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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 22-29
Lack of Interferon Consensus Sequence Binding Protein (ICSBP)
Transcripts in Human Myeloid Leukemias
By
Manuel Schmidt,
Stefan Nagel,
Jutta Proba,
Christian Thiede,
Markus Ritter,
Jeffrey F. Waring,
Frank Rosenbauer,
Dieter Huhn,
Burghardt Wittig,
Ivan Horak, and
Andreas Neubauer
From the Medizinische Klinik I, Universitätsklinik Carl Gustav
Carus, Dresden; Abteilung für Innere Medizin m.S.
Hämatologie, Virchow Klinikum der Humboldt Universität
Berlin, Berlin; Forschungsinstitut für Molekulare
Pharmakologie, Berlin; and Institut für Molekularbiologie,
Biochemie und Bioinformatik, Freie Universität Berlin, Berlin,
Germany.
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ABSTRACT |
Interferon consensus sequence binding protein (ICSBP) was
first identified as a transcription factor of the interferon (IFN)
regulatory factor family (IRF) which regulates expression of
IFN-dependent genes by binding to DNA at specific sites, IFN-stimulated
responsive elements. Analysis of ICSBP-deficient mice showed
hematologic alterations similar to chronic myelogenous leukemia (CML)
in humans and suggested a novel role for ICSBP in regulating
proliferation and differentiation of hematopoietic progenitor cells.
Here we show that ICSBP-mRNA expression is impaired in human
myeloid leukemias: 27 of 34 CML patients (79%) and 21 of 32 patients
with acute myeloid leukemia (AML) (66%) showed very
low or absent transcript numbers of ICSBP. In contrast, only 2
of 33 normal volunteers (6%) showed low transcription of ICSBP
(P < .0001 both for CML and AML values). The lack of
expression was not associated with lack of lymphatic cells, which
normally have been shown to express ICSBP at the highest level.
More detailed analysis showed an absence of ICSBP-mRNA also in
sorted B cells derived from CML patients. To analyze whether
ICSBP may be induced in leukemic cells, ex vivo
experiments using a known inducer of ICSBP, IFN- , were
performed. Ex vivo treatment of primary CML cells using IFN-
resulted in induction of ICSBP transcripts. Furthermore,
samples of CML patients during IFN- treatment were analyzed. In 11
of 12 CML patients ICSBP-mRNA was inducible upon in
vivo treatment with IFN-, but decreased with progression of CML.
Stable transfection of K-562 cell line with ICSBP led to no
difference in bcr-abl expression in vitro, although two
patients showed an inverse correlation between bcr-abl and
ICSBP in vivo. These data suggest that lack of ICSBP
may have an important role also in human myeloid leukemogenesis.
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INTRODUCTION |
INTERFERONS (IFNs), divided into type I
(IFN- and - ) and type II (IFN-), are cytokines regulating
antiviral activity, immune responses, and cell growth in mammals
through IFN-regulated genes. The expression of IFN-inducible genes is
regulated by IFN regulatory factors (IRFs), which bind to DNA sites
containing IFN-stimulated responsive elements (ISRE).1-3
IFN consensus sequence binding protein (ICSBP)4,5
is one member of the growing family of the IRFs.6-10
Although some are expressed in many different tissues, ICSBP is
preferentially expressed in cells of hematopoietic origin.
Specifically, highest ICSBP expression is detected in mature B
cells, while resting T cells and mature macrophages harbor relatively
low expression.11
It has been shown that mice with deleted IRF-1 and
IRF-2 do not reveal any gross abnormalities.12 In
human leukemias, deletion of IRF-1 has been
reported,13 but it has been questioned whether
IRF-1 was indeed the target gene of the deletion of chromosome
5q, which is common in myeloid disorders.14 Furthermore,
expression of IRF-1 and IRF-2 has been investigated in
chronic myelogenous leukemia (CML) and no abnormality was
detected.15 In contrast to these findings, mice with a null
mutation of ICSBP exhibit two prominent features: (1) enhanced
susceptibility to viral infections; and (2) granulocytic leukemia with
enlargement of lymph nodes, liver and spleen, similar to CML in
humans.16 Strikingly, there seemed to be a `dose-effect'
of ICSBP in that ICSBP / homozygous
mice were more prone to blastic transformation of the respective
myeloid proliferation compared to ICSBP+/
heterozygous mice.16 However, in contrast to CML in humans,
where the rearrangement of the c-abl gene into the
bcr-gene is observed in more than 90% of the
patients,17,18 no gross genomic alteration of the
c-abl gene was detected in the leukemic cells of the
ICSBP-null mice.16 These data pointed to
ICSBP as a potential tumor-suppressor gene and a role of
ICSBP in leukemogenesis.
To address the question whether ICSBP may play a role also in
human leukemic transformation, we investigated the transcriptional
level in human leukemias and normal hematopoietic tissues. Therefore,
we used a sensitive semi-quantitative polymerase chain reaction (PCR)
assay.
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MATERIALS AND METHODS |
Patient samples.
Patient samples were taken from a single institution (Virchow Klinikum,
HU Berlin, Germany). Ten to 20 mL heparinized peripheral blood (20
U/mL) were drawn after informed consent. All acute myeloid leukemias
(AMLs) exhibited blast counts above 75%, and acute
lymphoblastic leukemias had blast counts above 50%.
