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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4263-4268
Modulation of Granulocyte-Macrophage Colony-Stimulating Factor Gene
Expression by a Tumor Necrosis Factor  Specific Ribozyme in Juvenile
Myelomonocytic Leukemic Cells
By
Per Ole Iversen and
Mouldy Sioud
From the Department of Physiology, Institute of Basic Medical
Sciences, University of Oslo, Oslo, Norway; and the Institute for
Cancer Research, Department of Immunology, the Norwegian Radium
Hospital, Oslo, Norway.
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ABSTRACT |
The human cytokines tumor necrosis factor  (TNF) and
granulocyte-macrophage colony-stimulating factor (GM-CSF) both promote growth and survival of malignant cells from children with juvenile myelomonocytic leukemia (JMML). It has been postulated that TNF stimulates GM-CSF gene expression in an autocrine manner. We found here
that the specific inhibition of TNF gene expression by a catalytic
RNA molecule (ribozyme) also downregulated the expression of GM-CSF in
JMML cells. GM-CSF protein, GM-CSF-dependent colony formation, and
viability of JMML cells were reduced. The observed effect was specific,
because synthesis of interleukin-1 , another cytokine produced by
JMML cells, was not affected by the ribozyme treatment. The stimulatory
effect of TNF on GM-CSF gene expression in JMML cells probably takes
place at the transcription level, because the ribozyme treatment
decreased GM-CSF mRNA. No apparent toxicity of the ribozyme was
detected in normal bone marrow progenitor cells. Thus, the inhibition
of TNF gene expression in JMML cells by ribozymes may be a novel
therapeutic approach for this disorder.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
JUVENILE myelomonocytic leukemia (JMML),
also named juvenile chronic myelogenous leukemia or childhood chronic
myelomonocytic leukemia, is a myelodysplastic disorder affecting
infants and small children.1,2 Although the exact diagnosis
of JMML has been the subject of some controversy,2,3 all
epidemiological studies have shown a low incidence.2,4 The
aggressive nature of JMML is reflected in its resistance to
chemotherapeutic treatment5,6 and the fact that a
considerable number of patients relapse after bone marrow
transplantation.7,8
The molecular mechanism underlying the pathogenesis of JMML is
incompletely understood. JMML has been associated with
neurofibromatosis type 1 (NF1).9 NF1 patients typically
lack the NF1 tumor suppressor gene activity, thus possibly leading to
malignant transformation and uncontrolled proliferation of myeloid
cells as a consequence of a constitutive activation of the ras
oncogene.10-12 Interestingly, the hemopoietic cytokine
granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates
ras activation.13 A growing body of evidence shows
that GM-CSF is a stimulatory cytokine in JMML, because it both promotes
JMML cell growth14-17 and inhibits apoptosis of JMML
cells.18
Tumor necrosis factor (TNF) is another significant regulator in
JMML. Anti-TNF monoclonal antibodies (MoAbs) inhibit JMML cell
growth,17,18 TNF suppresses normal bone marrow
hemopoiesis,17 and the immunomodulatory cytokine
interleukin-10 (IL-10) downregulates both GM-CSF and TNF synthesis
in JMML cells, leading to a concomitant reduction in growth and
viability of these cells.19 Moreover, immunodeficient mice
engrafted with JMML cells produce high levels of human
TNF.20,21 Importantly, we recently demonstrated that, whereas a neutralizing human anti-TNF MoAb did not affect the disease progress in immunodeficient mice engrafted with JMML cells and
a human GM-CSF antagonist prevented dissemination of the disease, only
a combined blockade of GM-CSF and TNF eradicated the leukemic cells
in the mice.21 Possibly there is an autocrine loop
involving both TNF and GM-CSF in JMML.17 To clarify
this, we have now investigated the effect of an exogeneously delivered
TNF ribozyme on GM-CSF gene expression in JMML cells.
Ribozymes are RNA molecules with catalytic activity.22,23
Recent experiments indicate that ribozymes can be designed to cleave in
trans virtually any mRNA whose sequences are known.24-27 However, despite this significant progress of ribozyme engineering, there is still a need to optimize ribozyme function in the complex intracellular environment. In the present study, we examined if an
anti-TNF ribozyme could be delivered to primary JMML cells and
whether it could affect TNF and GM-CSF gene expression and, hence,
JMML cell growth and viability. Our data indicate that this novel
strategy is amenable for studying the effect of TNF on GM-CSF gene
expression in JMML cells and suggest that treatment of JMML patients
with ribozymes may be feasible.
