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Experimental Therapeutics |
Department of Cancer Biology, Lerner Research Institute, and Department of Radiation Oncology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
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ABSTRACT |
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-irradiation of Jurkat,
MOLT-4, CEM, and PBMC, all human T lineage-derived cells. Increased
Apo2L protein levels were found in MOLT-4 and Jurkat cells. Radiation
also activated the Apo2L death receptor (DR)5 (also called Apo2,
TRAIL-R2, or KILLER) in MOLT-4 cells, which harbor a wild-type p53. We
isolated 1152 bp of 5' flanking region of the Apo2L gene
and a shorter fragment of 716 bp, both of which showed promoter
activity driving the expression of a luciferase reporter gene; however,
the response to radiation in MOLT-4 cells was lost when only 430 bp of
5' proximal flanking sequence was maintained. Exogenous Apo2L induced
phosphatidylserine exposure on cell membranes, caspase 8 and caspase 3
activation, key markers of apoptosis, confirming that the Apo2L/DR5
pathway is functional in these cells. Bid, a Bcl-2 family protein also
known to contribute to receptor-mediated apoptosis, was also activated.
To determine whether Apo2L and DR5 were critical for radiation
signaling to apoptosis, we stably expressed a dominant negative
DR5
-receptor in Jurkat cells. Cell survival was significantly
augmented, indicating that increased Apo2L expression contributed to
radiation-induced apoptosis. Clonogenic assays demonstrated that
purified, recombinant soluble Apo2L enhanced the lethality of low,
therapeutic doses (1–2 Gy) of -irradiation. These data suggest that
production of Apo2L may cooperate synergistically with the cytotoxic
effect of radiation, and that combinations of Apo2L and radiation may
become a powerful tool in clinical therapy. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An alternative pathway to apoptosis could be mediated by surface receptor signaling, which depends on ligand-receptor interactions for cell death effector activity. These cell surface receptors are part of the TNF3 -receptor family and include Fas (also called CD95/Apo1), DR4 (also called TRAIL-R1; Ref. 2 ), and DR5 (also called Apo2/TRAIL-R2/TRICK2/KILLER; Refs. 3, 4, 5, 6 ) molecules (7) . Engagement of these receptors by the FasL (also Apo1L/CD95L), or the apoptosis-inducing ligand Apo2L (also TRAIL; Refs. 8 and 9 ) can lead to cell death. Expression of Apo2L at mRNA (8) and protein levels (10) was detected in many human tissues, particularly in those of hematopoietic origin. The receptors for Apo2L, DR4, and DR5 contain a cytoplasmic "death domain" capable of engaging the cell suicide apparatus through an adaptor molecule intermediate such as a Fas-associated death domain protein; (11) . Most importantly, it was reported that whereas many human tumor cell lines were sensitive to cell-surface or soluble Apo2L, normal cells were not. This apparent protection of normal cells from the cytotoxic effect of Apo2L is believed to be based on a unique set of DcRs; these cells either lack the DcR1 (also TRAIL-R3/LIT; Refs. 3, 4, 5 , and 12 ) or have a truncated DcR2 (also TRAIL-R4/TRUNDD; Refs. 13 and 14 ) so they are unable to signal, but compete instead for receptor-binding to Apo2L. An alternative view is that levels of an intracellular inhibitor of caspases, a FLICE-inhibitory protein (15) , may provide resistance in normal cells (16) . The activation of this receptor-mediated cell death pathway during cancer therapy is still not well understood.
The present experiments were designed to examine the role of Apo2L and
DR5 in -irradiation-triggered apoptosis. We present evidence that
radiation induces both Apo2L and DR5, with Apo2L induction being
detected only in T lineage-derived cells. Apo2L regulation is
mediated through 5' flanking sequences we have isolated and
characterized. Purified Apo2L induces caspase-dependent apoptosis, and
when combined with low doses of radiation, it significantly enhances
cell death, indicating that combinations of Apo2L and therapeutic
radiation may be useful in clinical cancer therapy.
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MATERIALS AND METHODS |
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(residues 1 to
268), as described (17)
. DR5 lacks the death domain,
and has been shown to function as a dominant-negative inactivating the
function of the endogenous DR5 (4)
. DR5 also contains a
FLAG epitope-tag which facilitates examination of its expression
levels. Transfected cells were selected in the presence of 1 mg/ml G418
(Life Technologies, Inc.)-containing medium and subsequently maintained
with 0.5 mg/ml of G418. Irradiation was performed as described previously (1) with a 137Cs source emitting at a fixed-dose rate of 2.8 Gy/min. The Fas response was examined using the Fas agonistic mAb (clone CH11; Panvera, Madison, WI) and Fas-Fc with enhancer (Alexis Corporation, San Diego, CA). All chemicals, unless specified, were obtained from Sigma Chemical Co. (St. Louis, MO).
RNA Analyses.
Total RNA was isolated from cells at various intervals post-irradiation
using the Trizol reagent (Life Technologies, Inc.). To determine
the steady-state levels of RNA, we used the RiboQuant system
(PharMingen, San Diego, CA) for RNase protection assay with multiprobe
template sets which allow simultaneous quantitation and
characterization of multiple RNA molecules, as described
(1)
. The hAPO-3 and hAPO-3C template sets (PharMingen)
were used for the T7 polymerase-directed synthesis of high specific
activity [32P]-labeled antisense RNA probes.
The hAPO-3 probe set contains the following genes:
(a) the receptors DR3 and
TNFRp55; (b) the ligands FasL and Apo2L;
(c) the adapters FLICE, Fas-associated death domain;
and RIP (receptor-interacting protein, which interacts with Fas), as
well as FAF (Fas-associated protein) and FAP (PNP1 protein tyrosine
phosphatase 1E). hAPO-3C contains the DR4, DR5, and DcR1
receptors for Apo2L. Both sets contain the housekeeping gene
L32 as an internal control.
To amplify DcR2, the sense 5'-catgggactttggggacaaa-3' and antisense 5'-gatagggagaggcaagcatc-3' primers were used as well as those for GAPDH with RNA isolated as above. The RT-PCR reaction was carried out as described (1) .
Promoter Activity.
By searching the National Center for Biotechnology Information using
the Basic Local Alignment Search Tool, we found that the Homo
sapiens chromosome 3, clone hRPK.44_A_1 (accession no.
AC007051), contained the whole Apo2L/TRAIL gene
(five-exon structure) as well as the 5' upstream promoter region. The
primers for the ApoP, ApoP/1152, 5'-gcctcgaggaatctgggaggtggagg-3',
ApoP/716, 5'-gcctcgagctgtccagcctaacacac-3', ApoP/430,
5'-gcctcgaggtgcctatgacagccagg-3', and ApoP/antisense,
5'-cgaagcttgatcctgtcagag tctgac-3' were used to generate, by PCR, the
fragments of the ApoP. The PCR reaction was done using the Expand High
Fidelity PCR System (Roche Molecular Biochemicals, Indianapolis, IN)
following the manufacturer’s protocol. MOLT-4 genomic DNA, isolated by
QIAamp Tissue Kit (QIAGEN, Valencia, CA), was used as the template. The
amplified 1152-, 716-, and 430-bp DNA fragments were subcloned into the
pGL2 plasmid containing the luciferase reporter gene (Promega, Madison,
WI), and their nucleotide sequence was confirmed by DNA sequence
analysis, as described (1)
.
The human ApoP activity was determined in MOLT-4 cells, as described (17) , after cotransfection with 1 µg of Apo2L luciferase reporter and 0.1 µg of pCMV-LacZ plasmid DNA. At 32 h after transfection with the DMRIE-C reagent (Life Technologies, Inc.), the cells were irradiated, and 4 h later, luciferase activity was determined and normalized to values of ß-gal. Each assay was performed in triplicate.
Apoptosis, Cytotoxicity, and Clonogenic Assays.
Phosphatidylserine exposure on cell membranes was determined with
FITC-Annexin V by flow cytometry as described (1
, 18)
using a FACScan and analyzed with CellQuest software (Becton Dickinson,
San Jose, CA) on mean values obtained from the cell population from
which debris were gated out. Caspase activity was measured as described
(1)
, using the IETD- and DEVD-pNA-derived
chromogenic substrates for caspase 8 and -3 activity, by
enzyme-catalyzed release of pNA monitored at 405 nm.
-irradiation and recombinant soluble Apo2L-treated cells
were seeded (2–3 x 104
cells) in
96-well plates. Cell viability was examined 24 h later with the
CellTiter 96 Aqueous One Solution Reagent (MTS; Promega) to measure
tetrazolium reduction, as indicated by absorbance at 490 nm using an
ELISA reader (Spectramax 340). All determinations were done in
triplicate.
For clonogenic assays, recombinant soluble Apo2L was added 16 h
before -irradiation to U-bottomed 96-well plates with each well
containing 4 cells/100 µl of medium. Cell colonies were counted 14
days later. Cellular clonogenicity was calculated by the formula:
% Clonogenicity = (ln x [96/Neg
Wells]) (plate density) x 100, where Neg Wells represent the
number of wells that have failed to grow to 50 cells (19)
.
MOLT-4 and Jurkat cells have a cloning efficiency of 40% and 66.5%,
respectively. Ps were determined by the t test
using Microsoft Excel.
Preparation of Recombinant Soluble Apo2L/TRAIL.
Escherichia coli BL21 strain was transformed with a
His6-tagged pET28b-TRAIL (residues 95 to 281), a
generous gift from Drs. E. S. Alnemri and S. M. Srinivasula
(4)
. Expression of the protein was induced with 0.4
mM
isopropyl-1-thio-ß-D-galactopyranoside for
2 h, and bacteria were pelleted and sonicated in a buffer
containing 50 mM Tris-HCl, (pH 8.0), 2
mM EDTA, 0.1% Triton X, and 100 µg/ml
lysozyme. The supernatant was collected after centrifugation at
14,000 x g for 15 min and used for
additional purification. The recombinant soluble Apo2L (used in Fig. 6
)
was then affinity-purified by His6-resin affinity
purification following the manufacturer’s instructions (Novagen,
Milwaukee, WI). Apo2L protein specificity was confirmed by SDS-PAGE and
Western blot analysis with an anti-Apo2L antibody (C19, Santa Cruz
Biotechnology, Santa Cruz, CA). For some experiments (Figs. 4
and 5)
a
commercial rhsApo2L was used, containing the extracellular domain of
Apo2L fused at the NH2 terminus to a FLAG-tag and
an 8-amino acid linker peptide. A combination of Apo2L protein and
enhancer have been used according to manufacturer’s specifications
(Alexis Corporation, San Diego, CA).
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RESULTS |
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-irradiation in PBMCs. Moreover, Apo2L expression
was also induced two-fold at 4 h post-irradiation in Jurkat cells
(Fig. 1)
. Similar results were obtained with another T cell-derived tumor cell
line, CEM-CM3, but not in several other human tumor cell lines (data
not shown). These data indicate that radiation-induced Apo2L expression
was observed in both normal as well as T cell-derived tumor cell lines.
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Activation of the Apo2L/DR5 Pathway by Ionizing Radiation in MOLT-4
Cells.
To further characterize Apo2L regulation, we next examined its
expression in MOLT-4 cells, which we have previously characterized for
the role of p53-dependent bax induction in apoptosis (1)
.
Control and irradiated MOLT-4 cells were examined at 2 to 8 h
after -irradiation, a time period during which no cytotoxic effects
were observed. Most significantly, we found that Apo2L mRNA was induced
significantly after 10 Gy of -irradiation (Fig. 2
A, top). There was no detectable Apo2L expression in untreated MOLT-4
cells or in those examined 8 h post-irradiation, indicating a
transient radiation-dependent regulation. Moreover, there was no
significant change in the expression levels of most of the other
transcripts, including the housekeeping gene L32, indicating
a specific change in expression levels of Apo2L. A time and
dose-response experiment indicated a transient increase in Apo2L
expression at all radiation doses tested (2–10 Gy; Fig. 2B
). However, the magnitude of this response, a 2.2- to
3-fold increase at the lower 2–4 Gy dose, was considerably less than
the 8.5-fold increase obtained after 10 Gy. Similarly, Fas was induced
up to eight-fold at doses as low as 2 Gy (at 8 h following
irradiation) compared with untreated cells. Treatment with 4 or 10 Gy
-irradiation resulted in higher induction levels with a more
substantial increase at earlier (4.5- versus 2.5-fold at
2 h) compared with later times (8 to 10.5-fold at 8 h; Fig. 2B
). These results indicate a time and dose-dependent
induction of both Apo2L and Fas, but with a
different kinetics of induction of the two genes.
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, bottom), with
no detectable DR4 and DcR1 present (data not shown). Following
irradiation, DR5 levels were increased in MOLT-4 cells, which harbor
wild-type p53, but not in Jurkat cells, which have a p53 mutation.
Because Apo2L was reported to also signal through DcR2, we examined its
expression by RT-PCR. There was no DcR2 expression in either MOLT-4 or
Jurkat cells, either before or after radiation treatment. In contrast,
under the same experimental conditions, DcR2 was readily detectable in
A549 lung carcinoma cells (Fig. 2C)
, as reported previously
(13)
, whereas the levels of GAPDH, an internal
control, were unchanged.
Immunoblot analyses, using anti-Apo2L antibodies, showed that Apo2L
protein levels were also up-regulated 2- to 3.5-fold in irradiated
Jurkat and MOLT-4 cells, respectively, a pattern of induction
consistent with that detected for RNA levels. This increase was
specific to Apo2L, as there were no changes in the levels of ß-actin
(Fig. 2D)
.
To determine the effect of promoter sequences on Apo2L regulation, we
isolated 1152 bp of 5'-flanking Apo2L DNA sequence upstream of the
Apo2L translation initiation site and created two deletion promoter
constructs: ApoP/716 and ApoP/430. Each construct was used to transfect
MOLT-4 cells, which were then either left untreated or subjected to 10
Gy of -irradiation. Compared with control, the expression of the
luciferase gene driven by ApoP/1152 was induced up to 2.3-fold by
irradiation (Fig. 3)
. Similarly, ApoP/716 showed a substantial, up to 1.8-fold, increase in
promoter activity in irradiated cells. In contrast, there was no
significant change in the expression of the luciferase gene driven by
ApoP/430 under similar conditions. These data suggest that the
5'-flanking Apo2L DNA sequence contained between nucleotides -1152 and
-430 is required for radiation-induced ApoP activity.
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. To examine the extent to which Apo2L
expression contributes to cell death, we stably expressed a FLAG-tagged
dominant-negative DR5 into Jurkat cells; these cells were chosen
because no other known death effectors are induced. In contrast,
multiple death effectors are up-regulated following irradiation of
MOLT-4 cells, such as Bax (1)
, and therefore it would be
difficult to dissociate the effect of Apo2L from that of other
death-promoting proteins.
If Apo2L expression had a role in apoptosis, then blocking the Apo2L
signaling pathway should have an impact on cell survival. Radiation
treatment induces phospatydylserine exposure on the cell membrane, a
relatively early marker of apoptosis (1
, 18)
. We found
that 27% of Jurkat cells were viable, as they did not stain with
Annexin V (which binds to phospatydylserine), 24 h after 20 Gy of
-irradiation. In contrast, 56% of Jurkat/DR5- cells survived
(Fig. 4A)
. Moreover, there was a marked difference in survival
between parental and DR5-expressing Jurkat cells as early as 12 h following irradiation (Fig. 4B)
. Thus 67, or 54%, of
Jurkat/DR5 cells survived, compared with 50, or 31%, of irradiated
parental cells examined at 12 or 24 h, respectively. These cells
expressed the DR5 receptor, as shown by the presence of the FLAG
epitope with which they were tagged (inset). Fas-Fc
pretreatment had no significant effect on survival of DR5/Jurkat
cells and only a slight effect on survival of Jurkat cells 12 h
after treatment. Furthermore, the Jurkat/DR5 cells were resistant to
the cytotoxic effect of Apo2L, but remained sensitive to killing by an
anti-Fas agonisitc antibody, indicating the specificity of DR5
function (Fig. 4C)
. Fas-Fc pretreatment had no significant
effect on survival of Apo2L-treated DR5 cells,
but prevented, as expected, the cell death of Fas-treated cells. These
results indicate that blocking the DR5-mediated Apo2L signaling has a
significant impact on cell survival and that -irradiation-induced
Apo2L expression contributes to the cell death of hematopoietic cells.
Apo2L Induces Apoptosis through Activation of Caspase 8, Bid, and
Caspase 3.
Apo2L can induce apoptosis in a variety of tumor cells. However, the
mechanism of Apo2L-induced apoptosis remains to be
clarified. Apo2L induced rapid apoptosis of Jurkat cells in a
time-dependent manner. Cells started to loose viability as early as
4 h after treatment, with most of cells being Annexin
V-FITC-positive by 12 h (Fig. 5A)
. To assess whether caspases were activated in our system,
we determined caspase activity in Apo2L-treated
cells. Apo2L induced effectively IETD- and DEVD-pNA cleavage activity,
corresponding to caspase 8 and caspase 3; this activity started at
2 h and reached a maximum at 4–6 h (Fig. 5B)
.
Moreover, Western blot analyses show that the procaspase 3 was
proteolytically cleaved to the p17 kDa fragment, known to be the
activated form of caspase 3. To address the pathway by which caspase 3
was activated, we examined in more detail caspase 8, an apical caspase
known to be the first caspase to be activated in receptor-mediated
apoptosis. Levels of procaspase 8 were decreased substantially by
4 h, with further time-dependent decrease in protein levels (Fig. 5C)
. These results, taken together with the increase in
IETD-pNA cleavage activity, indicate that caspase 8 is activated by
Apo2L. Finally we examined Bid, a Bcl-2 family member also
recently reported to be involved in receptor-mediated apoptosis and to
further caspase 3 activation (20
, 22)
. Bid levels were
significantly reduced upon Apo2L treatment, indicating processing of
the full-length Bid to a p15- or p13-kDa fragment not detectable under
our experimental conditions. Activation of caspase 8 and caspase 3
indicates a caspase-dependent apoptosis, with Bid most likely
contributing to the amplification of the caspase cascade.
Combination of Apo2L and Radiation Treatments Greatly Enhance
Lethality.
To further examine a direct contribution of Apo2L induction to cell
death, we affinity-purified recombinant, soluble Apo2L protein, which
was shown by SDS-PAGE to be present as a 28-kDa monomeric protein (Fig. 6A)
. Apo2L protein specificity was confirmed by Western
blotting with an anti-Apo2L antibody. Using a concentration of 2–5
µg/ml of Apo2L, we found that both MOLT-4 and Jurkat cells could be
effectively killed in a dose-dependent manner (Fig. 6B)
.