Cells and cell lines.
Cell lines U-937, Jurkat, and K-562 were obtained from the ATCC
(American Type Culture Collection; Rockville, MD) and BV-173 from the
DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
Braunschweig, Germany). Cell lines HL-60, Daudi and Raji were kindly
provided by Dr F. Schriever (Virchow Klinikum) and cell lines MOLT-4,
EHEB, and DHL4 were gifts from Dr I.G. Schmidt-Wolf (Virchow Klinikum).
All cell lines were maintained at 5% CO2 in RPMI 1640
medium with 1% glutamine (GIBCO-BRL Eggenstein, Germany) supplemented
with 10% fetal calf serum (FCS, GIBCO-BRL), 1%
penicillin/streptomycin (Biochrom KG, Berlin, Germany). Mononuclear
cells were separated from peripheral blood by centrifugation over a
Ficoll (Biochrom KG) gradient and were stimulated with 1,000 U/mL
IFN- (Intron A; Essex Pharma GmbH, München, Germany) or
IFN- (Boehringer Mannheim, Mannheim, Germany) for 6, 24, and 48
hours, respectively.
Separation of CD19+ B cells.
B cells were separated from peripheral blood of normal healthy
volunteers and CML-patients using MACS CD19 MultiSort Kit (Miltenyi
Biotec GmbH, Sunnyvale, CA) as recommended by the manufacturer. Purity
of some B-cell fractions was verified to be approximately 90% using
CD19-FITC antibodies (Dako Diagnostika GmbH, Hamburg, Germany) and
FACScan analysis (Becton Dickinson, Heidelberg, Germany).
RNA isolation and cDNA synthesis.
RNA was extracted from heparinized peripheral blood using the
RNAzol-kit (Paesel, Frankfurt, Germany) as recommended by the
manufacturer. One microgram of total RNA was heat denaturated at 90°C
for 5 minutes in the presence of 100 pmol random hexamers (Pharmacia,
Freiburg, Germany) and cooled on ice for 2 minutes. The RNA was reverse
transcribed in 20 µL final volume of 1X PCR buffer (Perkin Elmer,
Weiterstadt, Germany), 625 nmol/L of each dNTP (Boehringer Mannheim),
10 mmol/L dithiothreitol (GIBCO-BRL), 40 U RNasin (Promega, Madison,
WI), and 140 U SuperScript reverse transcriptase (GIBCO-BRL). The
reaction mixture was incubated for 10 minutes at room temperature
(25°C), 40 minutes at 42°C, and 5 minutes at 95°C.
ICSBP-mRNA expression analysis by PCR.
PCR was performed using 50 ng single-stranded cDNA in 25 µL 1X PCR
buffer, 200 µmol/L dNTP, 500 nmol/L of each primer,
and 0.625 U AmpliTaq DNA Polymerase (Perkin Elmer) under following
cycling conditions: 94°C for 2 minutes for denaturation; then 94°C
for 1 minute, 55°C (for -actin) or 61°C
(for ICSBP) for 1 minute, 72°C for 1 minute for 21 (for
-actin) or 27 cycles (for ICSBP),
followed by 90°C for 1 minute and 60°C for 10 minutes. The
sequences of the primers are as follows: -actin
sense primer, 5 -CCTTCCTGGGCATGGA GTCCT-3;
-actin reverse primer,
5-AATCTCATCTTGTTTTCTGCG-3, which results in a 407-bp PCR-product;
ICSBP sense primer, 5-CAGTGGCTGATCGAGCAGATTGA-3;
ICSBP reverse primer, 5-ATTCACGCAGCCAGCAGTTGCCA-3, which
results in a 360-bp PCR product. The products were electrophoresed on a
3% agarose gel. Gels were stained with ethidium bromide and
photographed. The gel-photos were scanned and integrated optical
densities (IntOD) were calculated using the ONE-Dscan 1.0 software
(Scanalytics, Billerica, MA). The ratio `IntOD
ICSBP/IntOD -actin' was then calculated. Analysis of normal
control samples suggested a value of 0.200 as cut-off-level.
Other reference genes, porphobilinogendeaminase (pbgd)
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
showed comparable results (data not shown).
Detection of the bcr-abl translocation and mRNA expression
analysis by PCR.
The PCR for detection of the bcr-abl translocation was
performed as described elsewhere.19 Quantitative
bcr-abl-PCR was performed using a competitive differential
quantitative PCR-assay which enabled us to determine the relative
amplification-equivalence points (EP) of bcr-abl and a
reference gene, pbgd, and to calculate the ratio `EP
bcr-abl/EP pbgd.'20
Construction of ICSBP expression vectors.
An expression-plasmid for hICSBP was constructed by cloning a
PCR-product of 1,372 bp into pCR3.1 (Invitrogen, NV Leek, The
Netherlands). PCR was performed using 50 ng single-stranded cDNA in 25
µL 1X PCR buffer, 200 µmol/L dNTP, 500 nmol/L of each primer, and
0.625 U Expand Long PCR System (Boehringer Mannheim) under following
cycling conditions: 94°C for 2 minutes for denaturation then 94°C
for 1 minute, 62°C for 1 minute 68°C for 1 minute for 34 cycles,
followed by 90°C for 1 minute and 60°C for 10 minutes. The
sequences of the primers are as follows: sense
5-GCGGCGAGACGGCGGCAGGA-3; reverse 5-GGCCACTGTAACAGGGAGATGGA-3. The
primers are based on the recently corrected sequence deposited at
GenBank/EMBL Data Bank (accession no. M91196).
