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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3379-3389
By
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.
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.
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.
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 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 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).
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.
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
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).
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 ( 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.
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 (
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.
Submitted August 7, 1997;
accepted December 18, 1997.
We thank H. Gallant and T. Potter for manuscript preparation.
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Abstract
Introduction
Abstract
8 to 10
.05
was considered significant in all analyses.
) 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%.
0.01, Spearman
correlation) (Fig 
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.
,