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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3379-3389
Expression of Apoptosis-Regulating Proteins in Chronic Lymphocytic
Leukemia: Correlations With In Vitro and In Vivo Chemoresponses
By
Shinichi Kitada,
Janet Andersen,
Sophie Akar,
Juan M. Zapata,
Shinichi Takayama,
Stanislaw Krajewski,
Hong-Gang Wang,
Xin Zhang,
Florencia Bullrich,
Carlo M. Croce,
Kanti Rai,
John Hines, and
John
C. Reed
From the Burnham Institute, Cancer Research Center, La Jolla, CA;
Eastern Cooperative Oncology Group, Brookline, MA; The Sidney Kimmel
Cancer Center/Thomas Jefferson University, School of Medicine,
Philadelphia, PA; The Long Island Jewish Medical Center; and the
MetroHealth Medical Center, Case Western Reserve University, Cleveland,
OH.
 |
ABSTRACT |
B-cell chronic lymphocytic leukemia (B-CLL) represents a neoplastic
disorder caused primarily by defective programmed cell death (PCD), as
opposed to increased cell proliferation. Defects in the PCD pathway
also contribute to chemoresistance. The expression of several
apoptosis-regulating proteins, including the Bcl-2 family proteins
Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, and BAD; the Bcl-2-binding protein BAG-1; and the cell death protease Caspase-3 (CPP32), was
evaluated by immunoblotting using 58 peripheral blood B-CLL specimens
from previously untreated patients. Expression of Bcl-2, Mcl-1, BAG-1,
Bax, Bak, and Caspase-3 was commonly found in circulating B-CLL cells,
whereas the Bcl-XL and BAD proteins were not present. Higher levels of the anti-apoptotic protein Mcl-1 were strongly correlated with failure to achieve complete remission (CR) after single-agent therapy (fludarabine or chlorambucil)
(P = .001), but the presence of only seven CRs among the 42 patients for whom follow-up data were available necessitates cautious
interpretation of these observations. Higher levels of the
anti-apoptotic protein BAG-1 were also marginally associated with
failure to achieve CR (P = .04). Apoptosis-regulating
proteins were not associated with patient age, sex, Rai stage, platelet
count, hemoglobin (Hb) concentration, or lymph node involvement,
although higher levels of Bcl-2 and a high Bcl-2:Bax ratio were
correlated with high numbers (>105/µL) of
white blood cells (WBC) (P = .01; .007) and higher levels of Bak were weakly associated with loss of allelic heterozygosity at
13q14 (P = .04). On the basis of measurements of apoptosis induction by fludarabine using cultured B-CLL specimens, in vitro chemosensitivity data failed to correlate with in vivo clinical response rates (n = 42) and expression of the various
apoptosis-regulating proteins. Although larger prospective studies are
required before firm conclusions can be reached, these studies show the
expression in B-CLLs of multiple apoptosis-regulating proteins and
suggest that the relative levels of some of these, such as Mcl-1, may provide information about in vivo responses to chemotherapy. In vitro
chemosensitivity data, however, do not appear to be particularly useful
in predicting responses in B-CLL.
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INTRODUCTION |
B-CELL CHRONIC lymphocytic leukemia
(B-CLL) represents the most common type of leukemia, with approximately
12,000 new cases annually and a prevalence of about 50,000 to 60,000 patients in the United States alone.1 In its classic form,
this neoplastic disorder is characterized by the gradual accumulation
in the patient of small mature B cells, most of which are
G0/G1-phase, nonproliferating cells and which
display typical B-cell surface markers (CD19, CD20) in addition to
CD5.2-6 B-CLL represents the quintessential example of a
malignancy caused by failed programmed cell death (PCD), as opposed to
altered cell-cycle regulation. In essentially all self-renewing
tissues, new cell production is normally offset by a commensurate
amount of cell destruction through PCD. Imbalances in the activities of
opposing genes that either promote or block physiological cell death
can therefore slow or halt the rate of cell turnover, creating a
selective survival advantage for a particular clone that permits
expansion, often at the expense of its normal neighbors.7-9
Most B-CLLs have been reported to contain high levels of the
anti-apoptotic protein Bcl-2.10-16 The mechanisms
responsible for the high amounts of Bcl-2 observed in more than 80% of
B-CLLs remain enigmatic, but only rarely do they involve rearrangements of the BCL-2 gene as a result of chromosomal translocations,
unlike the follicular B-cell non-Hodgkin's lymphomas (NHL), and may
entail BCL-2 gene hypomethylation in its promoter
region.11,17,18 Because overexpression of Bcl-2 is so
widespread in B-CLL, examination of the relative levels of this
anti-apoptotic protein have not been particularly helpful in predicting
outcome for patients with this disorder. In this regard, B-CLL remains
an incurable disease, perhaps attributable, in large part, to the
well-established association of chemoresistance and radioresistance
with defects in PCD. B-CLL not only prolong the
physiological life span of cells but also render them resistant to the
cytotoxic effects of essentially all currently available anticancer
drugs.19 The clinical course for patients with B-CLL can be
quite variable, with many patients enjoying normal age-adjusted
survival but others succumbing to their disease within 1 year of
diagnosis.2-6,20 Response rates to single-agent therapy,
such as the alkylating agent chlorambucil or the purine nucleoside
analogues fludarabine and 2-CdA, vary widely among
studies,2-6,20,21 with advanced Rai stage and older age
generally associated with worse outcome. However, the biologic basis
for the widely different therapeutic responses of B-CLL patients
remains largely unknown.
Consideration of Bcl-2 and other apoptosis-regulating proteins may
provide insight into the pathogenesis of B-CLL and could potentially
assist in predicting clinical outcome. Indeed, Robertson et
al22 reported an association between shorter survival and higher levels of Bcl-2 protein in a study of 33 B-CLL patients. However, Bcl-2 was not of prognostic value in some other investigations of B-CLL patients.15,23,24 In this regard, Bcl-2 is only
one member of a large family of apoptosis-regulating proteins, with some functioning akin to Bcl-2 as blockers of apoptosis and others as
promoters of cell death.7,25 For example, in a recent study of 38 patients with B-CLL, Bcl-2 mRNA alone was not predictive of
outcome, but higher ratios of mRNA encoding Bcl-2 relative to one of
its antagonists Bax were associated with progressive disease.23 High Bcl-2:Bax protein ratios in B-CLLs have
also been observed in previously treated patients, as compared with untreated patients.15,24 Additional studies have also
suggested an important role for the Bcl-2:Bax protein ratio in
determining in vitro sensitivity to cytotoxic agents but have not
correlated these results with clinical responses.10,13
In this report, we evaluated the relative levels of several Bcl-2
family proteins in 58 cases of typical CD5+ B-CLL,
including the anti-apoptotic proteins Bcl-2, Bcl-XL, and Mcl-1 and the pro-apoptotic proteins Bax, Bak, and BAD. Moreover, we
determined the expression of BAG-1, a protein that interacts with Bcl-2
and Bcl-XL and that enhances the ability of Bcl-2 to prevent apoptosis.26 Finally, the expression of a protease
intimately associated with apoptosis, Caspase-3, also known as CPP32,
was examined.27,28 This protease exists as an inactive
zymogen in cells but frequently becomes activated through proteolytic processing mechanisms during apoptosis, allowing it to cleave a variety
of protein substrates that contribute to the apoptotic demise of the
cell. The relative levels of pro-Caspase-3 are known to vary in normal
B cells, with apoptosis-prone germinal center B cells typically
containing high levels of Caspase-3 protein and long-lived mantle zone
B cells having little or none of this protease.29
Comparisons were made between expression of these apoptosis-regulating
proteins and both in vitro and in vivo responses to chemotherapeutic
drugs.
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MATERIALS AND METHODS |
Patient materials.
All 58 B-CLL specimens originated from previously untreated patients
enrolled in the Eastern Cooperative Oncology Group (ECOG) trial's Rai
stage (1 = stage 0; 22 = stages I/II; 20 = stages III/IV;
15 = unknown), with 53 representing patients enrolled by ECOG in the
intergroup study C9011. This trial initially set out to compare outcome
in B-CLL patients treated with chlorambucil or fludarabine or with a
combination of these drugs.30 The fludarabine plus
chlorambucil arm, however, was discontinued because of unacceptable toxicity. Eight of the patient specimens evaluated in this study were
derived from this arm and were not included in the correlations with
outcome. Another three patients initially enrolled on C9011 were later
deemed ineligible; thus, clinical follow-up data were available for
only 42 of the patients whose peripheral blood specimens were evaluated
for apoptosis-regulatory proteins. These patients display the following
characteristics: age (66 median, 60 to 73 interquartile range), sex (33 male; 9 female), hemoglobin (12.2 g/dL median, 10.3 to 14 interquartile
range), platelet count (150 median; 98 to 186 interquartile range),
white blood cells (WBC) (93.7K median, 49.3 to 169.7K interquartile
range), percentage lymphocytes (92% median, 83% to 95% interquartile
range), and incidence of involvement of the central nervous system
(CNS) (0/41), peripheral nervous system (PNS) (1/40), spleen (31/41),
liver (8/41), node (35/41), skin (1/41), gums (0/41), and mediastinal mass (4/41). One half of these 42 patients received fludarabine and one
half chlorambucil as their initial therapy. Clinical responses were
assessed as described,6 for assigning patients to complete responder (CR), partial responder (PR), and nonresponder (NR) categories, with NR also including patients who progressed while receiving therapy. All samples represented heparinized whole blood obtained before therapy, mixed 1:1 with either Iscove's or Dulbecco's modified essential medium (IMEM or DMEM) and shipped at ambient temperature by overnight mail with processing the next day. Pilot experiments determined that B-CLLs handled in this way remained more
than 95% viable and that relative levels of Bcl-2 and several of the
apoptosis-regulating proteins studied remained essentially unchanged as
compared with blood specimens processed immediately after removal from
the patient. Peripheral blood lymphocytes were purified from blood
specimens by Ficoll gradient centrifugation. Flow cytometric analysis
determined that all specimens contained more than 90% CD5+
CD19+ B cells.
Immunoblot assays.
Immunoblot assays were performed as described in detail elsewhere,
using the multiple antigen detection (MAD) immunoblotting method
previously developed in our laboratory.31 Briefly, lysates were prepared from B-CLLs, normalized for total protein content (12.5 to 50 µg per lane, depending on the experiment), and subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% gel), followed by transfer to nitrocellulose filters. The primary
antibodies employed represented rabbit polyclonal antisera raised
against either synthetic peptides (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak) or recombinant protein produced in bacteria (Caspase-3) or
were murine monoclonal antibodies (MoAbs) raised against recombinant proteins (BAG-1, BAD). The characterization and documentation of the
specificity of all antibodies have been reported
previously.32-37 Secondary antibodies consisted of
horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or sheep
anti-mouse IgG (Bio-Rad Laboratories, Richmond, CA). Detection was
performed by an enhanced chemiluminescence (ECL) method (Amersham,
Arlington Heights, IL), followed by colorimetric detection, using SG
substrate (Vector Laboratories, Burlingame, CA) as
described.31 Lysates from the t(14;18)-containing lymphoma line RS11846 were included on every blot as an arbitrary standard for
subsequent normalization of all results, which were quantified by
scanning densitometry. Forty of the specimens were analyzed two to
three separate times, with less than 20% deviation among the results,
implying that the method was reproducible. Comparisons of selected
B-CLLs with various concentrations of RS11846 cell lysates verified
that the immunoblot assay was operating within the linear range for
detection of all antigens studied when using 12.5 to 50 µg per lane
of B-CLL lysate. In five cases, only colorimetric data were obtained,
rather than ECL-based development of x-ray films, precluding
densitometric quantifications. In these instances, the intensity of the
bands was scored as either high or low compared with the RS11846
standard, with "high" representing band intensities approximately
50% or more of those obtained for this cell line. RS11846 cells were
determined beforehand to contain relatively high amounts of Bcl-2,
Mcl-1, Bax, Bak, BAG-1, and Caspase-3 compared with a variety of other
human tumor cell lines.31
In vitro chemosensitivity assay.
Cells were cultured at 2 × 106 cells per mL in IMEM with
20% heat-inactivated fetal calf serum (FCS), 1 mmol/L
L-glutamine, and penicillin/streptomycin in 24-well plates
(2 mL per well) without or with various concentrations of fludarabine
(10 8 to 104 mmol/L) (gift of Berlex
Laboratories, Richmond, CA) or 2-CdA (105 to
109 mol/L) (gift from Dennis Carson, San Diego, CA).
After 3 days, the percentage of cells with fragmented DNA typical of
apoptosis was determined by TUNEL assay,38 using terminal
deoxylnucleotidyl transferase (TdT), biotinylated UTP, and fluorescein
isothiocyanate-streptavidin as described previously.32 Data
were collected by flow cytometry, subtracting the background
fluorescence of cells subjected to the same procedure without TdT
enzyme addition. The percentage of cells having undergone spontaneous
apoptosis was subtracted to determine the net percentage of
drug-induced apoptosis. In some cases, cell lysates were prepared for
immunoblot analysis at various times after treatment of CLL cells in
vitro with fludarabine.
Statistical analysis.
Immunoblot and in vitro chemosensitivity data were compared with
clinical responses, Rai stage, laboratory studies, and various patient
characteristics. For specimens on which immunoblot analysis was
performed two or three times, the mean was employed. In cases in which
only colorimetric immunoblot data were available, these patient
specimens were omitted for analysis of continuous variables or included
as "low" equal to zero and "high" as higher than any of the
other measures for nonparametric analysis, with the exception of the
Bcl-2:Bax ratio, where they were again omitted from the analysis. The
inclusion of these data did not substantially move the median or
general weight of the data. Evaluations of the association of
immunoblot data with in vitro chemosensitivity data and spontaneous apoptosis TUNEL assay data were performed using the Spearman
correlation and Wilcoxon statistics. The association of clinical
response (CR, PR, NR) with apoptosis proteins and TUNEL assay data for the 42 patients with outcome data enrolled in C9011 was evaluated by
logistic regression. Associations between immunoblot data and clinical
response (CR v non-CR) were evaluated by Fisher's exact test,
dichotomizing at 1.0 with respect to immunoblot scores, or as
continuous variables by logistic regression. A P value of  .05
was considered significant in all analyses.
| |
RESULTS |
Expression of apoptosis-regulatory proteins in B-CLLs.
Using antibodies specific for Bcl-2, Bcl-X, Mcl-1, Bax, Bak, BAD,
BAG-1, and Caspase-3, we determined the relative levels of these
apoptosis-regulatory proteins in 58 cases of B-CLL by immunoblot assay.
Among these proteins, Bcl-2, Mcl-1, Bax, Bak, BAG-1, and Caspase-3 were
commonly expressed in B-CLLs, whereas the Bcl-X and BAD proteins were
not present at detectable levels. Figure 1shows representative immunoblot results for some of these apoptosis-regulating proteins. Note that the expression of all these
proteins is variable among B-CLL specimens. These blots also compare
the results obtained for a t(14;18)-containing NHL B-cell line RS11846
and an EBV-immortalized B-lymphoblastoid line BJAB. The Bcl-X and BAD
proteins were not detected in B-CLLs (Table 1).
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| Fig 1.
Representative immunoblot data for apoptosis-regulatory
proteins in B-CLLs. Detergent lysates from B-CLLs were normalized for
total protein content (25 µg per lane) and subjected to
SDS-PAGE/immunoblot assay (12% gels) using various antibodies and a
method for sequential detection of multiple antigens from the same
blot.31 Representative ECL results are shown for 2 groups
of patients (left/right). The t(14;18)-containing B-cell lymphoma line
RS11846 and a B-lymphoblastoid line BJAB are shown for comparison. Note
that BAG-1 was present as two proteins. Preliminary data suggest that
the larger of these may represent a phosphorylated version of the
protein (unpublished observations). Both bands were
combined for densitometric scanning analysis.
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Figure 2 shows the scanning densitometry
results for these B-CLL specimens, presenting the data as histograms
that indicate the relative frequencies of specimens with various
amounts of these apoptosis-regulatory proteins. The results were
arbitrarily normalized relative to the RS11846 cell line (assigned a
densitometry score of 1.0), included on all blots as an internal
standard. Previous studies suggested that the ratio of Bcl-2:Bax mRNA
or protein was associated with progressive disease or treatment failure in B-CLL15,23; we therefore examined the Bcl-2:Bax protein
ratio as well. For purposes of dichotomizing the data, we arbitrarily
set densitometry scores of greater than 1.0 relative to the RS11846
cell standard as "high" amounts of apoptosis-regulatory protein.
The rationale for this approach to dichotomizing the data is clear for
Bcl-2, because RS11846 cells contain a t(14;18) translocation that
activates the BCL-2 gene. These cells were also found to
contain relatively high levels of Bax, Bak, Mcl-1, BAG-1, and
Caspase-3, as compared with a variety of other hematopoietic and
nonhematopoietic cell lines.31 On the basis of this
arbitrary cutoff of 1.0 or greater, B-CLLs expressed relatively high
amounts of apoptosis-regulatory protein in the following proportions:
Bcl-2 34/57 (60%), Mcl-1 24/55 (44%), Bax 27/57 (47%), Bak 27/51
(53%), BAG-1 7/51 (14%), and Caspase-3 18/52 (35%) (Table 1).
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| Fig 2.
Histogram presentation of densitometry data for
apoptosis-regulatory protein expression. Histogram representations of
immunoblot score data, showing the number of patient specimens (y-axis)
versus score (x-axis). Note that for the Bcl-2:Bax ratio, one
patient's ratio was 11 (indicated here as 2.5 with an asterisk).
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In vitro chemosensitivity testing of B-CLLs.
In vitro chemosensitivity testing was performed for 42 patient
specimens. For these experiments, B-CLLs were cultured in the absence
or presence of various concentrations of fludarabine or 2-CdA for 3 days, determined to be the optimal time on the basis of pilot
experiments in which time-course analysis of drug-induced apoptosis was
performed. Both purine nucleoside analogues induced rapid apoptosis in
susceptible B-CLLs, making it possible to subtract spontaneous
apoptosis that occurred in cultures from drug-induced cell death.
Initially, attempts were also made to explore the sensitivity of B-CLLs
to chlorambucil, as the clinical trial C9011 entailed randomization of
patients to either fludarabine or chlorambucil monotherapy. However,
chlorambucil-induced apoptosis occurred with relatively slow kinetics,
necessitating that TUNEL assays be performed at 5 or more days, making
it difficult to distinguish spontaneous apoptosis from drug induced.
For this reason, chlorambucil in vitro sensitivity testing was
abandoned.
The dose-response curves for B-CLLs cultured with either fludarabine or
2-CdA demonstrated two clear types of cellular behaviors, as shown in
Fig 3. Some patients exhibited
concentration-dependent inductions of TUNEL positivity, with
IC50 values consistently between 107 and
105 mol/L for fludarabine and between 108
to 106 mol/L for 2-CdA. By contrast, another group of
patients' B-CLL cells failed to undergo apoptosis in vitro when
cultured with these drugs. Of the 42 specimens for which in vitro
chemosensitivity testing was performed, 29 (69%) were sensitive to
both fludarabine and 2-CdA, one (2%) was sensitive to fludarabine but
not 2-CdA, one (2%) was sensitive to 2-CdA, but not fludarabine, and
11 (26%) were resistant to both drugs in vitro. The co-sensitivity of
B-CLLs to fludarabine and 2-CdA was highly significant
(P < .0001; McNeman test). In vitro chemosensitivity did
not correlate with the levels of any of the apoptosis-regulatory
proteins examined in this study.
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| Fig 3.
Representative examples of chemoresistant and
chemosensitive B-CLL specimens. TUNEL data, expressed as a percentage
of cells with intact DNA (ie, TUNEL negative), shown for two B-CLL
specimens, which are representative of the chemoresistant ( ) and
chemosensitive ( ) phenotypes. B-CLLs were cultured with the
indicated concentrations of fludarabine or 2-CdA for 3 days before
TUNEL/flow cytometric analysis was performed. Note that the rates of
spontaneous apoptosis for these 2 B-CLL specimens were about 20%.
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Spontaneous apoptosis of cultured B-CLLs correlates with in vitro
drug sensitivity.
When placed into routine culture, B-CLLs remain in a
G0/G1-phase nonproliferative state and begin to
die by apoptosis over time. The rate of spontaneous apoptotic cell
death was determined for each B-CLL at 3 days after initiation of
cultures. Highly variable rates of spontaneous apoptosis were observed
among the 42 B-CLLs tested, within a range of 3% to 60%. The rate of
spontaneous apoptosis failed to correlate with any of the individual
apoptosis-regulatory proteins or with the Bcl-2:Bax ratio. However,
higher rates of spontaneous apoptosis were correlated with increased
percentages of drug-induced apoptosis, when examined as continuous
variables (r = .421; P  0.01, Spearman
correlation) (Fig 4). Similarly, using the
in vitro chemosensitivity results to dichotomize the specimens into
drug-sensitive (n = 29) and drug-resistant (n = 13) groups based on
an IC50 value of less than 105 mol/L for
fludarabine and an IC50 value of less than
106 mol/L for 2-CdA, a strong association was again
found between higher rates of spontaneous apoptosis and
chemosensitivity, with a median percentage spontaneous apoptotic cells
after 3 days in culture of 33% (interquartile range, 22% to 42%) for
chemosensitive B-CLLs versus 13% (interquartile range, 9% to 21%)
for chemoresistant specimens (P = .003; Wilcoxon test).
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| Fig 4.
Correlation of spontaneous with drug-induced apoptosis.
Rates of spontaneous and drug-induced (fludarabine) apoptosis were compared. Correlation by the Spearman method.
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Though spontaneous apoptosis rates for B-CLLs in culture failed to
correlate significantly with clinical response (n = 42), a tendency
was noted for better responses (CR or PR) among those patients whose
B-CLLs exhibited higher rates of apoptosis in vitro. For example, the
median percentage of B-CLLs having undergone apoptosis after 3 days of
culture was approximately 34% for the 26 patients who attained a CR or
PR, compared with only about 17% for the 16 patients who failed to
respond (NR).
Effects of fludarabine on expression of apoptosis-regulatory
proteins.
To explore preliminarily whether drug-induced changes in the expression
of apoptosis-regulatory proteins correlated with in vitro
chemoresistance or chemosensitivity, isolated lymphocytes from
specimens representative of 2 resistant and 2 sensitive CLL cases were
treated in vitro with 1 µmol/L fludarabine and at various times
thereafter (3 hours to 3 days) relative levels of specific proteins
were evaluated by SDS-PAGE/immunoblot assay as above. Among the
proteins tested, only Mcl-1 displayed changes in its relative levels
after exposure of B-CLLs to 1 µmol/L fludarabine undergoing
time-dependent decreases in both the drug-resistant and drug-sensitive
CLL specimens. Figure 5 presents a typical example at higher concentrations of fludarabine (10 to 100 µmol/L), reductions in BAG-1 protein levels were also seen beginning at about 1 day, but the high percentage apoptosis in cultures of drug-sensitive
cells made it difficult to determine whether there was any difference
between drug-sensitive and drug-resistant CLL cells (not shown). By
contrast, the relative levels of Bcl-2, Bax, and Bak were not
significantly altered by 1 to 100 µmol/L fludarabine treatment in
vitro, nor were the Bcl-X or BAD proteins induced in these leukemic
cells (Fig 5; and data not shown).
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| Fig 5.
Fludarabine induces declines in Mcl-1 protein levels in
B-CLLs in vitro. Representative immunoblot data are presented for CLL
specimens that exhibited in vitro resistance
(IC50 > 105 mol/L) (R) or sensitivity
(IC50 < 105 mol/L) (S) to fludarabine.
CLLs were cultured at 2 × 106 cells/mL with or without 1 µmol/L fludarabine, then lysed in 1% Triton X-100-containing
solution, and subjected to SDS-PAGE/immunoblot assay after
normalization of samples for total protein content (25 µg/lane). The
same blot was incubated with antibodies specific for Mcl-1 (top), BAG-1
(middle), and Bcl-2 (bottom).
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Comparisons of apoptosis-regulatory proteins with Rai stage and other
patient characteristics: Association of Bcl-2 and high Bcl-2:Bax ratio
with higher WBC.
The relative levels of Bcl-2 family proteins, BAG-1, and Caspase-3 in
untreated CLLs were correlated with Rai stage (0 v I/II v III/IV) and other patient characteristics, using data from
the 42 patients with outcome data. No significant correlations were observed between any of the apoptosis-regulatory proteins and Rai
stage, sites of disease involvement (CNS, PNS, spleen, liver, node,
skin, gums, mediastinal mass), age (65 v >65), or sex. Among the laboratory studies, including hemoglobin (9 v >9
g/dL), platelet count (<100K v  100K), and WBC (<50K
v 50 to 100K v >100K), only higher WBC correlated
with higher levels of Bcl-2 (r = .424; P = .01) and
higher Bcl-2:Bax ratios (r = .473; P = .007) as
determined by the Spearman method.
LOH at 13q14 is weakly associated with higher levels of Bak
expression.
Deletions at 13q14 represent the most common cytogenetic abnormality
associated with B-CLL.3 Recently, we have performed a
molecular analysis of allelic loss of heterozygosity (LOH) in the 13q14
area for these B-CLL specimens, using nine microsatellite markers that
detect polymorphisms in this chromosomal region.33 Comparisons of LOH at 13q14 with the levels of various apoptosis regulatory proteins demonstrated a weak association between higher levels of Bak (Fig 6). The median and mean
Bak densitometry scores, respectively, for B-CLLs with 13q14 LOH were
1.39 and 1.27 (interquartile range, .94 to 1.59) compared with .92 and
.86 (intraquartile range, .16 to 1.28) for B-CLLs without molecular
evidence of LOH (P = .03, Wilcoxon test). Other than higher
levels of Bak, however, LOH at 13q14 was not significantly associated
with any other apoptosis-regulatory proteins, Rai stage or other
study-entry characteristics of the patients, response to chemotherapy
in vivo or in vitro, or rates of spontaneous apoptosis.
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| Fig 6.
Higher Bak is associated with LOH at 13q14. Dot histogram
representation of Bak immunoblot scores compared with presence
(positive) or absence (negative) of LOH at 13q14.33
P value is indicated (analysis of variance). Bars = mean
immunoblot score.
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Correlations of apoptosis regulatory proteins with clinical response
to chemotherapy: Associations with Mcl-1 and BAG-1.
The levels of various apoptosis regulatory proteins were compared with
clinical response (CR, PR, NR) (Fig 7; Table
2). Mcl-1 protein levels were lower among patients who achieved a CR (median, 0;
range, 0 to .67), compared with those who experienced only a PR or NR
(median, 1.75; range, 0 to 5.36) (P = .03), when examined as
continuous variables and including only quantitative immunoblot data
(n = 37) (Fig 8, left). In addition to
continuous variable analysis, dichotomization of the Mcl-1 immunoblot
data into high (>1.0) and low (1.0) expression groups on the basis
of comparisons with an arbitrary standard (RS11846 lymphoma cell line)
also showed a significant association between higher Mcl-1 protein
levels and failure to achieve CR (Table 2). In this case, 0 of 18 patients with higher Mcl-1 levels achieved a CR compared with 7/22
(31%) with lower Mcl-1 levels (P = .01). Thus, higher levels
of Mcl-1 were significantly associated with failure to achieve CR among the B-CLLs examined here. These results were not biased by the type of
chemotherapy (chlorambucil v fludarabine) received by the
patients with low Mcl-1 levels, because 13 of 22 patients whose B-CLLs
contained low Mcl-1 received fludarabine compared with 7 of 18 who had
high Mcl-1 levels.
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| Fig 7.
Comparisons of apoptosis-regulating proteins with
clinical response. Relative levels of Bcl-2, Mcl-1, BAG-1, Bax, Bak,
and Caspase-3 (CPP32) was compared for B-CLL patients who achieved CR
or PR, or who had no responses or progressed while on therapy (NR).
Bars = mean scores.
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Table 2.
Comparisons of Clinical Response With Immunoblot, 13q14,
LOH, Apoptosis, and In Vitro Drug Sensitivity Testing Data
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| Fig 8.
Association of higher Mcl-1 and higher BAG-1 protein
levels with failure to achieve CR. Dot histograms compare Mcl-1 (left) with BAG-1 (right) protein levels for patients who attained a CR versus
those who did not (NR, PR, or progression). Bars = mean immunoblot
scores. P values are indicated (logistic regression).
|
|
A weak association between worse clinical response to chemotherapy and
higher levels of BAG-1 protein was also observed (P = 0.04) (Fig 8, right), when examined as a continuous variable. For example, median BAG-1 protein levels among patients who attained a CR were .42 (range, 0 to 1.2) compared with 0.74 (range, 0 to 1.4) for patients
with PR or NR (P = .04, based on univariate logistic regression analysis in which data represented continuous variables and
included only samples with quantitative immunoblot data [n = 32]). These results were not biased by the type of chemotherapy (chlorambucil v fludarabine), as 7 of 11 (64%) patients whose BCLLs
contained low BAG-1 received fludarabine, as compared with 12 of 26 (46%) which had high BAG-1 (P > .05).
Although the Bcl-2:Bax ratio was not significantly associated with
clinical response when examined as a continuous variable, dichotomization into "high" and "low" Bcl-2:Bax groups
using t(14;18)-containing RS11846 cells as an arbitrary standard showed
an unexpected association between lower Bcl-2:Bax ratios and failure to
achieve CR (Table 2). None of the other apoptosis-regulatory proteins
exhibited significant associations with CR versus non-CR when examined
by this dichotomization method, based on the use of RS11846 cells as an
arbitrary standard (Table 2).
| |
DISCUSSION |
The preponderance of evidence indicates that B-CLL is caused by
dysregulation of PCD. To date, relatively little is known about the
expression of various genes that control apoptosis in this disease. In
this report, we examined the relative levels in 58 B-CLLs of 8 proteins
involved in apoptosis regulation, including the anti-apoptotic proteins
Bcl-2, Bcl-X, Mcl-1, and BAG-1 and the pro-apoptotic proteins Bax, Bak,
BAD, and Caspase-3. Among these, Bcl-2, Mcl-1, BAG-1, Bax, Bak, and
Caspase-3 were commonly expressed in B-CLLs, whereas Bcl-XL
and BAD were not. It should be recognized, however, that our analysis
was limited to circulating peripheral blood B-CLLs and therefore cannot
address this issue of the potential dynamics of apoptosis gene
regulation in lymph node, bone marrow, or other tissue compartments.
These caveats notwithstanding, based on current dogma that the normal
counterpart of CD5+ B-CLLs represents a subtype of mantle
zone B-lymphocyte,3 it may be noteworthy that circulating
B-CLLs commonly expressed Caspase-3 and that roughly one half contained
relatively high levels of Mcl-1. Previous studies have shown that
mantle zone B cells typically do not express immunodetectable amounts
of either Mcl-1 or Caspase-3.29,34 By contrast, germinal
center B cells do express these proteins at high levels. The expression
of Mcl-1 and Caspase-3 in B-CLLs may therefore represent examples of
aberrant gene expression associated with the pathogenesis of B-CLL.
Moreover, the expression of Mcl-1 in B-CLLs appears to be substantial
in the sense that (1) Mcl-1 was present at levels equal to, or in excess of, RS11846 and other B-cell lymphoma lines of germinal center
origin in approximately 45% of cases of B-CLL; and (2) normal and
malignant germinal center B cells contain some of the highest levels of
Mcl-1 among a wide variety of human tumor cell lines and normal
tissues35 (unpublished data). It should be noted, however, that until more is known about the cell of origin for
B-CLL, the significance of Mcl-1 and Caspase-3 expression in B-CLLs
should be interpreted with extreme caution.
Overall survival for patients with B-CLL varies widely, making clinical
decisions about therapy difficult and prompting the search for new
prognostic markers that can provide reliable guidance for individual
patients.2-6,20,36 Once B-CLL patients progress to the
point that therapy is required, however, the extent and duration of
response become prognostically important. The ability to predict
clinical responses to conventional chemotherapy could therefore be
useful for selecting patients for whom innovative experimental
approaches should be considered37 and for balancing efficacy with quality-of-life issues. Although the number of cases of
B-CLL examined in this report was relatively small, our data suggest
that in vitro chemosensitivity testing of the type performed in this
study is not particularly helpful in predicting in vivo responses of
patients to monoagent therapy consisting of either fludarabine or
chlorambucil. One limitation of the study is that we were unsuccessful
in performing adequate in vitro studies of sensitivity to chlorambucil,
whereas one half of the patients were treated with this alkylating
agent. Nevertheless, when the analysis was limited only to those
patients who received fludarabine, as opposed to chlorambucil
(n = 21), again no statistically significant correlations were
obtained.
Multiple factors could potentially account for the discrepancy between
in vitro and in vivo responses to chemotherapeutic drugs, including (1)
differences in tissue compartments and local environmental signals that
affect chemosensitivity of B-CLLs in peripheral blood, bone marrow, and
nodes; (2) sampling bias introduced by basing analyses on peripheral
blood drawn at a single time; (3) differences between in vitro and in
vivo metabolism of drugs to active or inactive products; (4)
differences in the duration of treatment in vitro and in vivo and the
kinetics of responses measured in culture versus in the patient; and
(5) the possibility that clinical responses of B-CLL cells to
fludarabine or chlorambucil may reflect more than merely the efficiency
with which these drugs induce apoptosis, such as induction of immune
cell-mediated killing in vivo. Regardless, it is interesting that
B-CLLs exhibited two clearly different behaviors in vitro (ie,
sensitive v resistant) with respect to induction of apoptosis
by purine nucleoside analogues, implying either intrinsic or extrinsic
(influenced by tissue culture conditions) differences in these two
types of leukemia cells. Although these differences were not readily
explainable by examination individually of several apoptosis-regulating
proteins, it may be that development of more complicated statistical
models that simultaneously consider the contributions of multiple anti-
and pro-apoptotic proteins would have allowed such relations to be discerned. Thus, the data imply either that no one particular apoptosis-regulating protein is sufficiently dominant to account for
these two types of in vitro chemoresponses or that other
apoptosis-regulating proteins not examined in this report represent
important variables. Of course, these two possibilities are not
mutually exclusive.
The observation that higher rates of spontaneous apoptosis in culture
correlated with in vitro sensitivity to fludarabine and 2-CdA implies
that drug-sensitive B-CLL cells may be generally more easily induced to
undergo apoptosis, perhaps because of intrinsic differences in the
activities of apoptosis-controlling genes in these cells or differences
in their requirements for environment survival factors (eg, cytokines
or cell adhesion events), or both. Classic drug resistance mechanisms
thus seem unlikely to account for the poor in vitro apoptotic responses
to purine nucleosides observed for approximately one third of the
B-CLLs studied here, particularly, because the B-CLL specimens were all
derived from previously untreated patients. However, in the absence of
direct comparisons of apoptosis data with studies of drug uptake,
metabolism, and incorporation into DNA or RNA, we cannot be certain of
this interpretation. Nevertheless, given that many B-CLLs (as compared with normal B cells) have been reported to be resistant to apoptosis induced by a variety of stimuli, including anti-Fas antibodies and
TGF- ,39,40 it could be of interest in future studies to explore whether cross-resistance to several apoptotic stimuli can be
used to segregate B-CLLs into apoptosis-sensitive and -resistant subgroups and further to attempt correlating this information with
clinical outcome. Of note, unlike a previous study,41 we did not observe significant correlations between higher rates of
spontaneous apoptosis and lower Rai stage. Our finding that B-CLL
specimens that were resistant to fludarabine in vitro were also almost
uniformly cross-resistant to 2-CdA agrees with clinical experience.42
Deletions at 13q14 represent probably the most common cytogenetic
abnormality associated with B-CLL.33 Our comparisons of LOH
at 13q14 with apoptosis-regulatory proteins revealed a marginal association with higher levels of the pro-apoptotic protein Bak (P = .04). The Bak gene has been mapped to chromosome 1 in
humans (D. Tomei, personal communication), indicating that
LOH at 13q14 cannot directly explain this correlation. Until the
putative B-CLL suppressor gene located at 13q14 is identified, it seems
premature to suggest specific mechanisms that might account for the
observed association with higher Bak protein levels.
A major goal of this pilot study was to determine whether expression of
any of the apoptosis-regulatory proteins described in this report might
show promise as a predictor of clinical responses to chemotherapy in
previously untreated B-CLL patients. Our analysis of 42 patients points
to the need for further exploration of the anti-apoptotic
Bcl-2 family protein Mcl-1. Expression of Mcl-1 has not been studied
extensively in B cells, but it is induced in normal peripheral blood B
cells by interleukin-4 (IL-4), anti-IgM, phorbol ester, and the
combination of CD40 ligand and IL-13, all stimuli that prolong B-cell
survival in vitro.43 Mcl-1 expression is also induced in B
cells by the LMP-1 protein of Epstein-Barr virus (EBV).44
Conversely, stimuli that promote apoptosis of normal peripheral blood B
cells, such as transforming growth factor- (TGF-) and forskolin,
have been shown to down-regulate Mcl-1 protein levels.43
Compared with Bcl-2, the Mcl-1 protein has generally been less potent
as a cell death blocker in gene transfer experiments, but all these
studies have been limited to nonlymphoid cells, and hence do not
address the issue of cellular context.45-47 Like Bcl-2, the
Mcl-1 protein has been shown to be capable of heterodimerizing with
Bax, at least in vitro, and can neutralize Bax-mediated cytotoxicity in
yeast.47 As with Bcl-2, in vitro binding and yeast
two-hybrid experiments also suggest that Mcl-1 can potentially bind to
the anti-apoptotic proteins Bcl-2 and Bcl-XL, as well as
the Bcl-2 accessory protein BAG-147,48 (unpublished data). Thus, to a great extent, Mcl-1 can probably be
viewed as a substitute for Bcl-2. Unlike Bcl-2, however, the Mcl-1
protein appears to localize predominantly to endoplasmic reticulum and nuclear envelope with relatively less associated with mitochondrial membranes.49 Given that most B-CLLs contain high levels of
Bcl-2, it is tempting to speculate that the combination of Mcl-1 and Bcl-2 may provide more than additive protection against apoptosis induced by chemotherapeutic drugs in vivo. Alternatively, the in vivo
dynamics of Mcl-1 and Bcl-2 gene regulation in B-CLLs may create
situations in which either Bcl-2 or Mcl-1 but not both of these
anti-apoptotic proteins are present, thus affording B-CLLs that have
the capacity to express both genes a greater advantage, when confronted
with anticancer drugs. This situation would be analogous to normal B
cells in which the mantle zone B cells surrounding germinal centers in
the secondary follicles of lymph nodes have been shown to express high
levels of Bcl-2, but not Mcl-1, whereas a reciprocal pattern of Mcl-1
and Bcl-2 expression has been documented for germinal center B
cells.34
Higher levels of Mcl-1 protein were significantly associated with
failure to attain CR, as determined by logistic regression analysis
using continuous variable data (P = .001) and Fisher's exact
test, for which an arbitrary cutoff of 1.0 relative to the lymphoma
cell line RS11846 was used to dichotomize B-CLL patients into
"low" and "high" Mcl-1 groups (P = .01). Since
Mcl-1 protein levels were not significantly associated with Rai stage,
in vitro drug responses, LOH at 13q14, or other variables examined in
this report, Mcl-1 status may provide independent prognostic
information for patients with B-CLL. However, the sample size of this
study was insufficient to perform multivariate analysis. Moreover, the suggestion that high expression of Mcl-1 may correlate with failure to
achieve CR in B-CLL should be viewed only as a hypothesis that can now
be tested using additional independent data sets.
This caveat is also true for the BAG-1 data presented in this paper,
which demonstrated a more marginal but nevertheless statistically significant (P = .04) association with failure to attain CR.
An association between higher relative levels of BAG-1 and poorer in
vivo responses to chemotherapy is conceptually consistent with data
showing that BAG-1 can interact with Bcl-2 and enhance cellular resistance to apoptosis.26
Among the Bcl-2 family proteins evaluated in this report, only Mcl-1
displayed clear changes in its relative levels following exposure of
CLLs to clinically relevant concentrations of fludarabine in vitro. The
significance of this observation is unclear since similar drug-induced
declines in Mcl-1 protein levels were detected in B-CLLs, regardless of
whether they exhibited sensitivity or resistance to fludarabine in
vitro. Nevertheless, it would be interesting in future studies to
evaluate Mcl-1 protein levels in circulating CLL cells before and
immediately after initiation of therapy to explore whether it has
potential as a surrogate marker of clinical response.
In contrast to Mcl-1 and BAG-1, the association seen for lower
Bcl-2:Bax ratios and failure to achieve CR is paradoxical but was only
observed when data were dichotomized relative to the arbitrary standard
(RS11846 cells) and was not noted when immunoblot data were analyzed as
a continuous variable. Moreover, CART analysis50 failed to
detect a significant percentage cutoff for dichotomization of Bcl-2:Bax
immunoblot data that correlated with clinical response in this study
(data not shown). Thus, unlike some previous
studies,15,23,24 we failed to detect a correlation of
higher Bcl-2:Bax ratios with worse prognostic features, and the only
trend in these data was unexpectedly toward better response rates among
patients with higher Bcl-2:Bax ratios.
Taken together, therefore, the data presented in this pilot study of
previously untreated B-CLL patients demonstrate some of the
complexities of apoptosis-regulatory gene expression in this disease.
Although highly preliminary, the findings raise the possibility that
Mcl-1 and possibly BAG-1 may provide prognostic information about
clinical responses to chemotherapy. Additional independent studies
involving larger groups of patients are needed to explore this
hypothesis.
| |
FOOTNOTES |
Submitted August 7, 1997;
accepted December 18, 1997.
Supported by National Institutes of Health Grant No. U01-CA-60421 and
by an unrestricted educational grant (to S.K.) from Berlex
Laboratories.
Address reprint requests to John C. Reed, MD, PhD, The Burnham
Institute, Cancer Research Center, 10901 N Torrey Pines
Rd, La Jolla, CA 92037.
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 H. Gallant and T. Potter for manuscript preparation.
| |
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Abstract
Introduction
Abstract