Transfection and cloning of stable transfectans.
K-562 cells (1.2 × 107) were transfected with 20 µg of
control pCR3.1 (without insert) and pCR3.1 containing the coding
sequence for hICSBP by electroporation with a Gene Pulser II
(BioRad, München, Germany). Cells were selected with 0.5 mg/mL
geneticin (G-418; GIBCO-BRL) for 2 to 3 weeks and then cloned by
limiting dilution. Ten clones propagated from each transfection group
were screened by reverse transcriptase (RT)-PCR for ICSBP-mRNA
expression. Stable transfectans were maintained in culture medium with
0.2 mg/mL geneticin. Three different clones each were used for
stimulation experiments with 1,000 U/mL IFN- (Intron A; Essex
Pharma) or IFN- (Boehringer Mannheim).
DNA sequencing.
ICSBP-PCR products and ICSBP expression vectors were
verified by automated sequencing with the ABI Prism DNA Sequencer 377
(Perkin Elmer) using the ABI Prism Dye Terminator Cycle Sequencing
Ready Reaction Kit (Perkin Elmer) as recommended by the manufacturer.
Statistical analysis.
Differences in the ICSBP expression of various leukemias were
calculated by Fisher's exact test using Statistica 5.0 software
(StatSoft, Tulsa, OK).
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RESULTS |
Determination of ICSBP-transcript levels in human samples.
We used a sensitive and semi-quantitative RT-PCR-approach to study
transcription levels of ICSBP in human leukemias, as well as in
normal hematopoietic tissues. Data obtained using RT-PCR were confirmed
for several samples with the RNase protection assay (data not shown).
Due to the restricted availability of RNA amounts from leukemia
patients, RT-PCR was used for expression analysis in our further work.
To analyze the number of ICSBP transcripts, the relative
expression of ICSBP was compared to a reference gene,
-actin. For both of these genes, PCR protocols
were standardized such that the cycle number for each of these genes
ensured that PCR-amplification was in its exponential phase. The
optimal cycle number for ICSBP was determined to be 28 cycles,
and 22 cycles for -actin.
In each PCR, three positive controls were run as controls. The data
obtained showed reproducible results (coefficient of variation of 16
experiments for three different controls: CV = 12.7%, 9.9%, and
8.1%, respectively).
We also performed control experiments with two other reference genes
(pbgd and GAPDH) which showed comparable
results with the data obtained with -actin
(data not shown). Therefore, all other experiments were performed using
-actin as reference gene.
In addition, we diluted mononuclear cells from a normal healthy
volunteer with ICSBP-nonexpressing K-562 cells to determine the
accuracy of the semi-quantitative RT-PCR. As expected, no linear
correlation was found (Fig 1). However, the
resulting near exponential curve fitted well for a semi-quantitative
assay.
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| Fig 1.
ICSBP-transcript numbers in a dilution series of
mononuclear cells from a normal healthy volunteer with cells from
ICSBP-nonexpressing cell line K-562. ICSBP-mRNA levels
were detected by a semi-quantitative RT-PCR assay. Five different
dilutions were done and the percentages of normal cells
(ICSBP-expressing cells) were shown (0%, 10%, 50%, 90%, and
100%). The mean of three experiments is displayed (±SEM).
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Furthermore, we tested different cell lines with known expression
levels of ICSBP, which had been previously determined using
standard Northern blotting techniques.5 The results of the
RT-PCR approach were in keeping with these results: High
ICSBP-transcript levels were detected in cell lines Daudi and
U-937, whereas K-562, MOLT-4, and HL-60 exhibited no or low
ICSBP transcripts (data not shown).
Expression patterns of ICSBP in human leukemias.
To study expression patterns in primary human leukemias, we first
determined the level of ICSBP transcription in normal, healthy
individuals. In 2 of 33 (6%) normal individuals, a relative expression
below 0.2 (arbitrary value) was detected (Fig
2 and Table 1).Because ICSBP/ mice harbor a CML-like
disease, we next investigated blood samples from CML patients in
chronic phase, all of them bcr-abl-positive. Here, very low
transcript numbers of the ICSBP gene were found in most
patients (Fig 2 and Table 1). Twenty-seven of 34 (79%) CML patients
showed no or significantly impaired ICSBP-transcript levels
(P < .0001, Fisher's exact test).
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| Fig 2.
ICSBP-transcript numbers in peripheral blood from
normal healthy individuals and patients with different kinds of
leukemias. (A) Lack of ICSBP-transcript numbers in CML-patients
was detected by a semi-quantitative RT-PCR assay. Comparison of three
healthy normal blood donors (NB; lanes 1 through 3) with four CML
patients at diagnosis (CML; lanes 4 through 7) and three
hydroxyurea-treated CML patients (CML, lanes 8 through 10). The
expression level is displayed as integrated optical density (IntOD).
(B) As (A), except that ICSBP-transcript numbers of 33 normal
healthy blood donors, 34 CML, 32 AML, 11 CLL, 11 ALL, and 6 CMMoL
patients were compared.
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Table 1.
ICSBP-Transcript Numbers in Peripheral Blood
From Normal Healthy Individuals and Patients With Different Kinds of
Leukemias
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For further investigation of myeloid leukemias, samples from patients
with AMLs, all bcr-abl-negative, were analyzed. Only samples
with a blast count above 75% were investigated. In 21 of 32 (66%) of
these AML samples, ICSBP-transcript levels were low or absent
(P < .0001). Some AML patients harbored high to very high
levels of ICSBP-mRNA. Interestingly, these were samples derived
from patients with AML-M4 and M5, ie, monocytic differentiating
leukemias. In keeping with this, most patients with chronic
myelomonocytic leukemia (CMMoL), a disorder classified as
myelodysplasia and characterized through monocytic differentiation,
exhibited high ICSBP levels (Fig 2 and Table 1).
We also analyzed lymphatic neoplasias; none of 11 patients with chronic
lymphocytic leukemia (CLL) and 4 of 11 (36%) patients with acute
lymphoblastic leukemia (ALL) harbored low values of
ICSBP-transcript numbers (Fig 2 and Table 1). Out of these 11
ALL patients, all with blast counts above 75%, 3 exhibited high
ICSBP values. These samples were drawn from patients who
suffered from c-ALL, a B-cell-derived disorder. All of our samples
from CLL patients, in approximately 95% also a disease of B cells,
showed high levels of ICSBP transcripts (Fig 2). In keeping
with this, all tested B-cell lines (DHL-4, Raji, BV-173, EHEB, Daudi)
exhibited high, whereas T-cell lines (Jurkat, MOLT-4) showed low,
ICSBP values (data not shown).5
ICSBP expression can be induced by IFNs both in vitro and in
vivo.
ICSBP is inducible in vitro with
IFN-.4,5 Given that ICSBP levels were impaired
in CML samples, we first addressed the question whether ICSBP
may be inducible in vitro in
p210bcr-abl-positive CML cells. Therefore, leukemic
cells of a patient with bcr-abl-positive CML were obtained by
centrifugation over a Ficoll gradient and incubated in vitro
for 6, 24, and 48 hours using different IFNs (Fig
3). ICSBP-mRNA levels were low at
the beginning of the in vitro incubation and increased 3- to
10-fold during incubation with IFN-, but not with IFN-. These
data suggested that ICSBP may be downregulated in myeloid
leukemias, because in vitro cultivation with IFN- led to a
significant increase of ICSBP message.
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| Fig 3.
ICSBP-transcript numbers in mononuclear cells of
a CML patient during in vitro stimulation with IFN- and
IFN-. An initial control was obtained (lane 1); samples were
obtained after 6 hours (lanes 2, 5, and 8), after 24 hours (lanes 3, 6,
and 9) and after 48 hours (lanes 4, 7, and 10). No increase of
ICSBP-transcript numbers was seen without IFN (lanes 2 through
4) or with IFN- treatment (1,000 U/mL; lanes 5 through 7). Only
during IFN- treatment (1,000 U/mL) ICSBP-transcript numbers
increased significantly (lanes 8 through 10). The mean of three
experiments is displayed (±SEM).
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Since IFN- induced ICSBP in normal and malignant cells in
vitro, we asked whether ICSBP may also be induced after in vivo
treatment with IFN-, where a survival benefit has been shown in
large multicenter trials.21-23 Therefore, 12 patients were
investigated during follow-up. Indeed, ICSBP-transcript numbers
were significantly increased in 11 of them (91.7%) while patients were
receiving IFN-, but also decreased with progression of CML (Figs 4
and 5, Table
2).
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| Fig 4.
ICSBP-transcript numbers, increasing upon in
vivo treatment with IFN-, correlated with impairment of
bcr-abl-transcript ( ), leukocyte ( ), and lymphocyte ( )
numbers. The sample at diagnosis exhibits few ICSBP
transcripts, even with only 1% blast cells (lane 1). Samples during
IFN- treatment showed an increase of ICSBP (lane 2: after 3
weeks, bone marrow; lane 3: 6 weeks; lane 4: 36 weeks), and a decrease
of ICSBP after IFN- was withdrawn (lane 5: 68 weeks). The
mean of three experiments is displayed. All analyzed metaphases during
the patient's course were 100% Philadelphia-chromosome positive. The
relative equivalence points (EP) of bcr-abl and the reference
gene, pbgd, were determined by quantitative PCR.
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| Fig 5.
Increase of ICSBP-transcript numbers in a CML
patient during in vivo IFN- treatment, inversely correlated
with bcr-abl-transcript (), leukocyte (), and lymphocyte
() numbers. At diagnosis (lane 0), and during treatment with
hydroxyurea (lane 1: after 0.5 weeks; lane 2: 1 week) few ICSBP
transcripts were visible, although in this period the patient exhibited
only 0% to 3% blast cells. During early IFN- treatment
ICSBP-transcript numbers increased (lane 3: 15 weeks; lane 4:
15 weeks), but decreased with progression to blast crisis (lane 5: 22
weeks; lane 6: 31 weeks; lane 7: 37 weeks, blast crisis with 60% blast
cells; lane 8: 37 weeks, blast crisis, bone marrow). The mean of three
experiments is displayed (±SEM). The patient exhibited an additional
cytogenetic aberration, a monosomie 7, during blast crisis. BC, blast
crisis.
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In particular, in one patient, ICSBP levels were low at the
beginning (0% to 3% blast cells), increased during IFN- therapy,
and decreased during progression to blast crisis (60% blast cells)
(Fig 5). These results suggest that ICSBP expression is low in
chronic and blastic phase of CML; however, it may be induced in cells
of chronic phase by IFN-.
The detected changes in ICSBP-mRNA levels are not due to
variations in the blood differential.
To rule out the possibility that an effect of the peripheral blood
differential, especially lymphocyte numbers, accounted for the lack of
ICSBP transcripts in samples from CML patients at diagnosis and
under hydroxyurea, we compared blood counts from different CML patients
(Table 3). We found that in three of six
selected patients in chronic phase (Table 3, patients 2, 4, and 6),
peripheral blood counts and percentage of lymphocytes were near normal.
Still, ICSBP-transcript levels were significantly impaired or
absent, arguing against the possibility that lack of ICSBP
transcripts in CML was a result of changes in the blood differential.
In keeping with this notion was the comparison to CML patients under
IFN-: Three of five selected patients (Table 3, patients 7, 9, and
11) with high ICSBP-transcript numbers had comparable leukocyte
and lymphocyte counts as other patients with low ICSBP levels
and not receiving IFN-.
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Table 3.
Comparison of Blood Differentials
and ICSBP-Transcript Numbers in Selected CML
Patients Without or Under IFN- Treatment
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In addition, we compared ICSBP-transcript numbers with the
amount of leukocytes and lymphocytes in two patients (Figs 4 and 5)
during follow-up. The number of lymphocytes decreased in both patients
while the ICSBP message was upregulated. In one of these
patients the relative amount of lymphocytes remained the same while
ICSBP-transcript numbers decreased during a given period (lanes
3 through 6, Fig 5). The other patient never showed a cytogenetic
response during his clinical course, ie, all metaphases analyzed were
always 100% Philadelphia-positive (Fig 4), suggesting that an increase
of `normal,' Philadelphia-negative cells was not responsible for the
observed increase of ICSBP transcripts in this
patient.24
To investigate alteration of ICSBP expression in CML in more
detail, we analyzed ICSBP-transcript numbers in
CD19+ B cells, known to express ICSBP at the
highest level (Fig 6). Seven samples from
normal healthy individuals showed high ICSBP-mRNA levels
whereas two of three samples from CML patients without IFN- therapy
exhibited low ICSBP-transcript numbers (Fig 6, lanes 1 through 10).
Another sample, taken from a CML patient previously treated with
IFN-, had a high ICSBP level as did one of two samples from
CML patients currently under IFN- (Fig 6, lanes 11 through 13).
These data correlated favorably to our results with unsorted peripheral
blood cells.
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| Fig 6.
ICSBP-transcript numbers in CD19+ B
cells from normal healthy individuals and CML patients. High transcript
levels were detected in cells from normal individuals (lanes 1 through
7), low levels in two of three CML patients without IFN- (lanes 8
through 10) whereas another patient with high ICSBP levels had
received IFN- before (lane 11; IFN- was withdrawn 6 months before
sample was taken). One of two CML patients still under IFN-
exhibited low, the other high ICSBP levels (lanes 12 and 13).
The mean of three experiments is displayed (±SEM).
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All together these results made it unlikely that the observed lack of
ICSBP transcripts and the upregulation during IFN- therapy
in CML patients was due to changes in the blood differential, and
seemed mainly to be a phenomenon of aberrant ICSBP expression
in B lymphocytes.
No regulating effect of ICSBP- on bcr-abl-transcript
numbers was detected in vitro.
Since levels of bcr-abl transcripts have been correlated with
progression of the disease, we asked if there may be a inverse
association between bcr-abl and ICSBP expression. In
two CML patients we compared ICSBP-expression patterns with the
quantitative determination of the bcr-abl mRNA.20
We found an inverse correlation between transcript levels of
ICSBP and bcr-abl (Figs 4 and 5).
To evaluate the possible effect of ICSBP on bcr-abl
expression, we analyzed the relationship between ICSBP- and
bcr-abl-transcript numbers in mononuclear cells from a patient
with CML. Untreated cells and cells, which were treated in
vitro with IFN- or IFN- for 24 hours, showed no significant
differences in bcr-abl expression, though an increase of
ICSBP transcripts after incubation with IFN- was detected
(Fig 7A). To analyze a possible interaction
of ICSBP and p210bcr-abl in more detail, we
stably transfected the bcr-abl-positive cell line K-562 with an
ICSBP-expression vector. The K-562 cells stably transfected
with ICSBP were found to express ICSBP at high levels
(Fig 7B). In these cells no change of bcr-abl-transcript
numbers was detected compared to ICSBP-nonexpressing K-562
cells. Also, after incubation for 24 hours with IFN- or IFN-,
K-562 cells exhibited, as expected, nearly the same levels of
bcr-abl-transcripts (Fig 7B).
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| Fig 7.
Comparison of ICSBP- and
bcr-abl-transcript levels in vitro. (A) Mononuclear
cells from a patient with CML were incubated for 24 hours with no
cytokine (lane 1), IFN- (lane 2), or IFN- (lane 3). In
IFN--treated cells ICSBP-mRNA levels increased, but no
change was seen with bcr-abl-mRNA levels. (B) Stable
transfectans of ICSBP-expressing (lanes 1 through 3) and
nonexpressing K-562 cells (lanes 4 through 6) are compared. Incubation
for 24 hours with no cytokine (lanes 1 and 4), IFN- (lanes 2 and 5),
or IFN- (lanes 3 and 6) showed no differences in
bcr-abl-mRNA levels. Experiments were performed with three
different transfected clones each and the mean is displayed (±SEM).
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Although an inverse correlation was seen in vivo, our results
suggest no direct regulating effect of ICSBP on bcr-abl
expression, but an effect of bcr-abl on ICSBP
expression could not be ruled out and has to be further investigated.
| |
DISCUSSION |
Recently, ICSBP/ mice have been generated.
These mice showed no difference in size, behavior, and reproductive
ability as compared with normal littermates. However, in 100% of these
mice, a hematologic neoplasia resembling CML in humans was
observed.16 The most prominent features of these mice were
hepatosplenomegaly and enlargement of lymph nodes with infiltrations of
mature granulocytes, metamyelocytes, and bands. Blood films of these
mice showed leukocytosis with enlargement of immature cells and, in
keeping with CML in humans, transformation to blastic phase was
observed. Interestingly, there seemed to be a `dose-response' in that
homozygous mice were more prone to develop blast crisis as compared
with heterozygous mice.16 These data prompted us to
investigate the role ICSBP may play in human leukemia.
We used a semi-quantitative RT-PCR approach to determine
ICSBP-transcript numbers in different kinds of leukemia
samples, because no monoclonal antibody was available to study human
ICSBP-protein expression. The data presented here showed that
ICSBP transcripts are absent or significantly lower expressed
in peripheral blood cells in the vast majority of patients with myeloid
leukemias. In addition, in some patients with acute lymphoblastic
leukemia, mostly T-ALL, ICSBP transcripts were also found at
lower levels, but not in B-cell-derived disorders like c-ALL or CLL.
These data raise the possibility that loss of ICSBP expression
may play a significant role in human myeloid leukemogenesis. We do not
believe that the lack of ICSBP transcripts was simply due to
expansion of more immature progenitors in the investigated leukemias,
because in some of the investigated CML samples a nearly normal blood
differential due to treatment with hydroxyurea was observed, yet
ICSBP levels were still absent, in contrast to samples from CML
patients under IFN-, which had comparable blood counts but high
ICSBP-transcript numbers. In keeping with this, analysis of
CD19+ B cells, normally highly expressing ICSBP,
showed absence of ICSBP transcripts in CML samples.
Furthermore, ICSBP was inducible in the leukemic cells, both
in vitro and in vivo. These data suggest that
ICSBP is not genomically lost but rather downregulated in
myeloid leukemias, the mechanism of which remains to be determined.
Downregulation leading to gene-`silencing' has been described in
human cancer cells for MTS-1, a gene coding for an inhibitor of
cyclin-dependent kinases
(p16INK-4/CDKN2).25,26
Although patients with CML may respond beneficially to treatment with
IFNs, treatment with IFN-, in contrast to IFN-, remains
anecdotal.27-30 In our clinic CML patients were only
treated with IFN-, so we were unable to assess the effect of
IFN-, a known inducer of ICSBP,4,5 on
ICSBP expression in vivo. In several clinical samples,
although many CML patients showed low levels of ICSBP
transcripts at diagnosis, IFN- therapy led to an increase in
ICSBP message. These high levels were also detected in some
CD19+ B cells from CML patients under or after IFN-
treatment. The induction of ICSBP by IFN- has not been
described in vitro, in keeping with data obtained in this
study. One explanation for the missing induction of ICSBP
during IFN- incubation in vitro may be that the increase of
ICSBP transcripts in CML patients during IFN- therapy cannot
be found within a short period of time, and may take at least 1 to 3
months. Further, the observed upregulation of ICSBP during
IFN- treatment in vivo may also be an indirect effect
mediated by other factors or cells not present in the in vitro
incubation.
When the patients progressed to accelerated/blastic phase,
ICSBP-mRNA levels decreased significantly, even during
treatment with IFN-. Thus, this raises the possibility that low or
absent levels of ICSBP transcripts may be associated with
progression of myeloid leukemias, and upregulation may be of some
unknown benefit. The importance of some kind of ICSBP
-`dose-response' for myeloid cell differentiation may also be
supported through data from ICSBP knock-out mice, in that mice
with a complete loss of ICSBP progressed to blastic phase in
33% within 50 weeks of observation, whereas heterozygous mice showed
acute leukemia in only 9%.16
The data of this study make it unlikely that loss of ICSBP is
necessary for the development of all myeloid leukemias. However, the
majority of myeloid leukemias displayed decreased or absent expression
of ICSBP transcripts. The mechanism by which loss of
ICSBP may induce leukemias is unclear at present. IFNs play an
important role in the negative regulation of human hematopoiesis. In
keeping with this, the beneficial role of these cytokines in human
myeloproliferative diseases has been shown in recent years. There may
be direct repressing effects of ICSBP on downstream, positive
effector genes. Data obtained in two patients of this study showed that
upregulation of ICSBP-mRNA was inversely correlated with
downregulation of bcr-abl transcripts. However, data from the
ICSBP-transfected K-562 cells make a direct effect on
bcr-abl unlikely. Thus, the possible interaction between
ICSBP and transforming genes need to be determined, especially
a possible effect of bcr-abl on ICSBP expression. In
addition, the `homing' of myeloid cells could be negatively
influenced by the lack of ICSBP, ie, through the loss of
expression of certain adhesion molecules. Interestingly, IFN-
therapy may induce adhesion molecules, and this, at least in part, may
explain its beneficial effect in human CML.31 On the other
hand, IFN- induces negative cytokines in the human bone marrow
stromal cells, and this has been implicated in the therapeutic efficacy
of these drugs in treating CML patients.32,33
In summary, ICSBP loss induces myeloid leukemias in mice, and
this report describes that ICSBP-mRNA is very low or absent in
the majority of human myeloid leukemias. Together these data raise the
possibility that ICSBP may be a tumor-suppressor, but the exact
role ICSBP plays in hematopoiesis remains to be determined.
| |
FOOTNOTES |
Submitted June 3, 1997;
accepted October 2, 1997.
Supported by grants from the `Deutsche Forschungsgemeinschaft'
(Ne310/6-3 to A.N. and SFB 465 to I.H.).
Address reprint requests to Andreas Neubauer, MD, Medizinische Klinik
I, Universitätsklinik Carl Gustav Carus, Fetscherstraße 74,
01307 Dresden, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 17.34 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank C. Fieger and S. König for help with electroporation
procedures, Dr C. Brendel for FACScan assistance, Dr C. Busemann for
providing patient data, J. Laser for technical assistance, and Dr K.
Seeger for GAPDH primers. We are grateful to Dr F. Schriever
and Dr I.G. Schmidt-Wolf for providing cell lines, and to Prof Dr H.
Kleinkauf for helpful discussions.
| |
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C. Zhu, G. Saberwal, Y. Lu, L. C. Platanias, and E. A. Eklund
The Interferon Consensus Sequence-binding Protein Activates Transcription of the Gene Encoding Neurofibromin 1
J. Biol. Chem.,
December 3, 2004;
279(49):
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[Abstract]
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M. Schmidt, J. Bies, T. Tamura, K. Ozato, and L. Wolff
The interferon regulatory factor ICSBP/IRF-8 in combination with PU.1 up-regulates expression of tumor suppressor p15Ink4b in murine myeloid cells
Blood,
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[Abstract]
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A. Burchert, D. Cai, L. C. Hofbauer, M. K. R. Samuelsson, E. P. Slater, J. Duyster, M. Ritter, A. Hochhaus, R. Muller, M. Eilers, et al.
Interferon consensus sequence binding protein (ICSBP; IRF-8) antagonizes BCR/ABL and down-regulates bcl-2
Blood,
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[Abstract]
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R. Ren
Overriding BCR/ABL mitotic signal by ICSBP-induced differentiation
Blood,
December 15, 2003;
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T. Tamura, H. J. Kong, C. Tunyaplin, H. Tsujimura, K. Calame, and K. Ozato
ICSBP/IRF-8 inhibits mitogenic activity of p210 Bcr/Abl in differentiating myeloid progenitor cells
Blood,
December 15, 2003;
102(13):
4547 - 4554.
[Abstract]
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B. J. Barnes, M. J. Kellum, K. E. Pinder, J. A. Frisancho, and P. M. Pitha
Interferon Regulatory Factor 5, a Novel Mediator of Cell Cycle Arrest and Cell Death
Cancer Res.,
October 1, 2003;
63(19):
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[Abstract]
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J. Kochling, S. A. Konig-Merediz, R. Stripecke, D. Buchwald, A. Korte, H. G. von Einsiedel, F. Sack, G. Henze, K. Seeger, B. Wittig, et al.
Protection of Mice against Philadelphia Chromosome-positive Acute Lymphoblastic Leukemia by Cell-based Vaccination Using Nonviral, Minimalistic Expression Vectors and Immunomodulatory Oligonucleotides
Clin. Cancer Res.,
August 1, 2003;
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[Abstract]
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A. Burchert, S. Wolfl, M. Schmidt, C. Brendel, B. Denecke, D. Cai, L. Odyvanova, T. Lahaye, M. C. Muller, T. Berg, et al.
Interferon-alpha , but not the ABL-kinase inhibitor imatinib (STI571), induces expression of myeloblastin and a specific T-cell response in chronic myeloid leukemia
Blood,
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[Abstract]
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Z. Qian, A. A. Fernald, L. A. Godley, R. A. Larson, and M. M. Le Beau
Expression profiling of CD34+ hematopoietic stem/ progenitor cells reveals distinct subtypes of therapy-related acute myeloid leukemia
PNAS,
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[Abstract]
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M. Schwieger, J. Lohler, J. Friel, M. Scheller, I. Horak, and C. Stocking
AML1-ETO Inhibits Maturation of Multiple Lymphohematopoietic Lineages and Induces Myeloblast Transformation in Synergy with ICSBP Deficiency
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H. Tsujimura, T. Nagamura-Inoue, T. Tamura, and K. Ozato
IFN Consensus Sequence Binding Protein/IFN Regulatory Factor-8 Guides Bone Marrow Progenitor Cells Toward the Macrophage Lineage
J. Immunol.,
August 1, 2002;
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J. Nehyba, R. Hrdlickova, J. Burnside, and H. R. Bose Jr.
A Novel Interferon Regulatory Factor (IRF), IRF-10, Has a Unique Role in Immune Defense and Is Induced by the v-Rel Oncoprotein
Mol. Cell. Biol.,
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A. Kallies, F. Rosenbauer, M. Scheller, K.-P. Knobeloch, and I. Horak
Accumulation of c-Cbl and rapid termination of colony-stimulating factor 1 receptor signaling in interferon consensus sequence binding protein-deficient bone marrow-derived macrophages
Blood,
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[Abstract]
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H. R. Bose Jr.
Interferon Regulatory Factor 4 Contributes to Transformation of v-Rel-Expressing Fibroblasts
Mol. Cell. Biol.,
October 1, 2001;
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M. Deng and G. Q. Daley
Expression of interferon consensus sequence binding protein induces potent immunity against BCR/ABL-induced leukemia
Blood,
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[Abstract]
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C. Barthe, F.-X. Mahon, M.-J. Gharbi, C. Faberes, C. Bilhou-Nabera, A. Hochhaus, J. Reiffers, and G. Marit
Expression of interferon-{alpha} (IFN-{alpha}) receptor 2c at diagnosis is associated with cytogenetic response in IFN-{alpha}-treated chronic myeloid leukemia
Blood,
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[Abstract]
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M. Schmidt, A. Hochhaus, A. Nitsche, R. Hehlmann, and A. Neubauer
Expression of nuclear transcription factor interferon consensus sequence binding protein in chronic myeloid leukemia correlates with pretreatment risk features and cytogenetic response to interferon-{alpha}
Blood,
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[Abstract]
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D. W. Mullins, R. S. Martins, C. J. Burger, and K. D. Elgert
Tumor cell-derived TGF-{beta} and IL-10 dysregulate paclitaxel-induced macrophage activation
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M. Schmidt, A. Hochhaus, S. A. Konig-Merediz, C. Brendel, J. Proba, G. J. Hoppe, B. Wittig, G. Ehninger, R. Hehlmann, and A. Neubauer
Expression of Interferon Regulatory Factor 4 in Chronic Myeloid Leukemia: Correlation With Response to Interferon Alfa Therapy
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S. X. Hao and R. Ren
Expression of Interferon Consensus Sequence Binding Protein (ICSBP) Is Downregulated in Bcr-Abl-Induced Murine Chronic Myelogenous Leukemia-Like Disease, and Forced Coexpression of ICSBP Inhibits Bcr-Abl-Induced Myeloproliferative Disorder
Mol. Cell. Biol.,
February 15, 2000;
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F. Rosenbauer, J. F. Waring, J. Foerster, M. Wietstruk, D. Philipp, and I. Horak
Interferon Consensus Sequence Binding Protein and Interferon Regulatory Factor-4/Pip Form a Complex That Represses the Expression of the Interferon-Stimulated Gene-15 in Macrophages
Blood,
December 15, 1999;
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4274 - 4281.
[Abstract]
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M. Scheller, J. Foerster, C. M. Heyworth, J. F. Waring, J. Lohler, G. L. Gilmore, R. K. Shadduck, T. M. Dexter, and I. Horak
Altered Development and Cytokine Responses of Myeloid Progenitors in the Absence of Transcription Factor, Interferon Consensus Sequence Binding Protein
Blood,
December 1, 1999;
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[Abstract]
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L. Gabriele, J. Phung, J. Fukumoto, D. Segal, I-M. Wang, P. Giannakakou, N. A. Giese, K. Ozato, and H. C. Morse III
Regulation of Apoptosis in Myeloid Cells by Interferon Consensus Sequence-Binding Protein
J. Exp. Med.,
August 2, 1999;
190(3):
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W. Kantakamalakul, A. D. Politis, S. Marecki, T. Sullivan, K. Ozato, M. J. Fenton, and S. N. Vogel
Regulation of IFN Consensus Sequence Binding Protein Expression in Murine Macrophages
J. Immunol.,
June 15, 1999;
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H. Baurmann, S. Nagel, T. Binder, A. Neubauer, W. Siegert, and D. Huhn
Kinetics of the Graft-Versus-Leukemia Response After Donor Leukocyte Infusions for Relapsed Chronic Myeloid Leukemia After Allogeneic Bone Marrow Transplantation
Blood,
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[Abstract]
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E. R. Hofman, M. Boyanapalli, D. J. Lindner, X. Weihua, B. A. Hassel, R. Jagus, P. L. Gutierrez, and D. V. Kalvakolanu
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H. Cohen, A. Azriel, T. Cohen, D. Meraro, S. Hashmueli, D. Bech-Otschir, R. Kraft, W. Dubiel, and B.-Z. Levi
Interaction between Interferon Consensus Sequence-binding Protein and COP9/Signalosome Subunit CSN2 (Trip15). A POSSIBLE LINK BETWEEN INTERFERON REGULATORY FACTOR SIGNALING AND THE COP9/SIGNALOSOME
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N. Varela, C. Munoz-Pinedo, C. Ruiz-Ruiz, G. Robledo, M. Pedroso, and A. Lopez-Rivas
Interferon-gamma Sensitizes Human Myeloid Leukemia Cells to Death Receptor-mediated Apoptosis by a Pleiotropic Mechanism
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Abstract
Introduction
Abstract