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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1727-1737
Modulation of Caspase-8 and FLICE-Inhibitory Protein
Expression as a Potential Mechanism of Epstein-Barr Virus Tumorigenesis
in Burkitt's Lymphoma
By
Clifford G. Tepper and
Michael F. Seldin
From the Rowe Program in Genetics, Departments of Biological
Chemistry and Medicine, UC Davis School of Medicine, Davis, CA.
 |
ABSTRACT |
Ligation of the Fas receptor induces death-inducing signaling
complex (DISC) formation, caspase activation, and subsequent apoptotic
death of several cell types. Epstein-Barr virus (EBV)-positive group
III Burkitt's lymphoma (BL) cell lines have a marked resistance to
Fas-mediated apoptosis, although expressing each of the DISC components, Fas/ APO-1-associated death domain protein
(FADD), and caspase-8 (FLICE/MACH/Mch5). The apoptotic
pathway distal to the DISC is intact because ceramide analogs,
staurosporine, and granzyme B activate caspase-3 and induce apoptosis.
Fas resistance was not explained by the putative death-attenuating
caspase-8 isoforms. However, while Fas-activated cytosolic extracts
from sensitive cells were capable of processing both procaspase-8 and procaspase-3 into active subunit forms, resistant cell extracts did not
possess either of these activities. Accordingly, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed higher
transcript levels for the FLICE-inhibitory protein (FLIPL) in resistant cells and the ratio of caspase-8 to FLIPL
measured by competition RT-PCR analysis directly correlated with
susceptibility to Fas-mediated apoptosis of all cell lines. In
addition, modification of the caspase-8/FLIPL ratio by
caspase-8 or FLIPL overexpression was able to alter the
susceptibility status of the cell lines tested. Our results imply that
the relative levels of caspase-8 and FLIPL are an important
determinant of susceptibility to Fas-mediated apoptosis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
FAS (APO-1/CD95) RECEPTOR-mediated
apoptosis is an effector of activation-induced T-lymphocyte death,
peripheral T- and B-cell tolerance, and its physiologic importance is
underscored by defects that result in autoimmunity with aberrant
antibody production and lymphadenopathy in both mouse and
humans.1-10 Ligation of the Fas receptor initiates a death
signal by the formation of a death-inducing signaling complex
(DISC).11 Interactions between the homologous death domains
(DD) of Fas and the Fas/APO-1-associated death domain protein (FADD)
carboxyl-terminus promote the recruitment of procaspase-8
(FLICE/MACH/Mch5) by homophilic interactions between death effector
domains (DED) contained in the amino-terminus of FADD and the prodomain
of caspase-8.12,13 The resultant conformational change in
procaspase-8 leads to its activation and subsequent processing
resulting in the cleavage of the FADD-homology domain from the
ICE/CED-3-caspase-homology region.14 The former remains bound to the DISC, while liberated active caspase-8 initiates the
apoptotic proteolytic cascade. Dimerization of caspase-8 is required
for activation and yields an active tetrameric (p10/p18)2 complex.15 Major substrates for caspase-8 include the
zymogen forms of other caspases, such as caspase-6 (Mch2) and caspase-3 (Yama/CPP32 /Apopain), and the death substrate poly(adenosine diphosphate [ADP])-ribose polymerase
(PARP).13,16 The conversion of caspases to their active
forms and subsequent substrate cleavage are the quintessential events
comprising the "execution phase" of the apoptotic
pathway.17,18
The efficient triggering of Fas-mediated apoptosis suggests the
requirement for strict controls within this signaling pathway. Although
many cell types express the Fas antigen, few are constitutively susceptible to cell death triggered through it. Modulation of Fas
susceptibility appears to be a prominent feature of the immune system.
Resting T cells are initially resistant to Fas-mediated apoptosis, but
can be rendered sensitive by activation with sequential phytohemagglutinin and interleukin-2 treatments.19 The
initial resistance has been associated with a failure of caspase-8 to be recruited to the DISC by FADD.19 The downregulation of
Fas sensitivity through alteration in FADD and caspase-8 association has also been demonstrated in a Fas-resistant pre-B-cell
line.11 Furthermore, resistance can be induced in B
lymphocytes by antigen receptor cross-linking and is a characteristic
of the memory B-cell phenotype.20,21
A number of Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL)
cell lines also develop resistance to Fas receptor cross-linking after
having acquired a lymphoblastoid/group III, or activated, phenotype.22 In contrast, even though EBV-transformed
primary B lymphocytes typically possess a classic lymphoblastoid group III phenotype, they are acutely sensitive to Fas-mediated apoptosis showing that EBV infection does not independently lead to Fas resistance.22 Furthermore, the sensitivity of
Fas/APO-1-transfected EBV-negative BL cell lines to Fas ligation
indicates that Fas resistance is not a general characteristic of the
malignant B-cell phenotype.11,22 Because Fas resistance may
be critical to the persistence of Burkitt's lymphomas, we were
interested in elucidating the basis for this phenomenon.
Recently, viral inhibitors of Fas- and tumor necrosis factor (TNF)
receptor-induced cell death have been identified in several poxviruses
and  -herpes viruses, but not in EBV.23-25 These
DED-containing proteins behave as decoy molecules blocking the
interaction between FADD and caspase-8 essential for DISC assembly and
caspase-8 activation. These inhibitory proteins have been collectively
referred to as v-FLIPs, for viral FLICE-inhibitory proteins, and are
thought to participate in viral pathogenesis by attenuation of the host immune responses mediated through death receptors.25 Unlike cowpox crmA, baculovirus p35, and vaccinia SPI-2 (B13R) gene
products, but consistent with an inhibitory action proximal to
caspase-8 activation, v-FLIPs cannot block the apoptotic activity of
activated caspase-8.23-31
A cellular homologue of viral FLICE-inhibitory proteins (FLIP,
also referred to as I-FLICE/FLAME-1/CASH/CLARP/CASPER/MRIT/Usurpin) has
subsequently been identified by several groups.32-39 Stable transfection of cells with FLIP expression vectors protects them to
varying degrees from apoptosis mediated by the Fas, TNF, and TNF-related apoptosis-inducing ligand (TRAIL)
receptors.24,32,36,38 Alternative splicing leads to long
(FLIPL, 55 kD) or short (FLIPS, 28 kD) form variants.36 FLIPL has a very
similar structure to that of caspase-8 in that it contains two DEDs and
a caspase homology domain. However, it lacks the caspase active site
consensus pentapeptide and a tyrosine residue is substituted for the
highly conserved cysteine, rendering this a catalytically-inert
caspase. FLIPS, on the other hand, is truncated shortly
after the 2 DEDs and completely lacks the caspase homology domain. Both
FLIPs can interact with FADD and caspase-8 via the DED motifs and can
be recruited to the Fas DISC.32,36,38 The caspase domain
further contributes to the interaction with caspase-8. In addition,
similarly to procaspase-8, FLIP can serve as a substrate for activated
caspase-8, resulting in the generation of a C-terminally truncated
43-kD protein that possibly binds more tightly to caspase-8, preventing
further activation.36 FLIP has been implicated in the
regulation of the Fas pathway during T-lymphocyte activation and
probably contributes to blocking caspase-8 recruitment to the DISC
during the initial resistant phases.19,36 FLIP expression
has also been detected in melanoma specimens and has been associated
with the generation of Fas-resistance of several melanoma and chronic
myelogenous leukemia (ie, K562) cell lines.34,36 Because
FLIP was not detectable in nonmalignant melanocytes, its upregulation
may be an event critical to tumorigenesis.
In this report, we examined several potential mechanisms used by
EBV-positive BL cell lines for the establishment of Fas-resistance. To
this end, 3 classes of cell lines were used: (1) EBV-transformed primary B lymphocytes (ie, SKW6.4), (2) EBV-negative BL cells, and (3)
EBV-positive BL lines (groups I and III). This permitted examination of
two aspects of this model system without genetic manipulation, namely
the consequence of EBV infection on apoptosis and the influence host
cell type has on the outcome of infection. We report a strong
correlation between heightened FLIPL expression relative to
that of caspase-8 and the generation of the resistant phenotype. This
was strongly supported by our ability to reverse this phenotype by
overexpression of caspase-8. Furthermore, this is a tumor-specific
phenomenon, as lymphoblastoid cell lines (LCL) established from
EBV-transformed primary B cells did not display the same pattern of
FLIP and caspase-8 modulation.
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MATERIALS AND METHODS |
Cell lines and culture.
The cell lines used in this study include: (1) EBV-negative Burkitt's
lymphomas BJAB, Ramos, and ST486, (2) EBV-positive Burkitt's lymphomas
Akata, Daudi, Mutu-BL (groups I and III), Raji, Jijoye, and P3HR-1, and
(3) EBV-positive B lymphoblastoid SKW6.4. The susceptibility/resistance
of all cell lines to Fas-induced apoptosis and EBV status are indicated
in Fig 1. All cultures were maintained in
RPMI 1640 medium (GIBCO-BRL, Gaithersburg, MD)
supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine,
and 100 U/mL penicillin-100 µg/mL streptomycin at 37°C in a
humidified environment of 5% CO2 in air. Experiments using
anti-Fas IgM and staurosporine were also performed in this medium. For
treatment with C2-ceramide, the serum content was reduced
to 1%.
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| Fig 1.
Fas susceptibility and EBV status of cell lines used in
this study. Fas-mediated apoptosis was assayed with MTT. Triplicate
cultures of 100 µL (3 × 104 cells) were incubated with
anti-Fas IgM (250 ng/mL) for 24 hours at 37°C. MTT was then added
for an additional 2 hours followed by solubilization of the formazan
crystals. Absorbance was read at 570 nmol/L and percentage apoptosis
was calculated as previously described.40 Results are from
3 separate experiments and are expressed as mean percentage apoptosis ± standard deviation. Cell-surface expression of the Fas receptor was
determined by flow cytometry and the results indicate Fas-positive
( ) and Fas-negative ( ) cell lines. The EBV status (negative or
positive) of each cell line was obtained from the literature and later
confirmed in appropriate cell lines by LMP1 immunoblot
analysis.61,62
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Reagents.
The cytotoxic mouse anti-human Fas monoclonal antibody (anti-Fas; IgM,
clone CH-11), mouse anti-caspase-8 (clone 5F7, IgG2b), and rabbit
polyclonal anti-FLIP-CT antibodies were obtained from Upstate
Biotechnology, Inc (Lake Placid, NY). Mouse monoclonal anti-FADD (clone
1, IgG1) and anti-CPP32 (clone 19, IgG2a) antibodies were both
purchased from Transduction Laboratories (Lexington, KY). Polyclonal
goat anti-FLICE/Mch5 p20 (C-20) was obtained from Santa Cruz
Biotechnology, Inc (Santa Cruz, CA) and anti-ICE-LAP3 p20 (rabbit
polyclonal) was the kind gift of V.M. Dixit (Genentech, Inc, South San
Francisco, CA). MTT was purchased from Sigma (St Louis, MO), dissolved
in phosphate-buffered saline (PBS), and filter-sterilized.
C2-ceramide and staurosporine were purchased from
Calbiochem (La Jolla, CA). Stock solutions were made in dimethyl sulfoxide (DMSO) and stored at  20°C until use. Purified
granzyme B was supplied by Enzyme Systems Products (Livermore, CA). All polymerase chain reaction (PCR) primers were custom synthesized by
GIBCO-BRL.
Flow cytometry.
Cell-surface expression of Fas antigen was determined by FACScan flow
cytometric analysis as previously described.40 Briefly, cells were sequentially incubated with anti-Fas IgM (10 µg/mL) and
flouroscein isothiocyanate-conjugated goat anti-mouse immunoglobulins (Dako, Glostrup, Denmark) followed by fixation with
formaldehyde and analysis. Nonspecific binding of antibodies was
controlled for with each cell line by using a nonimmune monoclonal
antibody (clone P3, IgG) (generous gift of Dr Barton F. Haynes, Duke
University, Durham, NC).
Measurement of cell death and DNA fragmentation analysis.
Cell death was determined using an MTT-based assay as previously
described.40 Briefly, cell cultures in 96-well plates (3 × 104 cells/well in 100 µL) were exposed to
anti-Fas IgM (250 ng/mL) for 24 hours. MTT was then added to each well
and incubation was continued for another 2 hours followed by
solubilization of the resulting formazan crystals overnight and
measurement of absorbance at 570 nm. Apoptotic cell death was further
confirmed by the appearance of morphologic changes such as plasma
membrane blebbing, increased cell refractility, and nuclear
condensation. For DNA fragmentation analysis, DNA was isolated from 1 × 106 cells, purified by organic extraction,
ethanol-precipitated, and finally dissolved in 100 µL of Tris-EDTA
(pH 8.0). Twenty microliters of each sample was treated with DNase-free
RNase (35 µg/mL) for 30 minutes followed by electrophoresis through
1.2% ethidium bromide-agarose gels at constant current (20 mA) for 16 hours.
Immunoblot analysis.
Cell lysates were made in NP-40 lysis buffer (30 mmol/L Tris-Cl, pH
7.5, 1 mmol/L EDTA, 150 mmol/L NaCl, 1% NP-40) and protein content was
quantitated with a detergent-compatible assay kit (Bio-Rad, Hercules,
CA). Proteins (50 µg of lysate) were separated by
denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (Bio-Blot, Costar, Cambridge, MA). Membranes were blocked for at least 1 hour with 5% nonfat dry milk in Tris-buffered saline containing 0.05%
Tween-20 (TBST). Primary antibodies were diluted in blocking buffer
according to the manufacturers' protocols and subsequently incubated
with the blots for 2 hours at room temperature or overnight at 4°C. The membranes were washed three times with TBST and incubated with a
1:5,000 dilution of the appropriate horseradish peroxidase-conjugated anti-IgG in blocking buffer for 1 hour. After washing, the blots were
developed with enhanced chemiluminescence (Amersham, Piscataway, NJ) and exposed to BioMax x-ray film (Eastman-Kodak,
Rochester, NY).
Caspase activity assays.
Caspase-3-related protease activity in cell lysates was measured using
the ApoAlert CPP32 assay kit (Clontech, Palo Alto, CA). Briefly, 2 × 106 cells were treated with anti-Fas IgM (250 ng/mL, 3 hours), C2-ceramide (10 µmol/L, 16 hours), and
staurosporine (5 µmol/L, 16 hours), washed once with PBS, and lysed
in 50 µL of cell lysis buffer on ice. Nuclei were removed from the
lysates by centrifugation in a microcentrifuge for 30 seconds at
4°C. The pellet was discarded and the nuclei-free detergent extract
was used in the caspase assays. An equal volume of 2X reaction buffer
(containing 10 mmol/L dithiothreitol [DTT]) was then
added and reactions were initiated by the addition of the colorimetric
substrate DEVD-pNA (50 µmol/L final concentration) followed
by incubation at 37°C for 1 hour. The samples were then transferred
to 96-well plates and absorbance read with a plate reader at 405 nm.
Preparation of cytosolic extracts.
Untreated or anti-Fas-treated (1 and 2 hours) cells were harvested and
washed once in chilled PBS. The cell pellets were resuspended in
modified cytosolic extract preparation buffer (10 mmol/L HEPES, pH
7.5/10 mmol/L KCl/1 mmol/L DTT) and allowed to swell on ice for 15 minutes.41 The cells were lysed by aspiration through a
22-gauge needle 10 to 20 times and then centrifuged at 16,000g for 15 minutes at 4°C. The 16K supernatant was used for caspase processing assays.
In vitro caspase processing assays.
[35S]methionine-labeled procaspase-8 and procaspase-3
(Yama) were generated from pcDNA3-FLICE-HA and pcDNA3-Yama-AU1
expression vectors by coupled in vitro transcription/translation with
the TNT reticulocyte lysate system (Promega, Madison,
WI) driven by the T7 promoter. The mixture was
desalted by centrifugation through Bio-Spin columns (Bio-Rad). Caspase
processing activity contained in cytosolic extracts was examined by
combining 2 µL of the translated protein with 25 µg of extract in a
total volume of 10 µL followed by incubation for 2 hours at 37°C.
Reactions were terminated by the addition of Laemmli sample buffer
containing -mercaptoethanol. The samples were placed in a boiling
water bath and subjected to SDS-PAGE through 15% gels. The gels were
then fixed, dried, and exposed to BioMax x-ray film (Eastman-Kodak)
according to standard protocol.
RNA isolation and first-strand cDNA synthesis.
Total cellular RNA was isolated from 1 × 107 cells
using the TRIZOL reagent (GIBCO-BRL) according to the manufacturer's
protocol. First-strand cDNA was then synthesized from 5 µg of RNA
with the cDNA preamplification system (GIBCO-BRL) using SuperScript II reverse transcriptase (RT) and an oligo(dT) primer.
PCR analyses.
PCR reactions (50 µL) were performed according to standard protocol
with 2 µL of first-strand cDNA template, 10 µmol/L primers, 0.8 mmol/L dNTPs, and 0.5 U Taq polymerase. The amplification program was
as follows: 94°C for 2 minutes (1 cycle), 94°C for 30 seconds,
58°C for 30 seconds, 72°C for 90 seconds (30 cycles). The
following primer sequences were used for the detection of caspase-8/FLICE/MACH  and isoforms12:
MACH1(188).S (5'-TTG GAT CCA GAT GGA CTT CAG CAG AAA TCT
T-3'), MACH1(521).S (5'-AAG TGA GCA GAT CAG AAT TGA
G-3'), MACH1(575).S (5'-GAG GAT CCC CAA ATG CAA ACT GGA
TGA TGA C-3'), MACH1(914).AS (5'-ATT CTC AAA CCC TGC ATC
CAA GTG-3'), MACH1(930).AS (5'-GCC ACC AGCT AAA AAC ATT
CTC AA-3'). Amplification products were subsequently separated on
1.2% Tris-acetate-EDTA (TAE) agarose gels. The
sequences of the primers used for amplification of the FLICE caspase
homology domain were CASP8(649).S (5'-TTG TGG CAT ATG AGT GAA TCA
CAG ACT TTG GAC AAA G-3') and CASP8(1440).AS (5'-CAG CCG
GAT CCT CAA TCA GAA GGG AAG ACA AGT TT-3'). Full-length
FLIPlong was amplified with the FLIPL (383).S
(5'-GTA TAC ATA TGT CTG CTG AAG TCA TCC ATC AGG TTG-3') and
FLIPL (1822).AS (5'-GTG ACT CGA GTG TGT AGG AGA GGA
TAA GTT TCT TTC TC-3') primers.
CASP8/FLIP competition PCR (CFCP) analysis was performed by conducting
amplifications in the presence of primer pairs for both caspase-8
(caspase domain) and FLIPL using the same conditions described above. The amplified products were separated by agarose gel
electrophoresis, stained with ethidium bromide, and then analyzed by
scanning densitometry. The caspase-8/FLIPL ratio for each
sample was then obtained from the values for the relative band intensities.
Construction of caspase-8, FLIPL, and enhanced green
fluorescent protein (EGFP) mammalian expression constructs.
Full-length caspase-8 (CASP8[FL]) and FLIPL were
generated by high fidelity PCR amplification (Clontech, Palo Alto, CA)
from SKW6.4 and Raji cDNA templates, respectively, using the
CASP8(1)Kpn.S primer (5'-GAA CGG GGT ACC GCC ATG GAC TTC AGC AGA
AAT CTT TAT GAT-3') paired with the CASP8(1440).AS primer and the
FLIPL(383)SmtAKpn.S primer (5'-CGG GGT ACC GCC ATG
GCT GCT GAA GTC ATC CAT CAG GTT G-3') paired with the
FLIPL(1825)Xho.AS primer (5'-GTG ACT CGA GTT ATG TGT
AGG AGA GGA TAA GTT TCT TTC TC-3'). To maximize FLIPL protein expression, the forward primer was designed to mutate the
thymidine in the +4 position of the sequence to a guanine (ie,
resulting in conversion of Ser to Ala) in agreement with Kozak's
consensus sequence for optimal translation initiation. The resulting
amplification product was therefore designated FLIPL(SmtA). Gel-purified CASP8(FL) and FLIPL(SmtA) products were
TA-cloned into pCR3.1 (Invitrogen, Carlsbad, CA).
After determination of both sequence integrity and orientation, the
desired clones were excised by sequential NotI and KpnI
digestions and ligated into KpnI+NotI-digested pCEP4
(Invitrogen). These expression constructs were designated
pCEP4-CASP8(FL) and pCEP4-FLIPL(SmtA). The EGFP reporter
construct pCEP4-EGFP was generated by ligation of the NotI/KpnI EGFP fragment from pEGFP-N1 (Clontech) into
the same restriction sites of pCEP4. The vector pCEP4 was used for our constructs because it contains an EBV origin of replication (ori P) as
well as the strong cytomegalovirus (CMV) promoter. We
found the presence of ori P to be essential to obtaining easily
detectable protein expression in EBV-infected cell lines (data not shown).
Transient transfections and measurement of apoptosis.
Before transfection, BJAB, SKW6.4, Mutu-BL III, and Raji cells were
harvested, resuspended in twice the original volume of fresh complete
medium, and incubated for 18 hours. Cells were then harvested and
resuspended in fresh medium at a concentration of 2 × 107 cells/mL and placed on ice. Cotransfections with 1.5 µg of pCEP4-EGFP and 7.5 µg of either pCEP4-CASP8(FL) or
pCEP4-FLIPL(SmtA) were performed by electroporation (1,700 µF, 72  , 110 V) of 0.2 mL cells in 2-mm gap width cuvettes using a
BTX ECM-600 electroporator (Genetronics, San Diego, CA). The samples
were then diluted in 15 mL of prewarmed medium and incubated for 72 hours to permit recovery and maximum expression (ie, assessed by the
visualization of EGFP). For assessment of apoptosis in response to Fas
triggering, cultures were transferred into fresh medium containing 1%
FBS, rested at 37°C for 4 hours, and then left untreated or
challenged with anti-Fas IgM (500 ng/mL, clone CH-11) for 8 hours.
Percentage apoptosis of EGFP-positive cells was determined using
standard morphologic criteria (cytoplasmic condensation, plasma
membrane blebbing, increased refractility) by combined fluorescence and light microscopy with a Leica inverted phase/fluorescence
microscope (Leica Microsystems Inc, Deerfield, IL).
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RESULTS |
Apoptosis pathways distal to caspase-3 activation are intact in
Fas-resistant group III BL cell lines.
Previous studies by our group have shown that the synthetic ceramide
analog C2-ceramide effectively induced apoptosis of
Fas-resistant group III Mutu-BL cells. These results implied that even
though early events of Fas signaling were disrupted in this BL cell
line, components of this apoptotic pathway distal to caspase-3
activation were intact.40,42 To further characterize the
defect in the Fas pathway in resistant BL cells, they were exposed to
micromolar concentrations of the protein kinase inhibitor
staurosporine, also known to trigger caspase-3
activation.16,43 Staurosporine induced apoptosis to
different degrees in all cell cultures as shown by
high-molecular-weight DNA degradation and/or the generation of the
hallmark nucleosomal laddering pattern, which was very prominent in the
Ramos EBV-negative group I BL cell line
(Fig 2). In addition, immunoblotting of
whole-cell lysates confirmed the presence of comparable levels of
caspase-3 (Yama/CPP32/Apopain) and caspase-7 (ICE-LAP3/Mch3/CMH-1) in
sensitive and resistant cell lines
(Fig 3A).
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| Fig 2.
Induction of apoptosis in Fas-resistant BL cell lines by
staurosporine. Cells (1 × 106) were incubated with DMSO
vehicle alone (0 µmol/L) or exposed to 2.5 and 5 µmol/L
staurosporine for 16 hours and subsequently harvested for analysis of
DNA fragmentation as described in Materials and Methods.
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| Fig 3.
Downstream caspases are present and functional
in BL cell lines. (A) Immunoblot analysis for caspase-3 (Yama/CPP32)
and caspase-7 (ICE-LAP3). Whole-cell lysates (50 µg) were separated
on 15% SDS-PAGE gels under reducing conditions and immobilized on
nitrocellulose membranes. Caspase-3 and caspase-7 were detected with
anti-CPP32 and anti-ICE-LAP3 p20 antibodies, respectively, followed by
development with chemiluminescence. (B) Caspase-3 can be activated in
Fas-resistant BL cells. Cells (2 × 106; 5 × 105/mL) were left untreated (control, ) or treated with
anti-Fas IgM ( ), C2-ceramide (), and staurosporine
( ). Nuclei-free lysates were made from each sample and used in
DEVD-pNA cleavage assays. Controls for nonspecific protease
activity were obtained by preincubating one set of
staurosporine-induced cell lysates with the caspase-3 inhibitor
DEVD-fmk (5 µmol/L) for 30 minutes at 37°C before addition of the
substrate. Absorbance was read at 405 nm. Caspase activity is expressed
in units with 1 unit being the amount of enzyme activity liberating 1 pmol of pNA per minute. (C) Induction of caspase-3 activity in
resistant cell lysates by granzyme B. Cleavage assays were performed as
in (B) with naïve cell lysates of SKW6.4 and Mutu-BL III
cultures. Before performing the cleavage assay, lysates were
preincubated for 30 minutes at 37°C in the absence (, ) or
presence (+, ) of 5 U of granzyme B (GraB). Reactions performed
without cytosolic extract (buffer) showed the failure of granzyme B to
independently use DEVD-pNA as a substrate.
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These results implied that the apoptotic proteolytic cascade was
intact. To more directly confirm that caspase-3 and other proteases
with similar substrate specificity were processed and catalytically
active in the BL cytosol, we evaluated the ability of various apoptotic
cytosolic extracts to cleave the tetrapeptide substrate
DEVD-pNA. The highest caspase-3 activity was consistently observed in the detergent extracts from Fas-induced SKW6.4 and BJAB
cells, which contained 291 and 225 units total activity, respectively
(Fig 3B). As expected, Fas stimulation failed to generate appreciable
caspase-3 activity in Mutu-BL III cells. However, substantial caspase-3
activity could be generated in this cell line on exposure to
C2-ceramide (10 µmol/L) for 16 hours and approached that
present in Fas-stimulated BJAB cells. In fact, Mutu-BL III was more
responsive to this ceramide analog than both Fas-susceptible cell lines
tested. Although C2-ceramide did not activate caspase-3 as
effectively as anti-Fas IgM did in SKW6.4 and BJAB cells, this result
is consistent with our previous data showing Fas ligation to be the
more efficient inducer of apoptosis (Fig 3B).40,44 In
accordance with the DNA fragmentation data, caspase-3 activity could
also be demonstrated in cytosolic extracts from staurosporine-treated
Mutu-BL III cells (Figs 2 and 3B).
Because both of these agents involve as yet undefined events leading up
to caspase-3 activation, we also wanted to determine if cleavage
activity in resistant cell extracts could be generated by direct
proteolytic processing of caspase-3. To this end, naïve detergent extracts were preincubated with purified granzyme B followed
by DEVD-pNA cleavage analysis. Granzyme B was able to induce
approximately 214 and 258 units of caspase-3-like protease activities
in both susceptible and resistant cytosolic extracts, respectively (Fig
3C). Matched extracts preincubated in the absence of granzyme B
displayed a baseline, but minimal, activity. In the absence of
cytosolic extract, granzyme B was unable to cleave the substrate
eliminating the possibility that granzyme B was responsible for the
increased activity and consistent with DEVD not being a preferred
sequence motif for this enzyme (Fig 3C).45 Taken together,
these data clearly show that caspase-3 and related proteases can be
activated and functional in Fas-resistant BL cells and suggest that the
inhibition to the Fas pathway occurs proximal to caspase-3 activation.
DISC formation.
The fact that Fas-resistant group III BL cell lines could be induced to
undergo apoptosis by agents activating effector caspases implied the
presence of an inhibitory mechanism specific for the Fas pathway in
this system, specifically acting upstream of the activation of
caspase-3 and caspase-7. Therefore, it was critical to determine the
integrity of the Fas death-inducing signaling complex in the BL milieu.
Whole-cell lysates were examined by immunoblot analysis and
demonstrated that the adapter protein FADD (26 kD) and caspase-8 (55 kD) were both present in all cell lines examined
(Fig 4). We next wanted to determine if
cross-linking Fas on BL cells leads to the formation of a functional
DISC. This was examined indirectly with an in vitro caspase processing
assay. The pro-forms of caspase-8 and caspase-3 were in vitro
transcribed and translated in the presence of
[35S]methionine, incubated with 16K cytosolic extracts of
Fas-treated SKW6.4 and Mutu-BL III cells, and analyzed by SDS-PAGE and
autoradiography (Fig 5). This approach was
used rather than immunoblotting due to its higher sensitivity. As
expected, Fas-activated SKW6.4 extracts had the capacity to process
procaspase-8 (p55) into its active subunits (p18 and p10) as well as
the prodomain (p26) (Fig 5A). The p43 intermediate results from the
release of the p12 subunit after the initial caspase-8 cleavage event
occurring at Asp374. In addition, these extracts were able to convert
procaspase-3 (p32) into the p20 processing intermediate (representing
the prodomain and p17 subunit after cleavage of p12) and the active p17
and p12 subunits (Fig 5B). This ability clearly illustrated the
presence of endogenous active caspase-8 (p18/p10)2 in these
extracts. In addition, these extracts probably contained appreciable
amounts of the Fas DISC by virtue of their use of
35S-labeled caspase-8 as a substrate.14 In
contrast to the results above, extracts of anti-Fas-treated Mutu-BL
cells were unable to convert caspase-8 or caspase-3 into fully active
subunit forms or generate cleavage intermediates. This not only
indicates the absence of active caspase-8 subunits, but also the
failure of a functional DISC to have formed in response to Fas receptor
cross-linking on BL cells. Furthermore, the failure to generate the p43
caspase-8 processing intermediate argues against the presence of a
cellular counterpart of the cowpox crmA serpin/caspase
inhibitor, as p43 can be generated in crmA-overexpressing
cells.14
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| Fig 4.
DISC components are present in Fas-resistant cell lines.
Expression of FADD and caspase-8 was assessed by immunoblot analysis as
described in Materials and Methods.
|
|
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| Fig 5.
Caspase-8 and caspase-3 are not activated in
Fas-stimulated BL cells. Cytosolic extracts made from untreated (0) and
Fas-induced (1 and 2 hours) SKW6.4 and Mutu-BL III cells.
35S-labeled caspases were generated by coupled in vitro
transcription/translation driven by the T7 promoter of the pcDNA3
expression construct. Cleavage reactions were initiated by combining
labeled (A) caspase-8 or (B) caspase-3 with the cytosolic extracts (25 µg) and incubated at 37°C for 2 hours. Reactions were terminated
by the addition of Laemmli sample buffer and subjected to 15% SDS-PAGE
and autoradiography.
|
|
Fas-susceptible and -resistant BL cells have similar
caspase-8/FLICE/MACH isoform profiles.
The absence of caspase-8 processing activity in Fas-resistant Mutu-BL
III cells could have been due to the presence of death-attenuating caspase-8/FLICE/MACH isoforms associating with the DISC. This was a
particularly attractive hypothesis because these were originally cloned
from Raji BL cells and inhibited Fas-induced apoptosis after
transfection.12 An RT-PCR approach similar to that used for
their cloning was used to compare the various isoform transcripts present in Fas-sensitive and -resistant cell lines. In general, the
transcript encoding the full-length 1 isoform could be detected in
all cell lines analyzed and was represented by the 1,939-, 1,606-, and
1,556-bp products generated with the 188, 521, and 575 sense primers,
respectively (Fig 6). However, under the
PCR conditions used, only the third -specific primer combination (ie, 1,556-bp product) was able to yield a prominent amplification product in the Fas-resistant cell line Mutu-BL III. The other 2 caspase-8/FLICE/MACH isoforms, notably the 3 isoform, which could protect against Fas-mediated apoptosis, were not present (Fig
6).12 This would have appeared as a 1,300-bp amplification product using the third pair of PCR primers (ie, 575.S and 930.AS). Furthermore, this assay indicated that caspase-8/FLICE/MACH 1 expression in Fas-susceptible SKW6.4 cells was somewhat higher than in
resistant Mutu-BL. In addition, FLICE/MACH 3 and 4 isoforms lacking most of the ICE/CED-3 caspase homology domain were not only
detected in Fas-susceptible cell lines, but at higher levels than in
resistant cells. In conclusion, an inhibitory caspase-8 isoform (ie,
3) is not responsible for the observed lack of DISC activity and
consequential acquisition of Fas resistance in BL cells.
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| Fig 6.
 and Caspase-8 (FLICE/MACH) isoforms are expressed
in both Fas-susceptible and -resistant cell lines. First-strand cDNA
was prepared from total RNA isolated from the indicated cell lines.
Amplification reactions were performed with the indicated sense primers
complimentary to sequences shared by both and isoforms and
isoform-specific antisense primers. Products were then separated on
1.2% TAE-ethidium bromide agarose gels and photographed.
|
|
The equilibrium between caspase-8 and FLIPL levels
regulates susceptibility to Fas-mediated apoptosis in BL cells.
The results presented above suggested that cellular FLIP was a strong
candidate for mediating Fas resistance in this system: (1)
Fas-crosslinked BL cells lacked DISC activity despite the presence of
all its components and (2) the confirmation of the integrity of
apoptosis pathways distal to caspase-3 activation. Ten B-cell lines
were screened by RT-PCR for FLIPL expression (Fig 7A). All of the resistant EBV-positive
BL cell lines (ie, Mutu-BL III, Raji, Jijoye, P3HR-1) expressed the
mature transcript encoding the long form of FLIP (1,440 bp). The larger
amplification products, especially visible in the Jijoye cDNA sample,
were cloned, sequenced, and determined to be unprocessed
FLIPL transcripts (data not shown). However,
FLIPL was easily detectable in the two most Fas-sensitive
lines (ie, SKW6.4 and BJAB) as well. This result suggested that the
presence of FLIPL might not be the sole factor responsible
for conferring resistance on these cells. Moreover, it has been
demonstrated that FLIPL overexpression can lead to apoptosis via caspase-8 activation, while only moderate expression was
necessary for inhibition.33,35,36 RT-PCR analysis of the same samples performed with primer pairs specific for the caspase-8 caspase-homology domain (792 bp) showed higher caspase-8 expression in
the BJAB and SKW6.4 cell lines compared with all of the BL lines (Fig
7A). This implied that the equilibrium between caspase-8 and
FLIPL levels might regulate Fas-induced apoptosis. This was tested directly by performing caspase-8/FLIPL competitive
PCR (CFCP) analysis. We chose this method because it could
simultaneously compare the levels of transcripts for each protein in
each cDNA sample. A built-in endpoint for the reaction was provided by
limiting concentrations of dNTPs. If primers were the limiting reagent, cDNAs from both transcripts could theoretically be amplified equally regardless of their starting concentrations. All samples were subjected
to this analysis using the primer pairs for both caspase-8 and
FLIPL described above. Caspase-8 was the primary
amplification product in this assay system using cDNA samples from the
3 Fas-susceptible cell lines BJAB, ST486, and SKW6.4 (Fig 7B). This was
the case with the EBV-positive, but Fas-negative, group I BL cell lines as well. In contrast, amplification of cDNA templates from all 4 resistant group III cell lines yielded products for both caspase-8 and
FLIPL (Fig 7B). To provide a quantitative characterization of this apparent distinction between sensitive and resistant cell lines, we analyzed the ethidium-stained PCR products by scanning densitometry and calculated the caspase-8/FLIPL ratio from
the relative values obtained (Fig 7C). Considering only the
Fas-positive cell lines, the resulting graph was identical in
appearance to that in Fig 1 depicting percentage apoptosis in response
to Fas ligation. The results of the caspase-8/FLIPL
competitive PCR analysis indicate a strong correlation between a low
caspase-8/FLIPL ratio (ie,  5) and Fas resistance in group
III BL cells. Immunoblot analysis supported these results by showing
heightened FLIPL protein expression in group III BL cell
extracts compared with those of EBV-negative BL and nonmalignant SKW6.4
lymphoblastoid cell lines (Fig 7D). Although the Ramos cell line was
negative for expresssion of full-length FLIPL in these
experiments, a 12-kD species (p12) was observed. Presumably, this
represents the processed FLIPL carboxyl-terminal p12
subunit, as the antibody used in this analysis was specific for this
domain. However, the mechanism of its generation is unclear.
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| Fig 7.
FLIPL mediates Fas-resistance in EBV-positive
Burkitt's lymphoma cell lines. (A) FLIPL transcripts were
detected by RT-PCR analysis of total RNA samples. The amplification
product corresponding to the mature 1,440-bp coding sequence is
indicated. Higher-molecular-weight products representing unprocessed
transcripts were also detected and can be seen in the Mutu-BL III and
Jijoye lanes. FLICE/caspase-8 transcript levels were determined with
primers specific for the caspase-homology domain (792-bp). (B)
Caspase-8/FLIPL competitive PCR analysis. FLIPL
and FLICE/caspase-8 transcript levels were directly compared in the
same sample by performing RT-PCR reactions containing primer pairs for
both genes under conditions of limiting dNTPs. Gene-specific products
and their sizes are indicated on the right and left, respectively. (C)
The FLICE/FLIPL ratios were calculated from arbitrary
values obtained by scanning densitometry of the gel from (B) and are
graphically represented. Data were obtained from scans using 3 different exposures. Results are expressed as mean ± standard
deviation and are representative of at least 3 separate experiments.
(D) Direct comparison of FLICE/caspase-8 and FLIPL protein
expression. Immunoblot analysis was performed on a panel of
EBV-negative and EBV-positive BL cell lysates. The same membranes were
used for analysis of both FLICE/caspase-8 and FLIPL by
sequential antibody hybridizations. The 55-kD forms of both proteins
are indicated, as well as the FLIPL p12 cleavage product
predominant in the Ramos cell line.
|
|
Overexpression of caspase-8 renders group III BL cells susceptible to
Fas-mediated apoptosis.
To further validate our hypothesis that the equilibrium between
caspase-8 and FLIPL regulates Fas-mediated apoptosis,
particularly Fas-resistance in group III BL cell lines, we manipulated
the caspase-8/ FLIPL ratio by cotransfecting resistant and
susceptible cells with either caspase-8 or FLIPL expression
constructs, respectively, in combination with an EGFP construct.
Transient overexpression of FLIPL protected both BJAB and
EBV-positive SKW6.4 cells from Fas-mediated cell death as shown by a
65% and 58% reduction in apoptosis compared with that of
vector-transfected controls, respectively (Fig 8A). Importantly, overexpression of
caspase-8 in Fas-resistant Mutu-BL III and Raji cells resulted in
heightened susceptibility to Fas-mediated apoptosis, 25% and 29%,
respectively (Fig 8B). In addition, caspase-8 overexpression induced
spontaneous apoptosis of Mutu-BL III cultures, accounting for an
additional 25% cell death. These results support a
FLIPL-mediated mechanism of Fas-resistance in EBV-positive
BL cell lines, which can be overcome by increasing the expression of
caspase-8 and in turn the caspase-8/ FLIPL ratio.
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| Fig 8.
Susceptibility of BL cell lines to Fas-mediated apoptosis
can be regulated by caspase-8 and FLIPL. (A) Percent
apoptosis in Fas-sensitive BJAB and SKW6.4 cell lines cotransfected
with pCEP4-FLIPL (SmtA) plus the pCEP4-EGFP reporter
construct. After incubation for 72 hours, the transfected cultures were
either left untreated () or treated with anti-Fas IgM (500 ng/mL,
) for an additional 8 hours. Percentage apoptosis was measured in
300 EGFP-positive cells using a combination of fluorescence and phase
microscopy as described in Materials and Methods. (B) The Fas-resistant
BL cell lines Mutu-BL III and Raji were cotransfected with
pCEP4-CASP8(FL) and pCEP4-EGFP expression constructs. Transient
transfection and evaluation of Fas-mediated apoptosis was performed as
described in (A).
|
|
| |
DISCUSSION |
The results presented implicate the FLICE-inhibitory protein as a
causative factor in the resistance to Fas-mediated apoptosis displayed
by EBV-positive Burkitt's lymphoma cell lines. The ability of FLIP to
inhibit Fas signal transduction is apparently influenced by the level
of caspase-8 expression in that the magnitude of the
caspase-8/FLIPL expression ratio correlates directly with Fas susceptibility. Furthermore, the decrease in the
caspase-8/FLIPL ratio and the acquisition of Fas resistance
is not a general feature of all Burkitt's lymphomas, but only induced
upon EBV infection and subsequent progression from group I to the
lymphoblastoid/group III phenotype (Figs 1 and 7C). This is a response
specific to BL cells, as LCLs (eg, SKW6.4) established from EBV
transformation of nonmalignant B lymphocytes exhibit a high
caspase-8/FLIPL ratio and profound sensitivity to the
cytotoxic effects of Fas triggering. Because FLIPL can
block signals emanating from several death receptors, the EBV-induced
shift of the caspase-8/FLIPL ratio in favor of BL apoptosis
resistance represents an added survival advantage and novel mechanism
of viral tumorigenesis.
EBV also engages and initiates a variety of antiapoptotic mechanisms as
a result of latency gene expression in lymphoblastoid cells. LMP1 is
known to be critical to growth transformation and mimics proliferative
signals by the binding of p80 TNF receptor-associated factors/proteins
(TRAFs) and subsequent activation of NF-kappa B.46-48 As a
result, LMP1 can also induce resistance to TNF-- and p53-mediated
apoptosis by NF-kappa B-mediated transcriptional activation of the gene
for the A20 zinc finger protein.47,49 However, A20 does not
inhibit Fas-induced apoptosis.50 LMP1 also induces
upregulation of Bcl-2 by an NF-kappa B-independent mechanism providing
an additional survival advantage to LCLs and group III BL cell lines
under suboptimal growth conditions. It has previously been shown that
high LMP1 expression can also be toxic, suggesting a role for Bcl-2 in
antagonizing LMP1-mediated apoptosis.51,52 EBV also encodes
its own Bcl-2 homologue, BHRF1, which functions during the lytic cycle
to delay cell death induced by viral replication.53
However, Bcl-2- or BHRF1-transfected BJAB B cells were not protected
from Fas-mediated apoptosis.54
Although EBV gene expression demonstrably contributes to
immortalization, transformation, and/or oncogenicity, it is not clear how the virus imparts Fas resistance on BL cells. A search of the
protein and nucleic acid databases failed to identify homologues to
known viral or cellular inhibitors of receptor-mediated apoptosis encoded by the EBV genome. Instead, we propose EBV might arrest Fas
signal transduction by cooperating with a BL-specific protein to
transcriptionally modulate caspase-8 and FLIPL levels to
yield a low caspase-8/FLIPL ratio in a manner similar to
EBNA-3C augmentation of c-Ha-ras transformation of rat
fibroblasts.55 In this system, this is potentially the
primary mechanism of Fas signal attenuation because a potential
crmA-like inhibitor was ruled out by the ability of granzyme B
to activate caspase-3 activity in Fas-resistant cell extracts (Fig 3C).
Furthermore, the complete lack of caspase-8 activity in Fas-stimulated
resistant cells (Fig 5A) is consistent with the functioning of
FLIPL proximal to caspase-8 activation. In the presence of
a crmA-like protein, the initial cleavage at Asp374 generating
the p43 intermediate still would have occurred.14
Our data provide insight into a novel tumorigenic mechanism of EBV
involving the development of resistance to Fas-mediated apoptosis
through the antagonistic regulation of caspase-8 and FLIP expression.
This might be yet another example of how EBV functionally integrates
with its host machinery to ensure its survival. While EBV infection of
primary B lymphocytes consistently leads to a stable lymphoblastoid
phenotype, the result with Burkitt's lymphomas is not as
predictable.56 EBV-positive BL cells can be phenotypically
unstable during long-term passage due to their ability to repress the
expression of viral proteins (eg, LMP1), adhesion molecules, and class
I major histocompatibility antigens. Restriction of EBV gene expression
in BL is a common occurrence and may represent a mechanism of evasion
from immune system recognition, as well as contributing to resistance
to cytotoxic T lymphocyte (CTL)-mediated killing.57-59 The
activation of FLIPL and downregulation of caspase-8 might
contribute to this resistance by inhibition of the Fas component of
CTL-mediated killing. This may also be of critical importance in
EBV-associated B lymphomas that do not display a restricted pattern of
EBV gene expression occurring in immunocompromised
patients.60 In conclusion, we propose that death
receptor-induced cell death of EBV-positive group III BL cell lines is
inhibited via a novel mechanism, which uses FLIPL. Future
studies will be aimed at confirming the validity of this putative
mechanism and elucidating the mechanism by which the EBV-malignant
B-cell interaction modulates caspase-8 and FLIPL expression.
| |
ACKNOWLEDGMENT |
The authors thank Dr Maria Mudryj (UC Davis School of Medicine) for
careful reading of the manuscript and Dr Andrew P. Spicer (UC Davis
School of Medicine) for helpful advice regarding transfection. We are
also grateful to Dr Vishva M. Dixit (Genentech, Inc, South San
Francisco, CA) for the gifts of the BJAB cell line, ICE-LAP3 antibody,
and caspase-8 and Yama expression constructs. We also thank Drs Alan B. Rickinson (University of Birmingham, Birmingham, UK) and Jeffrey Sample
(St Jude's Children's Hospital, Memphis, TN) for supplying the
Mutu-BL cell lines.
| |
FOOTNOTES |
Submitted April 3, 1998; accepted April 23, 1999.
Supported by Grant No. AR41053 (to M.F.S.) from the National Institutes
of Health.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Michael F. Seldin, MD, PhD, Rowe Program in
Genetics, Department of Biological Chemistry, University of
California-Davis, 4303 Tupper Hall, One Shields Ave, Davis, CA 95616;
e-mail: mfseldin{at}ucdavis.edu.
| |
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ABSTRACT
INTRODUCTION