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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2914-2923
Fas/APO-1 (CD95)-Mediated Apoptosis Is Activated by
Interferon- and Interferon- in Interleukin-6
(IL-6)-Dependent and IL-6-Independent Multiple Myeloma Cell Lines
By
Helena Spets,
Patrik Georgii-Hemming,
Jan Siljason,
Kenneth Nilsson, and
Helena Jernberg-Wiklund
From the Department of Genetics and Pathology, Unit of Pathology,
University Hospital, Uppsala, Sweden.
 |
ABSTRACT |
A poor response to Fas-induced apoptosis is evident in some multiple
myeloma (MM) cell lines and primary cells. In this study, we have
examined the possibility to increase the sensitivity to Fas-induced
apoptosis by pretreatment of MM cells with interferon- (IFN-) or
interferon- (IFN-). Both IFN- and IFN- markedly increased
the Fas-induced apoptosis in all cell lines tested (U-266-1970, U-266-1984, and U-1958). In the U-266-1970 and U-1958 cell lines, pretreatment with either IFN- or IFN- also inhibited
proliferation in a dose-dependent manner. In contrast, IFN-
activation of the Fas death pathway in the U-266-1984 cells was not
accompanied by growth inhibition. Incubation with the IFNs increased
the Fas antigen expression in one of three cell lines but did not alter the expression of Bcl-2 or Bax. The IFNs are important regulators of
growth and survival in MM cells. Our results suggest that activation of
Fas-mediated apoptosis is a novel mechanism by which the IFNs exert
inhibitory effects on MM cells.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
MULTIPLE MYELOMA (MM) is a hematopoietic
malignancy characterized by a clonal expansion of malignant plasma
cells located to the bone marrow. The role of interleukin-6 (IL-6) as a
prominent growth factor for MM cells in vitro and in vivo has been well established,1-3 and we recently described the possibility
to dissociate the functions of IL-6 as a growth and survival factor in
IL-6-dependent and -independent MM cell lines.4 Also,
additional factors seem to operate to contribute to the prevention of
apoptosis in MM cells, including insulin-like growth factor-I
(IGF-I)5,6 and the bcl-2 family of
proteins.4,7 Several other cytokines have been reported to
be involved in the control of growth and survival of MM cells. Studies
on the effect of interferon- (IFN-) both in vitro and in vivo
have shown that high concentrations of IFN- can inhibit the growth
of MM cells,8-15 whereas low concentrations of IFN- may
stimulate MM cell proliferation.11,16,17 IFN- has also
been shown to induce cell death in MM cells, although in a recent study
a decreased sensitivity to Fas-induced apoptosis was reported as an
initial and transient effect of IFN-.18 IFN- has been
demonstrated to inhibit growth and induce death in MM cell lines and
primary MM cells.13,15 IFN- and IFN- have been
suggested to exert the effect on MM cells by interfering with the
signaling pathway of IL-6.19 Although downregulation of
IL-6 receptors in MM cells has been proposed as a plausible mechanism
for growth inhibition by IFN- and IFN-,20,21 a recent
study found no correlation of a reduced number of IL-6 receptors and
IFN-mediated growth inhibition in MM cell lines.22 This
finding suggests additional mechanisms responsible for the IFN effects
observed on MM cells.
Cross-linkage of the Fas (APO-1/CD95) antigen by its ligand FasL or
anti-Fas MoAbs triggers the cascade of signals for apoptosis in various
cell types.23 The Fas antigen is a member of the tumor
necrosis factor (TNF) receptor superfamily of proteins and is expressed
in many neoplastic and normal cells, including hematopoietic cell
lines, lymphoma cells, and activated normal T and B
lymphocytes.24-26 In a previous report, Moller et
al26,27 showed that normal and malignant murine plasma
cells only rarely express the Fas antigen. More recently, others have
demonstrated Fas expression on some human MM cell lines and
patient-derived primary cells. However, these studies show a poor
response to Fas-induced apoptosis in a majority of the cases and were
unable to establish a correlation between Fas antigen expression and
susceptibility to Fas-mediated apoptosis in MM.28-30 The
Fas antigen contains a cytoplasmic region, the death domain, essential
for the recruitment of the IL-1 -converting enzyme (ICE) family of
related caspases and induction of the signaling cascade mediating cell
death.31-33 However, the possible contribution of the
bcl-2 multigene family of survival antagonists and agonists to
inhibit or accelerate apoptosis by interfering with this signal cascade
of ICE family of related caspases is still essentially unknown.33,34
In several reports, IFN- and IFN- have been shown to activate
Fas-mediated apoptosis via upregulation of Fas antigen
expression.32,35 In the light of IFNs as potent regulators
of growth and survival in MM, we investigated the possibility of
activating Fas-mediated apoptosis in MM cell lines via IFN- and
IFN- and plausible molecular mechanisms underlying such an
activation. We selected three MM cell lines: the IL-6-independent
U-266-1984 cell line, previously suggested to be resistant to
Fas-mediated apoptosis29; its early passage IL-6-dependent
counterpart U-266-1970 cell line; and the IL-6-dependent U-1958 cell
line. These three MM cell lines have been demonstrated to vary in their
responsiveness to IFN- and IFN-.12,15
We show that pretreatment with IFN- or IFN- markedly augmented
the Fas-mediated apoptosis by anti-Fas MoAbs in the U-266-1984 cell
line as well as in the IL-6-dependent U-266-1970 and U-1958 cell
lines. This activation of Fas-mediated apoptosis seemed to act
independently of IFN- and IFN--induced growth inhibition and
upregulation of Fas antigen expression, which was only evident in one
of three MM cell lines. In addition, the IFN-activation of the
Fas-mediated apoptosis in MM cell lines did not alter the Bcl-2/Bax
ratio.
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MATERIALS AND METHODS |
Cell lines and reagents.
All MM cell lines were maintained in RPMI 1640 (Sigma Biosciences, St
Louis, MO) containing 10% fetal calf serum (FCS; GIBCO, Grand Island,
NY), glutamine, and antibiotics (100 IU/mL of penicillin and 50 µg/mL
of streptomycin). Cells of the U-266 cell line grows partly adherent
and partly in suspension.36 The phenotypic properties of
U-266-1970 (early) and U-266-1984 (late) passage U-266 cells were
described elsewhere.37 The IL-6-dependent cell lines
U-195838 and U-266-1970 were routinely grown on a layer of
the IL-6-producing human fibroblast line AG1523 (The Human Mutant
Genetic Cell Repository, Camden, NJ). Recombinant IL-6 (specific
activity, 0.67 to 2.0 × 106 U/mg) was purchased from
R&D Systems Europe Ltd (Abington, UK) and used at 20 to 100 U/mL.
Anti-Fas monoclonal antibodies (MoAbs) CH-11 (mouse IgM) and UB2 (mouse
IgG1) were purchased from MBL (Nagoya, Japan). CH-11 were used to
induce apoptosis, whereas the UB2 antibodies were used to analyze the
expression of Fas antigen. As a negative control for CH-11, we used
mouse IgM MoAbs (Dako A/S, Glostrup, Denmark). R-phycoerythrin
(RPE)-conjugated mouse IgG1 (Dako) and fluorescein
isothiocyanate (FITC)-conjugated mouse IgG1 were used as
negative control MoAbs and FITC-conjugated antirabbit IgG (Dako) served
as a secondary antibody in the flow cytometry analysis.
Assays for growth and apoptosis.
Exponentially growing MM cells were seeded in 0.2 × 106 to 0.4 × 10 6 cells/mL in 12-well
plates in RPMI 1640 containing 10% FCS. The U-1958 and U-266-1970
cells were incubated with rIL-6 (20 to 100 U/mL). rIL-6 was added to
the cultures at the initiation of the experiment and after 96 hours of
incubation. IFN- or IFN- was added to all MM cell lines at
concentrations ranging from 0 to 1,000 U/mL and incubated with
(U-266-1970 and U-1958) and without IL-6 (U-266-1984). Cells were
harvested at 96 hours for analysis of Fas, Bcl-2, and Bax expression.
Cell number and viability were determined by trypan blue exclusion. In
parallel cultures, cells were incubated for an additional 24 hours
without and with anti-Fas MoAbs (CH-11; 100 ng/mL) or isotype-matched
control IgM MoAbs (100 ng/mL) and then harvested for TUNEL analysis and
apoptotic morphology examination. As previously described, to
standardize the apoptotic assay and analysis, exponentially growing MM
cells from each cell line were induced by staurosporine treatment for 24 hours.4 For U-266-1984, 600 nmol/L of staurosporine was used, whereas in the U-266-1970 and U-1958 cell lines, 600 and 200 nmol/L, respectively, was used to induce apoptosis.
Flow cytometric analysis of Bcl-2 and Bax.
The cells were fixed in cold 2% paraformaldehyde-phosphate-buffered
saline (PFA-PBS) for 45 minutes on ice and were then permeabilized in
cold 70% methanol (MeOH) for 60 minutes. Cells were then washed and
incubated with 1° ab (FITC-conjugated anti-Bcl-2 or isotype control, FITC-conjugated IgG) for 30 minutes on ice or 1° ab
(anti-Bax or anti-Bax incubated with Bax-peptide for 2 hours as a
control) and 2° ab (FITC-conjugated antirabbit Abs) for 30 minutes.
After additional washing, the cells were resuspended in 1.0 mL and the mean fluorescence intensity (MFI) of Bcl-2 and Bax was determined by
flow cytometry (FACSort; Becton Dickinson, San Jose, CA).
Flow cytometric analysis of apoptosis by TUNEL staining.
Cells were fixed in cold 2% PFA-PBS for 45 minutes on ice and then
permeabilized in cold 70% MeOH for 60 minutes. The cells were then
washed and incubated with 100 µL terminal transferase buffer, 1 mmol/L cobalt chloride, 20 U terminal transferase, and 1 nmol
biotinylated-16-dUTP (Boehringer Mannheim, Mannheim, Germany) at
37°C for 30 minutes. The reaction was stopped by the addition of TB
buffer (300 mmol/L sodium chloride, 30 mmol/L sodium citrate) and
incubated at room temperature for 15 minutes. The cells were washed and
labeled with 50 µL of Streptavidin-RPE (Dako) diluted 1:30 in PBS + 2% FCS for 30 minutes on ice. For double-staining of Bcl-2 and Bax,
the procedure is described elsewehere. After additional washing, the
cells were resuspended in 1.0 mL and DNA fragmentation was determined
by flow cytometry (FACSort; Becton Dickinson).
| |
RESULTS |
IFN induces dose-dependent growth inhibition and augmentation of
Fas-mediated apoptosis in IL-6-dependent MM cell lines.
A considerable heterogeneity in MM cells in vitro and in vivo with
regard to Fas-antigen expression and anti-Fas-induced apoptosis have
been reported. Although MM cells in vitro and in vivo have previously
been demonstrated to express the Fas-antigen, anti-Fas-mediated apoptosis has only been demonstrated in a limited number of MM cell
lines and primary cells.28-30 In MM cells, IFN- and
IFN- have been reported to be important regulators of growth and
survival.11,12,15,17-19 Because IFN- and IFN- in some
lymphocytes have been shown to be potential inducers of Fas
antigen,32,35 we investigated the possibility of increasing
the Fas antigen expression and/or activating Fas-mediated
apoptosis in MM cells by IFNs.
We selected three MM cell lines: the IL-6-independent U-266-1984 cell
line, previously suggested to be resistant to Fas-mediated apoptosis,29 and the IL-6-dependent cell lines, U-266-1970
and the U-1958, all of which have different responses to IFN- and IFN-.3,38 We have previously demonstrated dose-dependent growth-inhibitory effects of IFN- in the IL-6-dependent U-266-1970 and U-1958, whereas the growth and viability of the IL-6-independent U-266-1984 cells were unaffected by treatment with
IFN-.15 As previously demonstrated by us and
others, both IL-6-dependent and -independent MM cells can be
growth-inhibited by IFN-.12,15,22
Exponentially growing U-266-1970 cells were preincubated with IFN-
(0 to 1,000 U/mL) or IFN- (0 to 1,000 U/mL) in culture medium
containing IL-6 (20 U/mL). At 96 hours, the number of viable cells was
determined by trypan blue exclusion. The apoptotic population was
analyzed by TdT-mediated dUTP nick end-labeling (TUNEL) technique after
24 hours of incubation without or with anti-Fas MoAb (CH-11; 100 ng/mL)
or with the addition of isotype-matched MoAbs (control IgM; 100 ng/mL).
The results show that Fas-induced apoptosis was augmented in U-266-1970
cells as a result of pretreatment with increasing concentrations of
IFN-. In U-266-1970 cells, pretreatment with IFN- (1,000 U/mL)
for 96 hours before the addition of CH-11 resulted in 55% of cell
death (Fig 1A). In contrast, no increase of
the cell death was recorded in cultures incubated with IFN- for 96 hours in the absence of CH-11 or with the addition of control IgM MoAbs
at concentrations corresponding to those of the anti-Fas MoAbs (Fig
1A). The cell death induced by CH-11 in exponentially growing
U-266-1970 cells was determined to be 13% as compared with the
spontaneous apoptosis recorded in the absence of CH-11 (6%; Fig 1A).
In keeping with previously published results,15 IFN-
induced a dose-dependent growth inhibition in the IL-6-dependent U-266-1970 cells, with no apparent effects on the viability (Fig 1B).
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| Fig 1.
(A) Cell death of U-266-1970 cells in the absence ( )
or presence ( ) of anti-Fas MoAbs (CH-11; 100 ng/mL) or control IgM
Abs (100 ng/mL;  ). U-266-1970 cells were preincubated for 96 hours
with IFN- in concentrations ranging from 0 to 1,000 U/mL before the
addition of anti-Fas MoAbs or isotype-specific control MoAbs and
harvested for TUNEL and flow cytometry analysis at 24 hours of
incubation. Vertical bars indicate the standard error of mean (SEM).
(B) Cell number and viability of U-266-1970 cells incubated with
IFN- at increasing concentrations (0 to 1,000 U/mL) in the presence
of IL-6 (20 U/mL). Cells were harvested at 96 hours and the number of
viable cells () and the percentage of viability of the total number
of cells () were determined by using trypan blue exclusion. Vertical
bars indicate the SEM.
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In parallel cultures, U-266-1970 cells were preincubated with
increasing concentrations of IFN- for 96 hours. The apoptotic population was analyzed with TUNEL and flow cytometry after an additional 24 hours of incubation without and with CH-11 or with the
addition of control IgM (Fig 2). The
preincubation with IFN- (1 to 1,000 U/mL) dose-dependently augmented
the Fas-induced apoptosis in U-266-1970 cells in a similar way as did
the pretreatment with IFN-. The population of apoptotic U-266-1970
cells, when pretreated with 1,000 U/mL of IFN-, was determined to be
75% with the addition of CH-11, whereas the addition of control IgM
induced only 8% of cell death (Fig 2A). In addition, increasing
concentrations of IFN-, without the addition of CH-11, could not
significantly increase the population of apoptotic U-266-1970 cells as
compared with the spontaneous apoptosis recorded in exponentially
growing cells (Fig 2A). In contrast to IFN--pretreated cells, the
growth inhibition induced by IFN- was accompanied by a small
reduction of the viability in U-266-1970 cells. At 96 hours, the
viability of U-266-1970 cells was decreased by 11% in cultures with
1,000 U/mL of IFN- as compared with exponentially growing cells (Fig 2B).
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| Fig 2.
(A) Cell death in the absence () or presence () of
anti-Fas antibodies (CH-11; 100 ng/mL) or control IgM (100 ng/mL; )
for 24 hours in U-266-1970 cells preincubated for 96 hours with IFN-
in concentrations ranging from 0 to 1,000 U/mL. Cells were harvested
and subjected to TUNEL and flow cytometry analysis. Vertical bars
indicate the SEM. (B) Cell number and viability of U-266-1970 cells
incubated with IFN- at increasing concentrations (0 to 1,000 U/mL)
in the presence of IL-6 (20 U/mL). Cells were harvested at 96 hours and
the number of viable cells () and the percentage of viability of the
total number of cells () was determined by using trypan blue
exclusion. Vertical bars indicate the SEM.
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In the IL-6-dependent U-1958 cells, a similar pattern of
dose-dependent IFN-augmentation of Fas-mediated apoptosis was recorded. The cell death of exponentially growing U-1958 cells in the presence of
CH-11 was calculated to be 30%, whereas pretreatment of U-1958 cells
with IFN- (1,000 U/mL) or IFN- (1,000 U/mL) and subsequent incubation with CH-11 resulted in 50% and 52% cell death,
respectively (Fig 3). However, in this cell
line, pretreatment with IFN- and IFN- (1,000 U/mL) led to a
moderate increase of the cell death, 33% and 24%, respectively, also
in the absence of CH-11, as compared with the cell death in
exponentially growing U-1958 cells (11%; Fig 3).
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| Fig 3.
Cell death in U-1958 cells in the absence or presence of
anti-Fas antibodies (CH-11; 100 ng/mL) or control IgM (100 ng/mL).
U-1958 cells were preincubated for 96 hours without or with IFN-
(1,000 U/mL) or IFN- (1,000 U/mL) before the addition of anti-Fas
MoAbs or isotype-specific MoAbs. At 24 hours, the cells were harvested
and subjected to TUNEL and flow cytometry analysis. Bars indicate the
SEM.
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IFN induces dose-dependent activation of Fas-mediated apoptosis also
in the absence of growth inhibition.
To evaluate whether the activation by IFNs was the result of growth
inhibition, the IL-6-independent U-266-1984 cell line was investigated
with regard to Fas-mediated cell death with and without pretreatment
with increasing concentrations of IFN- and IFN-. This cell line
is not growth inhibited by IFN-.15 The U-266-1984 cells
were incubated in the presence of increasing concentrations of IFN-
or IFN- (0 to 1,000 U/mL) before the addition of anti-Fas MoAbs
(CH-11; 100 ng/mL) or isotype-matched MoAbs (control IgM; 100 ng/mL).
The results show that IFN- augmented the Fas-induced apoptosis in
pretreated U-266-1984 cultures in the absence of growth inhibition. In
U-266-1984 cells pretreated with IFN- (1,000 U/mL) for 96 hours, the
population undergoing apoptosis with CH-11 was 21% as compared with
the cell death induced by CH-11 in exponentially growing cells (8%;
Fig 4). As determined by the TUNEL
analysis, the addition of control IgM (100 ng/mL) to
IFN--pretreated U-266-1984 cells had no effect on the cell death
(Fig 4). In contrast to the observed effects of IFN- in
IL-6-dependent U-266-1970 and U-1958 cells, growth was unaltered and
no significant decrease of the viability was recorded in the
IL-6-independent U-266-1984 cell line at 96 hours of incubation in
1,000 U/mL of IFN- (Fig 5A).
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| Fig 4.
Cell death in U-266-1984 cells in the absence or presence
of anti-Fas antibodies (CH-11; 100 ng/mL) or control IgM (100 ng/mL).
U-266-1984 cells were preincubated for 96 hours without or with IFN-
(1,000 U/mL) or IFN- (1,000 U/mL) before the addition of anti-Fas
MoAbs or isotype-specific MoAbs. At 24 hours, the cells were harvested
and subjected to TUNEL and flow cytometry analysis. Bars indicate the
SEM.
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| Fig 5.
(A) Cell number () and viability () of U-266-1984
cells incubated with IFN- at increasing concentrations (0 to 1,000 U/mL). Cells were harvested at 96 hours and the number of viable cells
was determined by using trypan blue exclusion. (B) Cell number ()
and viability () of U-266-1984 cells incubated with IFN- at
increasing concentrations (0 to 1,000 U/mL). Cells were harvested at 96 hours and the number of viable cells was determined by using trypan
blue exclusion.
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Also, pretreatment with IFN- augmented the Fas-induced apoptosis by
CH-11 in U-266-1984 cultures (Fig 4). In U-266-1984 cell cultures
pretreated with 1,000 U/mL of IFN-, the cell death was calculated to
be 31% when induced by CH-11 for 24 hours (Fig 4). The apoptotic
population was found to be essentially unaltered with the addition of
control IgM or without the addition of CH-11 to U-266-1984 cells
pretreated with IFN- (1,000 U/mL). In U-266-1984 cell cultures,
IFN- activation of the Fas-mediated apoptosis was accompanied by
dose-dependent growth inhibition and a reduced number of viable cells
(Fig 5B).
In view of the reported heterogeneity of primary cells and cell lines
of MM to Fas induced apoptosis, primary B-B4+ plasma cells
isolated from bone marrow aspirates of patients with MM responsive to
Fas-induced apoptosis were analyzed with regard to IFN--augmented
apoptosis. As an indication that our findings on the cell lines are
representative to the situation in primary MM cells, our results show
that IFN- may augment Fas-induced apoptosis also in selected cases
of primary MM cells (data not shown).
Expression of Fas/APO-1 (CD95) in IL-6-dependent and -independent
MM cell lines.
To examine the molecular mechanism underlying the activation of
Fas-mediated apoptosis, the MM cell lines representing two different
categories of IL-6 dependence and responsiveness to IFN were examined
for Fas/Apo-1 (CD95) expression. Because the Fas antigen in some
lymphocytes have been shown to be regulated by IFN-,32
the MM cells lines were examined for Fas antigen expression after 96 hours of incubation in increasing concentrations of IFN- (0 to 1,000 U/mL) by using anti-Fas MoAbs (UB2) and flow cytometric analysis. Fas
antigen was found to be expressed in exponentially growing cells of all
three MM cell lines. In the U-266-1970 and U-266-1984 cells, 87% and
84%, respectively, of the cells were Fas positive, whereas in the
U-1958 cell line, 55% of the cells were found to be positively stained
by the UB2 (Fig 6). The number of Fas
antigen expressing cells and MFI from each MM cell line after treatment
with 1,000 U/mL IFN- for 96 hours are also presented in
Table 1.
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| Fig 6.
Fas expression in the U-266-1970, U-266-1984, and U-1958
cell lines. Cells were incubated in the absence (U-266-1984) and
presence (U-266-1970 and U-1958) of IL-6 (20 U/mL). Cells were
incubated in the absence or in the presence of IFN- (1,000 U/mL) or
IFN- (1,000 U/mL) for 96 hours and subjected to flow cytometry
analysis using the UB2 antibody. The population of Fas-positive cells
was gated in the analysis by the use of mouse anti-IgG1 antibodies.
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IFN- did not significantly alter the number of Fas
antigen-expressing cells of the three MM cell lines (Fig 6 and Table
1). However, a 2.1-fold increase of the MFI within the
CD95+ cell population was observed with IFN- (1,000 U/mL) in the U-266-1970 cells as compared with exponentially growing
cells in culture medium with IL-6. A more moderate increase of MFI
could also be recorded in the U-266-1984 cell line (1.4-fold). The MFI
of CD95+ U-1958 was essentially unaffected by IFN-
treatment (Table 1). To evaluate whether IFN- could regulate Fas
antigen expression in MM cells, the U-266-1970, U266-1984, and U-1958
cells were also cultivated for 96 hours in culture medium containing
increasing concentrations of IFN-. As depicted in Fig 6 and Table 1,
and similar to the observed effects with IFN-, IFN- (1,000 U/mL) induced an 2.8-fold increase of the MFI of the U-266-1970 cells and a
moderate increase of the U-266-1984 cells (1.6-fold), although this
treatment did not significantly increase the number of Fas antigen-expressing cells in any of the MM cell lines studied.
Bcl-2 expression in IFN-induced/activated MM cell lines.
Because the delicate balance of Bcl-2 and Bax expression in some cases
seems to mirror the susceptibility to apoptosis,39 a
downregulation of Bcl-2 or/and an upregulation of Bax expression might
be a possible explanation for the IFN-induced augmentation of
Fas-induced death. We have previously reported on the expression of
Bcl-2 in human MM cell lines in which the U-266-1970 cell line has a
fourfold amplification of the gene.7 To evaluate whether the molecular mechanism underlying IFN sensitization to Fas-mediated apoptosis involves the regulation of members of the Bcl-2 family, the
MM cell lines were examined with regard to bcl-2 and
bax gene expression at 96 hours of IFN pretreatment.
Flow cytometric analysis was performed in U-266-1970 cells after
preincubation with IFN- and IFN- for 96 hours. The cells were
single-stained with anti-Bcl-2 or anti-Bax antibodies and/or double-stained with TUNEL and anti-Bcl-2 or anti-Bax antibodies. As
shown in Fig 7, no regulation of either
Bcl-2 or Bax protein could be shown in the U-266-1970 cells pretreated
with IFN- (1,000 U/mL) as compared with bcl-2 and
bax protein levels in exponentially growing control. However, a
slight downregulation of both Bcl-2 and Bax was recorded in the small
TUNEL-positive apoptotic population as compared with the TUNEL-negative
viable population of both control and IFN-induced cells.
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| Fig 7.
Expression of Bcl-2 and Bax in the U-266-1970 cells in
the absence or presence of anti-Fas MoAbs (CH-11; 100 ng/mL). Cells
were incubated in the presence of IL-6 (20 U/mL) without or with
IFN- (100 U/mL) for 96 hours. Cells were then subjected to
double-staining for TUNEL and Bcl-2 or Bax expression and flow
cytometry analysis. The population of
TUNEL+/Bcl-2+ and
TUNEL+/Bax+ cells was gated in the
analysis by the use of RPE-conjugated mouse IgG1/FITC-conjugated mouse
IgG1 and RPE-conjugated mouse IgG1/FITC-conjugated antirabbit
IgG, respectively.
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The viable population of the cells after IFN treatment, a later object
for Fas-induced cell death, was further investigated. Mean values of
the MFIs achieved from the viable population of the IFN-induced MM cell
lines single-stained with either anti-Bcl-2 or anti-Bax antibodies, as
well as the ratio between the MFIs of Bcl-2 and Bax, are presented in
Table 2. In the gated viable population of
IFN--treated U-266 cell lines, no significant regulation of Bcl-2
or Bax was found as compared with exponentially growing cells. IFN-,
on the other hand, induced both Bcl-2 and Bax upregulation in the
viable population of these cells.
Also, in the gated viable population of the U-1958 cell line,
expression of Bcl-2 and Bax was not significantly changed by treatment
with IFN- or IFN- as compared with exponentially growing control
cells.
Neither pretreatment with IFN- nor with IFN- downregulates the
Bcl-2/Bax ratio in viable cells of the three MM cell lines tested. The
Bcl-2/Bax ratio was unaltered when comparing IFN-induced cells to
exponentially growing cells of the three MM cell lines, one exception
being the IFN- induced U-266-1984 cells, in which a minor increase
of the ratio was recorded.
| |
DISCUSSION |
Some MM cell lines and primary MM cells express Fas antigen, but only a
few undergo apoptosis after Fas stimulation.28-30 Nagata and Golstein32 suggested that IFN- could
activate Fas-mediated apoptosis by the induction of Fas antigen
expression. In this study, we show that both IFN- and IFN- can
activate Fas-mediated apoptosis in three MM cell lines and that this
activation could not exclusively be explained by the upregulation of
Fas antigen expression or by IFN-induced growth inhibition.
Earlier studies have shown that MM cell lines can undergo apoptosis
after anti-Fas treatment, but the effect has, with few exceptions, been
small and variable.28-30 In addition, the magnitude of this
effect does not seem to correlate to levels of Fas or Bcl-2 expression.
Factors that activate normal B cells have previously been reported to
influence Fas expression and the sensitivity to Fas-mediated cell
death.40-43 In activated cells of B- and T-cell origin,
IFN- and TNF- have been shown to be potent inducers of Fas
antigen.44 IFN- has, in a similar way, been shown to potentiate Fas-induced apoptosis in CD34+ cells from
chronic myelogenous leukemia (CML) bone marrow.35 In MM,
IFN- and IFN- have been shown to inhibit growth and induce death.11,15,19 However, the biological effects of IFN-
in MM seem complex. Thus, IFN- may not only inhibit growth and
induce apoptosis,11,15,22,45 but may also stimulate growth
and survival.11,18,22
In U-266-1970, increasing concentrations of IFN- or IFN-
amplified the effect of anti-Fas antibodies in a dose-dependent manner.
Maximal amplification of Fas-induced apoptosis was reached after 96 hours. Other investigators have failed to show a sensitizing effect by
pretreatment of MM cells with IFN-. A reason for this can be
differences in the experimental protocol. In the report by Shima et
al,28 the cells were pretreated for 24 hours, which, in our
hands, has a very small effect on Fas-mediated apoptosis (data not
shown).
Both IFN- and IFN- inhibit the growth of U-266-1970 and U-1958
cells, whereas the U-266-1984 cell line was only growth inhibited by
IFN-. In U-1958 cells, treatment with IFN- or IFN- also induces cell death.15 In addition, the U-1958 cell line is
more sensitive to Fas-induced apoptosis than the U-266-1970 and
U-266-1984 cell lines, in which only a marginal effect of Fas MoAbs can
be recorded. Because both Fas and the IFNs induced cell death in U-1958
cells, we cannot exclude the possibility that the increased cell death
in Fas-stimulated U-1958 cells after pretreatment with IFN is purely
additive.
Augmentation of Fas-induced apoptosis could also be demonstrated in
primary B-B4+ plasma cells isolated from bone marrow
aspirates of patients with MM taken at diagnosis (data not shown).
Considering the well-known heterogeneity of MM, the role of
Fas-mediated apoptosis and the possibility of augmenting this apoptosis
pathway via IFN treatment of primary MM cells is a very important issue
that should be subject to further studies.
In contrast to the results obtained in this report, an earlier study
claimed that IFN- could inhibit Fas-induced apoptosis.18 However, a few crucial differences between the study by Egle et al18 and our work should be pointed out. The first is that
different panels of cell lines were used in these studies. This is
significant for two reasons, one being the well-known heterogeneity
between MM cell lines and the second being that the panel by Egle et
al18 also included Epstein-Barr virus (EBV)-positive cell
lines that do not represent true MM cell lines.46 Another
important difference is that the inhibitory effect of IFN- on
Fas-induced apoptosis reported by Egle et al18 was only
seen when IFN- was added simultaneously or up to 6 hours before the
addition of Fas antibodies. It is, of course, also possible that
IFN- may exert early and transient inhibitory as well as late
stimulatory effects on Fas-induced apoptosis.
To dissociate the direct effects of IFN- from indirect effects
mediated by growth inhibition, we tested the ability of IFN- to
activate anti-Fas induced apoptosis in U-266-1984 cells. IFN- does
not inhibit growth in U-266-1984 cells, but could nevertheless activate
Fas-induced apoptosis. IFN-, which does inhibit growth, can amplify
the effect of anti-Fas and can also, by itself, induce cell death, in
which the lowering of the number of viable cells at 1,000 U/mL of
IFN- was accompanied by a small increase in the number of
TUNEL-positive cells. The observed discrepancy between viability and
TUNEL staining could either be explained by methodological limitations
or by accounting a part of the cell death to necrosis.47,48
To investigate the mechanism behind the effect of the IFNs, we asked if
IFN- and IFN- could regulate either Fas, Bcl-2, or Bax
expression. Both IFN- and IFN- can upregulate Fas antigen in some
B cells.32,49,50 A twofold upregulation of Fas was seen
after stimulation with either IFN- or IFN- in U-266-1970, but
this effect was much less pronounced in the U-266-1984 cells and
nonexistent in the U-1958 cell line. The fact that all three cell lines
express high levels of Fas and that upregulation of Fas was obvious
only in U-266-1970 suggests that other mechanisms may be involved in
IFN activation of Fas-mediated apoptosis. Furthermore, IFN-induced
upregulation of Fas antigen expression is not predictive of Fas
function.18,50,51 The high expression of Fas antigen and
the low number of cells induced to death by apoptosis in the U-266 cell
lines after CH-11 treatment may rather point to either a high survival
capacity in MM cells or a defect/inactivated Fas signaling pathway.
This interpretation is consistent with earlier findings in
Fas-expressing MM cells in which no clear-cut correlation between the
level of Fas expression and sensitivity to Fas-induced apoptosis have
been found.28-30 IFN- has recently also been shown to
affect the expression and activity of several apoptosis related genes
of the ICE protease superfamily,51,52 thereby influencing Fas-mediated apoptosis. Different levels of caspase expression and
activation in the MM cells are therefore an attractive explanation for
the variability in the sensitivity to Fas-induced
apoptosis.
Bcl-2 can inhibit Fas-mediated apoptosis.32,53 Bax is a
proapoptotic protein that forms heterodimers with Bcl-2 and thus abrogates the effect of Bcl-2.54 All cell lines used in
this study express high levels of Bcl-2 protein as compared with cell lines carrying t(14;18) translocations.7 We investigated
the possibility that a downregulation of Bcl-2 or an upregulation of
Bax could explain the activation of Fas-mediated apoptosis by the IFNs.
However, our data suggest that the mechanism by which IFNs act to
activate Fas-mediated apoptosis does not involve a downregulation of
the Bcl-2/Bax protein ratio. Recently, Ossina et al52
showed that IFN- can induce expression of Bak, and Bak has been
suggested to play a role in Fas-mediated apoptosis.55 Further studies will elucidate the potential role of Bak and other members of the bcl-2 gene family in the IFN-mediated activation of Fas-induced apoptosis.
IL-6 was recently found to inhibit Fas-mediated apoptosis in the MM
cell line RPMI 8226.56 Although previously reported in some
MM cell lines,20 IFN- does not downregulate IL-6
receptors in U-1958 cells.15 It is, of course, still
possible that IFN- interferes with the IL-6 signaling pathway
downstream of the ligand-receptor interaction, as suggested
recently.22,52 We therefore performed a series of pilot
experiments on the U-266-1970 cell line with and without IL-6. However,
the effect of IFNs on Fas-mediated apoptosis was unaffected by the
presence or absence of IL-6 (data not shown).
The malignant cells in MM are characterized by a prolonged survival and
a slow proliferative activity, at least in the early stage of the
disease. This finding points to the importance of factors regulating
survival. Most studies on MM growth have focused on the role of IL-6,
which is considered to be the most important growth factor for MM
cells, regulating both proliferation and survival.19 In
this study, we focused on factors that can induce cell death in MM
cells, IFN- / and Fas. It has been known for some time that IFNs
can inhibit the growth of MM cells, and the mechanism implicated has
been a decreased IL-6 signaling. The results of this study points to
another mechanism by which the IFNs exert their inhibitory effect on MM
cells: activation of Fas-mediated apoptosis.
| |
FOOTNOTES |
Submitted December 29, 1997;
accepted June 17, 1998.
Supported by grants from the Swedish Cancer Society.
Address reprint requests to Helena Jernberg-Wiklund, PhD,
Laboratory of Tumor Biology, Department of Genetics and Pathology, Unit
of Pathology, University Hospital, S-751 85 Uppsala, Sweden; e-mail:
Helena.Jernberg_Wiklund{at}patologi.uu.se.
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.
| |
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Abstract
Introduction
Abstract