Download Role of Double-Stranded RNA-activated Protein

[CANCER
RESEARCH
57, 943-947,
March
I, 19971
Role of Double-Stranded RNA-activated Protein Kinase in Human
Hematological
Malignancies'
S. Basu, P. Panayiotidis,
S. M. Hart, L-Z. He, A. Man, A. V. HotThrand, and K. Ganeshaguru2
Department of Haematology. Royal Free Hospital School of Medicine. Rowland Hill Street. London NW3 2PF, United Kingdom (S. B., P. P.. S. M. H., L-Z H.. A. V. H.. K. G.J:
and Department of International Clinical Research, Ciba, Basel, Switzerland (A. M.J
ABSTRACT
The double-stranded RNA (dsRNA)-activated protein kinase (PKR) is
one of many genes induced by IFN. The PKR sequentially undergoes
autophosphorylation and activation on binding to dSRNA.Previous stud
ies have shown that PKR may be an important factor in the regulation of
viral and cellular protein synthesis. Recent studies suggest that PKR may
function as a tumor suppressor gene. The role of PKR in various human
leukemic cells was therefore investigated. PKR mRNA levels by reverse
transcription-PCR, protein expression by Western blot and FACSCaII
analysis, and activity by phosphorylation status were studied. The expres
sion ofa known inhibitor ofPKR, p58, was also investigated at mRNA and
protein levels. A total of 24 samples from normal mononuclear cells
(MNCs), 26 samples of acute lymphoblastic leukemia, 26 samples of acute
myelogenous leukemia, 32 samples of chronic lymphocytic leukemia, and
5 samples
of hairy
cell leukemia
was investigated.
Mean
mRNA
levels
were increased in acute lymphoblastic leukemia and acute myelogenous
leukemia and decreased in chronic lymphocytic leukemia compared to
normal MNCs. The mRNA levels in hairy cell leukemia were similar to
those of normal MNCs. PKR protein was detectable in normal MNCs and
leukemic cell extracts, and on FACSCananalysis, more than 70% of cells
stained positive for PKR. PKR activity was detectable in all samples
investigated and was enhanced 4—23-foldin the presence of the synthetic
dSRNA, poly(I)poly(C).
Protein
expression
of a known PKR inhibitor,
PS8, was barely detectable in normal MNCs and leukemic cells, with high
expression in the HeLa cell line. These findings provide no evidence to
support the hypothesis that PKR acts as a tumor suppressor in human
leukemic cells.
INTRODUCTION
The dsRNA3-activated protein kinase, PKR, also known as p68
kinase, DAI, ds-RNA-PK, is an IFN-activated serine/threonine kinase
and is 1 of more than 30 genes induced by IFN (1, 2). IFN in the
presence of dsRNA can inhibit protein synthesis in a cell-free system
made from rabbit reticulocytes (3, 4). An analogous enzyme has been
found in rabbit reticulocytes, various mouse tissues (p65 kinase), and
human peripheral blood mononuclear cells (5—8).In general, PKR is
activated by binding to dsRNA (9), although some single-stranded
RNA species due to their stem-loop structure can also bind and
activate PKR (10). Once activated PKR exhibits two distinct protein
kinase activities in the presence of ATP autophosphorylation and
phosphorylation of exogenous substrate of which the well-known
physiological substrate is the a subunit of the eIF-2 (11, 12). Other
substrates include the NF-scB inhibitor 1KB (13).
Evidence that PKR is implicated in the antiviral and antiprolifera
tive effects of IFN has been provided by expressing recombinant PKR
in murine cells and in Saccharomyces cerevisiae. Expression of hu
man PKR in murine cells mediated a partial resistance to encephalo
myocarditis virus growth (14), and in yeast it caused inhibition of cell
growth (15).
Two groups have reported the tumorigenic potential of murine
NIH3T3 clones expressing mutant, inactive forms of human PKR.
