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Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 764-773
Expression of the Wilms' Tumor Suppressor Gene, WT1, Is
Upregulated by Leukemia Inhibitory Factor and Induces Monocytic
Differentiation in M1 Leukemic Cells
By
Shirley I. Smith,
Dominique Weil,
Gregory R. Johnson ,
Andrew W. Boyd, and
Chung L. Li
From the Queensland Institute of Medical Research (QIMR), Royal
Brisbane Hospital, Herston, Queensland, Australia.
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ABSTRACT |
The Wilms' tumor gene, WT1, encodes a transcription factor
of the Cys2-His2 zinc finger type. The
functional significance of WT1 expression in leukemias, in
addition to tissues and cell lines of hematopoietic origin, has not
been determined. Using the murine myeloblastic leukemia cell line M1 as
a model for macrophage differentiation, expression of WT1 is
shown to be activated in M1 cells 24 hours after differentiation
induction by leukemia inhibitory factor (LIF). Upregulation of
WT1 in these cells is associated with cellular differentiation,
coinciding with expression of the monocyte/macrophage marker
c-fms, and the appearance of mature cells. WT1 isoforms lacking
the KTS insert are unable to be ectopically expressed in M1 cells.
Stable expression of the WT1 isoforms containing the KTS insert leads
to spontaneous differentiation of the M1 myeloblasts through the
monocytic differentiation pathway. These cells express c-fms,
in addition to the myeloid-specific cell surface marker Mac-1. Exposure
of these cells to LIF results in the rapid onset of terminal macrophage
differentiation, accompanied by apoptotic cell death. These results
show that the WT1 gene is an important regulator of M1 cell
monocytic differentiation in vitro, and suggests a potential
role for this gene in the molecular control of hematopoiesis.
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INTRODUCTION |
THE WILMS' TUMOR suppressor gene,
WT1, encodes a member of the Cys2-His2
zinc finger (ZF) family of transcription factors. WT1 possesses two
characteristics which indicate a role in the regulation of gene
transcription four consecutive ZF DNA-binding domains at its
carboxyl terminus, and a proline- and glutamine-rich transregulatory domain at its amino terminus.1 Other
members of this group include the mitogen-inducible early growth
response (EGR) proteins, such as EGR1,2,3 which shares
approximately 60% homology at the amino acid level with WT1 within the
ZF region.4
Two alternative splicing events within the WT1 gene lead to the
production of four distinct isoforms of WT1.5 Alternative splice I (ASI) results in either the presence or absence of 51 bp
(corresponding to exon 5 and encoding 17 amino acids) inserted between
the transregulatory and ZF regions. Alternative splice II (ASII)
results in either the presence or absence of 9 bp, encoding three amino
acids (KTS), inserted between the third and fourth ZF DNA-binding
domains. Splicing of exon 5 is thought to modulate the strength of
DNA-binding and/or transcriptional activity of +KTS
isoforms,6,7 while splicing within the ZF domain confers sequence-specificity of DNA binding. In vitro studies have
shown that isoforms which lack the KTS insert bind an EGR1-like
consensus motif (5 -GNGNGGGNG-3),8 and primarily act as
repressors of gene transcription from a number of promoters which
contain this sequence, such as the platelet-derived growth factor-A
(PDGF-A), macrophage colony-stimulating factor (M-CSF), retinoic acid
receptor- (RAR-), bcl-2, and c-myc
promoters.9-14 WT1 isoforms containing the KTS insert do
not bind the EGR1-like motif, but recognize other sequences whose
biological significance is yet to be characterized.15 In
addition, sequences have been defined to which both ±KTS forms of WT1
show affinity; however, their function is also
unclear.16,17 A role for WT1 outside the control of gene
transcription has been proposed with the discovery of an RNA
recognition motif (RRM) within its amino terminus,18 and
the observation that +KTS forms of WT1 may associate with components of
the mRNA splicing machinery within the nucleus.19,20
First implicated in the development of Wilms' tumor,21,22
an embryonal malignancy of the kidney,23 the WT1
gene has been found to play a vital role in the control of cellular
proliferation and/or differentiation within the genitourinary
system. WT1 is expressed in the early metanephric stem cells,
and increases upon cellular condensation around the ureteric bud and
induction of differentiation into epithelial structures, with
expression eventually confined to the podocyte layer of the
glomeruli.24 Mice null for WT1 exhibit a complete
abrogation in normal kidney development.25
The expression of WT1 in cells of hematopoietic origin has led
to speculation that this gene may also play a role in the molecular control of proliferation and/or differentiation within blood
cell development. WT1 is expressed in the spleen and
thymus,1,26 in addition to CD34+ hematopoietic
stem and progenitor cells (HSCs; HPCs),27 and leukemic cell
lines primarily of myeloid origin.28 Expression of
WT1 in the leukemic cell lines HL60 and K562 is downregulated upon induction of differentiation,29,30 and, in the case of K562 cells, antisense WT1 oligonucleotides have been shown to inhibit proliferation and induce apoptosis.31 WT1
is also expressed in phenotypically immature myeloid leukemias, such as
acute myeloid leukemia (AML) and blast crisis of chronic myeloid
leukemia (CML-BC).32 Genetic analysis has revealed the
mutation rate of WT1 in AMLs to be 15%, with all mutations
predicted to lead to truncation and subsequent disruption of the ZF
DNA-binding domain.33 Expression or mutation of WT1
in acute leukemia appears to be an indicator of minimal residual
disease (MRD) and poor prognosis,27,33-35 with a recent
study suggesting that WT1 is expressed in leukemic cells at a
level ten times higher than that found in normal hematopoietic cells.36
Leukemic cell lines provide useful model systems in which to dissect
the molecular mechanisms responsible for inducing hematopoietic cell
differentiation, and how blocks in these signals lead to disease onset.
The myeloblastic leukemia cell line M1 was generated from a spontaneous
leukemia which arose within the SL strain of mice37 and can
be induced to undergo terminal macrophage differentiation, coupled to
growth arrest and apoptosis of mature cells,38 by leukemia
inhibitory factor (LIF), interleukin-6 (IL-6), or oncostatin M
(OsM).39-41 In this study, we have examined the function of
the WT1 gene in the molecular control of M1 cell macrophage
differentiation. Our results suggest that the WT1 gene has an
important capacity to regulate monocytic differentiation within these
cells, and provides further evidence of a putative role for this gene
in normal hematopoietic cell differentiation, especially within the myelomonocytic lineage.
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MATERIALS AND METHODS |
Cells and cell culture.
The murine myeloblastic leukemia cell line M1 was maintained in
Dulbecco's modified Eagle's medium (DMEM; GIBCO-BRL, Grand Island,
NY) supplemented with 10% fetal calf serum (FCS) (QIMR, Brisbane,
Australia), and kept at 37°C in a humidified atmosphere with 10%
CO2. Viable cell numbers were determined by eosin dye exclusion upon counting in a hemocytometer. Recombinant murine LIF (a
gift from AMRAD Biotech, Melbourne, Australia) was titrated on M1 cells
for optimal differentiation-inducing properties, and added to
experimental cultures at a concentration of 1 ng/mL. Time-course
analyses in the presence or absence of LIF were performed by seeding
cells at a density of 0.1 to 1 × 105 cells/mL, except in
cultures for RNA or DNA extraction in which initial cell concentrations
were adjusted to give less than or equal to 1 × 106
cells/mL at the time of extraction. Clonogenic potential following exposure to LIF was assessed by colony formation in soft agar. After
thorough washing, 200 cells in 1 mL of Iscove's modified Dulbecco's
medium (IMDM; GIBCO-BRL) supplemented with 10% FCS (HyClone
Laboratories, Inc, Logan, UT) and 0.3% Bacto-Agar (DIFCO Laboratories,
Detroit, MI) were plated in 35-mm Petri dishes in the absence of
further stimuli, and kept at 37°C in a humidified atmosphere with 5%
CO2. Colony formation (>50 cells) was scored after 7 days, and the number of clonogenic cells in the original culture was
calculated based on the proportion of cells used in the colony assay.
Vector construction.
The 1.5-kb Sau3AI cDNA fragments encoding the four individual
murine WT1 isoforms were excised from the respective pCMV3-WT1 vectors
(obtained from Dr Jerry Pelletier, Montreal, Canada) and inserted
separately into the BamHI site of the retroviral vector pHEDMP1, enabling constitutive expression of the WT1 splice
forms from the Moloney murine leukemia virus (MMLV) 5-long terminal repeat (LTR). This retroviral vector is a derivative of pHEDMo (obtained from Dr Suzanne Cory, Melbourne, Australia),42
and was constructed by the exchange of the Cla I/EcoRI
MMLV 3-LTR fragment of pHEDMo with the equivalent fragment from the
murine myeloproliferative sarcoma virus (MPSV) 3-LTR obtained from
pMPZen.43 A control vector containing the 1.3-kb
BglII/BamHI cDNA fragment of the neomycin-resistance
gene, excised from pBR-neo, in place of WT1 was also
constructed. All vectors were purified on CsCl2 gradients,
and sequenced (as described below) to verify their identity and ensure
that translation start and stop codons were intact.
Mammalian cell transfection.
M1 cells were electroporated using a BioRad Gene Pulser (BioRad,
Hercules, CA). Briefly, 1 × 107 M1 cells were resuspended
in 0.8 mL of DMEM, and transfected at 280 V, 960 µF with 20 µg of
each pHEDMP1.WT1 vector combined with 2 µg of pSV2Neo
(10:1 wt/wt ratio, respectively), or 10 µg of pHEDMP1.Neo alone as a
control. All vectors were linearized with EcoRI before
electroporation. Geneticin-resistant populations of transfected cells
were selected on 400 µg/mL active G418 (GIBCO-BRL), and subsequently
maintained at a concentration of 200 µg/mL active G418. M1 cells
transfected with the individual WT1 splice forms were
designated (with reference to ASI and ASII, respectively): M1.WT1.1
(+/+); M1.WT1.2 ( /+); M1.WT1.3 (+/); and, M1.WT1.4 (/). M1
cells transfected with the neomycin-resistance gene were designated
M1.Neo. Integration of the transgene in M1 cells was detected by
Southern blotting, and expression of the WT1 transgene determined by both Northern and Western blotting, as described below.
Assay for differentiation.
Morphological differentiation of M1 cells was determined by counting
200 cells on Leishman's-stained cytospin preparations. The degree of
differentiation was enumerated based on the proportion of immature
blast cells to cells at intermediate monocyte and mature macrophage
stages.
Assay for chromosomal DNA fragmentation.
