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NEOPLASIA
From the Department of Cancer Biology, Lerner Research
Institute; the Department of Clinical Pathology; the Myeloma Program;
the Department of Radiation Oncology; the Taussig Cancer Center; and
The Cleveland Clinic Foundation, Cleveland, OH.
It has been reported that interferons (IFNs) may have antitumor
activity in multiple myeloma (MM). The mechanism for their effect on
MM, however, remains elusive. This study shows that IFN- Multiple myeloma (MM), currently an incurable
disease, is the second most common blood cancer. It is characterized by
the presence of malignant plasma cells predominantly located in bone marrow.1 Interferons (IFNs), a family of pleiotropic
cytokines, have been used for the treatment of MM alone or in
combination with other chemotherapeutic drugs.2-4 Despite
their clinical effectiveness for antitumor growth, how IFNs act on MM
is unclear.5 IFNs, which consist of type I (predominantly
Apoptosis is a genetically regulated cell death process. Cells undergo
apoptosis by default, and all the critical components for apoptosis are
compartmentalized within distinct subcellular organelles. Once
committed to death, the cell undergoes the relatively stereotypic
execution and degradation phases involving chromatin condensation,
phosphatidyl-serine externalization, and selective proteolysis by a
family of cysteine proteases, named caspases.7 It is
important to identify and characterize the precommitment signals that
engage the execution and degradation machinery, because these signals
hold promise for identifying novel pharmaceutical targets useful for
augmenting tumor cell death in cancer therapy. Mitochondria play a
central role in the execution process of apoptosis.8,9 Once the cells are committed to cell death, apoptogenic factors, such
as cytochrome c (cyt c)10-13 and
Smac/DIABLO,14,15 are released from mitochondria to
initiate the caspase cascade and thus may represent irreversible
commitment events. Cyt c acts as a cofactor to stimulate the complexing
of Apaf-1 (human homolog of Caenorhabditis elegans CED-4) with caspase
9.16,17 This complex then initiates activation of the
caspase cascade, which culminates in proteolytic targeting of key
intracellular proteins.18 Smac,14 once
maturated and released into cytosol, is able to interact with
inhibitors of apoptosis proteins to promote caspase activation. One
other important apoptotic event in many cell systems is the loss of an
electrical potential across the inner mitochondrial membrane,19 manifested by a reduction in mitochondrial
membrane potential ( The Bcl-2 family of proteins plays a pivotal role in regulating cyt c
release and apoptosis.8,20 This expanding family consists
of both anti-apoptotic molecules, such as Bcl-2 and Bcl-XL, as well as pro-apoptotic ones, such as Bax and Bid. Bcl-2 can block the
release of cyt c from mitochondria11,12 and prevent the
activation of caspase 3,21 whereas Bax and Bid can promote cyt c release from mitochondria and thus activate the caspase cascade.22-25 The interactions between pro- and
anti-apoptotic molecules seem to be the determining factors for cell
survival. Not surprisingly, most death modulators function by acting
through Bcl-2 family proteins to regulate cyt c
release.8,26 It has been recently reported that Bid could
induce cyt c release without MPT, whereas Bax induces cyt c
release and MPT.25 How Bcl-2 family proteins regulate MPT
and cyt c release is a recent focus of apoptosis research.
Of the many disparate death stimuli, the signaling pathway initiated by
tumor necrosis factor and the related Fas ligand (also Apo-1) is best
characterized.27 These cytokines are expressed as
membrane-bound ligands that can be cleaved to a soluble form. Engagement by these ligands of their respective receptors, followed by
recruitment of an adapter molecule, such as FADD (Fas-associated death
domain protein28,29) leads to direct activation of the caspase cascade. This activation is accomplished by recruitment of
caspase 8, followed by its proteolytic activation. Once activated, caspase 8 can proteolytically cleave Bid, with the truncated Bid targeting mitochondria and inducing cyt c release and further amplification of the caspase cascade.22-24,30 The
engagement of mitochondria through truncated Bid in receptor-mediated
apoptosis further supports the pivotal role of mitochondria in
apoptosis. Interestingly, Bcl-2 is a proteolytic target of caspases and
is converted in situ to a pro-apoptotic molecule13,31 to
promote cyt c release and apoptosis. We have recently described in MM 2 distinct stages of cyt c release during genotoxic stress, characterized by a limited initial cyt c release, sufficient to activate caspases, followed by a second one, characterized by a positive feedback amplification of cyt c release.13
In this study we examined the apoptotic pathways in MM following
treatment with IFNs. We show here that type I but not type II IFNs
induce typical apoptosis through activation of the Apo2 ligand (Apo2L,
also TRAIL) pathway and modulation of the Bcl-2 family of proteins in 3 MM cell lines and in plasma cells isolated from 10 patients with MM.
