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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1420-1426
NEOPLASIA
Thioredoxin prolongs survival of B-type chronic lymphocytic
leukemia cells
Joacim Nilsson,
Ola Söderberg,
Kenneth Nilsson, and
Anders Rosén
From the Department of Biomedicine and Surgery, Division of Cell
Biology, University of Linköping, Sweden; Department of
Pathology, Laboratory of Tumor Biology, University Hospital, Uppsala,
Sweden; Institute for Pathology and Molecular Immunology, University of
Porto, Portugal.
 |
Abstract |
Thioredoxin (Trx) is a ubiquitous protein disulfide oxidoreductase
with antioxidant, cytokine, and chemotactic properties. Previously, we
showed that Trx, in synergy with interleukin 1 (IL-1), IL-2, IL-4,
tumor necrosis factor  (TNF-), and CD40-ligation induced S-phase
entry and mitosis in normal B cells and B-type chronic lymphocytic
leukemia (B-CLL) cells. The viability of B-CLL cells stimulated by
these protocols is high, and it has been hypothesized that the
overexpression of Bcl-2 found in B-CLL protects the cells from
apoptosis in vitro and in vivo. In this study, we have analyzed the
response of cells derived from 12 samples of patients with B-CLL to
recombinant human Trx in spontaneous apoptosis, with special reference
to the Bcl-2 expression. Long-term cultures of B-CLL clones showed
significantly higher viability when supplemented with human Trx
(P = .031), also exemplified with clones surviving more
than 2 months. Short-term cultures of B-CLL cells exposed to 1 µg/mL
of Trx for 1, 5, or 12 days maintained expression or delayed
down-regulation of Bcl-2 compared with control cultures containing RPMI
1640 medium and 10% fetal calf serum only (P = .032, .002, .026, respectively). All B-CLL cells expressed constitutive Trx at
varying but low levels, in contrast to adult T-cell leukemias, which
overexpress Trx, as previously reported. We found that Trx added to
B-CLL cells increased in a dose-dependent fashion the release of
TNF-, which has been suggested to be an autocrine growth factor for
these cells. In conclusion, we have found that human recombinant Trx
induced TNF- secretion, maintained Bcl-2, and reduced apoptosis in
B-CLL cells.
(Blood. 2000;95:1420-1426)
© 2000 by The American Society of Hematology.
| |
Introduction |
B-type chronic lymphocytic leukemia (B-CLL) is the most
common leukemia of adults in western countries, accounting for 30% of
all cases.1-3 The diagnostic hallmark of B-CLL is an
accumulation of monoclonal CD5-positive B cells resting in the
G0/G1 stage of the cell cycle that express a
limited VH-gene repertoire. Typical B-cell surface antigens
are present along with low amounts of surface IgM/IgD.4,5
The expression of the proto-oncogene bcl-2 is up-regulated 1.5- to 25-fold in most cases of B-CLL.6,7 The Bcl-2 protein
overexpression may explain the relentless successive expansion of the
malignant clone despite a minimal proliferating cell fraction. Bcl-2
expression is enhanced by cytokines such as interleukin 4 (IL-4),
IFN- , IFN- , basic fibroblast growth factor, and
CD6-ligation.8-12 In contrast, glucocorticoids,
IgM-ligation, IL-10, or growth factor withdrawal leads to bcl-2
down-regulation and bax up-regulation.13-14 High
Bcl-2 to Bax ratios were found to protect against
apoptosis.12 It is not known by which mechanism B-CLL cells
proliferate, but physiological stimulation through membrane
receptor-ligation and cytokine ligand-receptor interactions will induce
mitogenic responses, although often weak.15-22 One of these
cytokines that contributed to S-phase entry and mitosis in synergy with
IL-2, IL-4, tumor necrosis factor (TNF-), and CD40-ligation was
thioredoxin (Trx).19
Trx is a ubiquitous redox-active and multifunctional molecule of
Mr 11 800, containing an active site motif with amino
acids Cys-X-X-Cys.23-25 Together with the selenoprotein
thioredoxin reductase (TrxR) and reduced nicotinamide adenine
dinucleotide phosphate (NADPH), it is a potent antioxidant
and protein disulfide oxidoreductase.26 Several
extracellular cytokine activities, such as IL-1-like factor, B-cell
stimulatory factor, IL-2R inducing factor, eosinophil cytotoxicity enhancing factor, and recently, chemotactic factor, have been attributed to Trx.19,27-30 In macrophages, it was found
that Trx is a potent costimulus of cytokine expression and
release.31 Overexpression of Trx has been observed in
neoplastic disease, protecting cells from oxidative
stress.23,32 In B-CLL cells, Trx expression was previously
found to be low but inducible.33
On the basis of the hypothesis that cognate interaction between B-CLL
cells and normal cells within bone marrow34 and peripheral lymphoid organs (eg, TH cells, follicular dendritic cells,
and monocytic cells) may initiate overexpression of cytokines,
including Trx, contributing to aberrant apoptosis regulation, we wanted to explore, in this study, the role of Trx in regulating Bcl-2 expression and in protecting the B-CLL cells from apoptosis. During the
course of the study, we found that Trx-stimulated autocrine TNF-
release in a dose-dependent manner and that B-CLL cells were rescued
from spontaneous apoptosis.