The onset of apoptosis in experimental cultures was determined by
assessing the degree of chromosomal DNA fragmentation within cells
isolated at specific timepoints. Cells (1 × 106) were
resuspended in 0.5% sodium dodecyl sulfate (SDS), 10 mmol/L EDTA, 50 mmol/L Tris-HCl, pH 8.0; and treated with 200 µg/mL proteinase K
(Merck, Darmstadt, Germany) for 1 hour at 50°C. Lysates were further
treated for 1 hour at 50°C with 150 µg/mL RNAase A (Sigma Chemical
Co, St Louis, MO), followed by electrophoresis on a 2% agarose gel in
0.5× TBE (1× TBE is 89 mmol/L Tris-borate, 2 mmol/L EDTA, pH 8.0) buffer.
DNA probes.
Probes for Southern and Northern blotting were prepared by restriction
enzyme digestion followed by purification on agarose gels. The 960-bp
Drd I cDNA fragment of murine WT1 was used to detect
expression of the WT1 transgene. Expression of the murine monocyte/macrophage marker c-fms was detected using the 1.5-kb cDNA BstXI cDNA fragment. Equal loading and quality of RNA
samples on Northern blots was monitored by probing with the 638-bp
Taq I cDNA fragment of murine  -actin. High specific-activity
probes were generated by random priming using Amersham's Megaprime DNA labelling system and [-32P]-dCTP (Amersham, Arlington
Heights, IL).
Genomic DNA extraction, Southern blotting, and hybridization.
Cells for DNA extraction (1 to 5 × 107) were
resuspended in 0.5% SDS, 1 mmol/L EDTA, 100 mmol/L Tris-HCl, pH 8.0, containing 250 µg/mL proteinase K (Merck), and left overnight at
65°C. DNA was purified by chloroform extraction and ethanol
precipitation. After digestion with appropriate restriction enzymes, 10 µg of DNA was electrophoresed on a 0.8% agarose gel in 1× TBE
buffer and transferred onto Hybond-N membrane (Amersham) using standard techniques. Filters were prehybridized in 5× SSPE (1× SSPE is 150 mmol/L sodium chloride, 10 mmol/L sodium phosphate, 1 mmol/L EDTA, pH
7.4), 5× Denhardt's solution, 1% SDS, 10 mmol/L sodium pyrophosphate, 100 µg/mL denatured sheared salmon sperm DNA at 65°C
for 5 hours. Denatured probe was added to the filters and hybridization
allowed to proceed overnight at 65°C, followed by washing under
stringent conditions to 0.5× SSPE, 0.1% SDS at 65°C. Filters were
exposed to X-OMAT AR film (Eastman Kodak, Rochester, NY) using
intensifying screens at 70°C.
RNA extraction, Northern blotting, and hybridization.
Total RNA was extracted from 0.5 to 1 × 108 cells, or
tissue samples, using guanidinium isothiocyanate, and purified by
ultracentrifugation through a CsCl2 cushion.44
Equal amounts (5 µg) of total RNA were run on a 1.2% agarose-400
mmol/L formaldehyde gel in 1× MOPS (20 mmol/L
3-(N-morpholino)propane-sulfonic acid, 5 mmol/L sodium acetate, 1 mmol/L EDTA, pH 7.0) buffer, and
transferred to Hybond-N membrane (Amersham) using standard
techniques. Filters were prehybridized and hybridized as described for
Southern blotting, but were washed to a stringency of 1× SSPE, 0.1%
SDS at 65°C. Filters were autoradiographed as previously described.
Protein extraction, Western blotting, and hybridization.
Cells for protein extraction (1 to 5 × 107) were
lysed in radioimmunoprecipitation assay (RIPA) buffer (0.1% SDS, 1%
sodium deoxycholate, 1% Triton X-100 (Sigma Chemical Co),
150 mmol/L NaCl, 1 mmol/L EDTA, 10 mmol/L Tris-HCl, pH
7.4) containing 100 µg/mL phenylmethylsulfonyl fluoride (PMSF), 2 µg/mL leupeptin, and 2 µg/mL aprotinin (Sigma Chemical Co) as
protease inhibitors. Equivalent amounts (20 µg) of protein lysate
were separated on an SDS-10% polyacrylamide gel, and transferred to
Hybond-ECL membrane (Amersham). An identical gel was stained with
Coomassie Brilliant Blue R-250 (BioRad) to confirm samples were loaded
equally. Cellular expression of murine WT1 was detected using the
cross-species WT (C-19) rabbit anti-human-WT1 IgG antibody (Santa Cruz
Biotechnology, Inc, Santa Cruz, CA), in combination with Amersham's
donkey anti-rabbit-IgG whole antibody conjugated to horseradish
peroxidase, as part of the enhanced chemiluminescence (ECL) Western
blotting analysis system (Amersham). Filters were exposed to BIOMAX MR
film (Eastman Kodak).
Reverse transcriptase-polymerase chain reaction (RT-PCR).
Total RNA (2 µg) was heat denatured at 70°C for 5 minutes, then
reverse transcribed in 1× Perkin-Elmer GeneAmp PCR Buffer II (Roche,
Branchburg, NJ) supplemented with 4.5 mmol/L MgCl2, 1 mmol/L dithiothreitol, 500 µmol/L of each deoxynucleoside
triphosphate (dNTP), 10 µg/mL random hexamers, and 0.4 U of
Inhibit-ACE (5 Prime  3 Prime, Inc, Boulder, CO) using 80 U of
MMLV reverse transcriptase (GIBCO-BRL) in a final volume of 40 µL.
Samples were incubated at 37°C for 2 hours. Aliquots (2 µL) of cDNA
were then used as template for subsequent PCR amplifications. PCR of murine WT1 was performed in 1× Perkin-Elmer GeneAmp PCR
Buffer II supplemented with 1.5 mmol/L MgCl2, 2.5%
formamide, 250 µmol/L of each dNTP, and 40 pmol of each primer using
1.25 U of Perkin-Elmer AmpliTaq DNA polymerase (Roche) in a final
volume of 40 µL. WT1 primers used in this study were directed
to the ZF region of the gene, and consisted of a 5 primer
5-CCCAGGCTGCAATAAGAGATA-3 (exon 7) and a 3 primer
5-ATGTTGTGATGGCGGACCAAT-3 (exon 10). WT1 PCR was performed
with an annealing temperature (AT) of 57°C for 25 cycles. The PCR
product was agarose gel purified and sequenced (described below) to
verify its identity. Amplification of the murine monocyte/macrophage
marker c-fms was carried out in 1× Perkin-Elmer GeneAmp PCR
Buffer II supplemented with 1.5 mmol/L MgCl2, 250 µmol/L
of each dNTP, and 10 pmol of each primer using 1 U of Perkin-Elmer
AmpliTaq DNA polymerase in a final volume of 25 µL. Primers used to
detect c-fms consisted of a 5 primer 5-CTTGCAGGAGGTGTCTGTGG-3 and a 3 primer
5-TTCTGACTCAGGACTTCAGGG-3, and PCR was performed with an AT of
61°C for 28 cycles. The amount and quality of cDNA added to the PCR
was monitored by amplification of murine -actin using the same
conditions as for c-fms except MgCl2 was added to a
final concentration of 2.36 mmol/L. Primers used to detect -actin
consisted of a 5 primer 5-GACATGGAGAAGATCTGGCA-3 and a 3 primer
5-GGTCTTTACGGATGTCAA-CG-3, and PCR was performed with an AT of 60°C
for 20 cycles. All products (5 µL) were run on 1.5% agarose gels in
1 × TAE (40 mmol/L Tris-acetate, 2 mmol/L EDTA, pH 8.5) buffer.
Flow cytometric analysis.
Expression of the cell surface marker Mac-1 was determined by
immunolabeling followed by flow cytometric analysis. Cells
(1 × 106) were resuspended in 50 µL of
phosphate-buffered saline supplemented with 5% FCS (QIMR) (PBS/FCS)
and incubated with 50 µg/mL murine IgG (Sigma Chemical Co) for 5 minutes on ice to block nonspecific Ig binding at Fc- receptor
sites. After washing in PBS/FCS, cells were incubated for 15 minutes on
ice with the biotinylated M1/70.15.11.5 monoclonal antibody which is
specific for the murine myeloid-specific marker Mac-1 present on
monocytes and macrophages. Cells were washed as before, and
Mac-1-stained cells further incubated with streptavidin-FITC conjugate
(Progen Industries Ltd, Brisbane, Australia) for 15 minutes on ice.
After final washing in PBS/FCS, cells were resuspended in 200 µL of
the same buffer containing 2 µg/mL propidium iodide (PI). Control
cells were stained with secondary conjugate or PI alone. Samples were
gated on live cells (as determined by forward light-scattering
properties and lack of PI staining) and analyzed for cell-surface
marker expression by examining 1 × 104 events on a
FACScan or FACS Vantage (Becton Dickinson, San Jose, CA).
DNA sequencing.
All DNA sequencing reactions were performed using the ABI PRISM Dye
Terminator Cycle Sequencing Ready Reaction Kit, followed by analysis on
an ABI PRISM DNA Sequencer Model 377, according to the manufacturer's
instructions (Applied Biosystems, Inc, Foster City, CA). Sequencing of
inserts cloned into the retroviral vector pHEDMP1 was performed using
primers directed to the vector, and consisted of a 5 primer
5-ACGTGAAGGCTGCCGACC-3 and a 3 primer 5-AGCCTGGACCACTGATATCC-3.
WT1 PCR products were sequenced with the primers used in the
original PCR as described above. Sequence comparisons were performed
using FastA default parameters45 within GCG (Genetics
Computer Group, Madison, WI).
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RESULTS |
Expression of WT1 is upregulated during LIF-induced macrophage
differentiation of M1 cells.
To determine whether levels of WT1 expression changed during M1
cell macrophage differentiation, cells were cultured in the presence or
absence of LIF and total RNA isolated at various timepoints. RT-PCR
analysis using primers directed to the ZF region of WT1 showed
that WT1 mRNA was undetectable in undifferentiated parental M1
cells after 25 cycles of PCR amplification (Fig
1A). However, expression of WT1 was
upregulated during M1 cell differentiation induced by LIF, with a
330-bp/321-bp PCR product evident after 24 hours of LIF exposure and
reaching maximal levels after 48 hours. This PCR product corresponds to
±ASII WT1 transcripts, encoding both ±KTS isoforms of WT1,
which migrate as a single band on a 1.5% agarose gel. High levels of
WT1 expression continued until the final timepoint (7 days),
and the level of WT1 expressed in differentiating M1 cells was
comparable to that seen in RNA taken from the kidney of a newborn
(1-day-old) mouse.
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| Fig 1.
Induction of WT1 expression during
LIF-induced macrophage differentiation of M1 cells. M1 cells were
cultured in the presence or absence of LIF (1 ng/mL) for the indicated
timepoints and assessed for: (A) expression of WT1,
c-fms, and -actin as determined by RT-PCR analysis of total
RNA extracted from the cells (or from newborn murine kidney as a
positive control), with the size and position of the
 X174/HaeIII molecular weight markers indicated to the left
of the gels; and (B) morphological differentiation as determined by
Leishman's staining of cytospin preparations of the cells and scoring
the proportion of blast cells to cells at intermediate monocytic and
mature macrophage stages. ( ), M1 +LIF; ( ), M1LIF.
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Upregulation of WT1 in M1 cells exposed to LIF was associated
with cellular differentiation as expression of this gene was coregulated with that of the monocyte/macrophage marker c-fms, the receptor for M-CSF (Fig 1A). After 28 cycles of amplification, c-fms expression in M1 cells, visualized as a 450-bp PCR
product following electrophoresis, was induced from basal levels after 48 hours exposure to LIF. WT1 expression during LIF-induced
differentiation of M1 cells also coincided with the appearance of cells
of differentiated morphology (Fig 1B).
As the WT1 primers used in this study were directed to the ZF
region of the gene, and ZFs are conserved motifs in numerous transcription factors, the PCR product obtained from the WT1
PCR was gel purified and sequenced to verify its identity. Sequence comparison using FastA45 showed this product to be 100%
homologous to the murine WT1 ± ASII ZF domain (data not
shown).
Constitutive ectopic expression of WT1 +KTS isoforms in M1 cells
induces spontaneous monocytic differentiation.
To ascertain whether the observed upregulation of WT1 during M1
cell differentiation induced by LIF was causally related to the
differentiated phenotype of M1 cells, stable cell lines expressing each
of the four individual isoforms of WT1 were established. WT1
constructs were cotransfected with pSV2Neo, and the control neomycin vector transfected alone, into M1 cells, and G418-resistant pools of cells were obtained for each construct. Control M1.Neo cells
were identical to the parental M1 cell line in culture. Upon
Leishman's staining of cytospin preparations M1.Neo cells exhibited
the typical blast morphology of M1 cells, with round, central nuclei
with prominent nucleoli and a high nuclear:cytoplasmic ratio (Fig
2). However, both M1.WT1.1 (+/+) and
M1.WT1.2 (/+) cells (transfected with transcripts which contain the
ASII insert encoding the +KTS isoforms of WT1) spontaneously exhibited
a mixed morphology in culture, consisting of nonadherent cells, in
addition to adherent cell types possessing a flattened, elongated
phenotype (Fig 2). Microscopic examination of M1.WT1.1 (+/+) and
M1.WT1.2 (/+) cells following Leishman's staining showed they had
been induced to differentiate to various stages along the monocytic differentiation pathway, giving rise to a heterogeneous population of
blast, intermediate, and mature monocytic stages (Fig 2). Mature stages
displayed irregularly shaped nuclei, a reduced nuclear:cytoplasmic ratio, vacuolation and reduced basophilia of the cytoplasm, and blebbing of the cytoplasmic membrane. However, in contrast to parental
M1 cells induced for terminal macrophage differentiation by LIF, these
cells did not undergo terminal differentiation and could be passaged
indefinitely as a persistent cell line in culture. Despite the obvious
changes in M1 cells transfected with transcripts encoding the +KTS
isoforms of WT1, it was noted that the G418-resistant pools of M1.WT1.3
(+/) and M1.WT1.4 (/) cells (transfected with transcripts
which lack the ASII insert encoding KTS) displayed little phenotypic
change. Both of these populations more closely resembled parental and
control cells in culture, and upon morphological staining (Fig 2).
Enumeration of the proportion of cell types within each population is
given in Table 1.
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| Fig 2.
Induction of spontaneous monocytic differentiation in M1
cells transfected with WT1 +KTS isoforms. M1 cells stably transfected with retroviral vectors containing cDNAs encoding individual WT1 isoforms were compared with parental cells or control M1.Neo cells generated by transfection with a retroviral vector encoding
neomycin-resistance. Differences in morphology were examined in culture
(original magnification ×200) and by Leishman's staining of cytospin
preparations (original magnification ×800).
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To determine whether the observed differences in phenotype between
cells transfected with either the +ASII or ASII splice forms could
be attributed to differences in the levels of expression of the
WT1 transgenes introduced into the cells, Northern analysis of
RNA from the various populations was performed. Parental and control
(M1.Neo) cells did not exhibit detectable levels of endogenous WT1 (Fig 3A). High levels of stable
ectopic expression of the WT1 transgene in M1.WT1.1 (+/+) and
M1.WT1.2 (/+) cells were observed, with the genomic and subgenomic
retroviral transcripts migrating at approximately 3.4 kb and 2.7 kb,
respectively (Fig 3A). In contrast, barely detectable levels of
exogenous WT1 expression were evident in M1 cells transfected
with the WT1.3 (+/ ) and WT1.4 (/) splice forms (Fig 3A), with
faint transcripts only appearing after a longer exposure of the
autoradiograph (data not shown), despite Southern analysis showing that
these cells did contain the WT1 transgenes (data not shown).
Differences in RNA expression between ±ASII populations also
corresponded to differences in the amount of ±KTS WT1 isoforms being
translated in these cells. Western analysis confirmed that M1.WT1.1
(+/+) and M1.WT1.2 (/+) cells were producing WT1 +KTS proteins of
approximately 54 kD and 52 kD, respectively, corresponding
to the presence or absence of the 17 amino acids encoded by the
alternatively spliced exon 5 (±ASI), whereas M1.WT1.3 (+/) and
M1.WT1.4 (/) cells did not express detectable levels of WT1
KTS isoforms (Fig 3B). Repeated experiments transfecting M1 cells
with the WT1.3 (+/) and WT1.4 (/) splice forms failed to
generate high-level stable expressors of these isoforms in vitro.
This indicated that the transgenes which lacked the ASII insert
encoding KTS were unable to be stably expressed in M1 cells to the same
levels as those which contained the ASII insert and, combined with an
observed fivefold decrease in transfection efficiency for these
transcripts (data not shown), suggests that high levels of expression
of WT1.3 (+/) and WT1.4 (/) transcripts may be detrimental to
M1 cells. However, these results confirmed that the spontaneous
induction of monocytic differentiation observed in M1.WT1.1 (+/+) and
M1.WT1.2 (/+) cells could indeed be attributed to the expression of
WT1 +KTS isoforms in these cells, and these populations were chosen for
further characterization.
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| Fig 3.
Stable ectopic expression of WT1 +KTS isoforms, but not
KTS isoforms, in M1 cells. (A) Northern blotting of total RNA
followed by hybridization with probes to WT1 and -actin,
with the 28S rRNA band shown below to illustrate equal loading of the
samples on the gel, and the position of the 28S and 18S rRNA bands
indicated to the left of the autoradiographs. (B) Western blotting of
protein lysates followed by hybridization with an antibody specific for WT1, with an identical gel stained for total protein shown below to
illustrate equal loading of the samples, and the size and position of
the protein molecular weight markers indicated to the left of the
autoradiograph.
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As M1.WT1.1 (+/+) and M1.WT1.2 (/+) cells appeared to be
morphologically more mature than parental M1 or control M1.Neo cells, they were evaluated for expression of cell surface myelomonocytic markers. Expression of c-fms mRNA was not detectable by
Northern analysis in parental M1 or control M1.Neo cells (Fig
4A). However, the 3.7-kb transcript for
c-fms was expressed in RNA from M1.WT1.1 (+/+) and M1.WT1.2
(/+) cells (Fig 4A), agreeing with the earlier finding suggesting
that WT1 and c-fms are coregulated during M1 cell
macrophage differentiation (Fig 1A). Flow cytometric analysis using an
antibody specific for the Mac-1 complex (or C3bi receptor) showed that
while M1 and M1.Neo cells were negative for cell-surface expression of
this marker, 61% of M1.WT1.1 (+/+) cells and 68% of M1.WT1.2 (/+)
cells now stained positive for Mac-1. These results demonstrated that
M1.WT1 +KTS cells, in addition to exhibiting morphological
characteristics of monocytic differentiation, displayed a number of
markers used to delineate the program of monocyte/macrophage differentiation in M1 cells.
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| Fig 4.
Expression of c-fms and Mac-1 cell surface
myelomonocytic markers in M1.WT1 +KTS cells. (A) Northern blotting of
total RNA followed by hybridization with probes to c-fms and
-actin, with the 28S rRNA band shown below to illustrate equal
loading of the samples on the gel, and the position of the 28S and 18S
rRNA bands indicated to the left of the autoradiographs. (B) Flow
cytometric analysis using a biotinylated antibody specific for Mac-1
followed by fluorescent detection with a streptavidin-FITC secondary
conjugate, with control cells stained with secondary conjugate alone.
(- - -), Control; (), Mac-1.
|
|
Exposure of M1.WT1 +KTS cells to LIF induces the rapid onset of
terminal macrophage differentiation accompanied by apoptotic cell
death.
As M1.WT1 +KTS cells were phenotypically more mature than M1 or M1.Neo
cells, their response upon exposure to LIF compared with parental and
control cells was investigated. Cells were cultured in the presence or
absence of LIF and isolated at the indicated timepoints. Cultures were
assessed for cellular proliferation (determined by counting viable cell
numbers), clonogenic potential (determined by agar colony formation in
the absence of LIF), and differentiation (determined by Leishman's
staining of cytospin preparations). Compared with untreated cells, upon
exposure to LIF M1 and M1.Neo cells underwent a limited phase of
proliferation (Fig 5A) coupled to a gradual
loss in the ability to form colonies in soft agar (Fig 5B). Loss of
clonogenicity in these cells could be attributed to an increase in
their differentiative state over the 5-day period (Fig 5C). In the
absence of LIF, M1.WT1.1 (+/+) and M1.WT1.2 (/+) cells proliferated
at a similar rate to parental M1 and control M1.Neo cells (Fig 5A).
However, treatment of these cells with LIF resulted in an immediate
suppression of proliferation, and a reduction in cell viability in
these cultures. Few clonogenic cells could be detected from these
cultures after 24 hours of exposure to LIF (Fig 5B). Examination of
Leishman-stained cytospin preparations showed that inhibition of
proliferation and colony formation in M1.WT1.1 (+/+) and M1.WT1.2
(/+) cells after exposure to LIF was coupled to the induction of
terminal macrophage differentiation of these cells. Starting from a
basal level of containing approximately 60% differentiated cell types
prior to LIF treatment, the proportion of differentiated cells within
these cultures rose to almost 95% after 24 hours of exposure (Fig 5C).
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| Fig 5.
Exposure of M1.WT1 +KTS cells to LIF results in the
rapid suppression of proliferation and clonogenicity associated with
the induction of terminal macrophage differentiation. M1 cell
populations were cultured in the presence or absence of LIF (1 ng/mL)
for the indicated timepoints and assessed for: (A) cellular
proliferation as determined by counting viable cell numbers on eosin
dye exclusion; (B) clonogenic potential as determined by colony
formation in soft agar in the absence of LIF; and (C) differentiation
as determined by Leishman's staining of cytospin preparations and
scoring the proportion of blast cells to cells at intermediate
monocytic and mature macrophage stages. (), M1 +LIF; (), M1
LIF; ( ), M1.Neo +LIF; ( ), M1.Neo LIF; ( ), M1.WT1.1
(+/+) +LIF; (), M1.WT1.1 (+/+) LIF; ( ), M1.WT1.2
(/+) +LIF; ( ), M1.WT1.2 (/+) LIF.
|
|
After terminal macrophage differentiation of M1 cells induced by LIF or
IL-6, mature cells undergo programmed cell death, or apoptosis,
indicated by the onset of chromosomal DNA fragmentation.38 As M1.WT1 +KTS cells exposed to LIF exhibited a decrease in cell viability, cultures were examined for the initiation of chromosomal DNA
fragmentation suggestive of apoptosis. Electrophoresis of DNA from
M1.WT1.2 (/+) cells exposed to LIF showed that DNA fragmentation was
induced in these cells 3 days after culture initiation (Fig 6). M1.WT1.2 (/+) cells left untreated,
and parental M1 cells untreated or exposed to LIF, for the same period
of time did not exhibit DNA fragmentation (Fig 6). The results obtained
for M1.WT1.1 (+/+) cells were identical to those which were obtained
for M1.WT1.2 (/+) cells, and control M1.Neo cells mimicked parental
M1 cells (data not shown).
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| Fig 6.
Exposure of M1.WT1 +KTS cells to LIF results in the
rapid onset of apoptotic cell death. M1 cell populations were cultured in the presence or absence of LIF (1 ng/mL) for the indicated timepoints and assessed for chromosomal DNA fragmentation by
electrophoresis of the extracted DNA on 2% agarose gels, with the size
and position of the X174/HaeIII molecular weight markers
indicated to the left of the gels.
|
|
| |
DISCUSSION |
This investigation provides evidence that the Wilms' tumor suppressor
gene, WT1, is a key mediator in the molecular control of
monocyte/macrophage differentiation in the murine myeloblastic leukemia
cell line M1. Although parental M1 cells do not express detectable
levels of WT1, LIF-induced macrophage differentiation of these
cells leads to the activation of WT1 expression after 24 hours,
and coincides with the upregulation of the monocyte/macrophage marker
c-fms and the appearance of mature cells (Fig 1). The notion that WT1 was playing a role in directing macrophage
differentiation within M1 cells was examined by ectopically expressing
WT1 isoforms in M1 cells. WT1 isoforms which lacked the KTS insert
could not be stably expressed in M1 cells (Fig 3). However, stable
ectopic expression was achieved with the +KTS isoforms of WT1, and
resulted in the spontaneous differentiation of M1 blasts along the
monocytic lineage. This gave rise to steady-state mixed populations of
cells, comprised of blasts, promonocytes, and monocytes (Fig 2 and
Table 1), which exhibited de novo expression of the
myelomonocytic markers c-fms and Mac-1 (Fig 4). Exposure of
M1.WT1 +KTS cells to LIF resulted in the rapid onset of terminal
macrophage differentiation (Fig 5), which was followed by apoptotic
cell death (Fig 6). The process of LIF-induced terminal
differentiation, growth suppression and cell death in M1.WT1 +KTS cells
was accomplished within 3 days, as opposed to 7 days for parental M1
cells exposed to LIF. This short-circuiting of the program of M1 cell
differentiation within M1.WT1 cells most likely represents the
completion of the normal LIF-induced pathway of macrophage
differentiation spontaneously initiated in these cells by the stable
expression of WT1 +KTS isoforms (Fig 7).
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| Fig 7.
A schematic representation of the program of LIF-induced
M1 cell differentiation (adapted and reprinted with permission from Selvakumaran et al)38 compared with that of
M1.WT1 +KTS cells, showing the upregulation or downregulation of key
genes involved in the cascade of events which mediate terminal
differentiation associated with growth suppression and induction of
apoptosis.
|
|
This study suggests that although WT1 +KTS isoforms are able to induce
monocytic differentiation within M1 cells, a second signal evoked by
LIF is required to complete the program of terminal macrophage
differentiation initiated by WT1 expression in these cells. Therefore,
in addition to the implications concerning the role of WT1 in
directing monocytic differentiation, the results of this study have
also revealed aspects of the signaling pathway which stems from the LIF
receptor complex. M1.WT1 +KTS cells provide a model which illustrates
that the monocytic differentiation program is a multi-step process that
can be arrested at intermediate stages, and uncoupled from terminal
differentiation normally associated with growth suppression and cell
death. This implies that divergent signaling pathways within M1 cells
are responsible for eliciting the separate responses induced by LIF
(Fig 8). One pathway involves the +KTS
isoforms of WT1 and the direct, or indirect, regulation of
monocyte/macrophage lineage-specific genes. The second pathway appears
to be responsible for the regulation of genes involved in the molecular
control of cell-cycle arrest and induction of cell death. The combined
cellular responses elicited by both of these pathwaysdifferentiation,
growth suppression, and apoptosistherefore fulfil the complete
program of terminal macrophage differentiation induced within M1 cells
by LIF (Fig 8).