Our data indicate that Apo2L induction is at least partially necessary
for caspase activation and the Bcl-2 and Bid cleavage-dependent
pathway. Our data further support the notion that Apo2L could become a
useful molecule for management of MM.
Cell culture, bone marrow plasma cell isolation, and
treatments
Clonogenic cell survival assays
Apoptosis assays Apoptosis was either measured by nuclear fragmentation and condensation examined by fluorescence microscopy after Hoechst 33258 staining or by examining phosphatidylserine exposure on cell membranes, using Annexin V, as described.13,33 Cells were stained with fluorescein isothiocyanate (FITC)-Annexin V (25 ng/mL; green fluorescence), simultaneously with dye exclusion (propidium iodide [PI]; negative for red fluorescence). This assay13,34 discriminates between intact (FITC /PI),
early apoptotic (FITC+/PI), and late
apoptotic cells (FITC+/PI+). Comparative
experiments were performed at the same time by bivariate flow cytometry
using a FACScan and analyzed with the CellQuest software (BD) on data
obtained from the cell population from which debris were gated out.
The caspase protease assays were performed as described,13,33 using the acetyl-Ile-Glu-Thr-Asp-pNA (IETD) and acetyl-Asp-Glu-Val-Asp-pNA (DEVD) p-nitroanilide (pNA)-derived chromogenic substrates for caspase 8 and 3 activity, by enzyme-catalyzed release of pNA monitored at 405 nm in a 96-well microtiter plate reader (Spectramax 340; Molecular Devices, Sunnyvale, CA). Caspase peptide inhibitors (z-VAD-fmk, benyloxycarbonyl-Val-Ala-Asp-fmk), or substrates were from BioMol (Plymouth Meeting, PA); all other chemicals were from Sigma (St Louis, MO), unless otherwise specified. To determine of the role of the Apo2L pathway, a FLAG epitope-tagged
dominant-negative Apo2L receptor, DR5 Immunoblotting Western immunoblotting was performed as described.13 Briefly, cells were washed and lysed in buffer containing 150 mM NaCl, 25 mM HEPES pH 7.4, 1% (vol/vol) NP-40, 0.25% (wt/vol) sodium deoxycholate, 1 mM EGTA, EDTA, and phenylmetholsulfonyl fluoride, 10 mg/mL aprotinin, leupeptin, and pepstatin. Cellular proteins from total cell lysates (10 µg/sample) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Schleicher and Schull, Keene, NH). The membranes were probed using immunoblot analyses with mAb to the human Bcl-2, and polyclonal antibodies (pAbs) to caspase 3 (Santa Cruz, Santa Cruz, CA), Apo2L/TRAIL (Santa Cruz), cyt c (Pharmingen, San Diego, CA), or Bid pAbs,22 followed by incubation with the secondary antibody conjugated to horseradish peroxidase for 1 hour at room temperature. Equal protein loading was confirmed by staining filters with Ponceau S and/or reprobing with actin mAb (Sigma). Immunoreactive bands were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech) and visualized by autoradiography.Mitochondrial isolation and membrane potential Cell fractionation was done as previously reported.36 Briefly, cells (1 × 106/sample) were washed twice with ice-cold phosphate-buffered saline then kept on ice for 20 minutes in buffer A, containing 20 mM HEPES-KOH (pH 7.2), 100 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, and protease inhibitors (1 mM phenylmetholsulfonyl fluoride and dithiothreitol, 10 mg/mL aprotinin, leupeptin, and pepstatin). The cells were treated at different time points and harvested at the same time for cell fractionation. The cell suspension was gently homogenized with a Dounce homogenizer (5-8 strokes), and the cell lysis was checked by trypan blue staining. The homogenate was centrifuged at 750g for 5 minutes, and the supernatant subjected to further centrifugation at 10 000g. This pellet, containing mitochondria, was designated P10. The supernatant was subjected to further ultracentrifugation at 100 000g for 45 minutes. The resulting pellet and supernatant, representing the endoplasmic reticulum and cytosolic fractions, were designated as P100 and S100, respectively. The purity of the cellular fractions was examined by Western blotting using -Calnexin, -PCNA, and mitochondrial cytochrome oxidase 1 (MTCO1) mAbs, as endoplasmic reticulum
(ER) membrane, nuclear, and mitochondrial markers,
respectively. Cyt c was not detectable in the P-100 fraction.
The mitochondrial membrane potential indicator DIOC6 (3) (2 µL of 2 mM stock solution in dimethyl sulfoxide) was added to a U266
cell suspension (4 × 105 cells/mL) in fresh RPMI 1640 medium (pH 7.2) and incubated at 37°C for 5 minutes. A change in
DIOC6 (3) fluorescence indicates a change in
RNA analyses Total RNA was isolated from U266 cells at various intervals after IFN treatment by using the TRIZOL reagent (GIBCO BRL). To determine the steady-state levels of RNA, the RiboQuant system (Pharmingen) for RNase protection was used, as described.33,38 The hApo-3C template set (Pharmingen), containing 10 complementary DNA probes, including the housekeeping gene product L32 as a control, was used for the T7 polymerase-directed synthesis of high-specific activity [32P]-labeled antisense RNA probes.
Type I but not type II IFNs induce typical apoptosis and loss of clonogenicity IFNs have been reported to be important regulators of growth and survival.6 Exponentially growing cultures of U266, an interleukin 6-dependent MM cell line resistant to ionizing radiation and Fas, were treated with IFN-, - , and - and examined for phosphatidylserine exposure on the cell membranes, indicative of
apoptosis. There was no change in the Annexin V staining at 6 and 24 hours following IFN- treatment compared with untreated cells.
However, 22% of cells showed Annexin V positivity at 48 hours, which
reached 37% at 72 hours and 45% at 96 hours following treatment with
2000 U/mL IFN- (Figure 1A). IFN-
and, more potently, IFN- induced Annexin V positivity in a
dose-dependent manner (data not shown). In contrast, under the same
experimental conditions, IFN- treatment did not induce Annexin
V-positive staining.
To confirm the above results, we further examined nuclear morphologic
changes by determining nuclear condensation and fragmentation, another
hallmark for apoptosis. Hoechst 33258 staining showed that 16% and
26% of cells displayed the nuclear condensation at 72 hours after
IFN- We also examined the response of another typical MM cell line,
NCI-H929. IFN- We further asked whether IFNs can induce apoptosis in plasma cells
freshly isolated from the bone marrow of MM patients. We found that
apoptosis was induced in patient-derived primary MM plasma cells by
type I IFNs. For 4 of the patient samples, the nonplasma lymphocytes
were also used as control. We found that only the samples containing
malignant (CD38+/CD45
IFNs induce Apo2L/TRAIL in MM We sought to determine next whether IFNs affect expression of known apoptosis-related genes and chose to examine components of receptor-mediated apoptosis.27 Control and IFN--treated (chosen because it had the most potent effect) U266
cells, examined by RNase protection at 4 to 24 hours, a time at which
no apoptosis was observed, revealed that Apo2L messenger RNA (mRNA) was
induced abundantly by IFN- (Figure
2A). In contrast, there were no
significant changes in expression levels of the Apo2L receptors DR5 and
DR4, or of Caspase 8 and Fas. Moreover, there was no detectable Apo2L mRNA expression in untreated cells. Western blotting revealed that
Apo2L was induced as early as 6 hours following type I IFN treatment
and a more pronounced expression at later times following treatment
(Figure 2B). In contrast, there was only a slight change in Apo2L
levels following IFN- treatment early on, with no additional change
at a later time. Similar analyses of patient-derived plasma cells
revealed that IFN-, at a concentration as low as 200 U/mL, was
effective, whereas a higher concentration of IFN- was required for
Apo2L induction (Figure 2C). Significantly, Apo2L induced apoptosis in
plasma cells derived from MM. Of these treatments, soluble Apo2L was
the most effective inducer of apoptosis (Table 1). Interestingly,
plasma cells isolated from one patient (No. 6), who received extensive
chemotherapy (13 cycles) but had a persistent tumor, were very
sensitive to Apo2L treatment. This finding suggests that Apo2L may be
useful for treating resistant or relapsing myeloma. Because the number
of patients is relatively small, a large-scale study will be needed in
the future to define a role for Apo2L in treatment of relapsing or
resistant MM. Nevertheless, these findings indicate that IFN- and
- induce Apo2L/TRAIL expression in the MM cell line U266 and in
freshly isolated plasma cells of MM patients.
IFN- and -, but not IFN-, treatment of U266 cells (Figure 3B). These data suggest that caspases were activated before the appearance of any morphologic changes associated with apoptosis. Similar to U266 cells, Western blotting showed that
type I but not type II IFNs induced caspase 3 cleavage in freshly
isolated plasma cells of MM patients (Figure 3C).
IFNs induce distinct levels of cyt c release from mitochondria We and others have reported that apoptosis in MM is associated with an increase in cytosolic cyt c levels.13,40 Examination of the distribution of cyt c in cytosolic and mitochondrial cellular fractions following IFN treatment of U266 cells revealed that cyt c was undetectable in cytosol isolated from untreated cells. Small amounts of cyt c were present in the cytosol by Western blotting at 16 and 24 hours following IFN- and - treatments, and these amounts
increased significantly at 48 hours. In contrast, there were no
significant levels of cytosolic cyt c present in the cytosol after
IFN- treatment even at 48 hours (Figure
4A). These results suggest that type I
IFN-induced apoptosis was associated with induction of a differential
cyt c release from mitochondria at 24 and 48 hours after IFN treatment.
To determine whether this differential cyt c release had a functional
significance, we examined next the   m, a critical
mitochondrial event implicated in apoptosis.19 The
m-sensitive lipophilic fluorochrome DIOC6
(3) indicated that there was no change in its intensity and bimodal
distribution at 24 hours. A subpopulation of these cells showed
decreased DIOC6 (3) staining, indicating that these cells
had a lower m. The proportion of cells displaying a
lower m significantly increased in IFN--treated
cells at 48 hours, reaching 76% at 72 hours (Figure 4B). Importantly,
the proportion of apoptotic cells at 72 hours following IFN-
treatment was only 26% (Figure 1). IFN- treatment was similar,
whereas IFN- had no effect on the DIOC6 (3) staining (data not shown). Altogether, these results indicate that the early cyt
c release from mitochondria took place before the reduction of
m, with the later, augmented cyt c release coinciding
with the reduction of m.
IFN- as low as 200 U/mL
induced Bcl-2 cleavage (Figure 5B, right panel). In addition, IFN-
but not - induced down-regulation of Bcl-xL. In
contrast, IFNs did not affect levels of any other Bcl-2 family
proteins, such as Bad, Bak, and Bax (Figure 5C). These results indicate
that some of these Bcl-2 family proteins are modulated in IFN-induced
apoptosis, with Bid and Bcl-2 cleavage corresponding to the early and
late stages of cyt c release.
IFNs induce apoptosis in MM through the Apo2L/TRAIL pathway The above results prompted us to further examine whether IFN-induced Apo2L expression might be directly involved in enhanced cytotoxicity against MM. Examination of the cellular response to Fas or Apo2L showed that, consistent with previous reports, U266 was insensitive to anti-Fas agonistic antibodies (Figure 6A). Strikingly, U266 cells were sensitive instead to Apo2L, which induced apoptosis at a concentration as low as 100 ng/mL (Figure 6A). RPMI-8226 cells were sensitive to both Apo2L and anti-Fas agonistic antibodies. Induction of apoptosis by Apo2L was specific because soluble Fc Apo2L receptor (Fc-DR5) was able to prevent Apo2L-induced apoptosis (data not shown). Furthermore, Apo2L but not Fas mAb, induced Bcl-2 and caspase 3 cleavage in U266 (Figure 6B). Freshly isolated plasma cells from a MM patient were also responsive to Apo2L, whereas the nonplasma cells from the same patient did not undergo apoptosis following Apo2L treatment (Table 1). Apo2L was also able to induce caspase 3 and Bcl-2 cleavage (Figure 6C).
To examine the Apo2L contribution to cell death, we stably expressed a
FLAG epitope-tagged dominant-negative Apo2L receptor, DR5
In an effort to characterize the molecule(s) responsible for
IFN-induced apoptosis of MM, we identified Apo2L as a critical determinant. Our data show that IFNs induce up-regulation of Apo2L, which is responsible, at least partially, for apoptosis. Apo2L mRNA was
induced dramatically as early as 4 hours after treatment of U266 cells.
We previously reported that the promoter region of Apo2L contains 2 IFN-stimulated regulatory elements, and IFNs can significantly induce
the promoter activity of this gene.41 Western blotting
confirmed that the Apo2L protein was induced as early as 6 hours and
continued to accumulate during IFN-induced apoptosis. The induction of
Apo2L is of functional significance because U266 cells were sensitive
to Apo2L but not Fas agonistic antibodies. Moreover, a
dominant-negative Apo2L receptor, DR5 We delineate here, for the first time, the signaling pathway for
IFN-induced apoptosis in MM (Figure 8).
Apo2L induction is followed by caspase 8 activation, Bid cleavage, cyt
c release, caspase 3 activation, and the subsequent mitochondrial
dysfunction. Bid was partially converted to the p15 and p13 fragments
at a stage prior to the appearance of apoptotic morphology but
coinciding with the initial cyt c release and caspase 3 activation. In
contrast, Bcl-2 cleavage was detected only at 48 hours, a time
corresponding to the amplification of cyt c release. It seems that Bid
and Bcl-2 cleavage are functionally linked to the initial and augmented cyt c release, respectively. It was recently demonstrated that Apo2L
could activate caspase 8 and downstream events of
apoptosis.28,29 It has been established that Bid is
cleaved during receptor-mediated caspase 8 activation, with the
truncated product being targeted to mitochondria and causing cyt c
release.22,23 Using isolated mitochondria from mouse
liver, we confirmed25 that the truncated Bid induces cyt c
release without jeopardizing the mitochondrial functions, as indicated
by maintenance of
Apo2L and FasL could use distinct pathways to induce apoptosis, because
Bid44 and caspase 845 knock-out mice are
resistant to Fas-induced apoptosis in hepatocytes but sensitive to
other types of death stimuli, including Apo2L. Our results show that U266 cells are sensitive to Apo2L but not to Fas agonistic mAb, further
suggesting that there are distinct apoptotic pathways activated by FasL
and Apo2L in our system. It was reported that, even though the Fas
receptor was also induced by IFNs, U266 cells were insensitive to FasL,
probably because of their intrinsic high level of
Bcl-XL.46 Our findings are consistent
with reports that, although U266 cells are resistant to Fas,
they are sensitive to IFN47 or
Apo2L.48 In fact, it has been reported that Fas-mediated apoptosis of MM cell lines required cotreatment with
IFN- IFNs play an important role in the immune system. One mechanism by which IFNs may achieve their role in immune surveillance could be through regulation of other cytokines, which, depending on the cell type, may induce cell cycle arrest or activate apoptosis. There are reports indicating that IFNs could also cause cell cycle arrest and activate other signaling pathways, such as mitogen-activated protein kinase50 or protein kinase C.49 Our data provide evidence that IFNs exert their profound effect by inducing apoptosis in MM and suggest that Apo2L may be an important mediator for these effects, although it may not be the sole molecule responsible for IFN-induced apoptosis. It has been reported that interleukin 6 (IL-6)51 and RNA-dependent protein kinase (PKR) may also be involved in regulation of IFN-induced apoptosis in MM. It remains to be determined whether PKR,52 IL-6 down-regulation,51 or other IFN-induced genes also have a role in apoptosis in our system, as our results do not exclude their possible involvement. There is a heterogeneous IFN response in different cell types, or even
within the same type of cells. We also examined other cell lines for
their sensitivity to IFNs, Apo2L, and Fas agonistic antibody. RPMI-8226
and NCI-H929, other typical MM cell lines, were sensitive to IFN- In summary, we delineated the signaling pathways of IFN-induced
apoptosis in MM and identified Apo2L as an important mediator, with
mitochondria, caspases, and Bcl-2 family proteins playing an important
role in this process. Importantly, we show that IFNs induce Apo2L in
plasma cells of MM patients. We also confirmed a previous
report58 that MM
(CD38+/CD45 |
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Abstract
Introduction
