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,
Departments of
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Interdisciplinary Oncology and
Biochemistry and Molecular Biology, and
Clinical Investigation Program, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL 33612
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 integrin-mediated adhesion to fibronectin
inhibits CD95-induced caspase-8 activation and apoptosis in hematologic
tumor cell lines. This adhesion-dependent inhibition of CD95-mediated
apoptosis correlated with enhanced c-Fas-associated death domain-like
IL-1-converting enzyme-like inhibitory protein-long
(c-FLIPL) cytosolic solubility compared with nonadhered
cells. Cytosolic c-FLIPL protein preferentially associated
with cytosolic Fas-associated death domain protein (FADD) and localized
to the death-inducing signal complex after CD95 ligation in adherent
cells. The incorporation of c-FLIPL in the death-inducing
signal complex prevented procaspase-8 processing and activation of the
effector phase of apoptosis. Adhesion to fibronectin increased
c-FLIPL cytosolic solubility and availability for FADD
binding by redistributing c-FLIPL from a preexisting
membrane-associated fraction. Increased cytosolic availability of
c-FLIPL for FADD binding was not related to increased
levels of RNA or protein synthesis. These data show that adhesion of
anchorage-independent cells to fibronectin provides a novel mechanism
of resistance to CD95-mediated programmed cell death by regulating the
cellular localization and availability of
c-FLIPL. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to mechanisms intrinsic to the cancer cell, extrinsic factors may also contribute to resistance to CD95-mediated apoptosis and provide a mechanism of immune evasion and tumor progression. Multiple myeloma is a hematologic malignancy characterized by bone marrow localization and metastatic dissemination throughout the skeleton. The bone marrow microenvironment may be a site of immune privilege for myeloma and other hematopoietic malignancies by blocking CD95-mediated apoptosis. At least two forms of tumor cell microenvironment interactions may influence tumor cell survival (14). The first form involves soluble mediators, such as ILs, that are secreted by nontumor stromal cells. IL-6 produced and secreted primarily by bone marrow stromal cells is an essential cytokine involved in the growth and survival of myeloma cells (15, 16, 17). IL-6 has been implicated in the resistance of myeloma cells to a variety of apoptotic stimuli, including cross-linking of the CD95 death receptor (18, 19, 20).
In addition to soluble mediators of protection, the tumor
microenvironment may also influence tumor cell survival through direct
cell contact. Tumor cells may adhere to adjacent cells or to the
extracellular matrix
(ECM)3 that comprises
the surrounding environment. Cell surface adhesion molecules, known as
integrins, interact with the ECM and may contribute to hematopoietic
tumor growth and survival (21, 22). Integrin receptors are
heterodimeric complexes of 
and subunits that interact with ECM
components or counter receptors of other cells (23).
Integrins influence a number of cellular functions, including
proliferation, differentiation, cytoskeletal rearrangement, migration,
and survival (24, 25). The survival-promoting effects of
integrin-mediated adhesion were first identified in studies
demonstrating that inhibition of extracellular contact by
integrin-specific Abs resulted in cell death of anchorage-dependent
epithelial and endothelial cells (26, 27). More recently,
studies demonstrated that direct contact between hematopoietic cells
and ECM components also confer a survival advantage to adhered cells
(21, 22). Because hematopoietic cells, unlike
anchorage-dependent cells, survive independently of direct contact,
these and subsequent studies suggest that direct contact between
hematopoietic cells and their environment may elicit antiapoptotic
events via mechanisms distinct from those observed in
anchorage-dependent cell models.
To investigate the potential role of bone marrow ECM in hematopoietic
tumor cell survival, we examined effects of cellular adhesion to
fibronectin (FN), an abundant bone marrow glycoprotein
(28), on sensitivity to CD95-mediated apoptosis. In this
report, we demonstrate that both CD95-mediated caspase activation and
apoptosis are reduced in hematopoietic cancer cells after
1 integrin-mediated adhesion. Moreover, we
show that adhesion to FN inhibits CD95-induced apoptosis by acutely
regulating cytosolic availability of c-Fas-associated death domain-like
IL-1-converting enzyme-like inhibitory protein (c-FLIP). c-FLIP is a
cellular protein that shares significant similarity with procaspase-8
and procaspase-10 and inhibits CD95-mediated programmed cell death
(29, 30, 31, 32, 33, 34, 35, 36, 37). Although the anti-CD95 signaling effects of
c-FLIP have been established in several cellular models
(33), delineation of the biochemical processes regulating
c-FLIP expression and function remains to be conclusively determined. A
number of recent reports have implicated specific signaling pathways in
the control of c-FLIP RNA expression (38, 39, 40).
In this report, we demonstrate a novel transcription- and
translation-independent regulation of c-FLIP-long
(c-FLIPL) protein levels after adhesion of
hematopoietic cancer cells to FN. Adhesion of hematopoietic cancer
cells to FN mediates the translocation of a preexisting pool of
c-FLIPL from a membrane-associated fraction to a
cytosolic fraction. We show that cytosolic
c-FLIPL preferentially associates with cytosolic
Fas-associated death domain protein (FADD) in adherent cells.
Furthermore, in adherent cells, c-FLIPL is
recruited to the death-inducing signal complex (DISC), blocking
procaspase-8 activation and apoptosis after CD95 cross-linking. These
data demonstrate that interactions between hematopoietic cancer cells
and their microenvironment regulate sensitivity to CD95-mediated
apoptosis through the cellular redistribution of the antiapoptotic
factor, c-FLIPL.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human U937 histiocytic lymphoma and RPMI 8226 multiple myeloma cell lines were originally acquired from the American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 (Mediatech-Cellgro, Seattle, WA), supplemented with 10 (U937) or 5% (8226/SH2) heat-inactivated FBS (Omega Scientific, Tarzana, CA), 100 µM L-glutamine, and 100 U/ml penicillin/streptomycin (complete medium; Life Technologies, Grand Island, NY). A clonal cell line isolated from the RPMI 8226 multiple myeloma cell line by limiting dilution, 8226/SH2, is characterized by high levels of CD95 expression and sensitivity to CD95-mediated apoptosis (12).
Apoptosis assays
Apoptotic cell death was analyzed by flow cytometry (FCM) using annexin V staining, as described previously (12). Cells were incubated on FN (40 µg/ml; Life Technologies)-coated NUNC immunoabsorbant dishes (Nalge Nunc International, Rochester, NY) or maintained in suspension in noncoated NUNC plates in serum-free RPMI 1640 for 2 h followed by three washes with complete medium (41). Cell populations were treated with 100 ng/ml CD95 cross-linking Ab, CH-11 (MBL International, Watertown, MA), in complete medium for 20 h. Where indicated, samples were pretreated with 1 µg/ml CD95 blocking Ab ZB4 (MBL International) for 60 min. Adhered cells were detached from FN by incubation with 5 mM EDTA/PBS for 2–3 min on ice and stained with annexin V-FITC for analysis by FCM using CellQuest software (BD Biosciences, Mountain View, CA). Apoptosis is reported as percentage of specific apoptosis: [(experimental apoptosis - spontaneous apoptosis)/(100 - spontaneous apoptosis)] x 100. Student’s t test was used to determine the statistical significance.
lp;&6qAnalysis of CD95 expression and CD95 Ab binding
CD95 surface expression was determined by FCM, as described previously (12). Data represent the median fluorescence (ZB4) above isotype control (ZB4 median fluorescence-isotype fluorescence). For semiquantitative analysis of Ab (CH-11) binding, cells were incubated with 50–1000 ng/ml CH-11 for 60 min, in suspension or after 24 h of adhesion to FN. Cells were washed three times with cold PBS, stained with goat anti-mouse-FITC secondary Abs, and analyzed by FCM.
Protein isolation and Western blot analysis
Cells were incubated for 2 or 24 h on FN or in suspension,
washed twice with ice-cold PBS, and incubated for 10 min at 4°C in
Triton X-100 lysis buffer (30 mM Tris-HCl (pH 7.5), 137 mM NaCl, 25 mM
NaF, 1% Triton X-100, 15% glycerol, 2 mM sodium orthovanadate, 25
µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM PMSF, and 10 µg/ml
pepstatin A) or whole cell lysis buffer (30 mM Tris-HCl (pH 7.5), 150
mM NaCl, 2% SDS, 5% 2-ME, 15% glycerol, 2 mM sodium orthovanadate,
and the same protease inhibitor mixture). Protein lysates were
quantitated with reagent (Bio-Rad, Hercules, CA), and 20–125 µg of
cellular lysates were separated on 12.5–15% polyacrylamide gels and
transferred to polyvinylidene difluoride membrane. Protein levels were
examined with antisera specific to caspase-8 (R&D Systems, Minneapolis,
MN), caspase-3 (kindly provided by H.-G. Wang, H. Lee Moffitt Cancer
Center, Tampa, FL), anti-FADD, CD95/Fas/Apo-1, Bcl-2, topoisomerase
II (topo II) (BD Transduction Laboratories, Lexington, KY),
Bcl-xL, c-FLIP-short
(c-FLIPS) (Santa Cruz Biotechnology, Santa Cruz,
CA), c-FLIPL (BD PharMingen, San Diego, CA), and
-actin (Sigma-Aldrich, St. Louis, MO) and visualized with Lumi-Light
chemiluminescence (Roche, Indianapolis, IN) or SuperSignal-Dura Light
(Pierce, Rockford, IL).
Cellular fractionation
Subcellular fraction was conducted using differential centrifugation (42). U937 cells (5 x 106 cells) were adhered to FN or maintained in suspension for 2 h. Cells were washed once in cold PBS and once in cold hypotonic buffer (10 mM HEPES (pH 6.9), 10 mM KCl, and protease and phosphotransferase inhibitor mixture as listed above). Cells were then harvested in 500 µl of hypotonic buffer, incubated on ice for 30 min, and lysed by Dounce homogenization (40–50 strokes). Lysates were centrifuged at 1,000 x g for 3 min at 4°C. The resulting pellet was subsequently washed three times with NTENT lysis buffer (10 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease and phosphotransferase inhibitor mixture). The resulting pellet was resuspended in lamillae buffer and represented the nuclear fraction. The supernatant isolated from the first centrifugation (after Dounce homogenizing) was further centrifuged at 14,000 x g for 30 min at 4°C. The resulting supernatant was was collected and stored (cytosolic fraction). The pellet was resuspended in NTENT lysis buffer and centrifuged at 14,000 x g for 30 min at 4°C. The resulting supernatant contained the heavy membrane fraction. The pellet was resuspended in lamillae buffer (cytoskeletal fraction). Proteins were separated by SDS-PAGE and analyzed by Western blot analysis.
Immunoprecipitation assays
U937 cells were adhered to FN or maintained in suspension for 2 h, then harvested in Triton X-100 buffer (as described above) and incubated with 2 µg of c-FLIP specific antisera (BD PharMingen) with 0.75 mg of protein lysates from FN adhered or suspension cells for 2 h. Samples were then incubated with 30 µl of protein A/G-agarose for 2 h (Santa Cruz Biotechnology). Immunoprecipitates were washed five times and separated on 12.5% SDS-PAGE gels. Western blot analysis was performed as described above.
DISC formation was analyzed after 2 h of cell adhesion. Cells adhered to FN or maintained in suspension (18 x 106) were incubated with or without with 2 µg/ml CH-11 for 60 min and harvested in Triton X-100 lysis buffer. Untreated postnuclear supernatants were incubated with 2 µg of CH-11 for 30 min. Lysates were then incubated with 50 µl of protein L-agarose for 2–4 h at 4°C (Santa Cruz Biotechnology). Immunoprecipitates were then washed four to five times and separated on 12.5% SDS-PAGE gel.
Caspase activity
Cells were incubated for 2 h on FN or in suspension before treatment with 100 ng/ml CH-11 (MBL International) for 24 h. To demonstrate specificity for CD95-induced apoptosis, samples were pretreated with 1 µg/ml CD95 blocking Ab ZB4 (MBL International) for 60 min. Caspase-3 and caspase-8 activity were determined by cleavage of p-nitroanilide (pNA)-conjugated caspase-specific tetrapeptides, Asp-Glu-Val-Asp (DEVD)-pNA and Ile-Glu-Thr-Asp (IETD)-pNA, respectively, as per the manufacturer’s instructions (BioVision, Palo Alto, CA). Substrate cleavage was determined by absorbance at 405 nm in a 96-well microtiter plate reader (Dynex Technologies, Chantilly, VA). Data are presented as fold control absorbance minus blank. Student’s t test was used to determine statistical significance.
RNase protection assay
Total RNA was isolated from 3–5 x 106 cells by TRIzol reagent according to the manufacturer’s protocol (Life Technologies). RNase protection assays were conducted using the BD PharMingen Riboquant hAPO-3b multi probe template as previously described (12). Protected RNAs were separated on a 5% polyacrylamide denaturing gel and quantitated with Image-Quant software (Molecular Dynamics, Sunnyvale, CA).
Metabolic labeling
Cells were washed twice and incubated in methionine-free RPMI medium (Life Technologies, Rockville, MD) for 60 min, then incubated with 0, 1, or 2 µg/ml cyclohexamide for 30 min (Sigma-Aldrich). Cells were then either maintained in suspension or adhered to FN in the presence of 100 µCi/ml [35S]methionine/cysteine for 2 h (EXPRE protein labeling mix; NEN Life Sciences, Boston, MA). After 2 h, cells were washed extensively with cold PBS and harvested in Triton X-100 lysis buffer (as described above). Incorporation of [35S]methionine was determined by TCA precipitation. Briefly, equal volumes of postnuclear supernatants were incubated with 300 µl of 10% TCA on ice for 10 min, centrifuged, and washed three times with cold acetone. Pellets were resuspended in 0.1% SDS and boiled for 1 min. Metabolic labeling (35S incorporation) was analyzed in an LS 6500 scintillation counter (Beckman Coulter, Fullerton, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD95/Fas/Apo-1 is a 45-kDa death receptor of the TNFR/nerve
growth factor receptor superfamily that initiates a relatively
well-characterized sequence of biochemical events leading to
apoptosis. To determine whether adhesion to FN affected death
receptor-mediated apoptosis, we compared CD95-induced apoptosis in the
U937 lymphoma and 8226/SH2 myeloma cell lines
when cells were either adhered to FN or maintained in suspension
culture. U937 cells were adhered to FN for 2 h before treatment
with CD95 cross-linking Ab (CH-11). CD95-mediated apoptosis was reduced
up to 91% when cells were adhered to FN compared with cells maintained
in suspension (Fig. 1
, A and
B; statistically significant at p < 0.05).
A similar effect was observed in 8226/SH2 myeloma
cells with 33.5 ± 8% specific apoptosis for cells in suspension
compared with 17.5 ± 13.5% (p < 0.05)
for cells adhered to FN. Pretreatment of both suspension- and
FN-adhered cells with the CD95-blocking Ab ZB4 completely inhibited
apoptosis mediated by CH-11, demonstrating CD95-specific apoptosis.
Similar inhibition of CD95-mediated apoptosis was observed when cells
were adhered for 24 h, demonstrating that the protective effect of
FN adhesion is not a transient event. Apoptosis after exposure to
soluble Fas ligand was also reduced in cells adhered to FN,
demonstrating that resistance was not an Ab-specific phenomenon (data
not shown).
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1-containing integrin receptors
(43). To determine the contribution of
1 integrins in FN-mediated resistance to
CD95-induced apoptosis, cells were incubated with 1-blocking
antisera before adhesion and CD95 cross-linking. The protective effects
of FN adhesion were reversed by incubation with the 1-blocking Ab
(Fig. 1
C), demonstrating that 1-mediated adhesion is
required for this antiapoptotic phenomenon. In contrast, the isotype
control Ab had no significant effect on FN-mediated resistance to CD95
cross-linking. These results demonstrate that
1 integrins are required for the protective
effects mediated by adhesion to FN. However, this does not exclude the
possibility that other adhesive matrices or
non-1 integrins may facilitate this
phenomenon. Adhesion to FN does not alter agonist Ab (CH-11) binding to CD95
To address the possibility that FN adhesion may
physically obstruct or reduce surface availability of CD95, we examined
the ability of CH-11 to bind FN-adhered cells. Flow cytometric analysis
of CH-11 Ab binding demonstrated that cellular adhesion to FN for
2 h did not significantly alter the ability of CH-11 to associate
with CD95 over a range of concentrations (50–1000 ng/ml) (Fig. 2). Thus, reduced association of agonist
Ab with CD95 was not a cause for decreased CD95-mediated apoptosis.
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CD95 cross-linking induces apoptotic signaling via a
conserved cytoplasmic protein-protein interaction domain known as the
death domain (1). Cross-linking of CD95 induces receptor
multimerization and recruitment of FADD/mediator of receptor-induced
toxicity-1 and procaspase-8/Fas-associated death
domain-like IL-1-converting enzyme/Mch5 to the death domain of
CD95 forming the DISC (44, 45, 46). DISC formation promotes
the proximity-induced proteolytic activation of procaspase-8 (47, 48), leading to cleavage and activation of the effector caspase,
procaspase-3, which cleaves key cytosolic and nuclear factors,
resulting in cell death (1). To investigate the effects of
FN adhesion on the CD95-mediated apoptotic cascade, we examined
activation of the initiator caspase-8, the effector caspase-3, and
mitochondrial perturbation after adhesion to FN. Adhesion of U937 cells
to FN for 2 h significantly reduced CD95-mediated procaspase-8 and
procaspase-3 processing and activation as measured by IETD (caspase-8)-
and DEVD (caspase-3)-specific activity (p <
0.05) (Fig. 3A). Similarly, FN
adhesion inhibited CH-11-mediated procaspase-8 and procaspase-3
cleavage and activity in the RPMI 8226/SH2
multiple myeloma cell line (Fig. 3B). These results
demonstrate that FN adhesion affects early, initiating events in the
CD95-mediated apoptotic cascade. Moreover, DiOC6
staining demonstrated that CD95-induced mitochondrial permeability
transition was reduced in FN-adhered cells compared with cells in
suspension (percentage of loss of membrane potential; suspension =
28 ± 1.44% DiOC6 negative; FN =
18.9 ± 1.21% DiOC6 negative; significant
at p < 0.05), corroborating the antiapoptotic
affects of FN adhesion (data not shown).
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C), indicating that alterations in CD95
receptor levels do not account for the antiapoptotic effects of
adhesion to FN. Examination of the DISC components, procaspase-8 and
FADD, revealed no significant changes in levels of these proteins
between cells adhered to FN for 2 or 24 h and those maintained in
suspension (Fig. 3C). These results indicate that altered
expression of these proteins does not contribute to the antiapoptotic
effects of FN adhesion. Similarly, Western blot analysis of cytosolic
lysates demonstrated that adhesion to FN had no effect on Bcl-2 or
Bcl-xL protein levels (Fig. 3C and
data not shown; Bcl-xL was not detectable in U937
cells). Together, these results demonstrate that in the face of equal
CD95, FADD, and procaspase-8 protein levels, FN adhesion inhibits the
activation of the initial proteolytic event in the CD95-mediated
apoptotic cascade. FN adhesion is associated with a reversible increase in cytosolic c-FLIPL
Because adhesion of hematopoietic cancer cell lines
reduced CD95-mediated procaspase-8 cleavage and activation without a
reduction in expression of procaspase-8, FADD, or CD95, we next
examined expression of c-FLIPL, a DISC regulatory
factor. c-FLIPL, a recently identified inhibitor
of DISC formation, competes with procaspase-8 for the death effector
domains of FADD (6, 49). Comparison of cytosolic extracts
of U937 and 8226/SH2 cells maintained in
suspension or adhered to FN revealed a significant increase in
c-FLIPL protein levels (Fig. 4, A and B).
Western blot analysis demonstrated that c-FLIPL
protein levels were elevated as early as 2 h after adhesion (Fig. 4A). In contrast to c-FLIPL, no
alterations in c-FLIPS protein levels were
observed in either the U937 or 8226/SH2 cell
lines (Fig. 4, A and B). Similarly, increased
levels of c-FLIPL protein were observed in
several additional hematopoietic cancer cell lines after adhesion to FN
(2 h) compared with cells maintained in suspension, including the 8226
(parental), mm1, and H929 multiple myeloma cell lines and the THP-1
leukemia cell line (data not shown).
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D). These
data suggest that FN adhesion acutely and reversibly regulated
c-FLIPL cytosolic protein levels. Furthermore,
cells released from FN regained sensitivity to CD95-mediated apoptosis
comparable to cells in suspension (Fig. 4E), demonstrating a
strong correlation between cytosolic c-FLIPL
levels and resistance to CD95-mediated apoptosis. These results are in
agreement with data demonstrating that exogenous expression of c-FLIP
in U937 cells blocked CD95-mediated apoptosis (50). c-FLIPL associates with FADD in cells adhered to FN and is recruited to the DISC after CD95 cross-linking
Previous reports using cotransfection experiments demonstrated
that c-FLIP interacts with FADD and may competitively inhibit the
association of FADD with procaspase-8 (6, 49). Therefore,
we compared the association of FADD and c-FLIPL
in suspension and FN-adherent cells. Immunoprecipitation with
c-FLIPL-specific antisera followed by FADD
Western blot analysis demonstrated interactions between FADD and
c-FLIPL in FN-adhered cells, but not in cells
maintained in suspension (Fig. 5A). Western blot analysis of
lysates pre- and post-c-FLIPL immunoprecipitation
demonstrated that both c-FLIPL and FADD were
depleted only in supernatants from cells adhered to FN, confirming the
association between c-FLIPL and FADD in adherent
cells (Fig. 5B).
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C),
demonstrating that c-FLIPL is recruited to the
DISC after CD95 stimulation only in cells adhered to FN. These results
indicate that adhesion of U937 cells to FN allows the recruitment of
c-FLIPL to the DISC, preventing procaspase-8
activation and inhibiting apoptosis. FN adhesion alters the intracellular distribution of c-FLIPL
Mechanisms regulating c-FLIP expression and activity are not yet
well defined. Several studies have recently demonstrated that the
activation status of leukocytes may affect c-FLIP RNA expression and
sensitivity to CD95-induced apoptosis (38, 51). Therefore,
we examined the effects of FN adhesion on c-FLIP RNA expression. RNase
protection assay demonstrated no significant changes in
c-FLIPL or c-FLIPS RNA
transcripts (Fig. 6, A and
B), indicating that transcriptional regulation is unlikely
to explain the reversible increase in c-FLIPL. To
assess the contribution of translational regulation, we examined the
effects of the protein synthesis inhibitor cyclohexamide on FN-mediated
c-FLIPL expression. Pretreatment of U937 cells
with concentrations of cyclohexamide that significantly inhibited
[35S]methionine and cysteine incorporation
demonstrated no significant change in c-FLIPL
accumulation after adhesion to FN. These data indicated that protein
synthesis is not the primary mechanism regulating
c-FLIPL accumulation (Fig. 6, C and
D).
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A). In contrast, Triton X-100
extracts from FN-adhered cells contained elevated levels of
c-FLIPL compared with cells in suspension (Fig. 7A). These results suggest that
c-FLIPL mobilizes from a relatively Triton
X-100-insoluble fraction to a more Triton X-100-soluble fraction after
adhesion to FN. To further characterize this phenomenon, we used
differential centrifugation to examine the effects of FN adhesion on
the intracellular localization of c-FLIPL.
Subcellular fractionation of U937 cells demonstrated that
c-FLIPL localized exclusively in the membrane
fraction in cells maintained in suspension (Fig. 7B). In
contrast, in cells adhered to FN for either 2 or 24 h,
c-FLIPL was increased in the cytosolic fraction
with a concomitant decrease in the membrane-bound fraction (Fig. 7B). These results indicate that, in nonadhered cells,
c-FLIPL is maintained in or associated with
organelle membranes. However, after FN adhesion,
c-FLIPL is released from the membrane-bound
fraction into the cytosol. Although the mechanism of mobilization and
the specific membrane localization of c-FLIPL
remain to be determined, the known activity of integrin signaling and
cytoskeletal remodeling suggest that specific signaling cascades or
physical alterations in the cytoskeleton may influence
c-FLIPL localization and availability to interact
with cytosolic factors such as FADD.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 integrin-specific adhesion to FN
confers resistance to CD95-induced programmed cell death in
hematopoietic cancer cell lines. Adhesion of hematologic cancer cell
lines inhibits CD95-mediated apoptosis by increasing the cellular
localization and availability of c-FLIPL, a known
inhibitor of CD95-mediated apoptosis.
Examination of specific steps of the CD95-mediated apoptotic cascade
demonstrated a reduction in CD95-mediated procaspase-8 cleavage and
proteolytic activity in cells adhered to FN compared with cells
maintained in suspension. CD95-mediated programmed cell death involves
death receptor cross-linking and propagation of the death signal from
the DISC. Multiple factors regulate DISC signaling from the
TNFR family of death receptors (29). One such regulatory
factor, c-FLIP, is a cellular homologue to the viral FLIP family of
proteins that confers resistance to CD95-mediated apoptotic signaling
(31, 34, 35, 36, 37, 38). c-FLIP is expressed as two predominant
splice variants, c-FLIPS and
c-FLIPL. Similar to the v-FLIPs,
c-FLIPS is composed of two death effector domain
(DED) motifs that share significant homology with the DEDs of
FADD, procaspase-8, and procaspase-10. c-FLIPL
contains the two amino-terminal DEDs of c-FLIPS
and an additional carboxy-terminal caspase-like domain; however, the
essential catalytic histidine and cysteine are replaced by arginine and
tyrosine, respectively, resulting in a proteolytically inactive enzyme.
Previous reports have demonstrated that exogenously expressed c-FLIP
associates with FADD and procaspase-8 in a DED domain-dependent manner
(33, 34, 35), suggesting that these interactions may reduce
recruitment of procaspase-8 to the DISC. Consistent with these
findings, recent reports demonstrated that exogenously expressed c-FLIP
associated with cross-linked CD95 and attenuated apoptotic signaling by
interfering with the transcatalytic activation of adjacent procaspase-8
molecules (56, 57). These reports demonstrate that
increased c-FLIP to procaspase-8 ratios lead to the inhibition of
CD95-mediated procaspase-8 processing and apoptosis. In this report, we
demonstrate that 1 integrin-mediated adhesion
to FN induces the release of c-FLIPL from a
preexisting membrane-associated compartment, allowing binding to
cytosolic FADD. Moreover, after CD95 cross-linking,
c-FLIPL localized to the DISC in adherent cells.
From these findings, we propose that FN adhesion blocks CD95-mediated
apoptosis by increasing cytosolic availability of
c-FLIPL, thereby facilitating association with
cytosolic FADD, inhibiting procaspase-8 activation and preventing cell
death (Fig. 8). Our proposed model of
adhesion-mediated mobilization of c-FLIPL from a
membrane-bound fraction to a cytosolic fraction is in agreement with
models showing that increases in the ratio of
c-FLIPL to procaspase-8 lead to the inhibition of
procaspase-8 cleavage and activation (56, 57). The
identification of alterations in c-FLIPL
cytosolic solubility as a regulatory mechanism of
c-FLIPL expression and function indicate that
cellular protein levels alone may not accurately predict cellular
sensitivity to CD95 stimulation.
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Recent studies have demonstrated that adhesion-induced
alterations in protein localization may play a significant role in
response to cytotoxic stimuli (43, 58, 59). Hazlehurst et
al. (43) recently demonstrated that the
nuclear redistribution of topoII from diffuse nuclear
localization to distinct punctate topo II clusters in FN-adhered
cells correlated with decreased topo II activity, DNA damage,
and programmed cell death by topo II inhibitors in hematopoietic cancer
cells. Similarly, the use of spheroid cell culture models examining the
effects of cell-to-cell contact on drug sensitivity of solid tumor
cells lines revealed that the proliferating outer cells of the spheroid
are more resistant to etoposide than cells grown on a monolayer
(59). This resistance correlated with a decrease in topo
II phosphorylation and a redistribution of topo II from the
nucleus to the cytosol. Together, these reports demonstrated that
sensitivity to topo II inhibitors is modulated by cell
adhesion-mediated redistribution of drug targets. Cellular adhesion has
also been shown to more directly regulate the localization of apoptotic
machinery. Gilmore et al. (58) demonstrated that the
pro-apoptotic protein Bax is maintained in the cytosol in adhered
mammary epithelial cells. After culture on polyhema-coated plates
(cells maintained in suspension), Bax undergoes a
conformational change exposing mitochondrial localization domain (BH3),
facilitating a reversible translocation of Bax from the cytosol to the
nucleus and the initiation of apoptosis. Specific signaling factors
associated with integrin activation have also been shown to promote
survival by regulating intracellular localization of apoptotic factors.
The apoptotic Bcl-2 family member Bad has been shown to be the target
of survival kinases (60, 61, 62). Serine phosphorylation of
Bad has been shown to suppress apoptosis by disrupting Bad/Bcl-2 or
Bad/Bcl-xL pro-apoptotic heterodimers
(60). Bad phosphorylation facilitated the recruitment of
14-3-3 chaperone proteins to Bad (61). The
phospho-Bad/14-3-3 complex is then shuttled to the cytosol, promoting
cell survival (60, 61, 62). These studies indicate that cell
adhesion can influence cell survival by regulating the localization of
key apoptotic effectors. In this study, we show that cell adhesion also
facilitates changes in the intracellular localization of
c-FLIPL, thereby inhibiting CD95-induced
programmed cell death. Together, these observations indicate that
interactions between cells and their environment may regulate
sensitivity to stress-induced apoptosis by controlling the distribution
of important drug targets and regulators of apoptotic machinery.
We propose that in myeloma and other hematopoietic malignancies, the bone marrow microenvironment provides sanctuary to tumor cells through a heterologous network of survival signals involving both cytokines and direct cell contact between cancer cells and adjacent stromal cells or the ECM. In support of this hypothesis, we previously demonstrated that the cytokine IL-6 confers resistance to CD95-mediated apoptosis in myeloma cells (20). In this report, we demonstrate that direct contact between tumor cells and the ECM component FN also confers resistance to CD95-mediated apoptosis. Taken together, these results indicate that the bone marrow microenvironment may provide a site of immune privilege through a network of antiapoptotic signals involving both soluble factors and direct contact with the ECM. This cellular protection against immune cytotoxicity may allow for tumor cell survival and progression.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William S. Dalton at the current address: Arizona Health Sciences Center, Office of the Dean, College of Medicine, University of Arizona, 1501 North Campbell Avenue, Room 2206, P.O. Box 245017, Tucson, AZ 85724-5017.
3 Abbreviations used in this paper: ECM, extracellular matrix; FN, fibronectin; c-FLIP, c-Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein; c-FLIPL, c-FLIP-long; FADD, Fas-associated death domain protein; DISC, death-inducing signal complex; c-FLIPS, c-FLIP-short; FCM, flow cytometry; pNA, p-nitroanilide; DED, death effector domain; MKK1, mitogen-activated protein kinase kinase; topo II, topoisomerase II; DEVD, Asp-Glu-Val-Asp; IETD, Ile-Glu-Thr-Asp.
Received for publication October 22, 2001. Accepted for publication December 21, 2001.
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41 integrin prevents apoptosis in B cell chronic lymphocytic leukemia: correlation with Bcl-2 and Bax. Leukemia 13:266.[Medline]
1 integrin-mediated adhesion correlates with drug resistance in U937 cells. Blood 98:1897.1) death signal. J. Biol. Chem. 273:4345.1 integrins regulates p27kip1 levels and contributes to cell adhesion mediated drug resistance (CAM-DR). Oncogene 19:4319.[Medline]
correlate with etoposide resistance in multicell spheroids and xenograft tumors. Cancer Res. 60:5747.This article has been cited by other articles:
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  K. H. Shain, D. N. Yarde, M. B. Meads, M. Huang, R. Jove, L. A. Hazlehurst, and W. S. Dalton {beta}1 Integrin Adhesion Enhances IL-6-Mediated STAT3 Signaling in Myeloma Cells: Implications for Microenvironment Influence on Tumor Survival and Proliferation Cancer Res., February 1, 2009; 69(3): 1009 - 1015. [Abstract] [Full Text] [PDF]
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 M. B. Meads, L. A. Hazlehurst, and W. S. Dalton The Bone Marrow Microenvironment as a Tumor Sanctuary and Contributor to Drug Resistance Clin. Cancer Res., May 1, 2008; 14(9): 2519 - 2526. [Abstract] [Full Text] [PDF]
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 L. E. Perez, N. Parquet, K. Shain, R. Nimmanapalli, M. Alsina, C. Anasetti, and W. Dalton Bone Marrow Stroma Confers Resistance to Apo2 Ligand/TRAIL in Multiple Myeloma in Part by Regulating c-FLIP J. Immunol., February 1, 2008; 180(3): 1545 - 1555. [Abstract] [Full Text] [PDF]
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W. Dalton and K. C. Anderson Synopsis of a Roundtable on Validating Novel Therapeutics for Multiple Myeloma. Clin. Cancer Res., November 15, 2006; 12(22): 6603 - 6610. [Abstract] [Full Text] [PDF]
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L. A. Hazlehurst, R. F. Argilagos, M. Emmons, D. Boulware, C. A. Beam, D. M. Sullivan, and W. S. Dalton Cell Adhesion to Fibronectin (CAM-DR) Influences Acquired Mitoxantrone Resistance in U937 Cells Cancer Res., February 15, 2006; 66(4): 2338 - 2345. [Abstract] [Full Text] [PDF]
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 N. Parquet, R. Nimmanapalli, C. Anasetti, M. Alsina, W. Dalton, and L. E. Perez Bortezomib Partially Overcomes TNF-Related Apoptosis Inducing ligand/Apo-2L (TRAIL/Apo-2L) Environment Mediated-Drug Resistance (EM-DR). Blood (ASH Annual Meeting Abstracts), November 16, 2004; 104(11): 2459 - 2459. [Abstract] [Full Text]
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T. Hayashi, T. Hideshima, A. N. Nguyen, O. Munoz, K. Podar, M. Hamasaki, K. Ishitsuka, H. Yasui, P. Richardson, S. Chakravarty, et al. Transforming Growth Factor {beta} Receptor I Kinase Inhibitor Down-Regulates Cytokine Secretion and Multiple Myeloma Cell Growth in the Bone Marrow Microenvironment Clin. Cancer Res., November 15, 2004; 10(22): 7540 - 7546. [Abstract] [Full Text] [PDF]
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 S. Shah and P. W. Sylvester Tocotrienol-Induced Caspase-8 Activation Is Unrelated to Death Receptor Apoptotic Signaling in Neoplastic Mammary Epithelial Cells Experimental Biology and Medicine, September 1, 2004; 229(8): 745 - 755. [Abstract] [Full Text] [PDF]
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 M. Fornaro, J. Plescia, S. Chheang, G. Tallini, Y.-M. Zhu, M. King, D. C. Altieri, and L. R. Languino Fibronectin Protects Prostate Cancer Cells from Tumor Necrosis Factor-{alpha}-induced Apoptosis via the AKT/Survivin Pathway J. Biol. Chem., December 12, 2003; 278(50): 50402 - 50411. [Abstract] [Full Text] [PDF]
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L. A. Hazlehurst, S. A. Enkemann, C. A. Beam, R. F. Argilagos, J. Painter, K. H. Shain, S. Saporta, D. Boulware, L. Moscinski, M. Alsina, et al. Genotypic and Phenotypic Comparisons of de Novo and Acquired Melphalan Resistance in an Isogenic Multiple Myeloma Cell Line Model Cancer Res., November 15, 2003; 63(22): 7900 - 7906. [Abstract] [Full Text] [PDF]
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 R. A. Screaton, S. Kiessling, O. J. Sansom, C. B. Millar, K. Maddison, A. Bird, A. R. Clarke, and S. M. Frisch Fas-associated death domain protein interacts with methyl-CpG binding domain protein 4: A potential link between genome surveillance and apoptosis PNAS, April 29, 2003; 100(9): 5211 - 5216. [Abstract] [Full Text] [PDF]
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 N. Mitsiades, C. S. Mitsiades, P. G. Richardson, V. Poulaki, Y.-T. Tai, D. Chauhan, G. Fanourakis, X. Gu, C. Bailey, M. Joseph, et al. The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications Blood, March 15, 2003; 101(6): 2377 - 2380. [Abstract] [Full Text] [PDF]
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 K. C. Anderson and W. S. Dalton Synopsis of a Research Roundtable Presented on Cell Signaling in Myeloma: Regulation of Growth and Apoptosis--Opportunities for New Drug Discovery Mol. Cancer Ther., December 1, 2002; 1(14): 1361 - 1365. [Abstract] [Full Text] [PDF]
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 D. G. Stupack and D. A. Cheresh Get a ligand, get a life: integrins, signaling and cell survival J. Cell Sci., January 10, 2002; 115(19): 3729 - 3738. [Abstract] [Full Text] [PDF]
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