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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4336-4343
By
From the Department of Cancer Biology, The Lerner Research Institute,
and Department of Hematology and Oncology, Cleveland Clinic Foundation,
Cleveland, OH; Section on Biomedical Chemistry, National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, Bethesda, MD.
We report an RNA targeting strategy, which selectively degrades
bcr/abl mRNA in chronic myelogenous leukemia (CML) cells. A
2
ANTISENSE OLIGONUCLEOTIDES (ODNs) have
the potential to function as highly selective therapeutic agents by
virtue of their ability to bind with unique nucleotide sequences in
mRNAs for disease-causing proteins, including those implicated in
cancer. Indeed, several antisense ODNs, which are designed to prevent the synthesis of different proteins necessary for cancer cell growth
and survival have been used in clinical trials.1 Chronic myelogenous leukemia (CML), a pluripotent hematopoietic stem cell disease, has been a frequent subject of antisense-based therapeutic approaches.1-4 CML cells contain the Philadelphia
chromosome (Ph1) generated from reciprocal translocation
of chromosomes 9q and 22q, in more than 90% of patients. The bcr/abl
fusion genes (b2/a2 and b3/a2) in Ph1 encode tyrosine
kinases implicated in the development of CML.5-7 The
presence of unique nucleotide sequences at the fusion site of bcr/abl
and the requirement of the encoded tyrosine kinase for cell survival
make CML an attractive target for antisense strategies. In this regard,
antisense ODNs directed at the breakpoint junction of bcr/abl mRNA or
at the translation start site of bcr mRNA have been shown to
selectively inhibit proliferation or survival of bcr/abl-expressing
cell lines and primary cells.8,9 Furthermore, antisense ODN
treatments in vivo suppressed development of CML-like disease in murine
models.10-13 These encouraging results have led to clinical
trials with antisense ODNs directed to bcr/abl mRNA for ex vivo
treatment of bone marrow.14
Despite progress, antisense approaches for CML remain controversial
because while hybridization-dependent, antisense mechanisms against
bcr/abl mRNA have been reported,8,15 others observed that
some ODNs clearly caused nonantisense or aptameric
effects.16,17 For instance, phosphorothioate-capped ODNs
had sequence-specific inhibitory effects against the kinase activity of
p210bcr/abl.16 Moreover, a significant obstacle
to the further development of antisense ODN therapeutics is that even
when an antisense effect is established, the molecular mechanism is
often unknown or speculative.
We have exploited the controllable endoribonuclease, RNase L, present
in a wide range of mammalian cell types, for the purpose of enhancing
the activity of antisense ODNs.18 RNase L is an endoribonuclease for single-stranded RNA that functions in the interferon-regulated, 2-5A system.19 A unique feature of
RNase L is that it is converted from a silent to an active form in
response to short 2 Cell culture and ODN treatments of cells.
The CML cell line, K562, containing a bcr/abl gene with a b3/a2 fusion
site (bcr exon 3 to c-abl exon 2) and the promyelocytic leukemia cell
line, HL60, were maintained in RPMI 1640 medium (GIBCO-BRL,
Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a moist atmosphere with 5% CO2
at 37°C. Primary cells were isolated from bone marrow of three CML patients in chronic phase by Ficoll-Hypaque density gradient
sedimentation. Analysis by reverse transcriptase-coupled polymerase
chain reaction (RT-PCR) showed a b3/a2 type of bcr/abl mRNA in bone
marrow from all three patients. In addition, normal bone marrow was
collected from two healthy individuals. Patient cells
(1 × 105) were placed in 1 mL liquid suspension
culture, Isocove's modified Dulbecco's modified medium (GIBCO-BRL)
supplemented with 2.5% human AB serum, penicillin, streptomycin, 20 U/mL of interleukin-3 (Sigma, St Louis, MO), 5 ng per mL
of granulocyte-macrophage colony-stimulating factor (GM-CSF) (Sigma)
and 50 ng/mL of stem cell factor (SCF) (Pharmingen, San
Diego, CA).24 Experiments were performed in duplicate or
triplicate. ODNs were added every 12 hours to the cells (about
1 × 105) in 1 mL of media/serum. The viable cell
numbers were determined by trypan blue dye exclusion assay in a
hemocytometer.
Chemical synthesis of 2-5A-antisense chimeras and control ODNs.
Chimeric phosphodiester ODNs for this study were synthesized and
purified using previously published procedures.25-29 The
generic structure of 2-5A-antisense is;
Sp5 Assay for targeted cleavage of RNA by purified RNase L.
A complete coding sequence cDNA for the p210bcr/abl in
vector pGEM 4Z (plasmid [0]p210) was a generous gift from Dr Owen
Witte (UCLA, Los Angeles, CA). Restriction fragments from
this plasmid were subcloned to produce two derivative plasmids encoding
the 5 p210bcr/abl kinase assays for oligonucleotide-treated cells.
ODN-treated K562 cells were harvested and washed twice with ice-cold
phosphate-buffered saline (PBS). Cell lysis, immunoprecipitation, and
assays of p210bcr/abl kinase activity were performed as
described.5 Briefly, protein samples (100 µg) of
100,000g centrifugations of cell lysates were incubated with
preimmune sera or anti-abl antisera (a kind gift of O. Witte) at 4°C
with gentle shaking for 16 hours. Protein A-sepharose beads (25 µL
packed volume) (Pharmacia, Piscataway, NJ) were added to
the protein samples and incubated for 2 hours at 4°C. The immune
complexes were harvested by centrifugation at 5,000g for 10 minutes at 4°C, washed, and phosphorylation was performed in the
presence of 10 µCi of [ RT-PCR.
Cells were harvested from 1-mL cultures and the total RNA was extracted
from untreated and ODN-treated cell lines or primary cells using RNazol
(Tel-Test, Friendswood, TX). RNA (3 µg) was treated with RNase free
DNase, extracted with phenol chloroform, and precipitated with ethanol.
The mRNA was used as template for cDNA synthesis by reverse
transcriptase using oligo (dT) primer. The cDNA was then diluted
serially with PCR buffer and cDNA at each dilution was amplified with
the use of primers to sense sequences in exon 3 of bcr
(5 Selection of target sites in bcr/abl mRNA and design of ODNs.
Two potential binding sites for ODNs in the b3/a2 form of bcr/abl were
selected for this study. The b3/a2 fusion site was chosen because it
offered a means of selectively targeting bcr/abl mRNA present only in
the malignant cells. The fusion site was in the center of these ODNs,
such that there would be 18 continuous complementary nucleotides to
bcr/abl mRNA. Therefore, bcr mRNA and c-abl mRNA contain only 9 nucleotides each of sequence complementary to the ODN. 2-5A-antisense
against the translational start site sequence was also synthesized
because of prior success in targeting this region of bcr/abl mRNA with
antisense.8,32 Both ends of the chimeric ODNs were
chemically modified to enhance their stability against enzymatic
degradation. A 5
Selective cleavage of the target RNA sequence in vitro.
To determine if the 2-5A-antisense molecules could direct RNase L to
cleave the translation start site or fusion site of bcr/abl mRNA, two
regions of the bcr/abl mRNA containing these sequences were synthesized
in vitro (Fig 1A). One RNA segment of 659 bases, referred to as 5
Effect of oligonucleotides on p210bcr/abl kinase activity
levels.
To determine whether 2-5A-antisense against the 5
Growth suppression of a CML cell line, K562, after treatment with
2-5A-antisense.
ODNs at concentrations of 2 µmol/L per treatment were added twice
daily to cultures of K562 cells, a CML cell line containing the b3/a2
type of the bcr/abl oncogene. Addition of 2-5A-antisense against the
translation start sequence,
SpA4-anti-bcr, substantially suppressed cell proliferation (Fig
3A). The cell doubling time increased from
1.7 to >4 days in the presence of SpA4-anti-bcr. In
contrast, the control ODN containing a disabled, dimeric, 2-5A moiety,
SpA2-anti-bcr, had no effect on K562 cell growth (Fig 3A).
Antisense lacking a 2-5A moiety, anti-bcr, as well as ODN with five
mismatches, SpA4-(M5)anti-bcr, had intermediate effects on
cell growth, increasing the doubling time to 3.0 and 2.4 days,
respectively. 2-5A-antisense to the b3a2 fusion site in bcr/abl mRNA,
SpA4-anti-b3a2, suppressed K562 cell proliferation, increasing the doubling time to 4.2 days (Fig
3B). In contrast, SpA4-(M6)anti-b3a2, with six mismatches in the antisense sequence, had no effect on the cell growth rate. A
2-5A-antisense to a b2/a2 fusion site, SpA4-anti-b2a2,
increased the doubling time slightly to 1.85 days, while anti-b3a2,
lacking 2-5A, increased the doubling time to 2.2 days. These findings demonstrate the ability of 2-5A-antisense to suppress proliferation of
CML cells in a sequence-specific manner. In addition, the enhanced anticellular activity of 2-5A-antisense chimeras, compared with ODNs
lacking 2-5A or containing inactive dimeric 2-5A, provided evidence
that recruitment of RNase L enhances the antisense effect.
2-5A-antisense ODNs selectively inhibit proliferation of CML cells.
The specificity of this approach was investigated by comparing the
effects of different doses of the ODNs on growth of K562 cells and HL60
cells, a promyelocytic cell line. After twice daily treatments of the
K562 cells for 4 days with 1 µmol/L of
SpA4-anti-b3a2 or
SpA4-anti-bcr, cell
numbers were 41% to 44% those of the untreated cultures, while 10 µmol/L treatments of the K562 cells reduced the cell numbers to about
10% of the control culture (Fig 4). In
contrast, 1 µmol/L ODN treatments had no effect on the HL60 cells. A
small (10% to 11%) inhibition of HL60 cell growth was observed with 2 µmol/L ODN treatments, and 10 µmol/L treatments reduced the cell
numbers to about 45% those of the control culture (Fig 4). The
concentrations of
SpA4-anti-b3a2 or
SpA4-anti-bcr required to
inhibit growth by 50% were 0.7 µmol/L and 7.5 µmol/L for the K562
cells and for the HL60 cells, respectively. Therefore, there was a
considerable (10-fold) specificity in inhibitory activity of the
2-5A-antisense ODNs for the K562 cells compared with the HL60 cells.
2-5A-antisense blocks proliferation and survival of primary CML
cells.
To determine the effect of 2-5A-antisense on the growth and survival
of primary leukemia cells, bone marrow aspirates from three CML
patients were analyzed. Chromosome analysis was performed on 20 cells
from each of the three patients' bone marrow. In cells from patients 1 and 3, Ph1 was present in all 20 cells analyzed, whereas in
patient 2, Ph1 was observed in 17 of 20 of the bone marrow
cells, which were analyzed (not shown). In addition, bone marrow cells
from all three patients contained the b3/a2 form of bcr/abl as
determined by RT-PCR (see below). Bone marrow cells of the three
patients were cultured and then treated with the ODNs. In every case,
treatments of 5 µmol/L of
SpA4-anti-b3a2 or
SpA4-anti-bcr caused cell
proliferation to cease after 24 hours (Fig 5A through
C). In contrast, bone marrow cells from two healthy individuals were resistant to the growth-suppressing effects of the ODNs (Fig 5D and data not shown).
2-5A-antisense caused a selective decrease in the levels of the
bcr/abl mRNA in the primary CML cells.
RT-PCR analysis was performed to determine if there was a specific
effect of the 2-5A-antisense ODNs on amounts of the bcr/abl mRNA.
Primary CML cells from patients 1 and 2 were treated twice daily for
2.5 days with ODNs before isolation of RNA (Materials and Methods). As
a reference, relative levels of
2-5A-antisense molecules complementary to either of two regions in the
bcr/abl mRNA, the translation start site or the fusion site, caused
bcr/abl mRNA to be selectively degraded by RNase L both in a cell-free
system and in intact CML cells (Figs 1 and 7). Selective targeting
of these specific regions in bcr/abl RNA was observed in cell-free
reactions containing in vitro synthesized RNA, 2-5A-antisense and
human RNase L (Fig 1). The addition of the 2-5A moiety to antisense
ODNs to bcr resulted in larger declines in p210bcr/abl
kinase activity levels than was obtained with the antisense sequence by
itself, thus implicating RNase L in the effect (Fig 2). Proliferation of the Ph1 positive cell line K562 and of primary bone
marrow CML cells was also suppressed by 2-5A-antisense treatment (Figs 2 to 6). Loss of the cell survival function associated with
p210bcr/abl could account for the substantial decrease in
the CML cell populations treated with 2-5A-antisense species to
bcr/abl.8,9 While the 2-5A-antisense ODNs directed to the
bcr/abl fusion site are expected to be specific for the bcr/abl mRNA,
the ODNs targeted to the translational start sequence might also cause
degradation of bcr mRNA. Therefore, the anti-CML effect of
SpA4-anti-bcr could be due
to a combination of targeting both the bcr and bcr/abl mRNAs. However,
the absence of an effect of
SpA4-anti-bcr on normal
bone marrow cells suggests that targeting bcr mRNA may have minimal
effects on normal cell survival.
We thank Roland Mertelsmann (University of Freiburg) for discussions
and encouragement, Gerald A. Hoeltge (Cleveland Clinic) for the
chromosome analysis, and to Owen N. Witte (UCLA) for the gift of
plasmid [0]p210 and anti-abl antisera.
Submitted December 18, 1997;
accepted August 3, 1998.
Address reprint requests to Robert Silverman, PhD, Department of Cancer
Biology, NB40, The Lerner Research Institute, Cleveland Clinic
Foundation, 9500 Euclid Ave, Cleveland, OH 44195; e-mail
silverr{at}cesmtp.ccf.org.
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,5
Top
Abstract
Introduction
,5
-actin mRNA, determined by
reverse transcriptase-polymerase chain reaction (RT-PCR). The
specificity of this approach was further shown with control
oligonucleotides, such as chimeras containing an inactive dimeric form
of 2-5A, antisense lacking 2-5A, or chimeras with altered sequences
including several mismatched nucleotides. The control oligonucleotides
had either reduced or no effect on CML cell growth and bcr/abl mRNA
levels. These findings show that CML cell growth can be selectively
suppressed by targeting bcr/abl mRNA with 2-5A-antisense for decay by
RNase L and suggest that these compounds should be further explored for
their potential as ex vivo purging agents of autologous hematopoietic
stem cell transplants from CML patients.
-32P] adenosine
triphosphate (ATP) (3,000 Ci/mmol). The RNAs were purified from 6%
polyacrylamide/8 mol/L urea gels for use in the cleavage reactions.
Reactions were performed in the absence or presence of ODNs (50, 100, or 200 nmol/L) with 50 nmol/L each of 5
30%) of the nontargeted RNA molecules present
in the same reactions. In addition, 2-5A-iso-anti-bcr, containing
reverse polarity antisense, was able to effect the selective in vitro
cleavage of 5
), anti-bcr (
),
SpA4-(M5)anti-bcr (
), or SpA2-anti-bcr (
)
or in (B) of SpA4-anti-b3a2 (
), anti-b3a2 (
),
SpA4-anti-b2a2 (
), or SpA4-(M6)anti-b3a2
(
). Cell numbers are the average obtained from duplicate wells.
16-fold decreases in levels of the bcr/abl mRNA in cells from patients 1 and 2, as estimated by comparison to the untreated controls
(Fig 