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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2200-2207
BCR/ABL mRNA and the P210BCR/ABL Protein Are
Downmodulated by Interferon- in Chronic Myeloid Leukemia
Patients
By
Fabrizio Pane,
Ilaria Mostarda,
Carmine Selleri,
Rossella Salzano,
Anna Maria Raiola,
Luigia Luciano,
Giuseppe Saglio,
Bruno Rotoli, and
Francesco Salvatore
From the CEINGE-Biotecnologie Avanzate, Dipartimento di Biochimica e
Biotecnologie Mediche and Divisione di Ematologia, Facoltá di
Medicina, Universitá Federico II, Naples; Dipartimento di Scienze
Biomediche e Oncologia Umana, Universitá di Torino, Italy.
 |
ABSTRACT |
The BCR/ABL hybrid gene plays a central role in the pathogenesis of
the chronic phase of chronic myeloid leukemia (CML). We used a very
sensitive quantitative reverse transcriptase-polymerase chain
reaction to investigate the levels of hybrid BCR/ABL mRNA in bone
marrow cells of 20 patients with Philadelphia positive (Ph+) CML treated with interferon- (IFN-) as a
single agent. Bone marrow samples were collected at diagnosis and at
hematologic remission induced by IFN-, or by hydroxyurea in case of
resistance to IFN-. The mean levels of BCR/ABL transcripts in bone
marrow mononuclear cells of patients who showed a complete hematologic response to IFN- were significantly reduced with respect to those at
diagnosis (48 × 103 v
168 × 103; P < .001), whereas no difference
was detected between the values at diagnosis and at hematologic
remission in patients resistant to IFN-. In cell culture
experiments, IFN- priming significantly reduced the levels of
BCR/ABL hybrid transcripts in a dose-dependent manner in Ph+ bone
marrow precursors obtained at diagnosis from patients who subsequently
responded to IFN- treatment (P < .005). No
downmodulation was observed in bone marrow precursors from patients who
subsequently proved to be IFN-resistant. These results indicate that
downmodulation of BCR/ABL gene expression could be one of the
mechanisms involved in the response of CML patients to IFN- treatment.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE BCR/ABL hybrid gene and its resulting
chimeric P210BCR/ABL protein play a crucial role in chronic
myeloid leukemia (CML).1 Experimental evidence indicates
that this hybrid gene may be the only genetic lesion in the chronic
phase of CML because its introduction into primitive murine cells
results in a syndrome similar to human CML.2 The BCR/ABL
hybrid gene sustains the abnormal expansion of myeloid committed
precursors3,4 that characterizes the chronic phase of CML.
The mechanisms of transformation by the P210BCR/ABL protein
have yet to be fully clarified. In vitro, the P210BCR/ABL
protein (1) increases the survival of hematopoietic precursors and cell
lines by blocking apoptosis5-7; and (2) enhances cell proliferation by MYC upregulation8; and (3) reduces
differentiation capacity by RAS activation.9 However, it is
not clear how these pathways affect the in vivo clinical
situation.10
Interferon- (IFN-) belongs to a cytokine family that exhibits
antiviral properties, immunomodulating effects, and antiproliferative activity on normal and transformed cells.11 These diverse
properties are mediated through binding to a high-affinity cell surface
receptor12 which, in turn, activates a variety of
intracellular signals and modulates gene expression.13 At
present, IFN- is the drug of choice in the treatment of CML patients
who cannot undergo allogeneic bone marrow
transplantation.14-16 However, the response to IFN- treatment is variable. The drug induces complete hematologic remission in up to 70% to 80% of patients after 6 to 12 months, but is
ineffective in 20% to 30% of cases. Moreover, in 40% to 60% of
patients, IFN- treatment significantly reduces the percentage of
Ph+ cells, an effect termed cytogenetic response,
which is maximal after 12 to 24 months of
treatment.15,17,18 However, the mechanism by which IFN-
controls the growth of Ph+ CML precursors is still unclear.
There is evidence that enhancement of immunosurveillance through the
induction of the expression of leukocyte function associated antigen-3
(LFA-3)19 and the reversal of deficient adhesion of CML
cells to stromal elements might be involved in the therapeutic effects
of IFN-.20 However, recent studies failed to reveal
LFA-3 induction by IFN-21 or to document any
antiproliferative effect of CML precursor binding to marrow
stroma.3
We and others showed that IFN- upmodulates membrane expression of
FAS receptor.22-24 Recently we have also shown that, in CML, FAS-mediated apoptosis is restricted to IFN- responder
patients.25 Furthermore, in KT-1 cells (a human cell line
derived from a CML patient), IFN- reduces the expression of the
BCR/ABL gene.26
In this report we show that in vivo and in vitro IFN- treatment
reduces the intracellular amounts of the BCR/ABL hybrid transcript and
of the corresponding P210BCR/ABL protein, and that, as in
the case of induction of FAS receptor expression,26 this
effect is restricted to IFN- responder patients.
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MATERIALS AND METHODS |
Patients.
After informed consent, 20 patients affected by CML were enrolled in
the study (Table 1). The diagnosis was
confirmed by the cytogenetic finding of the Ph chromosome and by the
molecular finding of the hybrid BCR/ABL gene. All patients were treated with IFN- as single agent according to the Italian Cooperative Study
Group (ICSG) CML protocol.15
Bone marrow samples were collected from the patients before the start
of treatment and when the patients were in complete hematologic
remission because of either IFN- (responders) or hydroxyurea after
IFN- withdrawal (nonresponders). The molecular analysis was
performed within a few days of sampling, without cell cryopreservation.
Our operational definition of the response to IFN- treatment after 6 months of the maximum tolerated daily dosage (6 or 9 million units)
was: (1) complete hematologic response: if white blood cell (WBC) <10 × 109/L, circulating immature hematopoietic precursors
<5% in the differential, platelet <500 × 109/L, and
spleen not palpable; (2) insufficient hematologic response (NR) if WBC
were >10 × 109/L, immature hematopoietic precursors
>5% in the differential, platelet >500 × 109/L, or
persistent splenomegaly.
Bone marrow cell separation.
Bone marrow was aspirated from the posterior iliac crest into syringes
containing 1:10 EDTA solution. Bone marrow mononuclear cells (MNC) were
isolated by Ficoll density gradient centrifugation at 800g for
30 minutes (Ficoll-Paque; Pharmacia Biotech, Sweden), and washed in
phosphate buffer saline (PBS).
Hematopoietic suspension cultures.
Bone marrow MNC isolated from 9 CML untreated patients were resuspended
within a few hours from withdrawal in Iscove's modified Dulbecco's
medium (IMDM) containing 20% fetal calf serum (FCS), and aliquots of 2 million cells were incubated for 24 hours in 24-well plates at 37°C
with increasing concentrations of IFN-, ranging from 20 to 1,000 U/mL, which encompasses the levels of IFN- reached in blood (50 to
100 U/mL) during the treatment of CML patients. A control culture
without IFN- was also prepared for each set of cells. IMDM and FCS
were purchased from Life Technologies Italia (Milan, Italy) and IFN-
from Hoffman La Roche (Basel, Switzerland).
Detection and quantitation of hybrid BCR/ABL mRNA.
Total RNA was extracted from bone marrow MNC using the acid guanidinium
thiocyanate and phenol-chloroform method.27 Any contaminating DNA detected was digested using 5 U of RNAse-free DNAse I
(Boehringer Mannheim Italia, Monza, Italy), and purified RNA was
re-extracted using the phenol-chloroform method.27 At least
40 µg of RNA was extracted from each sample, corresponding to 4 to 8 million cells. The reverse transcriptase-polymerase chain reaction
(RT-PCR) analysis was performed as described elsewhere.28 Briefly, 1 µg of total RNA extracted was prewarmed for 10 minutes at
60°C and then incubated for 60 minutes at 37°C in a 40 µL
reaction mixture containing 10 mmol/L Tris HCl (pH 8.3), 50 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L of each deoxyribonucleotide, 40 U
of RNAsin (Promega, Madison, WI), 2.5 mmol/L of an antisense primer, 5'-TGTGATTATAGCCTAAGACCCGGAG-3', that hybridizes to sequences of the
second ABL exon, and 100 U of MoMLV reverse transcriptase (BRL,
Bethesda, MD). A 20-µL aliquot of this solution was amplified by PCR
in a 100 µL final volume of a mixture containing 10 mmol/L Tris HCl
(pH 8.3), 50 mmol/L KCl, 2 mmol/L MgCl2, 0.2 mmol/L of each
deoxyribonucleotide, 2.5 U of Taq polymerase, 0.5 µmol/L of the same
antisense ABL primer, 5'-TGTGATTATAGCCTAAGACCCGGAG-3', and 0.5 µmol/L
of a sense primer, 5'-GAAGAAGTGTTTCAGAAGCTTCTCCC-3', complementary to sequences of BCR exons b1 and b2. The ABL
sequences were amplified in the remaining 20-µL aliquot of reverse
transcriptase reaction mixture, as a control of both RNA integrity and
the PCR reaction. Both amplification reactions consisted of 30 cycles with the following temperature cycle: 95°C for 30 seconds; 55°C for
30 seconds; 72°C for 30 seconds. PCR products were analyzed on a 2%
agarose gel containing ethidium bromide.
The absolute amount of BCR/ABL transcript was quantitated as previously
described.29,30 Briefly, 2 aliquots (500 and 250 ng,
respectively) of total RNA extracted from each sample were reverse
transcribed and the 2 cDNAs were PCR-amplified independently in 2 tubes, using a radiolabeled deoxynucleotide. The PCR conditions, ie,
the total number of amplification cycles and the reaction mixture, were
standardized to ensure a constant amplification efficiency throughout
all amplification cycles and to terminate the reaction during the
exponential phase of the amplification. Under these conditions there is
a log-log linear relation between the number of starting molecules of
BCR/ABL mRNA contained in the sample and the amount of PCR products
estimated by the radioactivity of the amplified bands, measured by
densitometric analysis using a computerized apparatus, the Molecular
Imager (Bio-Rad Laboratories, Hercules, CA), which is equipped with a
phosphor crystal screen and a laser screen-scanner. The absolute number
of BCR/ABL mRNA molecules was calculated by interpolating the amount of
PCR products with the titration curve obtained by amplifying, in
parallel with the samples, known amounts of a "synthetic" RNA
molecule whose sequence is identical to that of the BCR/ABL mRNA being
quantitated. The reduction (approximately 2-fold) of the number of
molecules calculated in the 250-ng aliquot of total RNA analyzed as
compared with the 500-ng sample is a control that the amplification
efficiency is constant in the samples being analyzed, and, hence, that
the assay is accurate. All samples that differed by more than 15% from
the results obtained with the 500-ng and 250-ng aliquots were reanalyzed.
The limiting number of amplification cycles up to which amplification
proceeds with a constant efficiency was previously calculated by
amplifying various sets of scalar dilutions from 500 to 5 ng of total
RNA by RT-PCR using a decreasing number of cycles (data not shown).
After each experiment, logarithmically transformed data were analyzed
by linear regression, and the highest number of cycles that ensured a
log-log linear relationship between RNA sample dilutions and amplified
products was used in the assays. The synthetic RNA used as a standard
was synthesized in vitro using a 2-step procedure, as previously
described.29
The RT and PCR assay conditions differed slightly from those used for
the qualitative assay: the aliquots of total RNA were incubated for 60 minutes at 37°C in a 20-µL reaction mixture containing 20 mmol/L
Tris HCl (pH 8.3), 50 mmol/L KCl, 5 mmol/L MgCl2, 0.5 mmol/L of each deoxyribonucleotide, 2.5 U of RNAsin, 0.75 µmol/L of
antisense ABL primer (see above), and 50 U of MoMLV reverse transcriptase; the reaction was stopped by heating at 95°C for 10 minutes. The PCR amplification of the cDNA obtained was performed in a
reaction mixture containing 20 mmol/L Tris HCl (pH 8.3), 50 mmol/L KCl,
2.5 mmol/L MgCl2, 0.1 µmol/L of both sense and antisense
primers (see above), 0.1 mmol/L of each deoxyribonucleotide, 2 mCi of
[32P] dCTP, and 2.5 U of Taq DNA polymerase. The
reaction was performed using a total of 20 cycles and the quantitative
frame, ie, the linearity range of the assay under these conditions was
between 1.2 × 106 and 6 × 103 molecules.
Southern analysis of the M-BCR region.
High-molecular-weight genomic DNA was extracted, using the standard
phenol/chloroform method,31 and the relative proportion of
Ph+ and Ph cells was assessed by
densitometric evaluation of Southern analysis of M-BCR.32
Briefly, for the identification of the BCR/ABL fused gene, 15 µg of
genomic DNA were digested BglII (New England Biolabs, Beverly,
MA), electrophoresed on 0.8% agarose gel, blotted on positively
charged nylon membranes (Hybond N-plus, Amersham, UK), and hybridized
to 50 ng of 32P-labeled 0.7-kb
HindIII-BamHI genomic probe encompassing exons 2 and 3 of the M-BCR region of the BCR gene.33 Densitometric analysis of autoradiograph bands was performed with the Molecular Imager. The ratio of rearranged band to germline band was used to
assess the relative proportion of Ph+ cells in the various
bone marrow samples.
Western blotting of the P210BCR/ABL protein.
Cell-culture pellets were lysed by incubation for 10 minutes at 4°C
with PBS-TDS buffer (PBS buffer containing 1% Triton X-100, 0.5%
sodium deoxycholate, 0.1% SDS). Samples were then centrifuged at
13,000g to remove cellular debris, and the protein
concentration of supernatants was determined by colorimetric assay
(micro-BCA; Pierce, Rockford, IL) according to the manufacturer's
specifications. Seventy milligrams of each whole-cell lysate, together
with molecular weight markers (Amersham, Little Chalfont, UK) were
analyzed by sodium dodecyl sulfate-polyacrylamide gel 7.5%
electrophoresis. After electrophoresis migration, the gel was soaked
for 30 minutes in a transfer buffer (125 mmol/L Tris, 960 mmol/L
glycine, 20% methanol) and the proteins were transferred on a 0.45-mm
nitrocellulose membrane using a semidry apparatus (Protean Mini-gel;
Bio-Rad Laboratories) for 40 minutes at 5.5 mA/cm2.
Block treatment of membranes was performed by overnight incubation in
TST buffer (10 mmol/L Tris pH 8.0, 0.15 mmol/L NaCl, 0.05% Tween 20) containing 1% nonfat dry milk.
Specific proteins blotted onto membranes were detected with a 2-step
procedure. First, the membrane was incubated overnight at 4°C with 10 µg/mL of anti-BCR/ABL antibody (Calbiochem, Cambridge, MA) and then
for 1 hour with a 1:1,000 dilution of anti-mouse horse radish
peroxidase-labeled secondary antibody (Amersham, Buckinghamshire, UK).
Visualization of the protein bands was obtained using a
chemoluminescent detection method (ECL Western Blotting Detection
System, Amersham) following the manufacturer's directions.
Statistical analysis.
The 2-tailed Student's t-test for paired data was used to
analyze the results of the assays of BCR/ABL transcripts in the samples
drawn at diagnosis and at remission in the responder and nonresponder
groups of CML patients. The results of the analysis of BCR/ABL hybrid
transcript levels in cell cultures were analyzed by the Friedman test,
a 2-way analysis of variance by ranks for matched samples testing the
hypothesis that the effects of the different concentrations of IFN are
the same in all sets of cultures. The statistical analyses were
performed using the StatView 4.51 software for the PowerPc Macintosh
computer (Abacus Concepts Inc, Berkeley, CA).
| |
RESULTS |
Intracellular amount of hybrid BCR/ABL mRNA after in vivo treatment.
The number of molecules of hybrid BCR/ABL mRNA was calculated by
quantitative RT-PCR of total RNA extracted from the bone marrow MNC
fraction. Because cytogenetic conversion, particularly at remission,
could lead to reduction in the number of BCR/ABL mRNA molecules due to
decreases in the proportion of Ph+ cells in the samples,
the relative proportion of Ph+ clonal cells was verified by
Southern blot, and the number of hybrid mRNA molecules for each sample
was adjusted to the proportion of clonal cells evaluated by Southern
blot analysis. Hence, the results are expressed as number of
transcripts per microgram of Ph+ bone marrow MNC total RNA.
Fourteen of 20 (70%) patients obtained a complete hematologic response
with IFN- treatment in a median time of 7 months (range, 5 to 24),
and were considered responders (Table 1). In the other 6 patients,
IFN- treatment was discontinued because of insufficient hematologic
response, and the patients were induced into hematologic remission by
hydroxyurea treatment. The number of hybrid mRNA molecules, and the
relative proportion of Ph+ cells were measured at diagnosis
and at remission, in both groups of patients.
Given the relatively short period of IFN- treatment (median, 7 months; Table 1), none of the responder patients had, at the end of the
study, a major or minor cytogenetic response (ie, relative proportion
of Ph+ cells <33% or between 66% and 33%,
respectively), which was evaluated by densitometric analysis of the
M-BCR; a slight reduction of Ph+ cells was detected in only
3 cases (Table 2; cases 1, 8, and 10). This
indicated that, at the end of the study, most of the hematopoietic
tissue was still clonal in both responder and nonresponder patients.
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Table 2.
Hybrid BCR/ABL Molecules in Ph+ Bone
Marrow Mononuclear Cells of IFN- Responder and Nonresponder CML
Patients Enrolled in the Study
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At diagnosis, the number of BCR/ABL hybrid transcripts/µg of total
RNA varied from patient to patient; however, there was no difference
between the mean values in the 2 groups of patients (168 × 103 and 144 × 103 molecules/µg
of total RNA, respectively; Table 2 and Fig
1). At the end of the study, in responder
patients the number of hybrid transcripts was significantly lower
(48 × 103 vs 168 × 103;
P < .01 at the Student's t-test for paired data),
whereas in IFN--resistant patients hybrid transcripts tended to
increase (Table 2 and Fig 1). During the course of treatment, 3 sequential bone marrow samples were collected from the same responder
patient (no. 1): the results of quantitative RT-PCR assay of the number of BCR/ABL transcripts paralleled the decrease of WBC and of peripheral blood myeloid precursors (Fig 2).
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| Fig 1.
Effect of IFN- treatment on the levels of hybrid
BCR/ABL mRNA in bone marrow mononuclear cells from CML patients with
complete or poor hematologic responses to therapy. For each patient,
values have been adjusted for the percentage of Ph+ cells.
At diagnosis, the amount of transcript was similar in the 2 groups of
patients; there was a significant difference (P < .01,
paired t-test) between the values at diagnosis and those at the
end of the study in the group of responder patients, whereas in
patients resistant to IFN-, there was a trend toward higher values
at the end of the study.
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| Fig 2.
Progressive decline of hybrid BCR/ABL mRNA molecules in
bone marrow mononuclear cells from a CML patient (no. 1 in Table 2) who
had a complete hematologic response after IFN- therapy.
BCR/ABL values are corrected for the percentage of Ph+
cells.
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Differential effects of IFN- on suspension cultures
of Ph+ bone marrow MNC. Comparison with hematologic
response to treatment.
The reduced number of hybrid transcripts in Ph+ bone marrow
MNC observed in responder patients could be because of either a direct
or an indirect effect of in vivo IFN- treatment. To clarify this
issue, Ph+ bone marrow MNC isolated from 9 untreated CML
patients were incubated for 24 hours in suspension culture with
increasing amounts of IFN- (from 20 to 1,000 UI/mL). At the end of
incubation, we used quantitative RT-PCR to determine the number of
transcripts in the total RNA fraction extracted from the
cells. In 6 patients who subsequently had a complete hematologic
response to IFN- treatment, there was a decrease of BCR/ABL
transcript number per µg of total RNA (P = .004 at Friedman
test; Fig 3). In an attempt to obtain a
rough evaluation of the corresponding intracellular P210BCR/ABL protein content, we analyzed the cell lysates
from this experiment with the Western blot technique. The decrease of
the P210BCR/ABL proteins was less pronounced than that of
mRNA levels, it was dose-dependent in 3 of 6 responder patients studied
and the greatest reductions occurred in the MNCs cultured with 1,000 U
of IFN- (Fig 4A and B). The differential
effect of IFN- on the BCR/ABL transcript and on the
P210BCR/ABL protein level could be explained by their
different half-lives.
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| Fig 3.
Dose response relationship between levels of BCR/ABL
transcripts and concentration of IFN- used in culture experiments.
Symbols and vertical lines are mean ± SD, respectively. Numerical
values and the results of the Friedman statistical analysis (see
Materials and Methods for details) are reported in the upper part of
the figure.
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| Fig 4.
Differential effects of IFN- priming on BCR/ABL
expression in bone marrow mononuclear cell cultures from CML
patients (A and B, IFN- responder patients; C,
IFN--resistant patient; D, a patient presenting in initial blastic
transformation). Bone marrow mononuclear cells were cultured for 24 hours with the indicated concentrations (U/mL) of IFN-.
Lower panels: Western blot for intracellular P210BCR/ABL
content, including P145ABL determination used as an
internal control.
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When the same set of experiments was performed on bone marrow MNC
isolated from 3 CML patients who subsequently proved to be resistant to
IFN- treatment, we found no variation in the intracellular levels of
both the hybrid transcript and the P210BCR/ABL protein
between untreated and IFN--primed cells (Figs 3 and 4C). No in
vitro IFN--induced downmodulation of BCR/ABL mRNA and of
P210BCR/ABL protein was observed in cultured bone marrow
MNC from an untreated patient who presented with CML in initial blastic
transformation (Fig 4D).
| |
DISCUSSION |
IFN- is a first-line drug in the treatment of CML and induces
hematologic remission in the majority of patients after 3 to 6 months
of treatment.14-16 Despite intensive efforts, the molecular mechanisms of the antiproliferative effects of IFN- in CML have not
yet been clarified.3 We and others have shown that IFN- upmodulates FAS-R expression on cell membrane of bone marrow
CD34+ cells from CML patients,22-24 and that
FAS triggering in the presence of IFN- inhibits the growth of these
cells.22 We also showed that only CD34+ cells
isolated from patients who were responsive to in vivo IFN- treatment
had a dose-dependent decrease of proliferation in methylcellulose colony assays when exposed to IFN- in the presence of a FAS
agonist.25 Interestingly, growth inhibition correlated to
increased apoptotic rate of these cells, and to posttranscriptional
downmodulation of the P210BCR/ABL protein. On the other
hand, growth of colony-forming units and apoptosis rate of
Ph+ progenitors isolated from CML patients who were
resistant to IFN- treatment remained unchanged.25
Moreover, a new cell line, KT-1, established from a Ph+ CML
patient in blastic crisis, is reported to be sensitive to both IFN-
and IFN- in vitro.26 Indeed, IFN- dose-dependently suppressed the growth of KT-1 cells both in suspension and in semisolid
cultures, inducing G1 cell-cycle arrest and apoptotic death. In
addition, Northern blot analysis showed that IFN- significantly reduces the expression of the BCR/ABL hybrid gene within 6 hours and
that this effect is more pronounced after 24 hours of IFN- exposure.26 Downmodulation of the BCR/ABL gene in the KT-1
cell line seems to be a specific effect of IFN-. When IFN- was
added to cultures, it suppressed the growth of KT-1, as did IFN-,
thus exerting a synergistic effect with IFN-. However, the growth arrest of KT-1 cells induced by IFN- was not associated with downmodulation of the BCR/ABL gene.26 These findings
suggested that IFN- and IFN- control the proliferation of KT-1
cells through different signal-transduction pathways, and that the
antiproliferative effect of IFN- may depend on downmodulation of
BCR/ABL expression. The latter mechanism could be the most plausible in
vivo, given the limited efficacy of IFN- in the clinical
setting.34-36
In the present study, we investigated the intracellular content of
BCR/ABL hybrid mRNA in Ph+ precursors during in vivo and in
vitro exposure to IFN-. The data regarding bone marrow MNC of CML
patients at diagnosis and at hematologic remission were normalized to
the percentage of Ph+ cells to avoid a bias because of
cytogenetic conversion. However, in only 3 of 14 cases that achieved
hematologic remission was there a limited reduction of Ph+
cell percentage. On complete hematologic response (after a median of 8 months of IFN- treatment), the number of intracellular BCR/ABL mRNA
molecules was reduced by a mean of 72% with respect to the number at
diagnosis. Interestingly, there was a clear correlation with the
hematologic response to IFN-: at variance with IFN- responders,
nonresponder patients did not show BCR/ABL downmodulation, rather, they
showed a trend toward enhanced intracellular levels of BCR/ABL mRNA. It
is unlikely that changes in cell composition of the bone marrow MNC
samples caused the decreases in transcript number in responder
patients, because both responders and nonresponders were in hematologic
remission at the end of the the study. The correlation between
variation of intracellular levels of BCR/ABL mRNA and hematologic
response to IFN- treatment suggests that downmodulation of the
BCR/ABL gene is an important mechanism of in vivo IFN- activity.
In principle, the decreased BCR/ABL expression observed in vivo in
responder patients could be attributed to an indirect effect of IFN-
treatment; however, we found a dose-dependent decrease of the number of
BCR/ABL mRNA molecules and of the corresponding P210BCR/ABL
protein in bone marrow MNC isolated from CML patients after 24 hours of
exposure to IFN- in suspension cultures. Thus, in an experimental
system closer to physiological conditions than the KT-1 cell line, we
provide evidence that the decrease in intracellular levels of BCR/ABL
mRNA during in vivo treatment is because of a direct interaction
between IFN- and Ph+ precursors. Remarkably, in vitro
effects of IFN- on BCR/ABL mRNA in bone marrow MNC correlated with
subsequent hematologic response. This suggests that response to IFN-
treatment is an intrinsic property of the Ph+ clonal precursors.
The data reported here indicate that the mechanism of action of IFN-
could rely on multiple and, probably, synergistic effects. The
P210BCR/ABL protein plays a crucial role in the
pathogenesis of the chronic phase of CML given its ability to reduce,
at least in vitro, the differentiation capacity8,37-40 and
the adhesion to the stroma of the Ph+
precursors,41-43 and to increase their proliferative
potential.44-45 Therefore, it is possible that the
IFN--mediated downmodulation of BCR/ABL expression exerts an
initial antiproliferative effect on Ph+ stem
cells directly, or alternatively indirectly, by restoring the
adhesion to stroma. Decreased intracellular levels of the P210BCR/ABL protein could enhance the
responsiveness of Ph+ progenitors to apoptosis and
specifically to FAS-related apoptosis. In fact, decreased intracellular
amounts of P210BCR/ABL protein results in reduced
resistance of CML cells to apoptosis induced by a variety of
agents.5-7 These mechanisms could account for the
hematologic response of CML patients to IFN- treatment, which is
necessary, although not sufficient, for a significant cytogenetic
conversion. It is still unknown whether the same mechanisms are also
important for complete reversion to Ph hematopoiesis,
which is induced by IFN- only in a small proportion of patients.
This study highlights the potential role of BCR/ABL downmodulation
induced by IFN- in the framework of the complex mechanisms by which
IFN- controls the chronic phase of CML. It also provides a potential
tool by which in vivo responsiveness to IFN- treatment can be
predicted. Recent articles have dealt with this issue using different
approaches.46-47 It is of considerable clinical relevance in terms of optimizing the therapeutic strategy (including bone marrow
transplantation timing) and in terms of cost effectiveness of the
treatment to be planned. Further experiments are necessary to identify
the intracellular signals involved in the downmodulation described,
which, in turn, may be important for the development of new therapeutic
strategies for CML.
| |
ACKNOWLEDGMENT |
We are grateful to Jean Gilder for editorial assistance.
| |
FOOTNOTES |
Submitted September 29, 1998; accepted May 23, 1999.
Supported in part by grants from AIRC (Milan), CNR P.F. Biotecnologie
(Rome), "Biogem" and "PRIN"MURST (Rome), and
Regione Campania.
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 Fabrizio Pane, MD, Dipartimento di
Biochimica e Biotecnologie Mediche, Facoltà di Medicina,
Università di Napoli Federico II, via S Pansini 580131 Napoli,
Italy; e-mail: fabpane{at}unina.it.
| |
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ABSTRACT
INTRODUCTION