice, respectively. Control animals received relevant vehicles.
Condition and weight was monitored daily, and animals were killed
when moribund.
The survival data were presented according to Kaplan and Meier and
survival distributions analyzed by the Mantel-Haenszel log rank statistics,
using Prism software (version 3.0; GraphPad Software, San Diego, CA).
Results
Inhibition of protein synthesis enhanced anthracycline-induced
AML cell death in vitro and in animal models of transplanted AML
Anthracyclines such as DNR are first-line drugs in the treatment of
AML and are believed to depend on protein synthesis to be
maximally effective.5-9 Contrary to expectations, we observed
more apoptosis in HL60 and NB4 AML cells exposed to DNR
and the protein synthesis inhibitor CHX than to DNR alone (Figure
1A,B). CHX intensified the DNR-induced conversion of procaspase3
to caspase3 (Figure 1C), the decrease of mitochondrial dehydrogenase
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
Figure 1. Protein synthesis inhibitor enhanced anthracycline-induced AML cell death, although suppressing death phenotype features in some cells. (A) HL60 cells
were treated for 6 hours with DNR (1.6 ␮mol/L) and CHX (3.6 ␮mol/L), alone or in combination, and scored for apoptosis (insets; see “Assessment of apoptosis”). (B)
NB4 cells were treated for 6 hours with DNR (5 ␮mol/L) and CHX (3.6 ␮mol/L), alone or in combination, and scored for apoptosis. (C) Extracts from HL60 cells treated
with DNR (8 ␮mol/L) for 2, 4, or 6 hours in the absence or presence of CHX (3.6 ␮mol/L, added 1 hour after DNR) were immunoblotted for caspase 3. (D-F) Flow
cytometric analysis (representative of 4 separate experiments) of HL60 cells treated for 4 hours with vehicle (D), 8 ␮mol/L DNR (E) or DNR ⫹ 3.6 ␮mol/L CHX (F). Note
that DNR ⫹ CHX converted twice as many cells as DNR alone to a compartment with decreased forward (FSC-H) and side (SSC-H) scatter. (G) Scoring of
morphologically evident apoptosis in IPC-81 cells treated for 5 hours with DNR (0.1 ␮mol/L) alone or in combination CHX (3.6 ␮M). (H) IPC-81 cells were treated for
6 hours with various concentrations of DNR alone or with CHX (3.6 ␮mol/L) and scored for apoptotic morphology. Note the lack of cell budding, nuclear fragmentation,
and chromatin condensation with DNR ⫹ CHX and with DNR more than ⬃1 ␮mol/L. The bars show apoptosis in cells incubated for 6 hours with DNR ⫹ CHX or CHX
alone, washed in excess medium, and incubated without DNR and CHX for another 12 hours before assessment of apoptosis. Note the high proportion of apoptotic cells
after washing. (I) As for panel G, except that cells were treated for 3 hours with DNR (0.1 ␮mol/L) and CHX (3.6 ␮mol/L), washed and further incubated for 9 hours (as
explained in panel H) before scoring of apoptosis. (J) HL60 cells received the protein synthesis inhibitors emetine (10 nmol/L), puromycin (180 nmol/L), rapamycin (10 or
100 nmol/L), or CHX (3.6 ␮mol/L) at various time points relative to DNR (1.6 ␮mol/L), and apoptosis scored 4.5 hours after DNR addition. (K) HL60 cells were treated for
6.5 hours with various concentrations of DNR (50-8000 nmol/L) in the absence (E) or presence (F) of CHX (3.6 ␮mol/L) and scored for apoptosis. (L) The accumulation
of apoptotic HL60 cells as a function of time after addition of DNR (8 ␮mol/L) alone (E), CHX (3.6 ␮mol/L) alone (䡺), or a combination of the 2 (F). CHX was added
1 hour after DNR. Apoptosis scores represent the means (⫾ SEM) of 3 to 6 experiments.
activity (data not shown), and the ultrastructural (Figure 2A-E) and flow
cytometric (Figure 1D-F) features of apoptosis.
The results obtained with the IPC-81 cells may explain the
discrepancy with at least some previous studies. In these cells,
CHX blocked signs of visible apoptosis induced by DNR
(Figure 1G,H). This was due not to protection against death but
rather to the induction of a “frozen” death type, because the cells
underwent massive conventional apoptosis after removal of
DNR/CHX (Figure 1H). In fact, a higher percentage of apoptosis was noted after washing of cells treated with DNR ⫹ CHX
than DNR alone (Figure 1I). We noted that the cells could
undergo “frozen” death also when exposed to concentrations of
DNR above 1 ␮mol/L (Figure 1H), indicating that CHX
aggravated an inherent tendency of these cells to become
blocked in certain aspects of apoptosis execution when exposed
to an overwhelming anthracycline challenge, as noted previously in other cell systems.30,31
That CHX acted via inhibition of protein synthesis was
supported by the similar efficiency of other general translation
inhibitors like emetine and puromycin. The inhibitors were most
efficient when given in the early preapoptotic phase after DNR
addition (Figure 1J). They were much less efficient when given
before DNR (Figure 1J; and data not shown). Rapamycin, which
counteracts mTOR action to decrease cap-dependent protein synthesis while enhancing internal ribosomal entry site (IRES)–
dependent translation,32 was inefficient (Figure 1J).
The presence of CHX from 1 hour after DNR addition served to
both sensitize the HL60 cells to respond to lower concentrations of
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
TRANSLATIONAL CONTROL BY ANTHRACYCLINES
2869
nuclear lamina and commencement of surface budding. In some
cells, the endoplasmic reticulum could still have some attached
polysomes even if the soluble polysomes had disaggregated
(data not shown). Disaggregated ribosomes remained numerous.
We conclude that because the protein synthesis machinery was
morphologically intact even in cells with clear signs of early
apoptosis, protein synthesis might occur in the late preapoptotic
and early apoptotic period of cell death.
To know whether CHX could enhance anthracycline-induced
AML cell death in vivo, we first studied irradiated rnu/rnu rats
transplanted with the IPC-81 cells. The combined treatment with
CHX and idarubicin (IDA) significantly prolonged survival
compared with IDA alone (Figure 3A). The transplantable
syngenic BNML model of leukemia, from which the IPC-81 cell
line is derived, has been extensively validated as a reliable
predictor for drug efficiency against AML in humans.34 In
addition, in this AML model, survival was enhanced by CHX,
whether combined with IDA (Figure 3B) or with DNR (Figure
3C). Finally, NOD/SCID/B2mnull mice transplanted with human
NB4 cells were studied. Again, a significantly prolonged
survival was observed when CHX was combined with DNR
(Figure 3D). The prolonged survival was due to decreased tumor
burden, because autopsied animals given either anthracycline
alone or CHX alone, had more AML infiltrate in spleen, bone
marrow, and lungs than animals given anthracycline ⫹ CHX
(not shown), even if the latter animals on the average had lived
longer after AML cell inoculation. In conclusion, CHX enhanced the death-inducing effect of anthracyclines on AML cells
in animals with deficient as well as with intact (BNMLtransplanted BN/Rij rats) immune system.
Anthracycline-treated preapoptotic AML cells had enhanced
relative synthesis of a subset of survival-associated proteins
Figure 2. Comparison of the ultrastructural properties of HL60 cells treated
with DNR alone and in combination with CHX. (A) Transmission electron
microscopic overview (left) and detail showing part of nucleus and perinuclear region
of control HL60 cell (incubated with vehicle for 5 hours). The inset (arrow; R) shows a
polysomal rosette. (B) Cell treated with CHX (3.6 ␮mol/L) for 4 hours. (C) Cell treated
with DNR (8 ␮mol/L) for 2.5 hours. Note beginning thickening of microvilli, perinuclear
accumulation of condensed chromatin and polysomes (inset). (D) Cell treated for
2.5 hours with DNR (8 ␮mol/L) ⫹ CHX (3.6 ␮mol/L, added 1 hour after DNR). Note
budding, separation of nuclear lamina from chromatin (arrows), and dispersal of
polysomes (inset). (E) Cell treated with DNR (8 ␮mol/L) for 5 hours. Note similarity of
features with cell in panel D. Panels A-E: scale bar: 5 ␮m for left subpanels, 0.3 ␮m
for right subpanels, and 0.18 ␮m for right subpanel insets. The cells in panels A, B,
and E are typical for the average cell in the population. The cells in panels C and D are
representative of the 10% of cell population with most advanced apoptosis.
DNR and accelerate the death in response to a maximally effective
(8 ␮mol/L) DNR concentration (Figure 1K,L). The synergistic
effects noted at 0.1 ␮mol/L DNR (Figure 1I,K) indicated that this
mechanism might be operating in clinical settings, because peak
plasma concentrations of DNR in patients with AML range from
0.3 to 5 ␮mol/L.33
By transmission electron microscopy, the DNR-induced and
DNR/CHX-induced HL60 cell death had similar morphology
when comparing similar stages of death (Figure 2A-E). The
death type was apoptotic with early margination of chromatin,
separation of the nuclear lamina and matrix, and swelling of
microvilli, followed by cell budding and hypercondensation of
chromatin. Disaggregation of polysomal rosettes (Figure 2D,E
right panels, including insets) occurred after separation of the
To estimate the effect of DNR and established protein synthesis
inhibitors on protein synthesis, HL60 cells were pulse-labeled with
[35S]methionine and the synthesis rate estimated as the ratio
between autoradiographic intensity and protein staining intensity of
SDS-gels of protein extracts from the cells (Figure 4A,B). The
percentage synthesis declined as a function of time after addition of
DNR (Figure 4A) but remained slightly higher than the percentage
of nonapoptotic cells under similar conditions (Figure 1L). This
suggested that translation was slightly enhanced in preapoptotic
DNR-treated cells, not completely compromised in apoptotic cells,
or both. Although CHX at 3.6 ␮mol/L led to strong inhibition of
protein synthesis, rapamycin, as expected from previous studies,35
inhibited the total synthesis by only 10% to 15% (Figure 4B;
data not shown).
To search for altered translation of specific transcripts, vehicleand DNR-treated HL60 cells were pulse-labeled with [35S]methionine, and individual protein spot intensity compared in 2DE gel
autoradiographs of the pI 4-5 sub-proteome. The DNR-treated cells
had more than 2-fold increased relative synthesis of ribosomal
protein P2 (rpP2, 3 spots labeled 1 in Figure 4C), protein disulfide
isomerase (PDI; spot 2), proliferating cell nuclear antigen (PCNA;
spot 3), and divalent cation tolerance homolog isoform 2 (CutA;
spot 4). Three protein spots (11, 12, and 13) decreased more than
2-fold in [35S]methionine labeling intensity.
The effects of DNR were probably translational rather than
transcriptional, because protein spots labeled during in vitro
translation had similar relative intensity whether the template
was RNA isolated from cells treated for 4.5 hours with vehicle
or with DNR (Figure 4D). Comparison of individual spot
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
intensity in cells and after in vitro translation revealed that the
synthesis of PCNA, PDI, rpP2, and, to a lesser extent, calreticulin may be translationally turned on even in nonstressed HL60
cells and that their synthesis was additionally increased in
DNR-stressed cells (Table 1). Conversely, proteins like MLC
had lower synthesis rate in intact cells than expected from their
in vitro synthesis rate (Figure 4C,D; Table 1).
The preferential synthesis of presumed survival proteins like
PCNA and PDI may explain why inhibition of protein synthesis
enhanced DNR-induced death.
Proteins involved in translational control are major targets in
anthracycline-treated cells
Figure 3. Prolonged survival of AML transplanted animals treated with protein
synthesis inhibitor in addition to anthracycline. (A) Survival of IPC-81 transplanted rnu/rnu rats treated with vehicle (E; n ⫽ 5), idarubicin (‚; IDA, 3 mg/kg;
n ⫽ 4), cycloheximide (F; CHX, 1.5 mg/kg; n ⫽ 3), or IDA ⫹ CHX (Œ; n ⫽ 5). The
IDA ⫹ CHX group had median survival time of 88 days compared with 58.5 days for
the IDA mono-therapy group (P ⫽ .0072). (B) Survival of syngenic BNMLtransplanted BN/Rij rats treated with vehicle (E; n ⫽ 12), IDA (‚; 1.5 mg/kg; n ⫽ 6),
CHX (F; 0.8 mg/kg; n ⫽ 6), or IDA ⫹ CHX (Œ; n ⫽ 6). The combination of IDA ⫹ CHX
(mean survival time, 37 days) significantly increased (P ⫽ .03) survival over IDA
alone (mean survival time, 26.5 days). (C) Survival of BNML rats treated with vehicle
(E; n ⫽ 12), DNR (‚; 1.5 mg/kg; n ⫽ 6), CHX (F; 0.8 mg/kg; n ⫽ 6), or DNR ⫹ CHX
(Œ; n ⫽ 6). The combination of CHX with DNR increased the median survival from
22.5 to 26 days (P ⫽ .0065). (D) Survival of NOD/SCID/B2mnull mice transplanted
with NB4 cells and treated with vehicle (E; n ⫽ 7), DNR (‚; 0.5 mg/kg; n ⫽ 5), CHX
(F; 5 mg/kg; n ⫽ 5), or DNR ⫹ CHX (Œ; n ⫽ 4). The combination of CHX with DNR
increased the median survival from 27 to 36.5 days (P ⫽ .0046). For panels A-D:
animal models, type and number of cells injected, and schedules are defined
under “AML animal models and estimation of the antileukemic efficacy of anthracyclines and CHX in vivo.”
That anthracyclines affect the translation of specific transcripts is a novel
finding. Switching from the usual cap-dependent to IRES-dependent
initiation of translation is seen in cells exposed to the mTOR activity
perturbing drug rapamycin, acting via decreased phosphorylation of
p70S6 kinase (p70S6K) and 4E-BP1.35,37 Phosphorylation of rpS6 via
p70S6K is considered necessary for efficient cap-dependent translational
initiation of ribosomal and other proteins with 5⬘-untranslated region
(UTR) polypyrimidine tracts,38 whereas phospho-4E-BP1 relieves the
tonic inhibition of general cap-dependent translation.39
We found that, like rapamycin, DNR not only decreased phosphop70S6K (P-p70S6K) but also led to degradation of p70S6K, suggesting that
DNR, unlike rapamycin, would lead to irreversible loss of p70S6K
activity. Likewise, DNR treatment led to more complete conversion of
the highly (␥,␤) to the less (␣) phosphorylated form of P-4E-BP1
(Figure 4F) than rapamycin treatment (data not shown). The effect of
DNR on P-p70S6K and P-4E-BP1 was not confined to HL60 cells and
high DNR concentrations, because significant effects were observed
already at 100 nmol/L DNR in IPC cells (Figure 4G).
To gain further insight into the effects of anthracyclines on
the protein synthesis machinery and other targets, protein
extracts from control and DNR/CHX-treated NB4 cells were
subjected to differential 16O/18O global peptide labeling, COFRADIC, and selective sorting of protein amino-terminal peptides.4,27 The method, unlike metabolic labeling, avoids exposing the cells to media with decreased amino acid content and
dialyzed serum, thus eliminating 2 factors that can influence the
protein synthesis machinery. All proteins identified in the NB4
cells (Table S1) were sorted ontologically into 49 biologic
processes, and 6% classified under translation. In the subgroup
of proteins whose N-terminal peptides were decreased after
DNR/CHX treatment, 34% had an assigned role in translation
(Figure 5A). This suggested that proteins involved in mRNA
translation were preferentially targeted by DNR.
The down-regulated proteins included the rapamycin receptor
FKBP1240 as well as eIF4H41 and eIF1A42 (Table 2), which support
cap-dependent and inhibit IRES-dependent initiation of translation.
These findings provide clues about how the action of rapamycin may be
blunted (Figure 1J) and suggest, in conjunction with the data of Figure
4F, that the machinery favoring cap-dependent rather than IRESdependent initiation of translation was severely damaged in DNRtreated cells. Both ribosomal protein L13a43 and hnRNP C1/C244 are
involved in the control of specific mRNA translation, and their
down-regulation may therefore alter the translational preference, as
observed in the DNR-treated cells (Figure 4C, Table 1). The eEF2
kinase (eEF2K) was strongly down-regulated, not only in the DNR/CHXtreated NB4 cells (Table 2) but also in HL60 cells, where it became
degraded after 4 to 5 hours’incubation with either 1.6 or 8 ␮mol/L DNR
(Figure 5B). Because eEF2 kinase inhibits elongation factor 2 (eEF2),45
its down-regulation is expected to enhance the efficiency of eEF2. The
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
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2871
Figure 4. DNR altered the control of AML cell protein
translation. (A) Total protein synthesis rate of HL60 cells
as a function of incubation with DNR (8 ␮mol/L) or CHX
(3.6 ␮mol/L) relative to vehicle-treated cells. Cells were
pulse-labeled with [35S]methionine during the last
15 minutes of the incubation. Synthesis is the ratio
between the labeling, determined by autoradiography of
SDS-PAGE gels of extracted proteins, and the protein
staining of the same gels (see right inset). The data
represent the means (⫾ SEM) of 4 separate experiments. (B) HL60 cells were treated for 2 hours with CHX
(3.6 or 36 ␮mol/L) or rapamycin (100 nmol/L), the last
0.5 hours of which was in the presence of [35S]methionine. Autoradiograms of SDS-gels (right panel) show
that CHX was a far more efficient protein synthesis
inhibitor than rapamycin in these cells. The left panel
shows protein staining of the same gels. (C) Autoradiogram of 2DE gels (pI 4-5) of extract from HL60 cells
treated with vehicle or 8 ␮mol/L DNR for 5 hours. A pulse
of [35S]methionine was given during the last 30 minutes
of the incubation. (D) Autoradiograms of proteins translated in vitro in the presence [35S]methionine with RNA
isolated from HL60 cells incubated with vehicle (left
subpanel) or 8 ␮mol/L DNR for 4.5 hours (right subpanel)
as template. (E) Autoradiograms of proteins from HL60
cells prelabeled with [35S]methionine for 90 minutes and
chased with unlabeled methionine for 6 hours in the
absence and presence of 8 ␮mol/L DNR. (F) HL60 cells
were treated with DNR (1.6 or 8 ␮mol/L) or rapamycin
(100 nmol/L) for the periods indicated; cell extracts were
immunoblotted and probed for P-Ser371-p70S6K, p70S6K,
P-Thr37/46-4E-BP1, 4E-BP1, as well as P-Ser2448mTOR, mTOR, and ␤-actin. (G) IPC-81 cells were
treated for 6.5 hours with various concentrations of DNR
(10-300 nmol/L), and extracts were immunoblotted and
probed as for (F). For panels C-E: The circled spots
correspond to proteins in Table 1. These proteins were
subjected to comparative quantitative analysis. Spots
found in all gels are circled with closed lines; spots found
only in panel B and E are circled with dashed lines. The
gels shown are representative of 3 to 5 experiments.
Table 1. Proteins affected by treatment with DNR
pI
Spot
number
Protein
abbreviation
Molecular weight
Observed
Predicted
Observed
Predicted
Relative spot intensity
Control,
in cellulo/in vitro
DNR,
in cellulo/in vitro
1
rpP2
4.48/4.39/4.3
4.42/4.37/4.31
13
12
5.0
26
2
PDI
4.75
4.76
58
57
⬎5.0
⬎20
3
PCNA
4.57
4.57
31
29
4.4
12
4
cutA
4.45
5.15
15
17
n.d.
n.d.
5
CALR
4.31
4.29
46
48
1.5
2.0
6
EF1Ba
4.50
4.50
25
25
0.89
1.8*
7
PSMA5
4.63
4.74
24
26
0.21
0.62
8
CTM
4.65
4.80
33
28
0.98
0.36
9
MLC
4.32
4.46
16
17
0.46
0.25
10
n.i.
4.62
n.d.
20
n.d.
0.12
0.11
11
n.i.
4.73
n.d.
15
n.d.
0.09
0.03
Cells were treated with vehicle or 8 ␮mol/L DNR for 4.5 hours, pulse-labeled for the last 30 minutes with [35S]methionine, and protein spots 1 to 11 in 2DE gels (see Figure 4
for details) analyzed for relative labeling intensity. Proteins translated from mRNA isolated from vehicle-treated (Ctr) or DNR-treated cells were similarly separated and
analyzed. The ratio between relative spot intensity in cells (in cellulo) and after in vitro translation (in vitro) is listed in the two rightmost columns.The data are averages from 3
separate experiments. Protein abbreviations refer to the following proteins (Swissprot accession numbers in parentheses). rpP2 indicates 60S acidic ribosomal protein P2
(P05387); PDI, protein disulfide isomerase/prolyl 4-hydroxylase ␤ subunit (NP_000909); PCNA, proliferating cell nuclear antigen (NP_002583); cutA, divalent cation tolerance
homolog isoform 2(NP_057005); CALR, calreticulin precursor (NP_004334); eEF1Ba, elongation factor 1Ba (P24534); PSMA5, proteasome endopeptidase complex, ␨ chain
(S17521); CTM, cytoskeletal tropomyosin (CAA28258); MLC, myosin light polypeptide 6 (P60662); n.d., not determined; and n.i., not identified.
*The value for EF1Ba is slightly underestimated due to cleavage of this protein (see Figure 6).
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GAUSDAL et al
Figure 5. COFRADIC N-terminal analysis revealed preferential down-regulation of proteins related to protein translation/RNA-binding in DNR/CHX-treated NB4
cells. (A) Gene Ontology-based analysis of the COFRADIC protein data according to biologic function. The inner ring (a) represents the biologic process relative values of all the proteins
identified in the analysis. The middle ring (b) represents the N-terminal down-regulated proteins, and the outer ring (c) represents the protein substrates producing stable cleavage
products. The pie chart visualizes a striking preferential down-regulation of proteins involved in translation (b). The chart was generated using the PIGOK analysis; http://128.40.158.133/
pigok.html. (B) HL60 cells were treated with 1.6 or 8 ␮mol/L DNR for the periods indicated, and extracts were immunoblotted and probed for eEF2K and eEF2.
level of eEF2 itself decreased only moderately (Table 2), mainly after
the first 4 to 5 hours of incubation with DNR (Figure 5B).
Several of the cleaved RNA-binding proteins (Table 3) may modify
the transport of mRNA and thereby its availability for translation,
whereas others, like hnRNP A046 and hnRNP U,47 have been incriminated in the control of translation of specific mRNAs. The proteinprocessing events noted after exposure to DNR could not be explained
by generalized proteolysis, because the 2DE protein pattern was quite
Table 2. List of peptides down-regulated more than 2-fold in NB4 cells treated with DNR (1.6 ␮mol/L) and CHX (3.6 ␮mol/L) for 8 hours
Protein description
Accession
no.
Start
End
EF2 kinase
O00418
2
9
Lysyl-tRNA synthetase
Q15046
2
25
Identified peptide
Fold downregulation
Ac-ADEDLIFR
⬎10
Ac-AAVQAAEVKVDGSEPKLSKNELKR
⬎10
⬎10
eIF4H, isoform 1
Q15056
2
10
Ac-ADFDTYDDR
60S ribosomal protein L13
P40429
2
12
Ac-AEVQVLVLDGR
5.9
Asparaginyl-tRNA synthetase, cytoplasmic
O43776
2
11
Ac-VLAELYVSDR
4.2
hnRNP C1/C2, isoform 1
P07910
2
12
Ac-ASNVTN⬍Dam⬎KTDPR
4.4
Ac-ASNVTNKTDPR
2.9
40S ribosomal protein S30
P62861
1
8
Ac-KVHGSLAR
2.5
60S ribosomal protein L30
P62888
2
17
Ac-VAAKKTKKSLESINSR
2.5
LSm3
P62310
2
22
Ac-ADDVDQQQTTNTVEEPLDLIR
2.5
eEF2
P13639
2
10
Ac-VN⬍Dam⬎FTVDQIR
2.4
eIF1A
O14602
2
12
Ac-PKNKGKGGKNR
2.3
Phenylalanyl-tRNA synthetase alpha chain
Q9Y285
2
12
Ac-ADGQVAELLLR
2.3
40S ribosomal protein S3a
P61247
2
8
Ac-AVGKN⬍Dam⬎KR
2.2
FK506-binding protein 1A/FKBP 12
P62942
2
14
Ac-GVQVETISPGDGR
2.1
In Tables 2 and 3, only peptides with corresponding parent proteins with functions related to mRNA-binding/processing and/or protein synthesis are shown. The sequence
of the identified peptide and its location within the parent protein is indicated Ac- denotes the ␣-N-acetyl group, and ⬍Dam⬎ indicates deamidation. The parent proteins are
referred to by description and UnitProt database accession number, The complete list of peptides is given in Table S1. Peptide identification was done using “Internal Protein
Index” databases (http://www.ebi.ac.uk/IPI/IPIhelp.html).
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Table 3. List of internally located ␣-N-acetylated peptides (formed by cleavage) exclusively found in the same DNR/CHX-treated NB4 cells
as in Table 2
Protein description
Accession
no.
Start
End
O60832
419
447
H/ACA ribonucleoprotein complex subunit 4
hnRNP A0
Q13151
74
81
hnRNP U, isoform long
Q00839
232
243
Identified peptide
Ac-YSESAKKEVVAEVVKAPQVVAEAAKTAKR
Site
EYVD_
Ac-GNTVELKR
HAVD_
Ac-GKTEQKGGDKKR
PAGD_
NAC, alpha polypeptide
Q13765
43
71
Ac-STQATTQQAQLAAAAEIDEEPVSKAKQSR
EEQD_
Poly(A) binding protein 2
Q86U42
109
125
Ac-PGDGAIEDPELEAIKAR
VEGD_
RNA-binding protein-39
Q14498
332
346
Ac-ASSASSFLDSDELER
ERTD_
Splicing factor U2AF 65 kDa subunit
P26368
129
146
Ac-GLAVTPTPVPVVGSQMTR
MTPD_
TAR DNA-binding protein-43
Q13148
90
98
Ac-ASSAVKVKR
DETD_
LSm3
P62310
7
22
Ac-QQQTTNTVEEPLDLIR
DDVD_
The amino acids preceding the identified peptide are indicated (site). The underlined Asp in the identified peptide for LSm3 indicates the processing site validated by
detection of an intermally located ␣-N-acetylated peptide. In Tables 2 and 3, only peptides with corresponding parent proteins with functions related to mRNA-binding/
processing and/or protein synthesis are shown.
Figure 6. Protein truncation and dephosphorylation
events detected by 2DE in DNR-treated HL60 cells.
(A,B) The boxed area shows the position (A) and detail
(B) of the methylosome subunit chloride conductance
regulatory protein (pICln) in Sypro Ruby-stained 2DE
gels (pI 3-10) with extract of control HL60 cells. (C) Like
panel B, except that boxed area is from gel with extract
from HL60 cells treated for 6 hours with 8 ␮mol/L DNR.
(D) Immunoblotting of pICLn on extracts of HL60 cells
treated with 1.6 ␮mol/L DNR for the times indicated.
Note truncation of pICLn from 29 to 27 kDa, starting after
3 hours of DNR exposure. (E) The spots circled with solid
lines show the MS-detected ribosomal protein P2 (rpP2)
and eEF1Ba (25 kDa). (F) After 6 hours with 8 ␮mol/L
DNR, a significant proportion of the eEF1Ba appeared as
a 17 kDa form (arrow), whereas rpP2 appeared as
3 spots of similar size but with different pI. (G and H)
MALDI analysis of the 25-kDa (G) and 17-kDa
(H) variants of eEF1Ba revealed a tryptic peptide
(1732.87), present only in the 17-kDa ⌬eEF1Ba. It was
identified by tandem mass spectrometry to have a predicted caspase cleavage site (DETD) at its C terminus.
(I-J) The relative distribution of eEF1Ba (circled) and
⌬eEF1Ba (arrow) in 2DE gels (pI 4-5) of extract from
blasts from patients with AML (M2, patient 5) treated in
vitro for 6 hours with vehicle (I) or 8 ␮mol/L DNR (J). The
gels were silver-stained.
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2874
GAUSDAL et al
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
Figure 7. The phosphorylation status of ribosomal protein P2 in AML cell lines and patient blasts. The effect of anthracycline in vitro and in vivo. (A,B) IPC-81 cells were
treated for 2.5 hours with 0.5 ␮mol/L DNR, the last 90 minutes of which with 32Pi. Proteins were separated by 2DE (pI 4-7) and silver stained (A) or autoradiographed (B). The
spots circled with a solid line were identified by MS analysis as rpP2. Note the lack of labeled phosphate in the most basic spot (right subpanel). (C) HL60 cells were
pulse-labeled with [35S]methionine for 5, 10, or 60 minutes and extracts subjected to 2DE (pI 4-5) and autoradiography. The circled spots show rpP2. Note the progressive but
slow shift toward the more acidic form as a function of time. (D) HL60 cells were prelabeled with [35S]methionine and chased for 6.5 hours with unlabeled methionine with
vehicle (left subpanel) or 8 ␮mol/L DNR present during the last 6 hours of incubation. The circled spots represent rpP2 from an autoradiogram of 2DE (pI 4-5) gel.
(E) Experiment as for panel D, except that gels were silver-stained and incubation time with DNR was 4 hours. (F) As for panel E, except that the phosphatase inhibitor calyculin
A (CCA; 0.1 ␮mol/L) was present during the last 3 hours of incubation. (G) Blasts from patients with AML (M5, patient 11) were treated for 6 hours in vitro with vehicle (left
subpanel) or 8 ␮mol/L DNR (right subpanel) and analyzed by 2DE (pI 4-5). The circled spots represent rpP2. (H) Blasts isolated from 11 patients with various AML classification
(M1, M2, M4, M5, M6), one with ALL, and one with LBL were treated in vitro with DNR (8 ␮mol/L, 6 hours). The percentage increase of nonphosphorylated rpP2 was determined
from the relative intensity of PP-rpP2, P-rpP2, and nonphospho-rpP2 in silver-stained gels like the one shown in panel G. (I) AML blasts were isolated from a patient (M4, patient
8) before (left subpanel) and 4 hours after (right subpanel) the onset of induction treatment with IDA and cytarabine and analyzed by 2DE (pI 4-5). Note the significant formation
of the monophosphorylated form of rpP2 after induction treatment.
similar for cells prelabeled with [35S]methionine and chased in the
absence or presence of DNR for 6 hours (Figure 4E).
In passing, it may be noted that cleavage of lamin B1, B2, the
lamin B receptor, and histone 2A (Table S1) could be related to the
early loosening of the nuclear lamina and chromatin hypercondensation seen in DNR-treated AML cells (Figure 2D,E).
The COFRADIC-based analysis was complemented by a 2DEbased approach, focusing on protein spots altered early in the
apoptotic process of DNR-treated HL60 cells. The earliest and
most consistent findings with both 1.6 and 8 ␮mol/L DNR were:
changed position of the methylosome subunit pICln as a result of
truncation (Figure 6A-D), appearance of 2 rows of spots (47 kDa,
pI 6.0/6.3/6.8; 33 kDa, pI 4.8/4.9/5.0/5.1; data not shown),
identified as hnRNP K and hnRNP C1/2, respectively, a basic shift
of rpP2 (Figure 6E,F), and de novo appearance of a spot (17 kDa, pI
4.53) resulting from cleavage of elongation factor 1Ba (⌬eEF1Ba)
at the caspase consensus sequence DETD154 (Figure 6E-H). The
DNR-induced appearance of ⌬eEF1Ba was noted also in blasts
from patients with AML (Figure 6I,J), indicating that it was not
restricted to HL60 cells.
The DNR-modified proteins detected by 2DE interact with
RNA (hnRNP K,48 C1/C244), modify RNA-binding proteins
(pICln49), or are components of the protein synthesis apparatus
(rpP2,50 eEF1Ba45), reinforcing the conclusion that protein synthesis is a major target in anthracycline-induced death.
The basic shift of the eEF2 binding rpP2 could be detected
already after 2 to 3 hours’ incubation with DNR (ie, before the
onset of morphologic indices of apoptosis), and dephosphorylation
of rpP2 was the most conspicuous effect of DNR on the 2DE
phosphoprotein pattern in DNR-treated cells metabolically labeled
with [32Pi] (Figure 7A,B; data not shown). Only the 2 acidic rpP2
spots were radioactive, indicating the basic spot as dephospho-rpP2
(Figure 7A,B). Although the phosphorylation rate of newly synthesized rpP2 was slow (Figure 7C) and unaffected by phosphatase
inhibitor (data not shown), calyculin A did inhibit the dephosphorylation of already phosphorylated rpP2 (Figure 7D-F). This suggested that phosphatase activation rather than kinase inhibition was
responsible for the net dephosphorylation of rpP2, but the enhanced
synthesis of rpP2 (Figure 4, Table 1) also contributed to the
accumulation of nonphospho-rpP2, because newly synthesized
rpP2 stayed unphosphorylated for a considerable period of time
(Figure 7C).
The dephosphorylation of rpP2 in response to anthracyclines was
found also in NB4 cells and mammary carcinoma MCF-7 cells (data not
shown). More importantly from the clinical point of view, when blasts
from 11 patients with AML, one patient with acute lymphoblastic
leukemia (ALL), and one with lymphoblastic leukemia/lymphoma
(LBL) were exposed to DNR in vitro, significant DNR-induced
dephosphorylation of rpP2 was observed in 10 of the 13 samples (Figure
7G,H). Because dephosphorylation of rpP2 was an early occurrence in
DNR-treated AML cells (Figure 7) it might be detected in AML blasts
from patients under chemotherapy, even if dying AML cells are
expected to be removed from the circulation early in the apoptotic
process.51 A basic shift of rpP2 was in fact detected in blasts recovered
from a patient with AML 4 hours after commencement of induction
chemotherapy (Figure 7I), lending further credibility to the notion that
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
modulation of the translational apparatus could occur early and be of
clinical relevance in human AML.
Discussion
We report enhanced synthesis of a subset of proteins, including the
ribosomal protein rpP2, cell survival proteins, such as PCNA, and
the ER chaperones PDI and calreticulin in AML cells treated with
anthracyclines. To our knowledge, specifically altered translational
control has not been described in anthracycline-treated cells before.
The effect of anthracyclines on protein synthesis was accompanied by altered phosphorylation, degradation, or limited proteolysis
(truncation) of proteins controlling specific mRNA translation,
cap-dependent initiation, or polypeptide elongation. We noted
massive perturbation of proteins required for cap-dependent translation, which is known to alter the mRNA preference for translation.37 Thus, the down-regulation of eIF1A will relieve its tonic
inhibition of IRES-directed initiation52 and disturb the assembly of
cap-dependent initiation factors.42 The degradation of the RNA
helicase eIF4H will prevent unwinding of secondary structure in
the 5⬘-UTR of mRNA required for cap-dependent initiation.53 The
dephosphorylation of 4E-BP1 will inhibit eIF4E and thereby capdependent initiation.39 Lack of p70S6K, as observed in the DNR-treated
cells, has traditionally been thought to inhibit cap-dependent translation
of proteins with polypyrimidine tracts in their 5⬘-UTR, like the
ribosomal proteins.54 A striking effect of DNR was enhanced synthesis
of rpP2, whose 5⬘-UTR has a 17 nucleotide long oligopyrimidine tract55
(REFSEQ: 00100456). This supports the notion that phosphorylation of
S6 by p70S6K is not required for efficient translation of mRNAs with a
oligopyrimidine tract.54
The high rate of protein synthesis in DNR-treated cells was
puzzling in view of the many proteomically detected hits on tRNA
synthetases and proteins involved in the elongation phase of
translation. It should be noted that the tRNA synthetases downregulated in DNR-treated cells are present in excess relative to the
need of the protein translational machinery,57 which may explain
why protein translation could persist despite tRNA synthetase
down-regulation. The control of elongation (for review, see Browne
and Proud45) has not been intensively studied in mammalian cells
and much must be inferred from yeast studies. The elongation
factor eEF1Ba, which became cleaved in DNR-treated AML cell
lines and patient blasts, was deleted in yeast without affecting the
total protein synthesis.58 This suggests that the cleavage of eEF1Ba
can occur and potentially fine-tune elongation without compromising overall elongation efficiency. The second step in elongation
depends on eEF2, which is complexed with the ribosomal stalk
proteins rpP1 and rpP259 and inhibited by phosphorylation catalyzed by eEF2K.45 The degradation of eEF2K in DNR-treated
AML cells will enhance eEF2 activity and thereby elongation.
Because dephosphorylation of rpP2 alters translational specificity
in yeast,60 it may do so also in AML cells. Dephosphorylation of
rpP2 occurred early after onset of DNR treatment in vitro and in
blasts from a patient with AML under induction therapy. This
supports that the dephosphorylation was preapoptotic because cells
are known to be cleared efficiently in vivo before onset of overt
apoptosis,61 even sometimes before the commitment to death.62
In addition, several proteins with roles in the control of translation of
specific mRNAs were modified in DNR-treated cells. One example is
the GAIT complex, which, via the constituent ribosomal protein L13a, is
down-regulated in the DNR-treated AML cells, can silence the translation of specific mRNAs by binding to their 3⬘-UTR.57 Another example
TRANSLATIONAL CONTROL BY ANTHRACYCLINES
2875
is the perturbation of primarily intranuclear proteins, including members
of the hnRNP family, which have roles in mRNA transport and control
of specific mRNA translation (Figure 5, Table S1). The high synthesis
rate of PDI in DNR-stressed cells may be related to modification of such
proteins, because its translational induction in hypoxic cells occurs
through the action of multiple originally nuclear RNA-binding proteins,
including members of the hnRNP family.63
The present study was initiated based on the assumption that the
proteins synthesized in anthracycline-treated cells were proapoptotic.6-9 The presumed prosurvival actions of the translationally
up-regulated PCNA and the ER-associated chaperone PDI64 call
this into question. That CHX enhanced rather than blocked
DNR-induced AML cell death further challenges this notion. That
anthracyclines when combined with CHX could lead to a nonconspicuous form of death in which the cell appeared “frozen,” can
explain some of the discrepancy with previous studies.65 Another
factor is the timing between the addition of anthracycline and
CHX. We found CHX to promote AML cell death most efficiently
when given after DNR, whereas most previous studies have
administered protein synthesis inhibitors before the anthracyclines.
Calreticulin is a marker for phagocytosis66 with selectively
increased surface presentation in anthracycline-treated tumor cells.51
The enhanced relative synthesis of calreticulin in our DNR-treated
cells might make more calreticulin available for surface presentation and facilitate AML removal in immune-competent animals.51
Thus questioning the relevance of data obtained in vitro or in
immunodeficient animal models of cancer. We found, however, that
protein synthesis inhibitor enhanced the anthracycline-induced
debulking of AML cells and prolonged survival also in the
syngenic rat BNML model of AML.
Although introduced as drugs more than 40 years ago, the
mechanisms of action of anthracyclines are still incompletely
known and controversial.33,67 Analysis of the proteome changes in
DNR-treated cells revealed some actions similar to those of more
recently discovered death pathways or drugs. The protein cleavage
pattern resembled the one in Jurkat T-cells treated with Fasactivator.27,68-70 Because the present data were obtained with CHX
present, they cannot be explained by anthracycline-induced synthesis of Fas and Fas-ligand,5 suggesting that an effector system
similar to that used by Fas was activated without Fas occupancy or
that preformed Fas or Fas ligand are externalized.71,72 Another
observation was cleavage of fatty acid synthase, which recently has
become a promising target for chemotherapy.73 Finally, DNR
mimicked known actions of rapamycin, such as dephosphorylation
of the major mTOR targets 4E-BP1 and p70S6K, which leads to
inhibition of cap-dependent protein synthesis. Rapamycin is under
study as an adjunct to conventional chemotherapy in selected
cancers.74-78 The mimicry by DNR of important rapamycin actions
and the down-regulation of the rapamycin receptor FKBP12 can
explain why rapamycin was inefficient as modulator of DNRinduced AML cell death in the present study.
In conclusion, the present study has pointed to several novel
actions of anthracyclines in AML cells that help explain the
efficacy of this drug, which still is the first choice in several
malignancies, including AML. More importantly, we discovered
altered translational preference for mRNAs in DNR-treated cells.
This effect was found at submicromolar concentrations of DNR,
which compares favorably with the peak concentrations
(0.3-5 ␮mol/L; most often 1-2 ␮mol/L) observed in serum of
patients treated with DNR.33 Furthermore, the synergy between
CHX and DNR in animal models of AML was observed with
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2876
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
GAUSDAL et al
clinically relevant concentrations (1-3 mg/kg/day) of DNR. Preapoptotic protein synthesis could enhance AML cell survival, which
suggests that it is a partially successful cell reaction to counteract
DNR-induced toxicity (ie, an “Achilles heel”) in DNR action.
Obviously, general protein synthesis inhibitors such as CHX cannot
be used for long-term treatment, although protracted administration
to humans has been reported in the past.79 But, by providing an
experimental system to test novel drug candidates for the ability to
inhibit the non–cap-dependent protein synthesis prevailing in
preapoptotic anthracycline-exposed cells, the present study should
stimulate the development of agents able to block protein synthesis
in the time interval after anthracycline administration when the
synthesis of cancer cell survival proteins is critical.
Acknowledgments
Expert technical assistance was provided by Erna Finsås, Nina
Lied-Larsen, Line Wergeland, Odd Harald Oddland, Monica Hals,
Kjetil Jacobsen, Kari Espolin Fladmark, Anne Nyhaug, Hans
Demol, and Magda Puype.
This work was supported by The Norwegian Cancer Society,
The Odd Fellow Medical Research Fund of Norway, the Fund for
Scientific Research–Flanders (Belgium), the Concerted Research
Actions of the Flemish Community (Belgium), Helse-Vest, and
the Norwegian Research Council (The Norwegian Centre for
Microarray Technology/The Norwegian Proteomics Center
[FUGE/PROBE]).
Authorship
Contribution: All authors contributed intellectually to the work.
G.G., B.T.G., and S.D. were involved in most aspects, although
E.M. was involved chiefly in animal experiments, P.V.D., K.G., J.V.,
and R.H. were involved mainly in proteomics and proteomicsrelated aspects, C.K. in ultrastructural assessment of apoptosis, and Ø.B. in selecting and characterizing patient AML blasts.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: S.O. Døskeland, Department of Biomedicine,
University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway;
e-mail: [email protected].
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2008 111: 2866-2877
doi:10.1182/blood-2007-07-103242 originally published
online January 8, 2008
Abolition of stress-induced protein synthesis sensitizes leukemia cells
to anthracycline-induced death
Gro Gausdal, Bjørn Tore Gjertsen, Emmet McCormack, Petra Van Damme, Randi Hovland, Camilla
Krakstad, Øystein Bruserud, Kris Gevaert, Joël Vandekerckhove and Stein Ove Døskeland
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