When injected into nude mice, both a point-mutated form of inactive
PKR
(catalytic
MATERIALS
study was supported by Hoffmann-La
whom
requests
for
reprints
should
Fax:
0171-794-0645;
E-mail:
abbreviations
used
are:
ds,
double
stranded;
PKR,
dsRNA-activated
protein
Lys
—@Pro)
and
a
blood,
15 bone marrow,
2 tonsils),
26 patients
with ALL (9 T-ALL, 11 B-lineage ALL), 26 patients with AML [FAB
patients
with
(27), 1 patient
M3, 8 patients
with M@, 1 patient
with
with M1, 3 patients
M4, 2 patients
with
M5,
with M2, 2
1 patient
with
secondary, 1 patient with biphenotypic, and 7 patients with AML and dyspla
sia), 32 patients with CLL (stage A, 17 patients; stage B, 6 patients; and stage
C, 9 patients),and 5 patientswith HCL. Diagnosis was based on cell mor
phology according to the FAB classification for acute leukemia and by im
munophenotyping and bone marrow histology for CLL and HCL. Disease
[email protected].
3 The
or
Patients. Peripheralblood or bone marrowwas collected from 24 patients
Roche, Basel, Switzerland.
be addressed.
—@Arg
AND METHODS
with normal MNCs (7 peripheral
18 U.S.C. Section 1734 solely to indicate this fact
I
Lys
The possibility that suppression of PKR through its underexpres
sion, dysfunction, or inhibition could contribute to the development of
human hematological malignancies was investigated by studying the
expression and activity of PKR in a wide range of leukemic cells.
classification
2 To
II,
(26).
Received 8/12/96; accepted 1/2/97.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
@
subdomain
six-amino acid deletion mutant form of PKR (catalytic subdomains V
and VI) produced rapidly growing tumors (16, 17). Although the exact
mechanism by which PKR exerts its tumor-suppressive action is not
known, it is believed that the inactive mutant PKR behaves as a
dominant negative inhibitor Of endogenous mouse p65 kinase and to
function as a tumor suppressor it needs functional PKR activity (17).
Conceivably under physiological conditions, mutations causing mac
tivation of PKR activity or factors inhibiting its expression may allow
normal cells to transform into tumors. PKR has also been associated
with tumor growth and antitumoral action (18, 19). It is known, for
example, that malignant embryonal carcinoma stem cells do not
express this kinase even after treatment with IFN (20).
The gene for PKR has recently been mapped to chromosome
2p2l—22.Chromosomal breakpoints involving 2p2l have been asso
ciated with lipoma and large cell lymphoma, while translocation and
altered 2p2l has also been reported in T-cell leukemia, myelodysplas
tic syndromes, and possibly acute myeloid leukemia (21, 22).
Certain viruses are known to down-regulate PKR to enable their
continued replication in a host (23). Purification and cloning of this
dsRNA-activated PKR inhibitor protein, p58. resulted in a cellular and
not a viral protein (24, 25). They also show that p58 is readily
expressed in some tumor cell lines, including HeLa. The mechanism
of PKR inhibition by p58 is unknown, but it is suggested that it may
block the autophosphorylation through direct interaction with PKR
stage in acute leukemia was further classified as presentation
kinase; MNC, mononuclear cell; ALL, acute lymphoblastic leukemia; AML, acute my
(not previously
treated with cytotoxic drugs) and relapse (or resistant) disease. In CLL the
stage of the disease was classified as described by Binet et a!. (28).
Samples were collected in preservative-free heparin and leukemic cells were
elogenous leukemia; CLL, chronic lymphocytic leukemia; HCL, hairy cell leukemia;
PHA, phytohemagglutinin; PFGE, pulse-field gel electrophoresis; eIF-2, eukaryotic mi
tiation factor 2; FAB, French-American-British;
f32M, f32-microglobulin; moab, mono
cionalantibody.
separated
by gradient
centrifugation
on Lymphoprep
(Nyegaard).
The MNCs
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PKR IN LEUKEMICCELLS
were washed twice with HBSS, and further purification with monocyte deple
over 48 h. The fractionated gels were transferred and probed in a manner
tion by plastic adherence and T-cell depletion by sheep RBC rosetting were
similar
carried out when required. In all samples the final leukemic/blast cell count
varied from 71 to 98% in acute leukemia, >70% in HCL, and >96% in CLL
described
using
May-Grunwald-Geimsa-stained
cytospin
Semiquantitative
Reverse
as described
was used for cDNA
Transcnption-PCR.
previously
synthesis
using a commercial
cellular
RNA was
ng of total cellular
kit (Boehringer
RNA
homologous
products,
to other
(3,M or (3-actin,
kinases.
which
For the semiquantitation
are constitutively
1.25 mM deoxyribonucleotides
expressed,
were also am
(Pharmacia),
100
bromide,
Dynamics
photographed,
Personal
and the negatives
Densitometer.
The results
scanned
were
@M
each of the
Protein
content
as a ratio
of
of the supematant
was esti
mated using a modified Lowry method (33). Total cell lysate or immunopre
cipitated
protein
from total lysate,
followed by 2.5
polyacrylamide
with and without
I p@g/ml poly(I)'poly(C),
@Ciof l'y-32PIATP incubation was subjected to a 10%
gel and electrotransferred
onto
nylon
membranes.
For total
protein studies, the blots after initial blocking were incubated with moab to
PKR (17) or p58 (24) at 4°Covernight followed by incubation with a
secondary antibody and detection using chemiluminescence (enhanced chemi
luminescence; Amersham). Blots from immunoprecipitated samples were au
toradiographed (Kodak S-OMAT), signal intensity was analyzed in a laser
densitometer
as
on each blot.
(Molecular Dynamics), and phosphorylation
purification
kit (Qiagen,
Inc.) and ligated
to SmaI-cut
mpl 8/19 vectors. Single-strand DNA from insert-containing plaques were
prepared and sequenced using the dideoxy termination method (Sequenase 2.0;
United States Biochemical Corp.).
Heparin Study. Since heparmnhas been shown to induce PKR in cell
heparmn-induced
PKR mRNA
levels were excluded.
PHAStudy. To studytheeffectof cell proliferation
on PKRexpression,
normal MNCs from two volunteers were cultured with PHA (I @zg/ml)
for 24
to 72 h, and PKR mRNA levels measured showed a sharp rise in PKR mRNA
levels at 24 h and a return to basal levels by 72 h.
Statistical Analysis. The mean values of the PKR:32M ratio or PKR:actin
ratio in different leukemias and their SDs were calculated and Student's
RESULTS
tion, cells were lysed in high salt buffer containing protease and phosphatase
for total cell protein.
scanned
and normal
unpaired t test was applied in each instance to calculate the significance
PKR:f3,Mor p58:f3-actin.
Protein and Phosphorylation. For Western blots and immunoprecipita
inhibitors
were
between the means.
using a Molecular
expressed
using a QlAquick-spin
artifactual
primers, and 4 units of Taq polymerase (Amersham). PCR was carried out on
a Hybaid thermal reactor with a I-mm denaturation at 94°C,1-mm annealing,
and 2-mm DNA synthesis at 72°C.The primers used with the annealing
temperature (T°)and cycle numbers are listed in Table I.
The PCR products were separated on a 2% agarose gel, stained with
ethidium
DNA samples
Autoradiographs
the data were quantitated
anticoagulant. Normal MNCs derived from two volunteers showed no differ
ence in PKR mRNA levels either at 0 h or after 18—24h of culture, and
of the PCR
plified and the products run on the same gel. When studies commenced on
PKR,the laboratorywas set upto studysemiquantitationof PCRproductswith
f3,M. With the developmentof other studies, includingthose with IFN, the
laboratory switched to amplifying j3-actin for quantitation. The reaction mix
ture for PCR in 50 @.dcontained 25 ng of cDNA, 5 pA of lOX buffer
(Amersham),
above.
extracts, our preliminary
study included a comparison
of PKR mRNA levels in
normal MNCs from blood collected
using either EDTA or heparmn as the
Mannheim).
The primers were designed from the published cDNA sequence, avoiding
regions
described
and for gene dosage,
Sequencing of PCR Products. Amplified PCR products were purified
Total
(29). Five hundred
above,
ized to control
slides.
Cell Cultures. Cell culturereagentswereobtainedfromLife Technologies,
Inc. All cell cultures were carried out in RPMI 1640 with 10% FCS at 37°Cin
a 5% CO1 incubator. PHA (1 j.@g/ml;
Wellcome) was used for treatment of cells
where indicated.
extracted
to the method
evaluated.
Flow cytometry analysis was performed on fresh cells fixed overnight at
4°Cin 0.25% buffered paraformaldehyde, incubated with either PKR or p58
moab, followed by FITC-conjugated secondary antibody, and analyzed on a
Becton Dickinson FACScan.
Gene Dosage, Rearrangement, and Translocation. High molecular
Specificity of the PCR Product. The specificity of the PKR
product was confirmed by (a) the predicted product size of 249 bp
being obtained on agarose gel; (b) Dde! restriction digest of the PCR
product yielded the expected fragment sizes of I55 bp and 94 bp as
predicted from the restriction map of the cDNA sequence; and (c)
amplification of genomic DNA yielded a product of400 bp which was
distinct from the 249-bp product of the cDNA, indicating the presence
of an intron between the selected primer sequence. The different
product size also helped to detect contaminating genomic DNA in the
RNA samples. The PCR products were also verified by blotting and
probing with labeled K- I PKR cDNA. Finally, PCR products from
one patient with normal MNCs, one patient with CLL and a low
mRNA level, and one patient with AML and a high mRNA level were
cloned and sequenced. No sequence abnormality in the region ampli
fled was detectable in any of these samples.
PKR mRNA Levels in Normal MNCs and Leukemic Cells. The
mean PKR:@7M mRNA ratio in seven normal MNCs was 0.42 ±0.23
[mean ± 2 (SD); range, 0.22—0.8]. The mean PKR value in 31
patients with CLL was 0.26 ±0.21 (range, 0.005—1.0).This mean
weight DNA from normal MNC and leukemic samples was subjected to two
level was lower than that of normal cells but this was not statistically
restriction
endonuclease
digests (Hindlll
and EcoRI), fractionated
on 1%
significant (P = 0.07). There was no apparent correlation among
agarose gels, transferred onto nylon membranes, and probed with [32PJdCTP
WBC count, previous treatment status, or stage of the disease in these
labeled K1 eDNA for PKR (30). To assess gene dosage, blots were simulta
patients with PKR mRNA levels. The mean PKR mRNA levels in
neously probed with a [32PJdCTP-labeledeDNA for the APC gene (34).
blast cells from 26 patients with AML was 0.77 ±0.39 (range,
For PFGE, whole cells were embedded in low melting agarose and sub
0.14—I.6). This mean level was significantly higher than that of
jected to two rare cutting restriction endonuclease digests (BssHII and BbrPI),
normal MNCs (P = 0.03). There was no correlation among FAB type,
fractionated
on a cooled PFGE system, with pulse time ranging from 25 to 60 s
WBC count, and the PKR mRNA levels. PKR mRNA levels were
significantly higher in relapsed/resistant disease (0.88 ±0.4) com
[email protected]'-gAACAgTgTgCATCgggggT-3'58'C3030AS5'-ATFCAgAAgCgAgTgTgCTg-3'p58S5'-TgCAgTACgAAggTgCTgAA-3'59'C2824AS5'-gATgAgCTCTfCAgC
Table I Primers and cycle
pared with presentation samples (0.52 ± 0.34, P = 0.05). PKR
mRNA levels in blast cells of 20 patients with ALL (0.87 ±0.61 with
a range of 0.03—1.8) were higher than those in normal MNCs
(P = 0.07). There was no correlation among PKR mRNA levels and
immunophenotype, disease status, or WBC count. The mean PKR
mRNA levels in the hairy cells of five patients with HCL was
0.46 ±0.26 (range, 0.02—0.7)and was not significantly different from
those in normal MNCs (P = 0.76; Fig. I).
PKR Protein Levels. PKRproteinlevels were studiedusing West
ern blot in two samples of normal MNCs, seven samples of CLL, and
two samples of AML cell extracts. The cell extracts were from
944
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PKR IN LEUKEMICCELLS
a
0. 11—0.38),respectively. There was a statistically significant eleva
tion in the p58 mRNA levels in CLL (P = 0.03) and none between the
AML (P = 0.4) or ALL (P = 0.7 1) groups and normal MNCs (Fig.
4). There was no difference in the p58 mRNA levels between disease
stages in CLL. In the cell line HeLa, p58 mRNA levels were consis
tently higher than those detected from most of the normal MNCs and
clinical samples (data not shown).
1.8
1.6
1.4
1.2
0
p58 Protein. In the seven CLL andtwo AML total proteinextracts
C,'
@
a
0.8
a.
(50;.Lg)studiedwith Westernblot,p58proteinwasbarelydetectable
and in most not detected. It was readily detected in the HeLa cell
extract (Fig. 2a). When studied with flow cytometry, p58-positive
cells ranged from 8 to 10% in two normal MNCs, 10—15%in four
0.6
8
0.4
Q
CLL,
and
0
HeLa
analyzed
10—20%
in
three
AML
samples.
Cells
from
the
cell
line
0.2
on
six
separate
occasions
showed
41—76%
positivity
for p58 (Fig. 2b).
0
CLI
AMI
ALL
HCL
Normal
DISCUSSION
b
1.6
1.4
1.2
This study shows that PKR mRNA levels are higher in cells
patients with acute leukemia (AML or ALL) and lower in
compared to normal MNCs. This seems paradoxical if PKR
tumor suppressor function, implying that rapidly proliferating
(i.e.,
ALL)
should
have
a lower
PKR
mRNA
level
which
0
@
@
from
CLL
has a
cells
would
allow more rapid protein synthesis. It was important therefore to
1
establish whether the cells in patients with acute leukemia ex
C,'
0
pressed abnormal, mutated, or dysfunctional PKR protein. The
0.8
representative PCR products that were sequenced failed to show
a.
0.6
any abnormality. The amplified fragment in this study was selected
from a region not homologous to other kinases but did not include
.80
0.4
the sequences from the kinase conserved regions used by Koromi
las et a!. (16) and Meurs et a!. (17) for their substitution and
0.2
deletion studies in nude mice. The existence of mutations outside
the amplified region of this study has therefore not been excluded.
0
Autophosphorylation
studies of PKR in representative leukemic
Presentation
Relapse
cells, however, failed to detect any functional abnormality. Since
Fig. I. PKR mRNA levelsas a ratioof PKR:f32Min CLL, AML ALL HCL, and
eIF-2 phosphorylation studies were not done, the possibility of a
normal MNC samples (a) and in presentation and relapsed AML cells (b). @,
AML with
dysplasia; bar, mean.
dysfunctional PKR cannot be totally ruled out.
Southern blot and PFGE analysis failed to show any gene ampli
fication or chromosomal abnormality in the samples studied. The
relapsed/resistant samples from AML showed higher PKR mRNA
representative samples showing normal, high, and low PKR mRNA
levels than those at presentation. This could reflect a selection phe
levels. PKR protein was detectable in all normal MNCs and leukemic
cell extracts studied (Fig. 2a). PKR protein was also studied with flow
nomenon as noted by Meurs et a!. (17). Two patients with AML had
a clone with 2p2 translocation associated with other abnormalities.
cytometry in 3 normal MNCs, 16 CLL, and 5 AML samples. Most
cells stained positive for PKR, ranging from 86 to 97% in normal
PKR is mapped to chromosome 2p2l—22.Their PKR values however
were 0.76 and 1.6 (i.e., high compared to normal MNC).
MNCs, 73—100%in CLL, and 75—95%in AML samples (Fig. 2b).
The possibility of inhibition of the expressed PKR protein and its
PKR Activity (Autophosphorylation).
PKR kinase baseline ac
activity were investigated by studying the PKR inhibitor p58. An
tivity was observed from 100 @g
of total protein immunoprecipitated
with PKR moab in two of two normal MNCs, six of seven CLL, and elevation in the mRNA levels of p58 in CLL cells with no statistically
two of two AML samples. In all samples, the baseline PKR activity
significant difference in the other leukemias from normal MNCs was
could be enhanced 4—23-fold after activation with 1 @ig/ml found. The elevated mRNA levels in CLL did not translate into
poly(I)-poly(C) in the different groups of samples. High baseline
increased p58 protein expression. Although high expression of p58
activity of PKR was observed in the control cell line Daudi, and this
protein can be detected in HeLa cells, its expression in leukemic cells
activity was further enhanced with poly(I)poly(C) (Fig. 3).
was either undetectable (using Western blots) or very low (using
PKR Gene Dosage. Gene dosage studies in a representativenum fluorescence-activated cell-sorting analysis). Furthermore, despite re
ber of samples from CLL (six samples), AML (three samples), and
ports that p58 inhibits autophosphorylation of PKR, this was not
ALL (three samples) showed no deletions of the PKR gene. Similarly,
observed in leukemic cells when treated with poly(I)poly(C). It could
no translocations were detectable in these samples by either Southern be concluded that PKR autophosphorylation activity is not inhibited
blotting or PFGE (data not shown).
by p58 in leukemic cells.
p58 mRNA Levels in Normal MNCs and Leukemia. The mean
The purity of cells in the CLL group was >96% and contaminating
p58:actin ratio in 21 normal MNC samples was 0.16 ± 0.30
cells were not a problem. In the case of the acute leukemias, the purity
(mean ±2 SD), with a range of 0.03—1.03.The p58:actin ratio in 32
varied from 71 to 98% leukemic cells. It is possible that contaminating
CLL, 9 AML, and 12 ALL samples was 0.44 ±0.24 (range, 0.13- residual nonleukemic cells may affect the final result. However, the
0.94), 0.47 ± 0.17 (range, 0.14—0.56), and 0.29 ±0.02 (range,
PKR mRNA
levels in the acute leukemias
were significantly
greater
945
0
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PKR IN LEUKEMIC CELLS
a
kDa
4@..
@
@-
—
PKR
@- .
66
@
.-.c,.
46
‘4
@m'
AML
CLL
Fig. 2. PKR and p58 protein expression by
Western blot (a). Lanes 1—4and 6 representative
of CLL, Lane 7 representative of AML, and Lane
8 representative of HeLa cell extracts. Left' size
markers. b, FACScan; MNC, normal MNCs.
‘p58
HeLa
ANt.
@@J@CL@L
.
@
b
¶PKR
CONT
P58
@
PKR
CONT
poly(I).poly(C)
@
+
P-PKR
-
+
..‘,
-
mRNA level at the time zone of peak DNA synthesis (36—60h after
PHA) and cell cycle. The initial increase in PKR mRNA level in
PHA-stimulated lymphocytes is not understood but can be attributed
+
I
to
multiple
gene
induction
by
PHA.
Additionally,
the
variable
PKR
mRNA levels in leukemic cells do not translate into significant
@
@
I -PKR
.
L. I
w@e@ic@@
Daudi
ow'-.
CLL
changesin PKRproteinexpression.
Thesefindingsdo notsuggesta
—
role for PKR in human leukemogenesis and do not provide evidence
for a tumor suppressor activity by PKR in these hematological ma
lignancies.
AML
Fig. 3. PKR activity (autophosphorylation) in imrnunoprecipitated Daudi. representa
tive CLL. and representative AML cell extracts with (+) and without (—) 10 @zWml
poly(I)poly(C). P-PKR, phosphorylation; l-PKR, immunoprecipitated PKR.
than those of the normal MNCs, and the lack of contaminating
nonleukemic cells would only have enhanced the statistical signifi
cance. Furthermore, there was no relationship between increased
purity of leukemic cells (71—98%)and increased mRNA levels for
PKR and p58. It should be noted that each group of leukemias was
compared to MNCs from normal samples and not to their equiv
alent normal cells.
Recent studies on PKR knockout mice show that they are phe
notypically normal and the induction of type I !FN genes by
poly(I)poly(C) was unimpaired. Additionally, injection of PKR°'°
mouse embryo fibroblasts into nude mice did not give rise to
tumors, and the study concluded that PKR does not play an
essential
role in growth
control
(35).
More
recently,
Haines
1.2
0.8
0
B
0
C
8
@0.6
8
aIt)
0.4
0I
et a!.
(36) report the inability of PKR to inhibit cellular proliferation in
human breast cancer. These observations raise doubts about the
role of PKR in growth control and tumor suppression.
The present findings show no abnormal structural or functional
changes in PKR in different types of human leukemia. The high and
low levels of PKR mRNA in acute and chronic leukemias, respec
lively, probably reflect their different proliferative rates, although in
the studies with PHA, there was no significant change in the PKR
8
0.2
AML
ALL
CLI
Normal
Fig. 4. p58 mRNA levels as a ratio of p58:actin in AML, ALL, CLL, and normal
MNCs. 0. bone marrow; •,peripheral blood; A, tonsils. Bar, mean.
946
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PKR IN LEUKEMIC CELLS
ACKNOWLEDGMENTS
Tumor-suppressor
We are grateful to Dr. A. G. Hovanessian (Pasteur Institute, Paris, France)
for advice and a generous supply of moab and cDNA for PKR, Dr. M. G. Katie
(University
of Washington,
Seattle,
function of the interferon induced double stranded RNA activated
protein kinase. Proc. NatI. Acad. Sci. USA, 90: 232—236,1993.
WA) for moab to p58, and Professor
M. J.
Clemens (St. George's Hospital Medical School, London, United Kingdom)
for helpful discussions.
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Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research.
Role of Double-Stranded RNA-activated Protein Kinase in
Human Hematological Malignancies
S. Basu, P. Panayiotidis, S. M. Hart, et al.
Cancer Res 1997;57:943-947.
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