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MATERIALS AND METHODS |
Donors and Cells
This investigation was approved by ethics committees, and parental
consent was given to study bone marrow samples collected from 3 children aged 1 to 4 with diagnosed JMML according to criteria defined
by the International Juvenile Myelomonocytic Leukemia Working
Group.2 Bone marrow cells from a healthy child (2 years) were used as a control.
Mononuclear cells were prepared from bone marrow by dextran
sedimentation of erythrocytes and using density centrifugation as
previously described.18 More than 99% of these isolated
cells from the JMML patients and about 50% of the cells from the
healthy child expressed surface receptors for GM-CSF and TNF, as
detected with MoAbs and flow cytometry (data not shown).
The Anti-TNF Ribozyme
Ribozyme synthesis.
A double hammerhead ribozyme (Rzd) having as cleavage sites a CUC and a
GUC corresponding to the codon for leu  60 and val +1,
respectively, within human TNF mRNA was synthesized by in vitro
transcription using DNA oligodeoxynucleotide and the T7 RNA polymerase
as described previously.26 Briefly, two overlapping half
deoxynucleotides containing the T7 promoter sequence and the sequence
coding for the ribozyme minigene were hybridized and then extented with
the Klenow fragment of DNA polymerase. After extension, the DNA
was gel-purified and then used as template for in vitro transcription.
The sequences of the overlapping primers are as follows: primer 1:
5 -TAATACGACTCACTATAGAAGATGATCTCTGATGAGTCCGTGAGGACGAAACTGCCTGGGCAAAAA-3; primer 2:
5-TGGAGGCGCTTTCGTCCTCACGGACTCATCAGCCCAATTTTTGCCCAGGC-3.
The sequence of the double ribozyme Rzd is as follows:
5-GAAGAUGAUCUCUGAUGAGUCCGUGAGGACgAAACUGCCUGGGCAAAAACUUCUUGGGCUGAUGAGUCCGUGAGGACgAAAGCGCCUCCUC-3. The hammerhead core sequence is underlined, and bold letters correspond to an adenosine linker. A noncleaving hammerhead ribozyme (Rzdm) was
made by deleting the G12 from the catalytic core of the ribozyme as
indicated by the lower case letter. The in vitro-transcribed ribozymes Rzd and Rzdm were gel-purified and their concentrations were
estimated by measurement of absorbency at 260 nm.
RNA substrates.
The 23 nucleotide (nt) target RNA
(5-GCCCAGGCAGUCAGAUCAUCUUC-3) corresponding to the
ribozyme site b (Fig 1A) was synthesized by in vitro transcription using the following deoxynucleotides: deoxynucleotide I:
5-GAAGATGATCTGACTGCCTGGGCTATAGTGAGTCGTATTA-3; deoxynucleotide II (T7 promoter): 5-TAATACGACTCACTATAG-3.
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| Fig 1.
(A) Schematic diagram of the ribozyme Rzd cleavage sites
and cleavage products. The closed arrows show the two sites of cleavage
(a and b), whereas the open arrow indicates the end of the translated
region (leu 157). (B) Cleavage of the 732 nt target TNF RNA with
(+) or without () ribozyme Rzd. The data represent the
PhosphorImager printout of 8% denaturing polyacrylamide gel. The
arrows starting from the top indicate the cleavage fragments no. 1, 3, and 2 depicted in (A), respectively.
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The 732 nt target RNA was synthesized by in vitro transcription using a
polymerase chain reaction (PCR)-amplified cDNA as template. Briefly,
total RNA prepared from endotoxin-stimulated (100 ng/mL) peripheral
blood mononuclear cells was reverse transcribed using a TNF specific
primer (5-TCGCCACTGAATAGTAGGGCGATTA-3). The cDNA was
PCR-amplified using a second primer specific for TNF and containing
at its 5 end the T7 promoter top strand
(5-TAATACGACTCACTATAGATGAGCACTGAAGCATG-3). The expected
amplified product (749 bp, including the T7 promoter sequence) was
purified and in vitro-transcribed using the T7 RNA polymerase. The in
vitro-transcribed 23 nt RNA substrate was gel-purified and
dephosphorylated, and the 5 end was labeled using
[ -32P]ATP and polynucleotide kinase. The 732 nt
substrate was internally labeled with [-32P]ATP during
in vitro transcription.