| |
Materials and methods |
B-CLL cells and patients
Blood was collected in heparinized tubes and diluted with an equal
volume of sterile phosphate-buffered saline (pH 7.2). Lymphocytes were
separated by density gradient centrifugation (Ficoll-Hypaque, Pharmacia, Uppsala, Sweden). Cells from the interface were carefully collected, washed 3 times with phosphate-buffered saline, and then
frozen and stored in liquid nitrogen. Cells from patients I-105, I-40,
I-90, I-276, I-397, and I-424 were collected at the University Hospital
in Uppsala, Sweden. The other patient samples were collected at the
Hematology Clinic, University Hospital, Linköping, Sweden. Table
1 shows the data for patients with CLL and
the phenotypes of the purified cells, which were analyzed by flow
cytometry with the use of Becton Dickinson reagents (San Jose, CA). The
majority was B cells, and NK and T cells were below 1% in most B-CLL
samples (range 0.02%-2.5%, mean = 0.86%) (Table 1). For
comparison, CD19-positive selected samples were analyzed from I-105
patient, showing identical results in Trx stimulation experiments. The
ethical committees at the University Hospitals approved this study,
including informed written consent from the patients.
Production of recombinant human Trx
Two different preparations of human recombinant full-length Trx were
used. A Trx expression plasmid, containing the complementary DNA
sequence for human Trx in the MP6 cell line was transferred into an
Escherichia coli host, BL21, and purified as previously described, by sonication, ultracentrifugation, and MonoQ liquid chromatography.35 Alternatively, a human trx
complementary DNA was subcloned into the pACA expression vector, and
the overexpressed protein was purified as described36 (also
commercially available from IMCO, Stockholm, Sweden). Trx is sensitive
to atmospheric oxidation and dimerizes easily. For maintenance of
biological activity, recombinant Trx was, therefore, reduced by
dithiothreitol-activated beads, ReductacrylTM reagent
(Calbiochem Novabiochem, San Diego, CA), and sterilely filtered
immediately before addition to cultures.
Analysis of apoptosis
FITC-labeled Annexin-V (Boeringer-Mannheim, Mannheim, Germany) for
detection of phosphatidyl serine exposed on the plasma membrane was
used, according to the manufacturer's instruction. Detection of
apoptosis was also performed by analysis of DNA fragmentation by
terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
(TUNEL) with the use of biotinylated dUTP, according to the
manufacturer's recommendation (Boeringer Mannheim). Alternatively, cells were stained with propidium iodide, according to the method of
Hotz et al,37 followed by FACS analysis for
detection of DNA loss. The viable cell number was assessed by counting
cells in triplicates in a Bürker chamber with the use of the
trypan blue dye exclusion method.
Flow cytometry and immunofluorescence microscopy
Immunofluorescence staining for microscopy or FACS analysis was
performed with 1 × 106 cells, which were washed,
fixed in 4% paraformaldehyde, and finally permeabilized with 0.1%
saponin, according to the method of Sander et al.38 In some
experiments, 70% methanol was used for permeabilization instead of
saponin, without affecting Bcl-2 fluorescence intensity. The following
antibodies were used: anti-Bcl-2-FITC (clone 124, Dako, Glostrup,
Denmark), mouse IgG isotype control FITC-conjugate (Dako),
anti-Trx39 monoclonal antibody (mAb), anti-TrxR
mAb,40 produced in our laboratory at Department of
Biomedicine and Surgery, Division of Cell Biology, Linköping
University, Sweden, anti-Bax (P19, Santa Cruz Biotechnology, Santa
Cruz, CA), in combination with FITC-labeled goat antimouse Ig,
F(ab)'2-fragments (Dako). Positive and negative cell lines
for each antigen were included as specificity controls for the
different antibodies.
Short-term and long-term cultures of B-CLL
B-CLL cells were cultured in RPMI 1640 medium with 10% fetal calf
serum (Gibco, Paisley, Scotland) plus antibiotics (Gibco) at a cell
density of 1 × 106 cells/mL at 37°C in 5%
CO2 atmosphere. Trx (1 µg/mL) was added into the RPMI
1640 medium containing 10% fetal calf serum. Long-term cultures were
fed once a week, inspected microscopically, and determined for
viability by trypan blue dye exclusion. Trx was also collected from
serum-free, 24-hour culture medium of the MP6 T-cell hybridoma
cells41 and used as an alternative source of Trx (25% of
MP6 supernatant) in some experiments as indicated. The stimulatory
activity of this 24-hour Trx-containing supernatant was completely
abolished by a preincubation with Sepharose-anti-Trx beads for 30 minutes at 4°C.19
TNF-, IL-6, and IL-1 determinations by enzyme-linked
immunosorbent assay
TNF-, IL-6, and IL-1 released into B-CLL supernatants were
determined by sandwich enzyme-linked immunosorbent assay techniques, using commercially available kits from R&D Systems Inc (Minneapolis, MA) and Mabtech (Stockholm, Sweden).
Statistical analysis
Statistical differences between Trx-treated and nontreated control
cultures of B-CLL clones were evaluated pair-wise by using Wilcoxon
signed rank sum test. All statistical evaluations were performed with
JMP version 3.2.5 (SAS Institute, Cary, NC) software and a Gateway
G6-300 PC microcomputer.
| |
Results |
Trx delayed Bcl-2 down-regulation and reduced apoptosis in B-CLL
cells
Our previous studies on in vitro cultures of B-CLL cells, in which
we established several B-CLL cell lines by Epstein-Barr virus
transformation, showed that addition of Trx or combinations of
Staphylococcus aureus Cowan I with IL-2/Trx to the cultures considerably increased the chances of obtaining cell
lines,22 probably by increasing viability and by allowing
the cells to enter the cell cycle.42 In considering the
improved cell culture viability, we asked whether exogenous Trx might
affect apoptosis and Bcl-2 expression in short-term cultures of B-CLL cells.
First, we analyzed the expression of Bcl-2 after 1, 5, and 12 days of
in vitro culture with human Trx added to 12 different B-CLL cell
samples. At the time of bleeding (day 0), Bcl-2 was found to be high
(median of 12 mean fluorescence intensities
(MFI) = 43.7, range 30.6-49.1) in all cells and reduced
to half (MFI = 20.4) on culturing in vitro over a 12-day period, in
agreement with previously published results.6 We found that
growth factor removal already led to Bcl-2 down-regulation after 24 hours in 7 of 12 samples, and, at day 5 and day 12, all B-CLL cultures expressed lower Bcl-2 levels. Trx-treated cell samples showed delayed
down-regulation and expressed minor albeit significantly more Bcl-2
protein compared with control medium in 9 of 12 cultured cells
(Table 2). The differences
were gradually increasing from 4.5%, to 7.0%, and to 11.5% on day 1, day 5, and day 12, respectively. Paired data were evaluated
statistically in Wilcoxon signed rank sum test, showing significant
differences between the two groups (P values day
1 = .032; day 5 = .002; day 12 = .026).
Representative flow cytograms from separate experiments at day 5 are
shown in Figure 1A (left panel), showing that the Bcl-2 expression in RD-1 B-CLL cells was not significantly changed after 5 days in the control medium. In cultures exposed to Trx (Figure 1A,
right panel), however, Bcl-2 expression was higher compared with
control culture with medium only. After 12 days, the Bcl-2 expression
levels were reduced to a high extent in control cultures without Trx,
but they were less reduced in cultures with Trx (Figure 1B),
represented by B-CLL samples I-40, I-276, and I-397. We also assayed
for apoptotic events in B-CLL cells by Annexin-V. Comparisons between
Trx-treated and medium-cultured cells revealed small but significantly
reduced percentages of apoptotic cells (median = 7.0%, n = 12) in
the Trx-treated group (P = .031). Apoptosis was also analyzed
by the TUNEL method for detection of DNA fragments, which occurs later
in the apoptotic process than the phosphatidyl serine expression, as a
rule. In one such experiment, 5 of 6 cell cultures showed significantly
less DNA fragmentation in the presence of recombinant Trx (1 µg/mL)
(data not shown).
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| Fig 1.
Bcl-2 expression.
A. Bcl-2 expression at day 0 and day 5. Right panel: cultures with
recombinant thioredoxin (rTrx) 1 µg/mL; left panel: cultures without
rTrx. B-type chronic lymphocytic leukemia (B-CLL) cells were obtained
from patient RD-1. B. Bcl-2 expression 12 days after onset of cultures
with Trx (Trx day 12, thick line) and without Trx (day 12, dotted line)
in comparison with Bcl-2 expression at the onset of experiment (day 0, thin line). Flow cytometry analysis was performed as described in
Materials and methods section. C = control mouse isotype IgG; counts = cell number; FL1-H = fluorescence intensity.
|
|
Trx promotes long-term survival of B-CLL cells
To find out whether the observed delay in Bcl-2 down-regulation and
the decrease in apoptosis could be sustained and affect long-term
survival, we extended the observation time and supplied exogenous
reduced Trx once a week to B-CLL cultures. Figure
2A and B show viable cell numbers (survival
curves) of 5 different B-CLL cells grown in RPMI 1640 with 10% fetal
calf serum with Trx and in their control cultures. The I-105 B-CLL
cells were followed for 68 days. Without Trx addition, the cells were
all dead after 12 days, in contrast to Trx cultures, which showed <40% reduction in cell numbers after 68 days (Figure 2A). Cells from
patients I-40, I-276, I-397, and I-90 (Figure 2B) were followed for 3 weeks. Addition of Trx had a significant survival advantage in 3 of the
4 B-CLL cultures. The percentage of viable cells at day 19 (last day of
observation in Figure 2B) was significantly higher in Trx-supplied
cultures (P = .031) (Table 3).
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| Fig 2.
A. Viability curves for I-105 cells cultured with and
without MP6-thioredoxin.
B. Survival curves for cells in vitro of 4 patients with B-type chronic
lymphocytic leukemia with and without recombinant thioredoxin or
MP6-thioredoxin.
|
|
Constitutive Trx expression is low in most B-CLL cells and does not
correlate with survival
The overexpression of Trx found in human adult T-cell leukemia and
certain other malignancies could not be found in B-CLL cells (Figure
3), showing fresh B-CLL cell samples
(before onset of in vitro culture). In addition to Trx, we analyzed, by
flow cytometry, Bcl-2 and the apoptotic protein Bax, which
heterodimerizes with Bcl-2, as well as TrxR, which maintains Trx in an
active and reduced state, and the anti-apoptotic protein Mcl-1, which was reported to play a role in B-CLL survival.43 The
expression of Trx was highly variable (Figure 3). KK-32 showed the
highest Trx expression but also the highest Bax expression. This B-CLL clone died rapidly in spontaneous apoptosis medium (Figure
4). The long-term survivor KF-101 was high
in Bcl-2 but low in Trx expression. In separate cultures, KF-32 cells
remained viable for a long time in the presence of 1 µg/mL of
recombinant Trx with a similar profile to Trx-treated I-105, I-40,
I-276, and I-397 cells shown in Figure 2 (data not shown). Thus,
constitutive Trx expression did not correlate with survival nor with
Bcl-2 expression. The correlation between high Bcl-2 expression levels (high Bcl-2 to Bax ratios) and degree of survival observed in various
B-CLL samples in vitro has been reported by several
groups.12,43,44 In this study, KK-32 showed the lowest
Bcl-2 to Bax ratio and died rapidly. KK-101, however, expressed the
highest Bcl-2 to Bax ratio combined with the highest observed viability
after 45 days in vitro (Figure 4).
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| Fig 3.
FACS analysis of thioredoxin, thioredoxin reductase,
Bcl-2, Mcl-1, and Bax expression of cells from patients with B-type
chronic lymphocytic leukemia (KK-32, KF-65, KF-101, and LO-157).
Mean ± SEM (vertical bars) is shown.
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| Fig 4.
Survival profiles of cells of 4 patients with B-type
chronic lymphocytic leukemia (B-CLL) after in vitro culturing without
thioredoxin.
The B-CLL cells were cultured in RPMI 1640 plus 10% fetal calf serum.
Viability was assessed by trypan blue dye exclusion.
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Trx induces TNF- secretion in B-CLL in a dose-dependent
manner
To approach an understanding of the mechanism behind the Trx-induced
enhancement of Bcl-2, we investigated Trx-treated B-CLL culture
supernatants for the presence of autocrine secreted cytokines. Recombinant Trx (1, 2, and 8 µg/mL) was added to B-CLL LS-109 cells
and cultured for 1 day. Figure 5 shows that
Trx significantly increased TNF- release compared with control
medium without Trx (P = .023, P = .001,
P = .042, respectively). Supernatants from 12 Trx-treated
cultures (1 µg/mL) and control cultures were analyzed for TNF-.
Ten of 12 Trx-treated cultures contained more TNF- than did control
cultures. The percentage of TNF- increase for these Trx-cultures
were +269%, +104%, +60%, +25%, +22%, +21%, +13%, +12%, +5%,
+3%, -11%, and -17% (median = 17%; P = .008). In
addition, IL-1 and IL-6 were found in Trx-stimulated cultures, but
no significant difference between stimulated and nonstimulated cultures could be found at the tested concentration of 1 µg/mL of
recombinant Trx (data not shown).
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| Fig 5.
Release of tumor necrosis factor (TNF-) in
response to increasing amounts of thioredoxin added.
106 B-type chronic lymphocytic leukemia LS-109 cells per mL
were cultured for 24 hours in RPMI 1640 with 10% fetal calf serum.
Culture supernatants were analyzed in a sandwich enzyme-linked
immunosorbent assay for TNF-. Mean values represent mean of 2 separate cultures each analyzed in triplicates with SEM. Wilcoxon
signed rank test P values were calculated.
*P < .05, *** P < .001.
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| |
Discussion |
The main finding of this study is that Trx added to B-CLL cells
significantly delayed Bcl-2 down-regulation and diminished the number
of apoptotic cells, resulting in prolonged survival. Trx also
stimulated autocrine TNF- release in a dose-dependent manner in
these cultures, which may partly explain the augmented viability.
The characteristic overexpression of the anti-apoptotic protein Bcl-2
found in B-CLL in vivo is well documented,6,7 and it is
not, as a rule, a result of t (14:18) translocation events, as
described for follicular lymphomas. Apoptosis can be mediated by
several factors, including oxidative stress.45 Bcl-2 does not appear to influence the generation of oxidative stress mediators (eg, hydrogen peroxide), but it does prevent oxidative damage to
cellular constituents, including lipid membranes.46 Several studies12,13 indicate that the mechanism behind the high
Bcl-2 level in B-CLL cells involves continuous input of external
signals generated via cytokine receptors. Bcl-2 expression, or rather the Bcl-2 to Bax ratio in B-CLL, was up-regulated by IL-4, IFN-, IFN-, TNF-, basic fibroblast growth factor, IL-8, and
CD6-ligation or down-regulated by IL-10.8-12,14,47 Growth
factor removal on explantation in vitro causes the external signals to
relax, leading to Bcl-2 down-regulation and apoptosis induction as
found in this study and by others.12 We found that growth
factor removal led to Bcl-2 down-regulation after 24 hours in 7 of 12 samples, and, at day 5 and day 12, all B-CLL cultures expressed lower
Bcl-2 levels. Supplementation of recombinant human Trx, however,
delayed to some extent the down-regulation of Bcl-2 in 9 of 12 cultures (Table 2) and resulted in a concomitant
drop in the percentage of apoptotic cells. After 1 day of Trx
treatment, a small but significant difference in Bcl-2 expression was
found: 4.5% higher than control, a value that increased to 7% and
11.5% on day 5 and 12, respectively. Also in the Annexin V analysis,
small (7%) but significant differences between Trx and control
cultures were found. Accumulating day-by-day effects of Trx may select
for survival enhancement of Bcl-2-expressing cells and add up to
viable cells as seen on day 19 in Figure 2 in which a more evident
difference between Trx and control cultures is seen. In view of the
biology of CLL and its chronic appearance and many times a slow
accumulation of leukemic cells in the blood of the patient, we think
that these tiny 1-day differences should not be overlooked.
During the course of this study, we found that Trx induced a
dose-dependent release of TNF-, a known growth factor for
B-CLL.17,18 In macrophages, extracellular Trx in synergy
with phorbol 12-myristate 13-acetate was previously shown by Schenk et
al31 to induce a release of cytokines IL1, IL6, IL8, and
TNF. The mechanism by which extracellular Trx acts is largely unknown,
but Trx may modulate membrane receptor thiols via membrane localized
protein disulfide isomerase. No specific receptor for Trx has been
identified hitherto nor has any active uptake of Trx over the plasma
membrane been described23,30 (Rosén and Lindquist,
unpublished observations). Direct modulation (reduction and activation)
by Trx of cystein-rich receptors has been described for the
interferon- receptor or ICAM-1.48,49 CD5 and CD6
membrane receptors, expressed on B-CLL cells, belong to the
cystein-rich scavenging receptor family.50 An alternative
explanation is that reduced extracellular Trx may transfer reducing
equivalents (electrons) via membrane bound TrxR, thus changing the
intracellular reduced or oxidized Trx balance. Intracellular Trx is a
direct inhibitor of apoptosis signal-regulating kinase 1 and is a
regulator of NF-kappa B activation.51,52
Has the Trx-induced survival of B-CLL cells in this study any in vivo
relevance? Since we found that the leukemic B-CLL cells themselves do
not overexpress Trx, in contrast to adult T-cell leukemia,28 we considered the possibility that Trx can be
supplied by other cells in close contact with the leukemic cells in the patient with B-CLL. Antigen-presenting cells residing in the
pseudofollicles of the lymph nodes of patients with B-CLL34
may contribute to Trx on the basis of suggestions from findings in mice
lymphoid tissue, revealing that interdigitating cells are highly
positive for Trx.53 We and others39,40,54
(Söderberg A, Sahaf B, Rosén A, et al [submitted]) have
found that Trx and TrxR are present in high quantities on plasma
membranes of antigen-presenting monocytes or macrophages. From other
systems, it is known that bone marrow stromal cells interact closely
with pro-B cells and may up-regulate Bcl-2 expression and prevent Bax
induction in pro-B cells55 and that bone marrow fibroblasts
will rescue plasma cells from apoptosis by sustaining a high Bcl-2
expression.56 Tötterman et al57 have
observed that, before recurrence of malignant B-cells, a transient peak
of increased T-cell numbers occurs in several patients. These T cells
may be potential Trx producers as suggested by our own previous
studies. Those studies19,58 showed that a T-helper cell
line, MP6, established by fusion between the leukemic cell line Molt4
and normal healthy blood donor T-helper cells, was the most potent Trx
producer and the best stimulator of B cells among a wide panel of cell
lines tested. We suggest that B-CLL cells maintain Bcl-2 expression and
survival in vivo through cell-cell contact with antigen-presenting
follicular dendritic cells that overexpress Trx or stimulated T-helper
cells that may lead to multiple but low-dose autocrine cytokine
release. Some of the B-CLL samples tested in this study
contained 1%-2% T cells; therefore, we compared in separate
experiments these samples with CD19 positively selected samples
(containing <0.2% T cells) for possible influence of T cells on the
outcome of Trx treatment. No difference in response was found.
We have shown in this study that the participation of the redox-active
molecule Trx is important for B-CLL cell survival in vitro for
extensive time periods and that Bcl-2 down-regulation was delayed by
Trx additions. Future work will focus on the in vivo cellular source
and expression pattern of redox-active proteins and their role in the
leukemogenic process.
| |
Acknowledgments |
We thank Ms Anita Lönn, Inga-Lill Scherling, and Ms Kerstin
Willander for skillful technical assistance. We express our
gratitude for help with clinical specimens of patients with B-CLL to
Drs Karin Karlsson, Gunnar Juliusson, Claes Malm, and Ilse
Christiansen, GSD.
| |
Footnotes |
Submitted June 12, 1999; accepted October 20, 1999.
Supported by funds from the Swedish Cancer Association, Stockholm,
Sweden, and the Portuguese Foundation for Science and Technology.
Reprints: Prof. Anders Rosén, Department of Biomedicine
and Surgery, Division of Cell Biology, University of Linköping, S-581-85 Linköping, Sweden; e-mail: AndRo{at}mcb.liu.se.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction