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Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2450-2460
Regulation of the c-jun Gene in p210 BCR-ABL Transformed
Cells Corresponds With Activity of JNK, the c-jun N-Terminal
Kinase
By
Gem S. Burgess,
Elizabeth A. Williamson,
Larry D. Cripe,
Sara Litz-Jackson,
Jay A. Bhatt,
Kurt Stanley,
Mark J. Stewart,
Andrew
S. Kraft,
Harikrishna Nakshatri, and
H. Scott Boswell
From the Walther Cancer Institute, and the Hematology/Oncology
Division, the Departments of Medicine, and Surgery, Indiana University
School of Medicine, Indianapolis, IN; and the Division of Medical
Oncology, University of Colorado Health Sciences Center, Denver.
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ABSTRACT |
Activity of the c-jun N-terminal kinase (JNK)
has been shown in hematopoietic cells transformed by p210
BCR-ABL. However, analysis has not been reported for
hematopoietic cells on the consequences of this activity for
c-jun promoter regulation within its distinctive proximal
8-base consensus CRE-like element, an element linked to JNK-mediated
increase in c-jun transcription. In the present study,
regulation of the proximal c-jun promoter was studied in murine
myeloid cells transformed by p210 BCR-ABL. Promoter regulation in p210
BCR-ABL transformed cells was compared with regulation of the promoter
in nontransformed interleukin-3 (IL-3)-dependent parental cells. The
composition of nuclear AP-1 proteins contained within cells with p210
BCR-ABL, and their binding to the c-jun promoter proximal
CRE-like element, was compared with the composition and binding of AP-1
proteins in IL-3-treated parental cells without p210 BCR-ABL. The
present analysis found fivefold increased c-jun transcription
occurring in p210 BCR-ABL transformed murine myeloid cells possessing a
corresponding magnitude of increased kinase activity of JNK, compared
with IL-3-stimulated parental cells. Augmented JNK activity was
accompanied by increased nuclear abundance of c-jun and
c-fos proteins that bound specifically to the proximal
c-jun promoter CRE element. Also, representative human leukemic
cell lines expressing p210 BCR-ABL and possessing abundant kinase
activity of JNK, when compared with parental cells that were deficient
in JNK activity, had increased c-jun and c-fos proteins. Finally, to show the relevance of these observations in model
systems, we studied blast cells from patients with Philadelphia chromosome-positive acute leukemic transformation, and observed comparable activities of JNK catalysis and c-jun/AP-1 protein relative to the cell lines that possessed p210 BCR-ABL and JNK activity. These studies provide a basis for investigating the set of
downstream genes which augmented c-jun/AP-1 activity enlists in
the process of transformation by p210 BCR-ABL.
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INTRODUCTION |
EXPRESSION OF THE nuclear-localized
product of the c-jun early response gene is required for
cellular transformation by the p21ras oncogene and for the cellular
proliferation and migration occurring during early embryogenesis, as
shown by a model of c-jun knockout in mice.1,2 In
addition, disruption of the cytoplasmic-to-nuclear signal transduction
cascade targeted for c-jun transcriptional control is also
embryo-lethal.3 Such disruption can be accomplished by
genomic knockout of the gene encoding the dual specificity serine/tyrosine kinase, SEK1. SEK1 is a major immediate upstream regulator of the serine/theonine kinase called c-jun N-terminal kinase (JNK) or stress-activated protein kinase (SAPK).4
SEK1 activates JNK by phosphorylating threonine and tyrosine residues within an amino acid sequence motif containing threonine separated from
tyrosine by a proline residue, TPY.5
Active JNK exerts its serine kinase function on substrate with
specificity for the N-terminal regions of c-jun (serines 63 and
73) and for ATF.5,6 JNK also phosphorylates a domain within
the Elk-1 (ets-related) transcription factor.7
Phosphorylation of these transcription factors by JNK augments their
interaction with the basal transcriptional machinery, and, hence,
enhances their transactivating potential on a collection of genes
relevant for cellular transformation. In the case of the phosphorylated c-jun protein, the most important promoter target may be that of the c-jun gene itself.8 JNK-mediated
posttranslational modification on the c-jun protein leads to
augmented c-jun/AP-1-driven c-jun transcription in a
positive autoregulatory loop.5,8
It has been proposed that upstream signals emanating from either p21ras
or p21rac, in their guanosine triphosphate (GTP)-bound active
forms, can activate JNK and thus lead to augmented
c-jun transcription.9-12 This function of oncogenic
p21ras was the basis for initial purification and characterization of
JNK in the laboratory of Karin,9 whereas the
JNK/SAPK enzyme was characterized by others for its stress
functions.13
Our laboratory observed strong activity of p210 BCR-ABL (the
nonreceptor tyrosine kinase oncogene product of the Philadelphia chromosome that is an initiating cause of chronic granulocytic leukemia) for maintaining the activation state of p21ras in its GTP-bound form.14 In consideration of this, it may not be
surprising that another laboratory observed high activity of JNK
conferred by p210 BCR-ABL.15 Most importantly,
nonfunctional c-jun mutants, expressed in cells along with p210
BCR-ABL, exerted dominant-negative activity for cellular
transformation by p210 BCR-ABL.15 Therefore, full
characterization of the mode of c-jun transcriptional
regulation in cells with p210 BCR-ABL, and the role of JNK in
establishing the content of nuclear AP-1 proteins, is important
to further understanding of the pathophysiology of chronic granulocytic
leukemia, CGL.
As part of a previous characterization of the consequences of JNK
activity within p210 BCR-ABL transformed fibroblasts, strong transcriptional activity of a 7-base consensus AP-1 binding
enhancer/promoter element derived from the collagenase gene was
observed, and was dependent on the cellular activity of p21ras, on
MEKK, an upstream regulator of SEK1, and on JNK itself.15
However, JNK-mediated increase in c-jun transcription is a
function of regulatory events on the distinctive 8-base consensus
CRE-like element of the proximal c-jun promoter, which is
capable of binding other AP-1 factors, including ATF. In
this report we studied the activity and regulation of the proximal
c-jun promoter in model myeloid cell lines that differ only in
expression of p210 BCR-ABL. In addition, the occurrence of high
activity of JNK, associated with abundant c-jun/AP-1 protein, was shown in primary hematopoietic cells in blast transformation of
Philadelphia chromosome-positive leukemia.
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MATERIALS AND METHODS |
Cells and cell culture.
The growth factor-independent murine myeloid cell line transformed
with an expression vector for p210 BCR-ABL, H7 Bcr-Abl.A54, has been
described previously.14 These cells were maintained in
McCoy's medium supplemented with 10% fetal calf serum (FCS). The
parental interleukin-3 (IL-3)-dependent cell line NFS/N1.H7 (H7) was
passaged in McCoy's medium plus 10% FCS supplemented with conditioned
medium from the WEHI-3 cell line as a rich source of IL-3. The human
chronic granulocytic leukemia blast cell line, K562, was obtained from
the Cell Depository of the American Type Culture Collection (ATCC,
Rockville, MD), as was the myelocytic leukemia cell line,
HL-60. The human factor-dependent cell line M07e was passaged in
Iscove's modified Dulbecco's medium (IMDM) plus 20% FCS
with the addition of granulocyte-macrophage colony stimulating factor
(GM-CSF). A transformed factor-independent variant of M07, arising as a
result of transfection with p210BCR-ABL, M07p210 BCR-ABL, was
used.16 It was passaged in IMDM plus 20% FCS. Blast cells
from the bone marrow of patients with blast transformation of
Philadelphia chromosome-positive leukemia were obtained at the time of
diagnostic testing after informed consent. These cells were diluted 1:4
with Hanks' balanced salt solution and layered onto Ficoll-Hypaque
(Pharmacia, Uppsala, Sweden). After centrifugation for 30 minutes at 1,200 rpm, the buoyant fraction was isolated and then washed
with phosphate-buffered saline (PBS) before processing. Those cells to
be assayed directly in kinase assays (see below) were washed in
ice-cold PBS with 1 mmol/L sodium orthovanadate (Na3VO4). The cell pellet was lysed in sample
buffer (20 mmol/L TrisHCl, pH 8.0, 137 mmol/L NaCl, 10% glycerol,
1 mmol/L phenylmethyl sulfonyl fluoride [PMSF], 1 µg/mL
aprotonin, 2 mmol/L EDTA, 10 µg/mL leupeptin, 1 mmol/L
orthovanadate, 1% Triton-X100). Bone marrow samples used for
analysis were restricted to those with greater than 80% blast cells in
the diagnostic aspirate, or with greater than 60% in the diagnostic
aspirate with further enrichment following mononuclear cell isolation
on the gradient.
Nuclear protein extraction and gel mobility shift analysis.
For nuclear protein isolation, cells were incubated in medium with
reduced serum content (2% FCS) overnight, either in the presence of
IL-3 (for H7 parental cells) or in the absence of IL-3 (H7
Bcr-Abl.A54). The method used for isolation was a modification of that
reported by Dignam et al, and has been described.17 Using
this method, nuclear proteins were also isolated from M07e and M07p210
BCR-ABL taken from their respective culture medium. For isolation of
DNA binding proteins from primary leukemia samples (mononuclear cells
containing >80% blast cells), a high-salt buffer containing 1%
NP-40 was used according to the method described.18,19 The
gel mobility shift reaction mixture contained 5 to 15 µg of the
nuclear protein extract, 0.1 to 0.5 ng of the proximal c-jun promoter ( 110/CTF site to +40 BamHI fragment)
end-labeled with 32P-ATP in a binding buffer (4 mmol/L
HEPES pH 7.9, 40 mmol/L KCl, 10 mmol/L MgCl2, 4% glycerol,
1 mmol/L dithiothreitol [DTT]) along with nonspecific
competitor DNA, polydIdC. Specific competitor DNA was added in 100- to
200-fold excess as required. Antibodies, when used, were added at 2 µg per reaction. Reaction contents were electrophoresed on 5%
polyacrylamide gels in 0.5× Tris borate EDTA buffer
at room temperature. In other gel mobility shift reactions, a consensus
7-base collagenase AP-1 binding site contained within a 36-bp
oligonucleotide was used, and was purchased from Promega (Madison,
WI).
Antibodies.
The following antibodies were used for supershifting (gel mobility
assays) or immunoblotting of proteins Western transferred onto
nitrocellulose filters after sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE): anti-c-jun/AP-1 (N), anti-JNK (C-17), anti-junB (N-17), anti-ATF-1, ATF-2 (C-19),
anti-c-fos (4), and anti-junD (329). These antibodies
were obtained from Santa Cruz Biotechnology Santa Cruz, CA).
Immunoblotting.
Cytosolic or nuclear proteins were resolved on 12% SDS polyacrylamide
gels and transferred to a nitrocellulose filter by a semi-dry Western
blotting method (BioRad, Hercules, CA). The filter was
preblocked in 5% blocking agent (BA) (BioRad) in TBS-T (Tris-buffered saline, 0.5% Tween 20) at 4°C for 16 hours. The blots were
incubated with the primary antibody at 0.5 µg/mL in TBS-T/5% BA at
room temperature for 2 hours. After extensive washing, the blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody at a dilution of 1:2,000 in TBS-T/5% BA at room
temperature for 2 hours. After further extensive washing the results
were visualized by enhanced chemiluminescence (ECL). The
blots were stripped according to the manufacturer's protocol (Amersham, Arlington Heights, IL) and subsequently
reprobed with the remaining antibodies.
Kinase assay of JNK.
Proteins (25 to 200 µg) were incubated with 2 µg of a GST
fusion protein of the N-terminal region of c-jun (amino acids
5-89) coupled to agarose beads. These reactants were brought to a final volume of 50 µL in a phosphotyrosine lysis buffer and incubated with
gentle rocking at 4°C for 5 hours to allow binding of endogenous JNK to substrate protein as described (solid-phase
assay).20 This mixture was washed extensively by pelleting
and resuspension, and the beads were finally resuspended in 10 µL
distilled water. Next, 20 µL of 2× kinase buffer containing 50 µCi/mL 32P- -ATP (1 µCi per reaction) was added, and
the reaction was incubated at 30°C for 20 minutes. In some
experiments, cell lysates were first immunoprecipitated with anti-JNK
coupled to protein A, and then immune precipitates were washed and
immune complex kinase assays were performed with substrate. The
reactions were stopped by adding 2× volume of Laemmli sample
buffer (BioRad) and the reaction contents were boiled and
electrophoresed on 12% SDS polyacrylamide gels. The gel was then dried
and subjected to autoradiography. Control reactions were performed with
GST-coated beads or with a mutant GST-c-jun (5-89) with
serines 63,73 converted to leucine.20
Transient transfections and reporter activity assays.
The c-jun promoter fragment 132/+170 cloned in front of
a CAT reporter vector was a gift of Michael Karin, PhD (University of
California San Diego).8 From this vector, the proximal
promoter element containing sequences at 110 bp 5 of the
CTF site to +40 was obtained by polymerase chain reaction (PCR) using
the HindIII-Pst I fragment as template.8
The PCR product was cloned into a non-TA tailed cloning vector (pNOTA;
Invitrogen). Subsequently, a 150-bp HindIII-Kpn I
fragment was used for directional cloning into the promoterless pXP2
luciferase reporter vector, to yield 5 CTF-c-jun
luciferase. A derivative vector with specific mutation of the AP-1 site
within 5 CTF -c-jun, converting the AP-1 consensus sequence from wild-type of GTGACATCA to an mAP-1 sequence of GATGCACCA, was prepared by a combinatorial PCR technique described
previously.17 The incorporated change was verified by
sequencing. Both parental and the p210 BCR-ABL transformed cell line
were transfected by methodology described.17 In brief, the
cells were resuspended in Ca2+/Mg2+-free PBS
containing 20 mmol/L HEPES. Vector DNA was added at 0.5 µg/106 cells for the luciferase vector plus the
constitutively-active RSV  -galactosidase to serve as an internal
control for transfection efficiency at 1 µg/106 cells.
The cell/DNA mixture was incubated on ice for 10 minutes, then exposed
to a pulse of 350 V at 500 µF, delivered over 10 to 13 ms (BioRad
gene pulser with capacitance extender). After further incubation on ice
for 10 minutes, the cells were resuspended in McCoy's medium plus 10%
FCS, and in the case of the factor-dependent parental cell line
additional supplementation by 5% WEHI-3 conditioned medium. The cells
were placed in incubation for 18 hours, and then cytosolic extracts
were prepared for measurement of luciferase activity using a Bechthold
luminometer according to methods recommended by the assay kit
(Promega). Data from three independent experiments are reported as
relative mean luciferase activity/-galactosidase × 100, from
equal numbers of cells of H7 parental or p210 BCR-ABL transformed
cells. Wild-type 5 jun luciferase activity in p210 BCR-ABL transformed cells was on average 10,000 to 20,000 luminometer units.
| |
RESULTS |
Correspondence between augmented kinase activity of JNK and
c-jun transcriptional activity in p210 BCR-ABL transformed
cells.
We have previously reported that myeloid cells transformed by p210
BCR-ABL overexpress c-jun mRNA compared with their
IL-3-stimulated parental cells, assayed by Northern blot
analysis.14,21 This increase in steady-state c-jun
mRNA in the cell line Bcr-Abl.A54, compared with IL-3-treated H7
parental cells, was on the order of fourfold to
fivefold.14,21 Therefore, we wondered whether JNK activity
and c-jun transcription rates might be correspondingly increased in the transformed cells, supporting their involvement in a
linear pathway to the accumulation of c-jun mRNA.
Activity assays for JNK were performed from the extracts of
IL-3-treated H7 parental cells and of Bcr-Abl.A54 cells
(Fig 1). There was significantly greater
incorporation of radioactive phosphate into the GST-c-jun
(5-89) substrate in reactions of Bcr-Abl.A54 cell extract compared with
H7 cell extract by a factor of fourfold to fivefold (Fig 1). Activity
of the kinase was absent or markedly reduced on the control substrate
GST-c-jun (delta 63, 73) (data not shown). By contrast to this
difference, the cellular abundance of JNK enzyme in the two samples was
identical (Fig 1, bottom).
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| Fig 1.
Increased kinase activity of JNK within cells transformed
by p210 BCR-ABL (Bcr-Abl.A54) compared with IL-3-treated parental
cells. (Top) Equal quantities (50 µg) of nuclear proteins isolated
from the respective cells growing in medium (in the case of H7 parental
cells including 5% WEHI CM as a source of IL-3) were placed in
side-by-side solid-phase JNK activity assays. The reaction contents
were then boiled and subjected to SDS-PAGE followed by autoradiography.
JNK reactions were also performed at a range of cytosol protein
concentrations with the same result. (Bottom) One hundred micrograms of
each cell lysate was immunoprecipitated with anti-JNK antibody, then
immunoblotted for JNK. The same result was obtained by direct
immunoblotting.
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Next we studied the consequences of these differences in JNK activity
as they might dictate c-jun regulation on the 5
c-jun proximal promoter element. In preparation for functional
studies of the importance of the c-jun/AP-1 binding site in
transcriptional assays, it was necessary to establish c-jun
binding to the promoter fragment in gel mobility shift assay
(Fig 2). Extract from Bcr-Abl.A54 cells was
found to form two complexes of distinct mobility with the radiolabeled
5 c-jun fragment, and these complexes were specific because they were totally competed by the unlabeled self-probe in
100-fold excess (Fig 2). A similar molar excess of a commercial 7-base
collagenase AP-1/TRE site oligonucleotide caused marked reduction in
band-shifts (Fig 2).
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| Fig 2.
Gel mobility shift reaction with 5 c-jun
probe shows two distinct complexes containing c-jun protein. A
gel mobility shift assay was performed with nuclear extracts (10 µg)
from Bcr-Abl.A54 cells. Cold-competition with nonradioactive self probe
(100×) or with nonradioactive commercial oligonucleotide containing a
7-base TRE was accomplished. Specific supershifting of the band-shift
complexes (a, b) was shown with c-jun antibody, but not by Sp1
or jun B antibodies.
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To confirm the presence of c-jun binding protein within the two
distinct band-shift complexes, antibody specific for c-jun was
added to the binding reactions to look for supershift or neutralization (Fig 2). Reproducibly, the c-jun antibody caused supershifting of both complexes and, additionally, near-total neutralization of the
faster mobility complex (complex b) (Fig 2). As a control, antibodies
against neither Sp1 nor junB effected these results.
Site-directed mutation within the 5 c-jun proximal
promoter was undertaken by PCR technology, and the fragment containing the mutant core element (GATGCACCA) was tested in gel mobility shift
reactions alongside the wild-type 5 c-jun fragment.
There was binding activity for c-jun/AP-1 by extract from
Bcr-Abl.A54 cells. The mutant probe radiolabeled to a comparable level
as wild-type probe did not elicit the expected band-shift
(Fig 3).
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| Fig 3.
The 5 c-jun promoter fragment containing a
mutation disrupting the AP-1 consensus fails to bind
c-jun/AP-1. Side-by-side mobility shift reactions were
performed with the same nuclear extract from Bcr-Abl.A54 cells and
5 c-jun probes (150-bp inserts) labeled to a comparable
activity. (A) On the left, the wild-type c-jun probe formed two
band-shift complexes, which were cold-competed with nonradioactive
self-probe, and were neutralized by c-jun antibody. (B) On the
right, the gel mobility shift performed with the 150-bp 5
c-jun probe containing a mutated AP-1 core sequence failed to
bind c-jun. A faint band-shift complex was formed that was
competed with nonradioactive self-probe that also lacked AP-1
consensus. This faint band-shift was not affected by the c-jun
antibody dilution that completely neutralized c-jun/AP-1
complexes in the companion reaction.
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Next, a series of transcriptional assays involving transient
transfection of the 5 c-jun luciferase versus the mAP-1
5 c-jun luciferase reporter vectors were performed to
compare AP-1 transacting capacity for the c-jun promoter within
Bcr-Abl.A54 cells versus H7 parental cells (grown in IL-3). There was
30-fold activation of the 5 c-jun luciferase vector
above background control in the Bcr-Abl.A54 cells, and the AP-1 site
mutant reporter vector was essentially inactive
(Table 1). By contrast,
5-c-jun-mediated transcriptional activity within H7
parental cells was fivefold lower than in Bcr-Abl.A54 when both values
were controlled for -galactosidase activity, which was exactly
comparable within the two cell types (Table 1).
Composition of AP-1 proteins binding to the proximal CRE element
of 5 c-jun in cell lines differing in p210
BCR-ABL. It was of interest to distinguish the binding
interactions occurring on the jun promoter in cells transformed
by p210 BCR-ABL compared with IL-3-dependent parental cells. Gel
mobility shift was performed involving side-by-side analysis of
extracts from H7 parental cells compared with Bcr-Abl.A54 cell extract
(Fig 4). Repeatedly, it was observed that
Bcr-Abl.A54 cell extracts were unique in their content of the double
band-shift complex containing a unique faster mobility complex (complex
b) that was supershifted and largely neutralized with antibodies
specific for c-jun and for c-fos (Fig 4). As noted
previously, antibody to junB did not cause any supershifting, which is consistent with the results of other immunoblot experiments for the absence of junB protein in these extracts (data not
shown). On the other hand, addition of an antibody against ATF2 (known by immunoblot experiments [see below] to be a major constituent of
the extracts) was not effective in supershifting either mobility shift
band, indicating ineffectiveness of this antibody preparation (Fig 4).
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| Fig 4.
Gel mobility shift experiment involving 5
c-jun probe and comparing nuclear extracts of H7 parental cells
grown in IL-3 versus Bcr-Abl.A54 cells. Equal quantities of nuclear
extract from the two cell lines were placed in a gel mobility shift
reaction as noted above, without or with the addition of antibody
against c-jun or c-fos, or against ATF-2 or jun
B. Another reaction was performed with each extract along with the
inclusion of 100-fold excess of nonradioactive 5 c-jun
DNA (self) probe to establish specificity of the band-shift. Note
absence of complex (b) in H7 parental cell extracts, and the
supershifting/depletion of complex (b) in Bcr-Abl.A54 cells extracts by
either antibody against c-jun or c-fos.
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By contrast to this result with extract from Bcr-Abl.A54 cells,
parental H7 cell extract had somewhat lower overall binding to the
radiolabelled 5 c-jun probe. This extract was
characterized by complete absence of the faster mobility band-shift
complex that had been easily neutralized by the c-jun and
c-fos antibodies (Fig 4). In addition, the single band-shift
complex formed by H7 cell extract was poorly affected in terms of
neutralization or supershift by inclusion of antibodies against
c-jun and c-fos, suggesting deficiency within this AP-1
band-shift of antigenically recognizable c-jun and
c-fos proteins (Fig 4).
Immunoblot experimentation was undertaken to confirm the content of
c-jun and c-fos and to investigate the presence of
ATF-2 within these extracts. Equal amounts of nuclear extract from H7 parental cells, either IL-3-deprived or IL-3-deprived and
-restimulated, and from Bcr-Abl.A54 cells were subjected to
electrophoresis by SDS-PAGE and then immunoblotting
(Fig 5). It was observed that Bcr-Abl.A54
cells had equal amount of ATF-2 protein compared with IL-3 stimulated
H7 cells (Fig 5).
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| Fig 5.
Comparative immunoblot of nuclear protein extracts from
H7 parental cells versus Bcr-Abl.A54 cells shows increased
c-jun and c-fos in cells transformed by p210 BCR-ABL.
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On the other hand, Bcr-Abl.A54 cells had greater quantities of
c-jun and c-fos than H7 cells subjected to IL-3
stimulation (Fig 5). Interestingly, IL-3 stimulation of H7 cells
increased the abundance of c-fos and ATF-2, as previously
reported,22 but IL-3 stimulation did not increase, and
actually reduced somewhat, the amount of detectable c-jun
protein (Fig 5). Considering the composition of the band-shift
complexes formed on the 5 c-jun promoter described
above, and also taking into consideration the results of other
investigators,23,24 it is likely that the slower mobility
band-shift (complex a) from extracts of Bcr-Abl.A54 as well as the sole
complex from H7 cells may be composed of c-jun and ATF-2,
whereas the faster mobility complex is likely to be uniquely composed
of c-jun and c-fos.
Human acute leukemia cells containing the Philadelphia
chromosome have strong constitutive kinase activity of JNK and
contain abundant c-jun/AP-1 that binds to DNA. The
scientific relevance of the above findings depends on the existence of
a similar state of control that exists in primary human leukemia cells
containing the BCR-ABL gene in a Philadelphia chromosome
(Ph+). The human K562 is a standard cell line model derived
from a patient with Ph+ CGL in blast phase. We studied the
activity of JNK within K562 as compared with a "naive" leukemic
cell line, HL-60, not possessing a known tyrosine kinase oncogene of
the class of BCR-ABL as a negative control
(Fig 6).
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| Fig 6.
Comparative analysis of human leukemia cell lines
differing in expression of p210 BCR-ABL for JNK activity. (A) Equal
quantities of cell lysate from HL-60 versus K562, a p210 BCR-ABL
transformed cell line, were placed into solid-phase JNK activity assay
or were immunoblotted for expression of c-jun. (B) Equal
quantities of cell lysate from M07e cells growing in GM-CSF versus
M07p210 cells were placed into solid-phase JNK activity assay or were
immunoblotted for expression of c-jun, c-fos, ATF-2, or
JNK. Note absence of difference in abundance of JNK (isoform 1) or
ATF-2 between cells, but major increase in c-jun (2.8-fold by
densitometry) and c-fos (2.4-fold) proteins in M07p210 that
mirrors increase in kinase activity of JNK.
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K562 had strong constitutive JNK activity, but HL-60 did not possess
measurable activity (Fig 6A). K562 had abundant c-jun protein
but HL-60 had only a very small amount of c-jun (Fig 6A). Similarly, we compared the activity of JNK within M07p210 BCR-ABL transformed cells versus GM-CSF-dependent parental M07e cells as a
stricter control for possible cell lineage background effects independent of p210 BCR-ABL. Again, there was more than fourfold to
fivefold greater JNK activity within p210 BCR-ABL-containing cells
(Fig 6B). There was a corresponding twofold to threefold greater amount
of c-jun and c-fos proteins accompanying p210 BCR-ABL and its augmented JNK activity (Fig 6B). On the other hand, like the
situation that obtained in the comparison of IL-3-treated H7 versus
Bcr-Abl.A54, the two cell lines had exactly the same amounts of ATF-2
(Fig 6B). Further, in the case of the related M07e/M07p210 BCR-ABL
paired cell lines, we showed that differences in the absolute abundance
of the JNK enzyme could not explain the magnitude of difference in JNK
activity and the resulting c-jun protein expression because JNK
(the JNK1 isoform) was expressed equally (Fig 6B).
JNK activity assay was also performed on cell lysates from patients
with acute leukemic blast transformation associated with the
Philadelphia chromosome. Cell lines were included as controls: HL-60
cell lysate was lacking in JNK activity, K562 had strong JNK activity
(Fig 7). However, kinase activity of JNK
within sample no. 2224 from a patient with a Ph+ acute
leukemia was even greater than activity within K562 (Fig 7). Another
distinct (no. 2116) sample from a patient with Ph+ acute
lymphocytic leukemia also showed strong kinase activity of JNK at a
level equal to K562 (data not shown). In contrast to the constitutive
kinase activity of JNK observed in these samples was absence of
activity within a series of "good prognosis" acute leukemia cases
studied (Cripe et al, in preparation; see also below).
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| Fig 7.
JNK activity assay on lysates of cell lines and a
leukemic blast crisis bone marrow sample from a patient with a
transformed myeloproliferative disorder containing a Philadelphia
chromosome. Both the human cell line model of acute leukemia formed in
the presence of a Philadelphia chromosome (K562) and the primary
patient sample containing Philadelphia chromosome show strong
constitutive JNK activity. Another Ph+ acute leukemia
(not shown) also demonstrated a similar level of activity.
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The antibody against human c-jun is poorly active in supershift
reactions. Therefore, we wished to more accurately determine relative
levels of c-jun and c-fos composing an AP-1 species
that is capable of binding DNA, within the extracts of the K562 cell line, the patient sample no. 2271 (relapse specimen from same patient
as sample no. 2224), and also within the murine model cell line,
Bcr-Abl.A54. A gel mobility shift reaction was performed with the
7-base collagenase TRE, which binds only to c-jun/c-fos AP-1 (as opposed to binding also with jun/ATF)
(Fig 8). Another gel mobility
shift reaction was simultaneously set up with an oligonucleotide probe
representing the consensus CTF box-binding CTF/NF-1 factor site (Fig
8A). This was used as a binding control for a constitutive CTF factor
whose abundance is not expected to differ significantly among these
extracts. We observed relatively equal binding by extracts from these
distinct BCR-ABL-containing cells to the radiolabeled CTF probe (Fig
8A, right). By contrast, there was a definite increase in
c-jun/AP-1 within extract from patient sample no. 2271 (relapse
specimen no. 2224) compared with the positive control human cell line
K562 and Bcr-Abl.A54 (Fig 8A). This result was confirmed in another
independent experiment, and specificity of the binding reaction was
shown by competition of the binding with 100-fold excess of
nonradioactive collagenase TRE DNA (Fig 8B).
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| Fig 8.
Gel mobility shift reactions using whole cell
extracts from cell lines and patient samples prepared on a small scale
by high-salt extraction demonstrate c-jun/AP-1 binding
activity. (A) Extracts from the Ph+ model cell lines
K562, Bcr-Abl.A54 (the murine cell line expressing p210 BCR-ABL), and
patient sample no. 2271 were placed in gel mobility shift reactions
with commercial oligonucleotide for the 7-base collagenase TRE (left)
or a CTF/NF-1 oligonucleotide, which was used to control for protein
loading by assay of a constitutively abundant transcription factor.
Note relative abundance of c-jun/AP-1 binding activity. (B)
Increased c-jun/AP-1 binding activity present in the patient
sample is specific. On the left, another gel mobility shift reaction
was performed with the 7-base collagenase TRE(AP-1) oligonucleotide
including an extract, also prepared by the same high-salt extraction
protocol, from a cell line (T47 breast cancer) known to be deficient in
c-jun/AP-1 activity (HL-60 lysates could not be prepared by
this protocol because of extensive autodigestion that could not be
effectively inhibited). On the right, specificity of binding by extract
from patient sample no. 2271, as well as from lysate of patient no.
2306 (known to harbor constitutive JNK activity), are shown to be
specific because of cold-competition of their band-shift complexes
formed with the collagenase TRE oligonucleotide by nonradioactive
collagenase TRE at 100-fold excess. Note that the intensity of
band-shift complexes formed is proportionately reduced after decay of
the probe subsequent to labeling.
|
|
Unlike the situation in cell lines that are matched in a
parental:progeny relationship such as H7:A54 and M07e:M07p210,
comparison of patient samples implies a distinctive cellular
background. Other features of the blast cells might also be relevant to
the distinction of c-jun/JNK activity levels, irrespective of
the presence of p210 BCR-ABL signaling from upstream. Relevant
questions on this point include whether all leukemic cells may have
upstream signaling pathways activated that facilitate JNK-mediated
c-jun expression, but pathway output is simply limited by JNK
enzyme levels. To answer this question, leukemic samples with known
activity of JNK in solid-phase assay, and whose levels of c-jun
protein were independently established, were studied for JNK expression by immunoblot and immune complex kinase assay after immunoprecipitation (Fig 9).
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| Fig 9.
Variations in JNK activity between different leukemia
samples is not explained by the absolute level of cellular enzyme
expression. Lysates from leukemic samples registering negative (nos.
2099, 2127, 2137) or positive (no. 2135) in the solid-phase JNK assay
were immunoprecipitated and immunoblotted to show the presence of JNK
protein in the lysates. Half of each immunoprecipitate was placed in an
immune complex kinase JNK assay. Note the inactivity of
immunoprecipitated JNK1 within no. 2099, despite the presence of a
similar protein amount as no. 2127. Also note the apparent absence of
immunoprecipitable JNK in the HL-60 cell line.
|
|
Leukemia samples studied included the sample no. 2135 from an
NK-phenotype ANLL FAB-type M2 without p210 BCR-ABL. The blast cells of
this marrow sample demonstrated strong JNK activity and very abundant
c-jun protein, at a level comparable with Ph+ blast
crisis cells (no. 2271) (see below). Also included were samples no.
2127, 2099, and 2137, which showed reduced content of JNK enzyme by
direct immunoblotting, and which were negative for kinase activity of
JNK in the solid-phase assay (not shown). These samples were
immunoprecipitated with anti-JNK and half of the immunoprecipitate was
immunoblotted and the other half was placed in immune complex kinase
assay. Immunoprecipitates of the control cell lines were also
immunoblotted (Fig 9).
All of the immunoprecipitated samples had demonstrable expression of
JNK1 except HL-60, which was lacking in JNK (Fig 9). However, the
relative abundance of JNK as detected by immunoblot was not the primary
determinant of JNK activity in the aliquot subjected to kinase assay.
Immunoprecipitated samples no. 2099 and 2127 had comparable levels of
JNK1, but, among these, sample no. 2099 did not demonstrate JNK enzyme
activity (Fig 9). On the other hand, sample 2127 had greater JNK enzyme
activity than sample 2137, whose content of JNK1 protein was greater
still (Fig 9). These results show that different leukemic samples
contain variable levels of JNK enzyme, but the enzyme activity is
largely independent of the quantitative level of JNK protein.
Importantly, we also determined in an independent immunoprecipitation
experiment that cell samples from the Ph+ leukemia case
with strong JNK activity presented above (nos. 2224/2229/2271) had
exactly the same content of JNK1 as did sample no. 2099, whose JNK
activity was profoundly diminished (Fig 9, and data not shown).
Immunoblotting was also performed to confirm the abundance of
c-jun within extract from Ph+-patient samples nos.
2224 and 2271 (relapse specimen), and the unrelated leukemia sample
characterized by constitutive JNK activity (no. 2135)
(Fig 10). On the other hand, there was an
absence of c-jun protein within the JNK inactive patient sample
no. 2099, as well as in the negative control cell line, HL-60 (Fig 10).
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| Fig 10.
Western blots prepared from SDS-PAGE electrophoresis of
primary leukemia cell sample extracts were blotted for expression of
c-jun protein. c-jun protein was shown in samples known
to possess constitutive JNK enzyme activity.
|
|
| |
DISCUSSION |
Activation of a p21ras pathway downstream of p210 BCR-ABL, a
nonreceptor tyrosine kinase oncogene that is the best characterized of
a class of leukemia oncogenes, has received much
attention.14-16,25-27 However, it is less well understood
what number of consequences static p21ras activation has for cellular
homeostasis. One of these consequences of p21ras activity is a
transformation program that has been suggested to involve the activity
of JNK, the N-terminal c-jun kinase, and, as a consequence,
upregulation of c-jun within cells.
Previously, JNK was identified as an effector in the p21ras pathway of
transformation initiated by p210 BCR-ABL.15 In consequence of BCR-ABL-induced JNK activity, abundant AP-1 transactivating capacity was identified in cells, which was inhibited by
dominant-negative versions of p21ras, MEKK, JNK, as well as by
dimerization-defective and DNA-binding domain-defective versions of
c-jun.15 However, several of these events were
studied in fibroblasts, and the relative consequence of JNK activity
differences imparted by p210 BCR-ABL on c-jun gene regulation
in myeloid cells was not reported. In addition, it has not been
reported whether these changes documented in model cell lines are also
observed in clinical cases of acute leukemia associated with the
Philadelphia chromosome.
The results of our investigation suggest a scenario by which stable
expression of pBCR-ABL and constitutive upregulation of JNK activity
results in augmented c-jun expression. Augmentation of the
enzyme activity of JNK within murine myeloid cells in the presence of
stable overexpression of p210 BCR-ABL acutely affects increased
transcription of the c-jun gene, and binding to the proximal
CRE element of the c-jun gene with more c-jun and
c-fos proteins follows (Fig 5). When this novel AP-1 complex
composed of c-jun and c-fos is abundant, 5
c-jun transcription, which is highly dependent on this binding,
is upregulated fourfold to fivefold (Table 1). Devary et
al23 and Herr et al24 have also demonstrated
that the proximal CRE element of 5' c-jun is unique in its
binding of c-jun/c-fos AP-1, and is the site responsive to signals conferred by JNK.
In the model systems we studied, parental cells without the oncogenic
p210 BCR-ABL and growing under the influence of IL-3 or GM-CSF had
lower JNK activity, which was associated with a lower stable
concentration of c-jun and c-fos (Figs 5 and 6). Recently, stimulation of factor-dependent cell lines with cytokines was
noted to acutely activate JNK and p38/RK MAP kinases, but in each case
the level of activation was low and quite transient.28,29 In addition, whereas ATF-2 is a substrate for p38 RK, c-jun is not.30 In keeping with these results, c-jun is not
an abundant transcript induced by IL-3 or GM-CSF in factor-dependent
myeloid cell lines.14,21,31-34 These data raise the
question of what components of AP-1 are present in the "normal"
growth factor-dependent cells without the high level kinase activity
of JNK that characterizes p210 BCR-ABL, when they are growth factor
stimulated.
In our model systems, ATF-2 was an abundant jun/ATF factor
present in extracts of IL-3-treated H7 cells or GM-CSF-treated M07e
cells, as determined by immunoblotting. Unfortunately, none of the ATF
antibodies we tested (ATF-1,-2 antibodies from various suppliers) was
effective in supershift/neutralization analysis of gel mobility-shift
bands, so that the participation of this protein in DNA binding could
not be addressed. Also, poor recognition of the sole band-shift complex
in the IL-3-stimulated H7 parental cells by c-jun antibody
suggests that another jun family member may be in a dimeric
complex with ATF-2 to the exclusion of c-jun (Fig 4).
Immunoblot experiments not shown here suggest that the most abundant
jun family protein in these IL-3-treated parental H7 cells is
junD (data not shown). On the other hand, junB has been
found in abundance in IL-3-stimulated FDC-P cells.32
It might be queried whether, in addition to static p21ras activation,
another mechanism exists that promotes such strong kinase activation of
JNK in the presence of p210 BCR-ABL. Recently, the rac-specific guanine
nucleotide exchange factor, vav, was found to be activated by tyrosine
phosphorylation, leading to rac-dependent JNK
activity.34,35 This occurs when active GTP-bound rac binds to MEKK1.36 When p210 BCR-ABL is expressed in myeloid
cells, vav is known to be heavily tyrosine-phosphorylated and bound to p210 BCR-ABL, which would be the initiation point of such a
cascade.37 In contrast to the vav-rac mechanism downstream
from p210 BCR-ABL, a mechanism by which active, GTP-bound p21ras
directly initiates the JNK cascade is less well defined but may also
involve MEKK1 binding.38 In addition, a Grb-2 adaptor
interaction can activate the Ste-20 homologue, hematopoietic-specific
kinase 1 (HPK1) to initiate MLK3 and SEK1 activity en route to JNK in a
parallel pathway to p21ras.39
Additionally, our data show that heightened JNK activity, increased
c-jun expression, and c-jun/AP-1 binding to DNA also
characterized primary leukemic blast cells from patients with
Ph-mediated leukemias (Figs 7-10). Indeed, such
leukemias may exhibit a state of resistance to induction chemotherapy
regimens containing the oxidative damage-inducing drug adriamycin,
because of the participation of gene products transcriptionally
upregulated in a c-jun/AP-1-dependent manner which buffer
oxyradicals, and which also mediate glutathione conjugation and export
of oxyradical metabolites and xenobiotics. Among these genes/gene
products are metallothionein IIA; -glutamyl cysteine synthase, the
rate-limiting enzyme for de novo synthesis of
glutathione40-45; as well as the enzymes,
glutathione-S-transferase and quinone reductase.46-50
In keeping with this hypothesis, we have measured the pools of reduced
and oxidized glutathione in the paired murine cell lines H7 versus
Bcr-Abl.A54, and have observed significant augmentation of the cellular
content of total glutathione in A54 cells with p210 BCR-ABL (data not
shown). The origin of this adaptive response may relate to the cellular
requirement to counteract peroxidative byproducts downstream of active
p21ras.51 Interestingly, it was recently observed in model
cell lines that the anti-apoptotic mechanism conferred by p210 BCR-ABL
exists upstream of, and protects against, mitochondrial release of
cytochrome C and activation of caspases, and may partly involve
bcl-xL.52 It has been observed by others that intracellular
thiol pools are additive with bcl protein levels in the capacity of
upstream "buffering" and protection of mitochondrial
integrity.53,54
Another aspect of our data is that certain acute leukemic blast cell
samples, in addition to those that express p210 BCR-ABL, may also
exhibit constitutive kinase activity of JNK and abundant levels of
c-jun protein. Our preliminary experience in examining approximately 20 cases of adult acute myeloid leukemias on diagnostic marrows obtained before treatment shows that relapsed and secondary leukemias commonly exhibit constitutive JNK activity, whereas de novo,
"good prognosis" acute myeloid leukemias occurring in young
adults are more likely to lack JNK activity, and to fail to express
c-jun protein (eg, see sample no. 2099, Figs 6 and 9). The
feature distinguishing acute leukemic samples that display constitutive
JNK activity and c-jun protein versus those samples that lack
c-jun protein in the absence of constitutive JNK activity cannot be explained simply on levels of JNK enzyme(s) expression. Rather, we hypothesize that the majority of acute leukemias which exhibit constitutive kinase activity of JNK may be those that emit
strong upstream tyrosine kinase signals, or those that contain activated small ras/rho-family G proteins. On the other hand, we have
preliminary evidence that dominant suppression of JNK may account for
absence of JNK activity in some samples (not shown).
In summary, a pathway from the tyrosine kinase oncogene p210 BCR-ABL to
JNK-mediated c-jun transcriptional expression in myeloid leukemias is demonstrated here. Constitutive activation of the JNK
pathway in a range of myeloid leukemias may confer important functional
characteristics that determine prognosis. We are currently pursuing
this hypothesis.
| |
FOOTNOTES |
Submitted November 24, 1997;
accepted June 1, 1998.
E.A.W. and L.D.C. contributed equally to this work.
Supported in part by a Leukemia Society of America Translational
Research Grant, and a Merit Review Award from the Department of
Veterans Affairs, and by Grant No. DHP-124 from the American Cancer
Society to H.S.B.; and by National Institutes of Health Grant No. R01
CA 42533 to A.S.K. L.D.C. and H.S.B. receive support from the Joseph
and Gloria Boone Leukemia Research Fund of the Indiana University
Foundation.
Address reprint requests to H. Scott Boswell, MD, Indiana University
School of Medicine, Medical Research Four Bldg, Room 202, 1044 W Walnut
St, Indianapolis, IN 46202.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract