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Division of Oncology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Materials and Methods |
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RNA Extraction and Semiquantitative RT-PCR.
Total RNA was extracted from NB cell lines using the Qiagen RNeasy Kit
(Valencia, CA). One µg of total RNA was reverse transcribed using the
SuperScript Preamplification System (Life Technologies, Inc.). PCR was
carried out according to a previously described protocol
(13)
. PCR primers were designed to bracket cDNA sequences
that cross an intron-exon boundary in genomic DNA. Primers for
TRAIL-R1, TRAIL-R2, TRAIL-R3, and
TRAIL-R4 have been previously described, and their
specificity has been confirmed (14)
. Primer sequences
specific for TRAIL, human c-FLIP,
Caspase-2, -3, -8, -9, and -10, and
GAPD are available on request. All PCR primers were
biotinylated at their 5' ends. The housekeeping gene GAPD
was coamplified as an internal standard control as previously described
(13)
. Each PCR sample was analyzed on a nondenaturing 6%
polyacrylamide gel. DNA was transferred to a nylon membrane (Hybond N+;
Amersham) and immobilized by UV cross-linking. Detection of
biotin-labeled DNA on X-ray films was performed according to the
Southern-Light Protocol (Tropix, Bedford, MA; Ref. 13
).
Assessment of Cell Viability and Apoptosis.
NB cell lines were seeded into 96-well plates at a density of
5 x 103
cells per well and
cultured in 10% serum containing RPMI medium. After 24 h, cells
were treated either with recombinant soluble human TRAIL (200 ng/ml;
UBI, Lake Placid, NY) according to the manufacturer’s instructions,
with CHX (1 µg/ml; Sigma) alone, or simultaneously with TRAIL (200
ng/ml) + CHX (1 µg/ml). The caspase inhibitors zVAD-fmk
(Enzyme Systems Products, Livermore, CA) and zIETD-fmk and zLEHD-fmk
(Calbiochem) were dissolved in DMSO and used at a final concentration
of 50 µM in culture medium where indicated. Each
condition was performed in triplicate. Cell cultures were maintained
for indicated treatment times. A colorimetric MTT assay was then
performed as previously described (15)
. For study of dose
dependency, cells were treated with increasing concentrations of TRAIL
alone (0, 3, 6, 12.5, 25, 50, 100, and 200 ng/ml) or in the presence of
CHX (1 µg/ml). Cell lines were considered sensitive to TRAIL-induced
cell death if there was <70% viability after 24 h of TRAIL
treatment at a concentration of 200 ng/ml. To assess specific
apoptosis, the extent of DNA fragmentation in TRAIL-treated cells was
detected by TUNEL with the Apoptosis Detection System (Promega,
Madison, WI) and was also quantitated (optical density, 405 nm)
using the Cell Death Detection ELISA (Roche Molecular Biochemicals,
Indianapolis, IN), which detects cytoplasmic histone-associated DNA
fragments. In addition, apoptotic cell death was confirmed by staining
of TRAIL-treated cells with annexin V and propidium iodide (Roche
Molecular Biochemicals) according to the manufacturer’s instructions.
Apoptosis was then monitored by flow cytometry analysis with a FACScan
using CELLQuest software (Becton Dickinson, Mountain View, CA).
Western Blot Analysis.
Cells were rapidly lysed in 800 µl/dish NP40 lysis buffer [1% NP40,
20 mM Tris (pH 8.0), 137 mM NaCl, 0.5
mM EDTA, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 0.15 units/ml aprotinin, 20
µM leupeptin, 1 mM sodium vanadate]. Samples
normalized for total protein content were separated by SDS-PAGE,
electroblotted onto nitrocellulose, and immunostained. Anti-caspase-8
monoclonal antibody (UBI), anti-cFLIP (Alexis Biochemicals, San Diego,
CA), and anti-TRAIL-R2 (Santa Cruz Biotechnology) were used according
to the manufacturer’s instructions. Detection of immunocomplexes was
conducted using an ECL chemiluminescence system (Amersham Corp.,
Arlington Heights, IL).
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Results |
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. TRAIL and TRAIL-R1 were not expressed in any of the NB
cell lines. TRAIL-R4 was expressed at high levels in only one cell line
and at lower levels in four additional cell lines. TRAIL-R2 and
TRAIL-R3 were expressed at various levels in almost all of the cell
lines, which suggests that they are the primary TRAIL receptors used by
NB cells. Protein expression of TRAIL-R2 essentially correlated with
mRNA expression levels (Fig. 1B)
.
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). For comparison, Jurkat T cells were also exposed to TRAIL. Only 1 of
18 cell lines was susceptible to TRAIL-induced apoptosis (defined as a
survival rate of <70% after 24 h of treatment with TRAIL). The
same experiment was performed in the absence and presence of CHX,
because inhibition of protein synthesis has previously been shown to
sensitize cancer cell lines to CD95L/Fas-induced apoptosis
(16)
. Coexposure to TRAIL+CHX enhanced TRAIL-induced
cytotoxicity in four additional NB cell lines. However, 13 NB cell
lines were still resistant to TRAIL-mediated apoptosis even in the
presence of CHX. TRAIL-induced cell death in sensitive NB cells showed
typical morphological signs of apoptosis, such as rounded cell bodies
and nuclear condensation, as observed by phase contrast microscopy
(data not shown). DNA strand breaks were confirmed by TUNEL labeling
(data not shown), and DNA fragmentation was quantitated using a cell
death ELISA (Fig. 3A)
. Exposure of phosphatidylserine on the outer leaflet of
the plasma membrane of apoptotic cells was demonstrated by annexin
V-fluorescein isothiocyanate staining and flow cytometric
analysis (data not shown).
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apoptosis was maximal at 100 ng/ml TRAIL for CHP902 cells. Jurkat cells
appeared more sensitive to apoptosis induced by TRAIL than CHP902 cells
(IC50 = 6 ng/ml TRAIL versus
IC50 = 50 ng/ml TRAIL). Studies on the
kinetics of induction of apoptosis by TRAIL shown in Fig. 3C
indicated that apoptosis was induced rapidly in Jurkat cells (maximum
at 8 h), whereas 24 h was needed for maximal induction of
apoptosis in CHP902 cells. Time and dose dependency of TRAIL-induced
apoptosis was similar in the other four TRAIL-sensitive NB cell
lines.
Caspase Dependency of TRAIL-induced Apoptosis in NB.
Because caspase activation is an important step in CD95/Fas-mediated
apoptosis and has also been shown to play a role in TRAIL-induced
apoptosis, we tested the susceptibility of TRAIL-sensitive NB cell
lines to apoptosis in the presence of the broad spectrum caspase
inhibitor zVADfmk and a specific caspase-8 and caspase-9 inhibitor
(Fig. 3A)
. TRAIL-induced apoptosis in each of the
TRAIL-sensitive NB cell lines tested was completely blocked in the
presence of zVADfmk and almost completely inhibited in the
presence of the specific caspase-8 inhibitor Z-IETD-FMK. In contrast,
the caspase-9 inhibitor Z-LEHD-FMK had only a minor inhibitory effect
on the cytotoxic effects of TRAIL. Thus, activation of caspase-8 seems
to be essential for TRAIL-induced apoptosis in NB cells, whereas
activation of caspase-9 appears to be rather redundant. Caspase-8
activation in response to TRAIL treatment in NB cell lines could
directly be demonstrated by cleavage of the protein in a Western blot
assay (data not shown).
Expression of cFLIP and Caspase-2, -3, -8, -9, and -10 in NB Cell
Lines.
Because critical involvement of caspases in TRAIL-induced apoptosis has
been demonstrated before, we examined the expression profile of
caspases and of the caspase-8 inhibitor cFLIP in our panel of NB cell
lines. RT-PCR of 18 NB cell lines revealed high expression of cFLIP in
10 cell lines and various expression levels of caspase-2, -3, and -9 in
all of the cell lines. Interestingly, caspase-8 and caspase-10 mRNA was
detected in only 5 of 18 cell lines (Fig. 1A)
. The
expression pattern of caspase-8 and cFLIP was confirmed on protein
levels by Western blot analysis with specific antibodies (Fig. 1B)
.
Correlation of Caspase-8 and Caspase-10 Expression and
TRAIL-induced Apoptosis.
Most interestingly, a clear correlation of caspase-8 and caspase-10
expression and susceptibility to TRAIL-induced apoptosis became
apparent: all of the 5 TRAIL-sensitive NB cell lines expressed
caspase-8 and caspase-10 on mRNA levels, whereas all of the 13
resistant cell lines did not express any caspase-8 or caspase-10. On
the other hand, no clear relation between TRAIL receptor expression,
c-FLIP expression, or expression of any other caspase examined was
evident.
Demethylation by 5-ADC Restores Caspase-8 and Caspase-10 Expression
and TRAIL Sensitivity.
Treatment of all of the TRAIL-resistant NB cell lines with the
methyltransferase inhibitor 5-ADC resulted in dose-dependent
restoration of caspase-8 and caspase-10 mRNA expression. Figure 4A
shows SY5Y cells as a representative
example. However, the induction of caspase-8 mRNA was weak in CHP134,
NMB, OAN, and NGP cells. Treatment of TRAIL-sensitive SK-N-AS
cells with 5-ADC further increased expression levels of caspase-8 and
caspase-10 in these cells. Interestingly, sensitivity to TRAIL-induced
apoptosis was also restored in SY5Y and IMR5 cells after
treatment with 5-ADC (Fig. 4B)
. After cells were treated
with 5-ADC, we demonstrated induction of caspase-8 protein and
cleavage of caspase-8 in the presence of TRAIL+CHX. These data suggest
that epigenetic changes such as methylation of the gene effect the
expression of caspase-8 and caspase-10 and are a major
determinant of TRAIL sensitivity in NB cells.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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We show in our study that 5 of 18 NB cell lines are susceptible to TRAIL-induced apoptosis. Our results demonstrate that TRAIL sensitivity in NB is primarily regulated by expression of caspase-8 and/or caspase-10, rather than at the receptor level. Caspase-8 activation can be seen within minutes after the addition of TRAIL to sensitive cells, suggesting it may be one of the proximal compounds in the signaling pathway (14) . Activation of caspase-10 and caspase-3 has also been reported in response to TRAIL treatment (5 , 14) . Caspase-8 and Caspase-10 are closely related genes that have both been mapped to 2q33-34, suggesting that the two genes have evolved by tandem duplication (18) . Thus, it is possible that both genes are involved in the same gene rearrangement, mutational event, or epigenetic change. Our data suggest that the mechanism leading to the loss of caspase-8 and caspase-10 expression in NB is DNA methylation rather than deletion or recombination. Promoter hypermethylation is an important pathway for repression of gene transcription in cancer cells (19) . Some tumor suppressor genes such as p16, VHL, and MLH1 have been found to harbor promoter hypermethylation associated with loss of protein expression in cancer cells.
The fact that TRAIL-induced apoptosis in our study can be completely
inhibited by the pancaspase inhibitor zVADfmk and largely by the
specific caspase-8 inhibitor Z-IETD-FMK suggests an essential and
nonredundant role of caspase-8 in TRAIL-mediated apoptosis of NB cells.
Thus, the TRAIL receptor-signaling pathway appears to be similar to
that identified for FAS and TNF receptors. The direct
correlation of caspase-8 and caspase-10 expression with sensitivity of
NB cells to TRAIL-mediated apoptosis is indicative of an early apical
block of TRAIL-induced apoptosis attributable to a lack of caspase-8
and/or caspase-10. Caspase-8 knockout studies indicate that this enzyme
is essential for apoptosis initiation by FAS, TNFR1, and DR3, but not
for activation of the NF
B or JNK/AP-1 pathways by these
receptors (20)
. Studies with a mutant human Jurkat T
leukemia cell line that is deficient in caspase-8 confirm the
requirement for this caspase in FAS-mediated apoptosis but show only
partial attenuation of TNF-induced cell death (21)
. Thus,
it appears more likely that the lack of caspase-8 expression is more
important for the failure of NB cells to commit cell death than the
lack of caspase-10 expression. However, the data obtained with the
specific caspase-8 inhibitor cannot rule out the possibility that the
downstream TRAIL signaling pathway can be triggered alternatively by
activation of caspase-8 or caspase-10. Additional experiments with
dominant negative forms of caspase-8 and caspase-10 are needed to
elucidate the role of both caspases in TRAIL signaling.
In our study we found coexpression of TRAIL-R2 and TRAIL-R3 in the majority of cell lines. No expression of TRAIL-R1 and restricted expression of TRAIL-R4 transcripts does not suggest a significant regulatory role of these receptors in human NB. The lack of TRAIL-R1 might contribute to the TRAIL resistance of most NB cell lines, because it has been suggested for other cancer cell lines (22) . However, the TRAIL-sensitive cell line CHP902 did also not express TRAIL-R1, which suggests rather that this receptor might not be needed for mediating the TRAIL signal in NB cells. The presence of mRNA encoding the "protective" TRAIL-R3 receptor did not correspond to resistance or sensitivity to TRAIL-induced apoptosis. In agreement with this, TRAIL-R3 and TRAIL-R4 have been detected in a number of other transformed cell lines that are sensitive to TRAIL-mediated apoptosis (23) . Our data suggest that additional extracellular as well as intracellular factors such as caspase expression might modulate the previously proposed "decoy" model. However, the existence of additional TRAIL receptors as well as of soluble decoys has to be considered. As previously reported for other cell types, addition of the protein synthesis inhibitor CHX to four TRAIL-resistant NB cell lines rendered them sensitive to TRAIL, indicating that intracellular apoptosis inhibitors with a short half-life, such as cFLIP, may also be involved in the protection of NB cells from the cytotoxic effects of TRAIL. In our study 7 of 13 TRAIL-resistant cell lines demonstrated high expression of cFLIP on mRNA and protein levels. Although this correlation is not as convincing as the correlation of caspase-8 expression with TRAIL sensitivity, cFLIP might also contribute to the TRAIL resistance of NB cell lines. The role of cFLIP is controversial because in some cell types its overexpression can induce apoptosis (12) , and resistance to apoptosis could also not be correlated with protein expression of the caspase inhibitor cFLIP in melanoma cell lines (24) .
Induction of apoptosis via death receptors has been shown to represent one possible mechanism by which chemotherapy agents act on tumor cells (25) . Deficient expression or activation of caspases may account in part for the failure of many current anticancer therapies. NB cells may respond to cytotoxic agents only if they express a minimal level of caspase-8 and/or caspase-10. Our present results regarding the lack of caspase-8 and caspase-10 expression in a large subset of NB cell lines provides an explanation for the finding that these NB cell lines are resistant to TRAIL-mediated apoptosis. Our data may also explain the resistance of some NB cells to drug-induced apoptosis and thus have important consequences for additional attempts to manipulate tumor cell death. High levels of inactive, uncleaved caspase-2 and caspase-3 have recently been associated with poor survival in patients with acute myelogenous leukemia and acute lymphoblastic leukemia (26 , 27) . However, lack of caspase-8 and/or caspase-10 might not be the sole factor responsible for conferring resistance to TRAIL-induced apoptosis on NB cells. Regulation of the TRAIL system in NB is most likely also conferred by multiple agonistic and inhibitory receptors, a complex intracellular signaling mechanism involving a number of signaling adapters and activation of inhibitory molecules. On additional elucidation of the regulatory mechanisms of the TRAIL system, treatment with recombinant soluble TRAIL, possibly in combination with 5-ADC, may be an interesting, novel therapeutic approach for NB that deserves additional investigation.
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FOOTNOTES |
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1 Supported by the Wolfson Young Investigator
Award (to A. E.), the Jeffrey Miller Neuro-Oncology Research Fund (to
M. A. G.), the National Institutes of Health Grant NS 34514 (to
G. M. B.), and the Audrey E. Evans Endowed Chair (to G. M. B.). 
2 To whom requests for reprints should be
addressed, at The Children’s Hospital of Philadelphia, Division of
Oncology, ARC Room 902-D, 3516 Civic Center Boulevard, Philadelphia, PA
19104. Phone: (215) 590-2817; Fax: (215) 590-3770; E-mail: brodeur{at}email.chop.edu
3 The abbreviations used are: TNF, tumor necrosis
factor; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R,
TRAIL receptor; cFLIP, cellular FLICE-like inhibitory protein; NB,
neuroblastoma; 5-ADC, 5-aza-2'-deoxycytidine; RT, reverse
transcription; CHX, cycloheximide; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; TUNEL,
TdT-mediated dUTP nick-end labeling.
Received 4/ 5/00. Accepted 12/27/00.
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REFERENCES |
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B.. Immunity, 7: 831 1997.[Medline]
B and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain.. Immunity, 7: 813-820, 1997.[Medline]
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) or
presence (
) of CHX (1 µg/ml). Cell survival was determined after
24 h by MTT assay. Data points are the mean of triplicate samples
(error bars, ± SD) and represent the percentage of
survival compared with untreated control cells (CHX only treated
cells). The representative results shown are from one of three
different experiments.
,
Jurkat +TRAIL. C, kinetics of TRAIL-induced apoptosis in
Jurkat and NB cells. Jurkat and CHP902 NB cells were incubated with
recombinant TRAIL (200 ng/ml) in the presence and absence of CHX for
the time periods indicated. Cell survival of TRAIL-treated cells in the
percentage of untreated control cells and of TRAIL+CHX-treated cells in
the percentage of cells treated with CHX alone was measured by MTT
assay. Data are the means of triplicate samples; SDs were <10%.
Similar data were obtained in three independent experiments. 
,
Jurkat+TRAIL+CHX.
; CHX-only treated cells). C, restoration of
caspase-8 protein expression. Representative Western blot showing
protein expression levels of caspase-8 in TRAIL-resistant SY5Y cells
before (Lane 1) and after (Lane 2)
treatment with 3 µM 5-ADC for 5 days. Lanes
3 and 4: cleavage of caspase-8 in 5-ADC-treated
cells in response to 200 ng/ml TRAIL with or without 1 µg/ml CHX,
respectively.