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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 981-989
Fas-Mediated Modulation of Bcr/Abl in Chronic Myelogenous
Leukemia Results in Differential Effects on Apoptosis
By
Carmine Selleri,
Jaroslaw P. Maciejewski,
Fabrizio Pane,
Luigia Luciano,
Anna Maria Raiola,
Ilaria Mostarda,
Francesco Salvatore, and
Bruno Rotoli
From the Division of Hematology, CEINGE and Department of
Biochemistry and Biotechnology, Federico II University Medical School,
Naples, Italy; and the Department of Internal Medicine, University of
Nevada, Reno, NV.
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ABSTRACT |
Fas-R is expressed constitutively in CD34+ cells of
patients with chronic myelogenous leukemia (CML); Fas-R triggering
results in decreased proliferation rate due to apoptosis of clonogenic cells. We have already shown that  -interferon (IFN-) enhances Fas-R expression on CML progenitor cells, thus increasing their sensitivity to Fas-R agonists. Although it appears that IFN- can
prime CML cells for the effects of Fas, the response to IFN- in vivo
is not a constant feature in CML patients. We studied the mechanisms of
Fas-mediated apoptosis in 11 patients suffering from CML in chronic
phase and tried to see whether there was a correlation between in vitro
inducibility of apoptosis in CD34+ CML cells after Fas-R
triggering and the clinical response to IFN-. After priming with
IFN-, Fas triggering resulted in in vitro suppression of
hematopoietic cell growth in seven of eight patients who had optimal
hematologic response to IFN-; in the same conditions, no inhibitory
response to Fas-R agonist was observed in cells from three of three
patients who proved to be poor responders to IFN-. In responders to
IFN-, Fas-R agonist induced dose-dependent apoptosis of
CD34+ cells; this effect was associated with a decrease
in the bcr/abl protein level. In cells derived from patients
with a poor response to IFN-, the rate of apoptosis in culture
remained unchanged in the presence of Fas-R agonist and no
bcr/abl downmodulation was observed. Finally, we measured
bcr/abl mRNA by quantitative reverse-transcriptase polymerase
chain reaction (RT-PCR) and found that decreased bcr/abl
protein after Fas triggering was not associated with decreased amounts
of specific mRNA, a finding which is consistent with a
posttranscriptional regulation of the bcr/abl protein
expression. It appears that Fas-mediated downmodulation of p210
bcr/abl restores susceptibility to apoptosis of CML cells; in
addition, in vitro studies on CML cells may predict response to IFN-
treatment.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
RELATIVE RESISTANCE to apoptosis is
thought to be one of the features of malignant cells. Many
chemotherapeutic agents exert their anticancer activity through
induction of apoptosis,1-4 and inhibition of this process,
eg, through a blockade of the apoptotic pathway, can produce resistance
to these agents.5-8 In chronic myelogenous leukemia (CML),
the bcr/abl translocation and the resulting chimeric protein
appear to play a central role in the pathophysiology of the
disease.9,10 The ultimate effect of the bcr/abl
product seems to be enhanced cell survival rather than a higher
proliferation rate. The bcr/abl protein seems to exert
antiapoptotic activity in CML cells, as shown in several experimental
systems,9-18 with some discordant results.19
Under normal circumstances, programmed cell death may be induced by a
variety of exogenous signals; it may also be the result of an intrinsic
program through a totally endogenous pathway. Immune-mediated killing
mechanisms also involve apoptosis, induced by tumor necrosis factor
(TNF), interferons (IFNs), Fas-ligand, or the perforin/granzyme
pathway.20-22 We have shown that Fas-receptor (Fas-R) is
expressed on hematopoietic progenitor cells from CML patients and that
Fas triggering transduces apoptotic signals to both
normal23,24 and CML cells.25-27 Expression of
Fas-R can be induced by TNF- or IFN- , as well as by IFN- on
normal and malignant cells.23,24,28-36 In vitro induction
of programmed cell death in CML cells by Fas indicates that the
bcr/abl-mediated resistance to apoptosis is not absolute and
can be overcome. Fas-R triggering may cause downmodulation of
bcr/abl17-19 or may exert other influences
downstream in the sequence of events leading to
apoptosis.16 Because resistance to chemotherapy can evolve
through inhibition of drug-mediated apoptosis, it is possible that CML
cells can also resist or acquire resistance to Fas-induced cell death.
This process may be related to cell selection in the face of the
pressure exerted by the immune system; such a hypothesis is supported
by the observation of development of resistance to IFN-37,38 or to a graft-versus-leukemia39-41
effect in CML patients.
In view of the in vitro observation that IFN- upmodulates Fas-R
expression on CML cells,25-27 and of the variability of the in vivo response to IFN- treatment, we hypothesized that
Fas-mediated apoptosis may be related to modulations of the
bcr/abl protein level. Therefore, we studied the mechanisms of
apoptosis induced by Fas-R triggering on CML cells and attempted to
establish a relationship between inducibility of apoptosis in vitro and
clinical response to IFN- in vivo.
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MATERIALS AND METHODS |
Patient specimen collection.
Bone marrow (BM) samples were obtained after informed consent from 28 patients with CML. In 16 of them, we evaluated only the expression of
CD95 on CD34+ cells; in 12 patients, complete in vitro
studies could be performed. Of the latter, 11 patients were in chronic
phase; eight were studied before starting IFN- treatment and three
during IFN- treatment; one patient was studied in blastic crisis.
The diagnosis of CML was confirmed by the cytogenetic finding of
the Ph chromosome and by the molecular finding of rearranged
bcr/abl. Patient characteristics are presented in Table 1. Our
operational definition of the response to IFN- treatment was as
follows: optimal response = complete hematologic recovery after 1 month of treatment at the maximum tolerated dose (6 or 9 million units
[MU]); poor response = persisting or increasing
leukocytosis (> 80.000) after 1 month of treatment at the maximum
tolerated dose (6 or 9 MU), leading to IFN- treatment discontinuation. Karyotypic response was evaluated after 1 year of the
therapy according to the proportion of residual Ph+
metaphases: no response (Ph+ 100%), minimal response
(Ph+ 99% to 66%), minor response
(Ph+ 66% to 35%), and major response
(Ph+ <33%).
BM cell separation.
BM was aspirated from the posterior iliac crest into syringes
containing Iscove's modified Dulbecco's Medium (IMDM) supplemented 1:10 with heparin (O'Neil & Feldman, St Louis, MO). Mononuclear cells
(BMMNC) were isolated by density gradient centrifugation using
lymphocyte separation medium (Organon, Durham, NC). After washing with
Hanks' Balanced Salt Solution (HBSS), cells were resuspended in IMDM
supplemented with 5% fetal calf serum (FCS). HBSS, IMDM, and FCS were
purchased from Life Technologies, Gaithersburg, MD.
Separation of CD34+ cells.
In some experiments, purified CD34+ cells were used for the
analysis of Fas-R expression or for colony assay. CD34+
cells were separated using affinity columns (Cellpro, Bothel, WA).
Briefly, nonadherent BM cells were incubated at room temperature with
streptavidin-conjugated murine antihuman CD34 IgM, washed with
phosphate-buffered saline (PBS), and applied to an affinity column
containing biotin-coated beads; after mechanical disruption, the
CD34+ fraction was eluted with PBS. An aliquot of the
eluted cells was stained with phycoerythrin
(PE)-conjugated anti-CD34 HPCA-2 monoclonal
antibody (MoAb) (Becton Dickinson, Mountain View, CA) for
purity assessment; the average purity of the separated cell population
was 87% for all purifications (70% to 95%).
Flow cytometry analysis.
A fluorescein isothiocyanate (FITC)-conjugated (Fab ) fragment of
a murine antihuman CD95 (clone UB2; Amac, Westbrook, ME) was used to
determine the expression of Fas-R on BM cells. For two-color analysis,
PE-conjugated MoAb to CD34 (clone UCH11, Becton Dickinson, Mountain
View, CA) was used in combination with FITC-conjugated CD95 MoAb.
Proper isotypic controls were used in all experiments. BMMNC were
resuspended in 100 µL of PBS (Life Technologies, Grand Island,
NY) containing 2% FCS, incubated with 20 µL of MoAb,
washed three times with PBS, and analyzed by flow cytometry (Epics,
Coulter, Miami, FL). Fas expression on CD34+ cells was
analyzed in a uniformly set blast gate.
Hematopoietic colony assay and suspension cultures.
Isolated CD34+ cells were plated in methylcellulose (Stem
Cell Technologies, Vancouver, Canada) at a concentration of 1 × 103 cells/mL of medium (35-mm dishes; 1 mL of medium/dish).
The growth factor cocktail consisted of 10 ng/mL interleukin-3 (IL-3),
50 ng/mL granulocyte colony-stimulating factor (G-CSF), 50 ng/mL GM-CSF, 20 ng/mL stem cell factor (SCF), and 2 U/mL erythropoietin (EPO) (Amgen, Thousand Oaks, CA). Human recombinant IFN- (Hoffmann La Roche, Basel, Switzerland) and anti-Fas MoAb (clone
CH11, Amac) was used at a concentration range of 20 to 1,000 U/mL and
at 1 µg/mL, respectively. All cultures were performed in duplicate. Suspension cultures were performed in 24-well plates in IMDM containing 20% FCS and growth factors at the concentrations described above. All
experimental procedures were performed in endotoxin-free plastic ware.
DNA fragmentation assay.
DNA fragmentation was measured after extraction of low molecular weight
DNA from a constant number of cells as previously described.24,25 Briefly, 2 × 106 cells
were resuspended in 900 µL 1X TRIS-EDTA buffer and lysed with 25 µL
20% sodium dodecyl sulfate (SDS). High molecular weight DNA fraction
was precipitated for 6 hours in the presence of 5 mol/L NaCl and
pelletted by high-speed centrifugation; the fragmented DNA was
extracted from the aqueous phase with phenol and chloroform, and
precipitated with ethanol. After resuspension in water, DNA was
electrophoresed using 1.5% agarose gel and visualized by ethidium bromide staining.
Terminal deoxynucleotidyl transferase (TdT) assay for quantitation
of apoptotic cells.
To estimate the number of cells undergoing apoptosis, cultured
CD34+ cells were washed in PBS, fixed with 4%
paraformaldehyde, and cytocentrifuged onto siliconized slides.
Apoptotic cells were identified using the TdT method (Apotag; Oncor,
Gaithersburg, MD). Endogenous peroxidase was first quenched with 0.5%
hydrogen peroxide, and the cells were permeabilized using equilibration buffer supplied by the manufacturer. The 3 OH ends of degraded DNA were reacted with TdT and labeled with digoxygenin-uvidine triphosphate (UTP) for 30 minutes. After washing with
PBS, slides were reacted with peroxidase-conjugated antidigoxygenin
MoAb, washed, and developed with 3.3-diaminobenzidine
tetrahydrochloride (Pierce, Rockford, IL). Stained cells were counted
using a light microscope.
Detection and quantitation of bcr-abl hybrid mRNA by reverse
transcription-polymerase chain reaction (RT-PCR).
Total RNA was extracted from a constant number of total BM CML cells
using the acid guanidinium thiocyanate and phenol-chloroform method.42 RT-PCR analysis was performed as previously
described.43 Reverse transcription was performed with
the antisense primer 5-TGTGATTATAGCCTAAGACCCGGAG-3, which
hybridizes to sequences of the second abl exon, and 100 U of
Moloney murine leukemia virus reverse transcriptase (BRL, Bethesda,
MD). For the amplification, the antisense primer (see above) and the
sense primer 5-GAAGAAGTGTTTCAGCTTCTCCC-3, complementary
to sequences of bcr exons b1 and b2 were used. PCR products
were analyzed on 2% agarose gel containing ethidium bromide.
The absolute amount of the bcr/abl transcripts was quantified
using a noncompetitive PCR technique.44,45 Briefly, the
technique consists in the reverse transcription followed by PCR
amplification of two aliquots (500 and 250 ng, respectively) of total
RNA extracted from each sample. The conditions of the PCR reaction
ensured a constant amplification. The reaction was terminated during
the exponential phase of amplification allowing a log-log linear
relation between the number of starting molecules of bcr/abl
mRNA and the amount of PCR products. The limiting number of
amplification cycles up to which each amplification proceeds with a
constant efficiency was calculated by amplifying scalar dilutions from
500 to 5 ng of total RNA by RT-PCR using a decreasing number of cycles.
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 sample RNA dilutions and amplified
products was used in the assays. The absolute amount 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
with the same sequence as that of the type of bcr/abl mRNA to
be quantitated, as previously described.44,45 The reaction was repeated for a total of 18 cycles and the quantitative frame, ie,
the linearity range of the assay under these conditions, was between
1.2 × 106 and 6 × 103 molecules.
Western blotting of bcr/abl protein.
Total BM cells derived from patients with CML were cultured in
suspension without or with IFN- and Fas triggering. After 48 hours,
cells were washed in PBS and lysed in PBS-TDS (PBS containing 1%
Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) for 10 minutes at
4°C. Cellular debris was removed by centrifugation, and the protein
concentration of supernatants was determined by colorimetric assay
(micro-BCA; Pierce, Rockford, IL) according to the manufacturer's specifications. A total of 70 µg of each whole-cell lysate, together with molecular weight markers (Amersham, Little Chalfont, UK) were
fractionated by 7.5% and 10% SDS-polyacrylamide gel electrophoresis (PAGE) for p210 bcr/abl and actin, respectively. Equal protein loading was assessed by Coomassie-blue staining of SDS-PAGE gels. Gels
were equilibrated in transfer buffer (125 mmol/L TRIS-base, 960 mmol/L
glycine, 20% methanol) and electrically transferred to polyvinyl
difluoride membrane filters (Millipore, Bedford, MA). Membranes were
blocked in TBST-3% milk (10 mmol/L TRIS-HCL pH 8.0, 150 mmol/L NaCl,
0.5% Tween-20, 3% nonfat dry milk) for 1 hour at room temperature and
incubated with 10 µg/mL of mouse anti-bcr/abl MoAb
(Calbiochem, Cambridge, MA) or rabbit anti-actin polyclonal serum
(Sigma, St Louis, MO) in TBST-0.5% milk overnight at 4°C. The
reaction was developed by incubating filters with horseradish
peroxidase-conjugated goat antirabbit antibodies (BioRad, Richmond,
CA) and ECL (Amersham, Little Chalfont, UK) according to the
manufacturer's specifications. Bands of 210 and 145 kD correspond to
human bcr-abl and c-Abl proteins, respectively. Densitometer
analysis of films was performed using a scanning densitometer (GS 300;
Hoefer Scientific Instruments, San Francisco, CA).
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RESULTS |
Expression of Fas antigen on BM cells from CML patients.
The expression of CD95 on CD34+ BM cells was studied in a
global population of 28 CML patients. On average, 25.5% ± 22%
CD34+ BM cells from CML patients expressed CD95, as
compared with 8.4% ± 6% in 40 normal controls. When the CML
population was dissected according to cytoreduction by IFN-
treatment, the figure was 25.3% ± 16% in 21 patients with optimal response to IFN- (P = .01 v
normal controls) and 11.3% ± 6.7% in seven patients with poor
response (P = .26 v normal controls). When analyzed
according to the cytogenetic response, the figure was 19.7% ± 14%
in 8 patients with a major (<33% residual Ph+ mitoses)
response and 22.0% ± 16% in 20 patients with absent, minimal, or
minor response (P = .25). In 11 of these patients, it
was possible to perform Western blotting for bcr/abl
expression, apoptosis assays, and methylcellulose cultures
(Table 1).
IFN-- and Fas-mediated apoptosis of BM progenitor
cells derived from patients with CML in chronic phase. Correlation with
bcr/abl product cell content.
In 7 of 11 patients tested, the addition of Fas agonist resulted in
enhancement of apoptosis, as demonstrated by agarose gel electrophoresis of low molecular weight DNA extracted from total BM
cells (Fig 1B and
2B) or by the Tdt assay (Table 1, Figs 1C, 2C, 3A and B). Western blot performed on
cell extracts derived from the same cells using identical protein
concentration for each sample showed that this effect was associated
with downmodulation of the bcr/abl protein (Fig 1A). The
downmodulation, which was not caused by proteolysis, as p145 c-abl
and actin proteins remained unchanged, was enhanced by in vitro
addition of IFN-; the enhancement was dose-dependent in three cases
and occurred only at the highest IFN- concentration (1,000 U/mL) in
four cases (data not shown). Addition of the partially blocking
anti-Fas MoAb ZB4, serving as a Fas antagonist, significantly reduced
the effect of IFN- + Fas triggering on apoptosis, as well as on
downmodulation of bcr/abl (Fig 2). These seven patients all
showed a complete hematologic response after IFN- treatment. In a
single patient (Table 1, no. 8), who also showed an optimal response to
IFN-, the effect of Fas triggering in vitro was only marginal. By
contrast, in 3 of 11 patients, Fas triggering failed to induce
apoptosis in IFN--treated cells, as shown by characteristic DNA
laddering in agarose gel electrophoresis and quantitative Tdt assay
(Fig 3B), and no change in bcr/abl expression was observed;
these patients had a poor response to IFN- treatment and were
switched to other types of treatment. In the only case of lymphoid
blastic crisis studied, we could not document any effect of Fas
triggering in vitro (Table 1, no. 12).
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| Fig 1.
Effects of Fas triggering by anti-Fas MoAb CH11 on p210
bcr/abl protein expression and on susceptibility to apoptosis
of total and CD34+ BM cells from a representative patient
with CML in chronic phase who had optimal response to IFN-. (A)
Immunoblotting of p210 from total BM cells (upper) and Coomassie-blue
staining to document equal protein loading (lower). P145 c-abl
and actin proteins are used as controls for constant protein loading
and absence of degradation. (B) Agarose gel stained with ethidium
bromide after electrophoresis of low-molecular-weight DNA extracted
from a constant number of total BM cells. Cell lysates and
low-molecular-weight DNA were obtained from the same plates for both
(A) and (B) experiments. (C) In situ TdT assay of CD34+
cells treated as indicated; bars represent the mean number of apoptotic
cells determined in triplicate experiments ± standard error of mean
(SEM). Statistical analysis (paired t-test) showed P < .05 for control versus Fas triggering alone and after addition of
any IFN- concentration and for Fas triggering versus IFN 1,000 U/mL + Fas triggering. Total BM and CD34+ cells
were cultured for 48 hours in the presence of the indicated concentrations of IFN-. MoAb CH11 was used at a concentration of 1 µg/mL.
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| Fig 2.
Effects of Fas blocking by MoAb ZB4 on Fas-mediated
regulation of p210 and apoptosis of total and CD34+ BM
cells from a CML patient in chronic phase who had optimal response to
IFN-. Total BM and CD34+ cells were cultured for 48 hours in the absence or presence of Fas triggering (by MoAb CH11)
and/or Fas blocking (by MoAb ZB4), without or with IFN-
(1,000 U/mL). For (A), (B), and (C), see legend to Fig 1. (D), (E), and
(F) show apoptotic CD34+ CML cells stained positively
with peroxidase (black cells) after the indicated treatment. In (C),
bars represent the mean number of apoptotic cells determined in
triplicate experiments ± SEM. Statistical analysis (paired
t-test) showed P < .05 for untreated cells versus Fas
triggering + IFN- and for Fas blocking versus Fas blocking + IFN- + Fas triggering, while there was no statistical difference
between untreated and Fas blocking.
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| Fig 3.
Comparison of apoptotic effect of Fas triggering on
CD34+ BM cells from representative CML patients in
chronic phase who showed optimal (A) or poor (B) response to IFN-
treatment. Values (mean ± SEM of triplicate measurements) represent
percentage of apoptotic CD34+ BM CML cells evaluated by
the Tdt assay. CD34+ cells were purified from CML BM and
cultured in the presence of IFN- and MoAb CH11. MoAb CH11 was used
at a concentration of 1 µg/mL. TdT assay was performed as described
in Materials and Methods. Each experiment was performed in triplicate.
Statistical analysis (paired t-test) showed P < .05 only in the responder group, for control versus IFN- 100 and 1,000 U/mL; control versus Fas triggering; any IFN- concentration
versus IFN- + Fas triggering.
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Posttranslational modulation of bcr/abl expression mediated by Fas
triggering.
Decreased expression of the bcr/abl protein after Fas
triggering could be related to inhibition of transcription, to
decreased stability of bcr/abl mRNA, or to posttranslational
regulation. To clarify this issue, we determined the level of
bcr/abl mRNA by a quantitative mRNA assay. In total CML cells
susceptible to Fas-mediated apoptosis, the bcr/abl mRNA level
was not influenced by Fas-R triggering (Fig
4A and B). Nevertheless, when identical amounts of protein extract
derived from total CML cell cultures from the same patient were
blotted, Fas-triggered samples showed markedly decreased levels of
bcr/abl protein (Fig 4C), suggesting a posttranslational
regulation.
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| Fig 4.
Effect of Fas triggering on bcr/abl expression
(mRNA and protein) from a CML patient in chronic phase who had optimal
response to IFN- and showed in vitro susceptibility to Fas-mediated
apoptosis. Quantitative RT-PCR: (A) densitometry and (B) agarose gel.
(C) Immunoblotting of p210 from total BM cells. Fas triggering induced downregulation of p210, but not of bcr/abl mRNA.
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Differential effects of IFN- and Fas-L on
hematopoietic colony formation of patients with CML. Relation with
clinical response to IFN-.
In previous studies, we showed that the activity of Fas triggering on
hematopoietic progenitor cells derived from patients with CML is
related to the levels of Fas-R induced by IFN-.25 When
proliferation of CD34+ CML progenitor cells was studied in
methylcellulose colony assays, the inhibitory effect of Fas triggering
on hematopoietic colony formation paralleled the induction of apoptosis
by Fas triggering. We looked for a possible correlation between the
differential susceptibility of CML cells to Fas triggering, as assessed
by a Fas-mediated inhibition colony assay, and the hematologic response to IFN- therapy. BM cells were treated with increasing
concentrations of IFN- to induce Fas-R expression, while the
concentration of the Fas triggering agent was kept constant. BM cells
derived from patients with an optimal response to IFN- showed higher
expression of Fas-R on CD34+ cells (Table 1) and a higher
baseline sensitivity to Fas triggering than those derived from patients
with a poor response (Table 2). The
suppression of colony formation by IFN- was dose-dependent in good
responders, while poor responders showed virtually no IFN--mediated
hematopoietic inhibition. Finally, in patients with an optimal response
to IFN-, there was synergism in the hematopoietic suppression by
addition of Fas triggering to IFN--treated samples; no synergism
was observed in patients who failed to respond to IFN- therapy
(Table 2). In parallel experiments, we evaluated whether there was a
correlation between rate of apoptosis induced by Fas triggering in
vitro and hematologic response to IFN-
(Table 3). CD34+ CML progenitor
cells derived from good responders to IFN- were suceptible to in
vitro Fas-mediated apoptosis, while CD34+ cells derived
from poor responder patients were resistant to Fas triggering, in
agreement with the data obtained in methylcellulose cultures.
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Table 2.
Effect of Fas Triggering on CFU-C Growth
From CD34+ BM Cells of CML Patients Sensitive or
Resistant to IFN-
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Table 3.
Effect of Fas Triggering on Apoptosis of
CD34+ BM Cells From CML Patients Sensitive or Resistant
to IFN-
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DISCUSSION |
High response rates to IFN-, evidence of a graft-versus-leukemia
effect in transplanted patients, and the favorable effect of donor
lymphocyte infusion in cytogenetic relapse of transplanted patients all
imply that immunologic mechanisms play an important role in the
pathophysiology of CML. We previously showed that Fas-R is expressed on
CD34+ cells derived from CML patients and that it can be
further upmodulated by IFN-. Triggering of Fas-R induces inhibition
of proliferation of these cells and its effect is potentiated in the
presence of IFN-.25-27 In the current study, we expand
these observations and describe a possible mechanism by which IFN-
and Fas-L may act on the proliferation and survival of CML cells.
Our results show that CD34+ cells derived from CML patients
who showed an optimal cytoreducing effect after IFN- therapy undergo apoptosis upon Fas-R triggering, while those derived from patients with
a poor response to IFN- are almost completely resistant to
Fas-mediated killing. Based on the reported antiapoptotic activity of bcr/abl, we hypothesized that the differential effect of
Fas-R triggering could be related to modulation of bcr/abl
protein expression in CML cells. Western blotting confirmed this
hypothesis: in seven of eight samples susceptible to Fas-induced
apoptosis, we observed a decrease of the bcr/abl hybrid
protein, whereas lack of apoptotic response to Fas-agonist was
associated with unchanged bcr/abl protein level. The mechanism
of the Fas-mediated bcr/abl downmodulation is, at present, only
a matter of speculation. In principle, the differential effects of the
Fas agonist could be due to the variability of Fas-R inducibility by
IFN-. However, because there was no difference in the effect of
IFN- alone on the proliferative capacity of CML cells between the
two groups of patients and our previous studies showed that the effect
of IFN- on Fas-R upmodulation is reproducible in all patients
studied, we conclude that an intrinsic mechanism related to the
intracellular transduction pathway of Fas must be responsible for the
inability of Fas agonists to induce apoptosis in patients with a poor
response to IFN-.
Our results are in agreement with other reports showing that
downregulation of bcr/abl produces decreased resistance of CML cells to apoptosis induced by various agents.17-19 The
presented experiments show that the decrease in bcr/abl protein
levels caused by Fas triggering is related to a posttranscriptional
modulation, as the levels of bcr/abl mRNA were not affected by
Fas triggering in cells susceptible to Fas-mediated apoptosis. The
lower levels of bcr/abl after Fas-R triggering cannot be due to
unspecific degradation of cellular proteins by proteases activated in
the process of Fas-mediated apoptosis, as documented by the constant amount of p145 c-abl and actin protein levels (Fig 1A).
A correlation between susceptibility to in vitro Fas-mediated
inhibition and in vivo response to IFN- has several implications. We
have analyzed the hematologic response to IFN-, which can be
evaluated in a few months' time and is considered necessary, although
not sufficient, for a major cytogenetic response, which, in turn, is
associated with a better survival. Cytogenetic response analysis was
precluded by the fact that poor responders to IFN- were switched to
other treatments. Enhanced Fas-R expression on CD34+ CML
cells of patients with optimal cytoreduction by IFN- may be related
to the in vivo action of several cytokines, such as IFN-, IFN-,
and TNF-, endogenously released as a part of the natural response to
the expansion of the leukemic clone, and further suggests the
involvement of the Fas-R/Fas-L system in the immunologic regulation of
CML progenitor growth. In addition, although IFN- may exert a direct
effect on hematopoietic progenitor cells, its inhibitory effects may
also be mediated by other factors, such as Fas-L supplied by T
lymphocytes. In patients with Kaposi's sarcoma, IFN- is effective
only if the number of CD4+ T cells is greater than
500/µL, implying that at least in that disease, an efficient immune
system is required for the antitumor effect of IFN-.46
In CML, the situation is even more complicated; in some patients, a
poor response to IFN- may be due to an intrinsic inability
(resistance) of the CML cells to respond to Fas-L. At present, it is
unknown whether such a defect is a specific feature of some subset of
CML or whether the ineffective Fas-R transduction pathway in CML cells
may develop as a secondary phenomenon. Indeed, the pressure exerted by
the immune system may select for cells resistant to Fas-mediated
apoptosis. In patients with blast crisis, we have seen a spectrum of
Fas-R patterns: while in some patients, CML blasts showed a high level
of Fas-R, in others, no Fas-R expression was detected (personal
unpublished data, October 1997). In the case of lymphoid
blastic crisis reported here, the bcr/abl protein level did not
decrease upon Fas triggering, and we could not demonstrate Fas-mediated
colony inhibition and induction of apoptosis. The inability to induce
apoptosis via the Fas pathway in the reported case, as well as in other
patients in blastic crisis (manuscript in preparation),
suggests that different resistance mechanisms may operate in
transformed cells. In acute myeloid leukemia, a correlation between
Fas-R expression and response to initial induction chemotherapy has
been reported; however, there was no correlation between inducibility
of apoptosis and level of Fas-R expression.47-50 Because it
is likely that the transduction pathways involved in apoptosis are
convergent, ie, common effector mechanisms may be used,3,51-55 resistance to Fas-mediated killing may be
associated with resistance to apoptosis induced by a variety of agents
acting downstream.4,8 Interruption of the apoptotic
response in CML cells may be related to properties of the p210
bcr-abl hybrid protein; however, several putative effector
genes such as phosphatidylinositol-3 kinase,56
Shc,57 GRB-2,58 p21RAS,59
p160BCR,60,61 Myc,62 and C-Myb63
may also play a role in this process. Therefore, apoptosis could result
either from signals counterbalancing the apoptotic drive or from a
blockade in the apoptotic machinery. Even under normal circumstances,
TNF or IFNs have been shown to induce upregulation of Fas-R on normal
hematopoietic progenitor cells and subsequent Fas-R triggering results
in apoptosis.23,24
Our study was designed to investigate the regulation of proliferation
and survival of CML progenitors rather than the utility of IFN- and
Fas triggering in the elimination of the Ph+ clone. Further
studies, some of which are currently in progress in our laboratories,
are needed to assess whether exposure to IFN- followed by Fas
triggering can be of any help for eradicating the disease or for
purging purpose.
| |
FOOTNOTES |
Submitted September 22, 1997;
accepted March 18, 1998.
Supported in part by Associazione Italiana Ricerca Cancro (A.I.R.C.),
Milan, Associazione Italiana contro le Leucemie (AIL), Rome, and MURST,
Rome, Italy.
Address reprint requests to Jaroslaw P. Maciejewski, MD, Department of
Internal Medicine, University of Nevada, Reno, Howard Medical Bldg 320, Reno, NV 89557-0046.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr Luigi Del Vecchio (Immunohematology Service, Cardarelli
Hospital, Naples) for flow cytometry analysis.
| |
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Abstract
Introduction
Abstract