In vitro hammerhead ribozyme cleavage activity.
Cleavage reactions were performed at 37°C in buffer containing 50 mmol/L Tris-HCl (pH 7.4) and 10 mmol/L MgCl2 for 1 hour. Cleavage products were separated by electrophoresis on an 8%
polyacrylamide gel containing 7 mol/L urea and visualized by a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Transfection experiments.
The cells were transfected with cationic liposomes (DOTAP; Boehringer
Mannheim, Mannheim, Germany) at a concentration of 25 µg/mL either
alone or complexed with the test molecules as described previously.27 To study the kinetics of uptake of our
ribozyme into the JMML cells, we transfected cells simultaneously with two batches of liposomes: some containing the ribozyme Rzd and some
containing an irrelevant oligonucleotide 5 labeled with fluorescein. The concentration of the oligonucleotide versus the ribozyme was 1 to 5. The uptake rate into these cells was analyzed with
flow cytometry (FACScan; Becton Dickinson, Mountain View, CA).
Colony Assay, Determination of Cell Viability, and Cytokine
Production
Cells from the JMML patients or the healthy child (105
cells per plate) were cultured for 2 weeks either in methylcellulose or
agar.18 The cells were incubated in liquid culture (RPMI medium) with liposomes containing the desired oligonucleotide for
approximately 10 to 15 hours before they were plated. Colonies (>40
cells) were then scored, plucked, and stained
supravitally.28
To examine cell viability, JMML cells or normal bone marrow progenitor
cells were grown in liquid culture for specified times before they were
stained with propidium iodide and analyzed with flow
cytometry.18
From the liquid cell cultures we collected samples to measure the
protein concentrations of GM-CSF, TNF, and IL-1 using specific
enzyme-linked immunosorbent assay (ELISA) kits (sensitivity, >0.5
pg/mL; R&D Systems, Minneapolis, MN).
We also measured the mRNA for GM-CSF, TNF, and granulocyte
colony-stimulating factor (G-CSF) in cells from the liquid cultures and
using an RNAse protection assay.29 Gels were quantified with a PhosphorImager. Values are expressed relative to the GAPDH signal, with corrections made to incorporate different specific activities of the probes.
Recovery of the Ribozyme Rzd From Agar Sectors
We added 400 µL TE buffer and 400 µL phenol:chloroform:isoamyl
alcohol mixture to each agar sector. Samples were shaken vigorously and
then spun for 10 minutes at 4°C. The top aqueous phase was carefully removed, 40 µL of 3 mol/L sodium acetate (pH 7.0) was added, and then the RNA was precipitated by 2.5 vol of ethanol at
70°C for 12 hours. After precipitation and washing with 70% ethanol, each RNA pellet was dissolved into 10 µL water and then tested for cleavage.
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RESULTS |
In Vitro Cleavage of Ribozyme Rzd
A ribozyme (Rzd) directed to site a and b, as shown in Fig 1A, was
synthesized by in vitro transcription, and its in vitro cleavage activity was determined using the 732 nt target TNF mRNA.
Because the ribozyme is directed to two different sites, cleavage at
site a and/or site b should generate 5 different cleavage fragments. To visualize the smallest fragments, numbered as 1, 2, and
3, the cleavage products were analyzed by 8% denaturating polyacrylamide gel. Figure 1B shows that the ribozyme Rzd cleaved the in vitro-transcribed target RNA at the expected sites. The cleavage products with high molecular weights were not separated from
the target RNA in our electrophoresis conditions.
Ribozyme Uptake by JMML Cells and Diffusion Within the Agar Plate
To determine the uptake rate of the ribozyme Rzd into JMML cells, we
simultaneously transfected the cells with two batches of liposomes, one
containing Rzd (1 µmol/L) and one with an irrelevant oligonucleotide
(0.2 µmol/L) labeled with a fluorescent molecule, and then we
analyzed the samples with flow cytometry.
Figure 2 shows that, by 12 hours, almost
all the cells had taken up the fluorescent oligonucleotide, and it
remained with the cells throughout a 72-hour period during which we did
our measurements.
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| Fig 2.
Ribozyme uptake kinetics in JMML cells. The cells were
transfected with two types of liposomes, one containing the ribozyme
Rzd (1 µmol/L) and one containing a 20-nucleotide fluorescein-labeled
oligonucleotide (0.2 µmol/L). Samples were taken at various time
points and cell-associated fluorescence was analyzed with flow
cytometry. Values are the means ± SEM from triplicate measurements of
JMML cells taken from 1 patient and representative of the 2 other
patients.
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In the next set of experiments, we have analyzed the diffusion of the
cationic liposomes containing Rzd within different sectors of an agar
plate after 2 weeks of incubation with JMML cells
(Fig 3A). Five agar sectors were excised
from the plate, and the Rzd from each agar sector was extracted. Half
of the recovered material from each sector was assayed for cleavage
using a short substrate corresponding only to site b. It is evident
from Fig 3B that significant and comparable amounts of cleavage
activity were recovered from all sectors, suggesting a homogeneous
distribution of the ribozyme Rzd within the entire agar plate.
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| Fig 3.
(A) Schematic diagram of a wedge of an agar plate. Sector
1 represents the center, whereas sector 5 represents the periphery of
the plate. (B) Cleavage of the short substrate (site b) by the
recovered ribozyme Rzd from each agar sector (1 through 5) as shown in
(A). The arrow points to the cleavage products, whereas ()
represents a negative control.
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Toxicity
To examine possible toxic effects of ribozyme Rzd, we studied colony
formation of normal bone marrow progenitor cells receiving GM-CSF and
stem cell factor (10 ng/mL) and cocultured with Rzd (1 µmol/L). No
decrease in either the number of colonies scored or the phenotypes of
the colonies was observed after 2 weeks
(Table 1). In one experiment, ribozyme Rzd
(1 µmol/L) was added to the plates after 1 week. However, this extra
addition did not affect colony formation by normal bone marrow
progenitors. No apparent toxicity was observed when using Rzd at
concentrations up to 10 µmol/L (data not shown).
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Table 1.
No Inhibition of Colony Formation of Normal Bone Marrow
Progenitor Cells by Treatment With the Anti-TNF Ribozyme Rzd
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JMML cell viability in liquid culture upon incubation with the
ribozymes Rzd or Rzdm is shown in Fig 4. An
approximately 40% decrease in viability could be detected after 72 hours with Rzd (1 µmol/L). No significant activity of the Rzdm (1 µmol/L) was detected. This finding indicates that the effect of Rzd
is due to its cleavage activity rather than to its antisense
effect.26
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| Fig 4.
Decrease in viability of JMML cells supplemented with
ribozyme Rzd. JMML cells were kept in liquid culture only (none), with
only liposomes (DOTAP), or with liposomes containing the ribozymes Rzd
or Rzdm. After 72 hours of incubation, the cells were harvested and
their viability was assessed with flow cytometry after staining with
propidium iodide. Results are the mean + SEM based on
triplicate measurements from 1 patient and are representative of the 2 other patients.
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Notably, normal bone marrow progenitor cells survived the presence of
ribozyme Rzd for a 24-hour period in liquid medium only, and survival
was prolonged to 72 hours when G-CSF (10 ng/mL) and stem cell factor
(10 ng/mL) were added (data not shown).
Cytokine Production by JMML Cells Receiving Exogenous Ribozyme
The effects of 48 hours of incubation with the test molecules Rzd or
Rzdm (1 µmol/L) on the synthesis of TNF, GM-CSF, and IL-1 by
the JMML cells are shown in Table 2. The
data indicate that both the constitutively produced TNF and GM-CSF
were inhibited by Rzd. This inhibition was specific, because no effect
was observed with the IL-1 production, another cytokine implicated
in the pathogenesis of JMML.30 Relative to the production
of cytokines in cells without addition of Rzd, the inhibitory effect of
ribozyme Rzd was more marked for TNF (~70%) than for GM-CSF
(~60%).
The inhibitory effect of ribozyme Rzd on TNF gene expression was
further established by a decreased mRNA levels for TNF (~60%)
and, importantly, for GM-CSF (~30%), whereas the level of mRNAs for
G-CSF was unaffected by this treatment (Fig
5).
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| Fig 5.
Ribozyme Rzd inhibits synthesis of mRNAs for TNF and
GM-CSF. JMML cells were incubated alone (none), with only liposomes
(DOTAP), or with the ribozymes Rzd or Rzdm for 48 hours before the
cells were collected and total RNA was extracted. Cytokine mRNA levels
were then estimated with an RNAse protection assay. Results are
expressed relative to the mRNA level of the internal GAPDH standard,
and representative data from 1 of the 3 patients are shown. ( )
GM-CSF; ( ) TNF; ( ) G-CSF.
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Colony Formation
To examine the effect of ribozyme Rzd on the proliferative capacity of
JMML cells, we counted the numbers of colonies formed by plating JMML
cells in the presence or absence of the test molecules Rzd and Rzdm (1 µmol/L) under semisolid conditions using methylcellulose. As can be
seen, for example in patient no. 3, the number of colonies was reduced
from approximately 100 to approximately 15 after treatment with
ribozyme Rzd (Fig 6A). On the average,
ribozyme Rzd exerted a marked inhibition on JMML cell proliferation,
leading to a reduction of approximately 60% in colony numbers (Fig
6A). No further inhibition was observed with ribozyme Rzd added extra
(5 µmol/L) after 1 week (data not shown). The mutated ribozyme Rzdm
also reduced JMML colony formation, albeit less markedly than ribozyme
Rzd. Exogenous supplement of either TNF (10 ng/mL) or GM-CSF (10 ng/mL) not only abolished the inhibitory effect of ribozyme Rzd, but markedly enhanced JMML colony formation (Fig 6B), again indicating the
specificity of the ribozyme treatment.
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| Fig 6.
Inhibition of colony formation of JMML cells supplemented
with ribozymes. JMML cells were plated for 2 weeks either alone (none),
with only liposomes (DOTAP), or with ribozymes (Rzd/Rzdm). (A) The
ribozyme Rzd markedly reduced JMML cell colony formation, whereas the
ribozyme mutant Rzdm had less pronounced effects. (B) Exogenous supply
of either GM-CSF (10 ng/mL) or TNF (10 ng/mL) abolished the
inhibitory effect of Rzd and profoundly increased JMML cell colony
numbers. Triplicate measurements were made from each of the 3 patients,
and the obtained median values were used to calculate the mean + SEM shown. Note the different abscissa values in (A) and
(B). ( ) Patient no. 1; () patient no. 2; () patient no. 3.
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DISCUSSION |
Methodological Considerations and Toxicity
Efficient use of ribozymes to regulate gene expression requires
sufficient delivery of these molecules into the cells and that the
molecules are resistant to degradation within the cells. JMML cells
transfected with liposomes containing an oligonucleotide tagged to a
fluorochrome remained fluorescently labeled from 12 hours
posttransfection and throughout our experimental period. These cells
were simultaneously transfected with liposomes containing the ribozyme
Rzd. In addition, we could recover significant catalytic activity in
samples collected from agar sectors with JMML cells that had been
transfected with liposomes and ribozyme Rzd before plating. We
therefore believe that substantial amounts of ribozyme Rzd were taken
up by the JMML cells; thus, any limited delivery or lack of stability
of ribozyme Rzd did apparently not influence our data. For possible
clinical use, further experiments are needed to establish the delivery
and stability of ribozyme Rzd in patients.
We could not attribute any toxic effects to ribozyme Rzd when studying
colony formation or survival of normal bone marrow progenitor cells.
Ribozymes targeted to mouse TNF inhibited endotoxin-induced TNF
gene expression when administered intraperitoneally in mice and these
mice survived, whereas their littermates receiving endotoxin, but not
ribozyme, all died.31 The use of ribozyme Rzd might be
safe, but this needs to be verified during in vivo conditions.
Anti-TNF Ribozyme Inhibits Both TNF and GM-CSF Gene Expression
in JMML Cells
The downregulation of the TNF gene expression by ribozyme Rzd offers
a novel way of interfering with cellular processes in which TNF
plays an important regulatory role. By selectively inhibiting TNF
gene expression in JMML cells, our data indicate that a direct loop of
regulation exists between the cytokines TNF and GM-CSF. Indeed, the
ribozyme Rzd did decrease both TNF and GM-CSF gene expression at the
level of both protein and mRNA.
We found that, whereas the mutant ribozyme Rzdm did not affect JMML
cell survival (Fig 4), it partly inhibited JMML colony formation (Fig
6A). The survival assay was performed with cells cultured in liquid
medium. Possibly partially transfected cells could still secrete TNF
protein, which could act in a paracrine way on other cells. It is
possible that TNF gene expression has to be downregulated below a
certain threshold for apoptosis to occur, and this threshold might be
reached with the ribozyme Rzd, but not by its mutant form Rzdm. The
inhibitory effect of Rzdm on JMML colony formation suggests that an
antiproliferative effect among JMML cells might not require a strong
inhibition of TNF gene expression. At any rate, Rzdm reduces TNF
protein and the clonal growth of JMML cells to an intermediate level,
thus confirming the linkage between TNF and the clonal growth in
JMML. In addition, the diffusion of cytokines within the semisolid
methyl cellulose medium might be slower than in liquid medium, so that
a rescue of transfected cells by some cell-secreted TNF might take
place.
The effect of ribozyme Rzd was specific, because no effect on the
production of IL-1 was observed. The reduction of GM-CSF mRNA by Rzd
treatment suggests that the effect of TNF on GM-CSF gene expression
is mediated by either a transcriptional mechanism or alternatively
through catabolism of GM-CSF mRNA. In line with the former, we recently
showed that the triplex-forming oligonucleotide GM3 directed against a
binding site for the transcription factor NF- B within the GM-CSF
gene promoter inhibited GM-CSF gene expression.29 Interestingly, we then also noted that neutralizing TNF with an
anti-TNF MoAb resulted in a complete inhibition of the constitutive nuclear expression of NF-B proteins in JMML cells.29
Similar to the GM-CSF gene promoter, the TNF gene promoter also
contains binding sites for NF-B, and NF-B regulates TNF gene
expression.32 Collectively, our previous observations and
the present findings raise the intriguing possibility that TNF, in
addition to its possible direct growth-promoting action in JMML cells,
stimulates GM-CSF gene expression. This observation would explain our
recent finding that both TNF and GM-CSF needed to be blocked to
completely eliminate the leukemic cells that had been transplanted into
immunodeficient mice.21 In contrast to monospecific
antibodies, the ribozyme approach raises the possibility of designing
an RNA molecule with many target sites within the same target mRNA, as
demonstrated in this study, or with many target RNA specificities. A
single ribozyme, for example, directed against the TNF and GM-CSF
could possibly be designed.
Although the exact molecular mechanism underlying the abnormal growth
of myeloid cells in JMML patients is not known, dysregulated GM-CSF
stimulation probably plays a major role.14-18 It is
unlikely that any mutation of the heterodimeric GM-CSF receptor itself is responsible for the malignant process.33 Previous work
has suggested that events downstream of GM-CSF receptor activation, such as mutated ras activation, or a disturbed activation of
intracellular signaling molecules such as JAK2/STAT5 or adaptor
proteins such as Shc might explain the GM-CSF hypersensitivity in JMML
cells.2 However, more than 50% of JMML patients have a
normal ras gene.2,3,34 Our findings with the
ribozyme Rzd support the hypothesis that TNF might play an important
role in the pathogenesis of JMML by its uncontrolled stimulation of
GM-CSF gene expression.
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ACKNOWLEDGMENT |
The authors thank Drs H. Hasle and F. Wesenberg for the supply of JMML
cells; Dr A.F. Lopez for the generous gift of TNF, GM-CSF, and stem
cell factor; Dr E.B. Smeland and H.B. Benestad for critical reading of
the manuscript; and L. Aarseth for technical help.
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FOOTNOTES |
Submitted October 23, 1997;
accepted July 10, 1998.
Supported in part by The Norwegian Cancer Society, The European
Communities (Biotech program), and by the Norwegian Radium Hospital.
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.
Address reprint requests to Per Ole Iversen, MD, Department of
Physiology, PO Box 1103 Blindern, 0317 Oslo, Norway.
| |
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Abstract
Introduction