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| Fig 8.
A schematic representation of events in the M1 cell
nucleus which stem from the LIF receptor complex, highlighting the
involvement of WT1 +KTS isoforms in the direct, or indirect,
regulation of lineage-specific genes, and the suggestion of a separate
LIF-induced pathway of programmed cell death. The role of WT1 KTS
isoforms in this pathway, and the potential for interactions between
±KTS isoforms of WT1, is unclear.
|
|
However, the data contained in this report indicate that WT1 may play a
role in both pathways. The observation that endogenous WT1 KTS
isoforms are also upregulated during LIF-induced M1 cell differentiation (Fig 1), combined with their potential to regulate genes involved in both cell survival (repressing bcl-2 and
M-CSF expression) and cell death (activating p21/WAF1
expression),14,12,46 suggests that these isoforms may
mediate the progression toward growth suppression and cell death in
M1.WT1 +KTS cells (Fig 8). The inability to establish cell lines that
stably express high levels of KTS isoforms, as described in this
report for M1 cells but observed in additional cell lines by other
investigators,47-49 suggests that these isoforms exert
potent growth suppressive effects. Recent work by Murata et
al,50 using an IPTG-inducible expression system in
which expression of the WT1.3 (+/) transcript induces cell-cycle
arrest and apoptotic cell death in M1 cells, also supports this
concept. Therefore, the apparent dissociation of the differentiation
and growth suppression pathways in M1.WT1 +KTS cells may at least in
part be due to the requirement for both ±KTS isoforms to act
cooperatively to induce the complete program of M1 cell
differentiationa requirement that is only fulfilled in +KTS cells
upon upregulation of endogenous KTS isoforms of WT1 by exposure to
LIF.
This investigation has demonstrated a number of key observations: (1)
expression of WT1 is upregulated during LIF-induced M1 cell
macrophage differentiation; (2) +KTS isoforms of WT1 have the potential
to direct lineage-specific gene expression and induce monocytic
differentiation in M1 cells; (3) exposure of M1.WT1 +KTS cells to LIF
induces rapid growth suppression and cell death, possibly through the
upregulation of endogenous KTS WT1 isoforms; and (4) ±KTS isoforms
of WT1 have distinct roles in mediating differential effects within M1
cells during induction of macrophage differentiation by LIF. The role
of WT1 expression in normal HSC and HPC populations within the
bone marrow,27 and how disruption of its function may lead
to the onset of leukemia, is not yet understood. The demonstration of
distinct biological actions of ±KTS isoforms in M1 cells, namely
induction of either differentiation or cell death, may identify new
approaches in the understanding of how WT1 may contribute
leukemogenesis.
The abolition of WT1's transregulatory properties through mutation and
truncation of the ZF DNA-binding domain has the capacity to severely
disrupt normal hematopoiesis.33 The potential for ±KTS
isoforms of WT1 to induce cellular differentiation coupled with growth
suppression would be lost, leading to the uncontrolled proliferation
and persistence of leukemic blasts, and may explain why these leukemias
behave in a more aggressive fashion.33 Overexpression of
WT1 in HSCs may be another mechanism by which these cells
become leukemic,36 although it has yet to be determined
whether certain isoforms of WT1 are preferentially affected.
WT1 splice form expression, at least at an mRNA level, appears
highly conserved throughout a number of normal tissues
studied5; therefore, an imbalance in the expression of WT1
isoforms in these cells could conceivably have important implications
for normal development. The ability of WT1 to
self-associate,51,52 combined with the observation that
+KTS WT1 isoforms are able to modulate the subcellular localization and
activity of KTS isoforms (despite both isoforms localizing to
distinct regions within the nucleus),19,20 highlights this
as an area for further investigation. The use of cell lines, such as
M1, which can be induced for single-lineage differentiation and
programmed cell death may provide useful model systems with which to
dissect the function of individual WT1 isoforms in the molecular
control of hematopoiesis.
| |
FOOTNOTES |
Deceased.
Submitted September 18, 1997; accepted November 10, 1997.
Supported by the Leukaemia Foundation of Queensland, the Queensland
Cancer Fund, and the University of Queensland, Brisbane, Australia.
S.I.S. was a Queensland Cancer Fund John Earnshaw Scholar. D.W. was a
post-doctoral fellow of the University of Queensland.
Address reprint requests to Shirley I. Smith, Queensland
Institute of Medical Research, Post Office, Royal Brisbane Hospital, Herston Rd, Herston, Queensland, 4029, Australia.
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 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Jerry Pelletier who provided the murine
WT1 cDNA splice forms; Dr Suzanne Cory for providing the
retroviral expression vector pHEDMo; Drs Elizabeth Algar, Melissa
Little, and Doug Hilton for helpful discussion; and Shan Li Liu for
expert technical assistance.
| |
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Licht J,
Little M:
Two N-terminal self-association domains are required for the dominant negative transcriptional activity of WT1 Denys-Drash mutant proteins.
Biochem Biophys Res Commun
233:723,
1997[Medline]
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Abstract
Introduction
Abstract