To further examine the possible clinical effects of Apo2L, clonogenic
assays were next performed on cells treated with radiation or Apo2L
alone or the two agents used in combination. We choose a 10- to
20-times lower concentration of Apo2L protein, which did not have any
detectable cytotoxic effects. Thus, MOLT-4 and Jurkat cells were
treated with 0.1–0.2 µg/ml of Apo2L for 16 h, then irradiated
with a dose of 1–2 Gy. The radiation survival of MOLT-4 and Jurkat
cells, which contain a functional or mutated p53, respectively, is
shown in Fig. 6
. Low concentrations of Apo2L resulted in only a slight
reduction in clonogenic survival of MOLT-4 and Jurkat cells. Clonogenic
survival decreased slightly, however, in MOLT-4 cells, from
40 ± 4.3 to 33 ± 3.5% following 1 Gy
of -irradiation (Fig. 6C)
. In contrast, combined
treatment with radiation and Apo2L reduced the clonogenic survival from
34.5 ± 3.2% to 20 ± 2.2%
(P < 0.5) and from 35.9 ± 4.8% to 16 ± 2.8% (P < 0.01), respectively, for the two Apo2L concentrations. For Jurkat
cells, survival was reduced from 66.5 ± 4.7% to
41.3 ± 3.7% or 16.8 ± 1.8% when 1 or
2 Gy of -irradiation were used (Fig. 6D)
. Use of
radiation and 0. 1 µg/ml of Apo2L resulted in a decreased survival
from 55.9 ± 8% to 29.5 ± 2.6% or
7.3 ± 1.7% (P < 0.01) for
the 1- or 2-Gy dose, respectively. When higher concentrations of Apo2L
(0.2 µg) were used, survival decreased from 52.2 ± 6.8% to 22.9 ± 4.8% or 6.7 ± 0.9%
(P < 0.01) at the two radiation doses. These
data indicate a statistically significant (t test) decrease
in clonogenic survival of MOLT-4 and Jurkat cells treated with a
combination of Apo2L and radiation as compared with radiation alone.
These results indicate a synergistic interaction between Apo2L and
radiation in reducing clonogenic survival of these hematopoietic tumor
cell lines.
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DISCUSSION |
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significantly prevented radiation-induced cell death,
demonstrating the important contribution of Apo2L/DR5 expression to
radiation-induced apoptosis. This finding is consistent with a
recent suggestion that radiation kills lymphocytes by a Fas-independent
mechanism (23)
. Apo2L induction in T cells could be most
important because radiation could selectively kill tumor cells, which
express only the DR5 and/or DR4 receptors, but not normal cells, which
also express DcRs or FLICE-inhibitory protein. To elucidate the mechanism of Apo2L regulation, we isolated and characterized the 5'-flanking region of the human Apo2L gene, which showed promoter activity in irradiated MOLT-4 cells. The consensus sequences for several transcription-regulatory factor-binding motifs were identified to be present in ApoP/1152, but not the ApoP/430 promoter region, including sites for OCT-1, SP-1, GATA1–3, and C/EBP. These sites, located in the promoter region between nucleotides -1152 to -716 (OCT-1, SP-1, GATA1-3), and -716 to -430 (C/EBP) could mediate the radiation-caused transcriptional induction of Apo2L. In fact, a DNA-binding activity was reported in several tumor cell lines following irradiation for both OCT-1 and SP-1 (24) . The exact role of these elements for the radiation response needs to be investigated further.
A functional role for Fas in the radiation response has been reported
in T cells from animals with defective expression of Fas or FasL
function, which showed reduced sensitivity to radiation-induced
apoptosis (25)
. Fas was induced in our system too,
consistent with its up-regulation, in a p53-dependent manner, following
treatment with DNA-damaging and oxidant agents of a number of
hematopoietic and solid tumor cell lines (21)
. However,
MOLT-4 cells were resistant to Fas treatment (500 ng/ml for 24 h,
data not shown). Moreover, in Jurkat cells, which harbor mutant p53,
Fas was not induced by radiation, there was no detectable FasL mRNA
present, and Fas-Fc pretreatment had no effect on radiation-induced
cell death. Most importantly, DR5 protected Jurkat cells from
radiation and Apo2L-induced, but not Fas-induced, cell death,
indicating that the Fas/FasL system is not involved in
radiation-induced apoptosis in our system, although it is clearly
important for regulation of apoptosis in some other biological systems.
This finding is consistent with reports that radiation kills
lymphocytes by a Fas-independent mechanism (23)
and that
chemotherapy-induced apoptosis is not dependent on Fas/FasL
interactions (26
, 27)
. In fact, various human lymphoid
cell lines differ in their sensitivity toward induction of apoptosis by
Apo2L and Fas (28)
, an observation we have extended to
myeloid
cells.4
Radiation induces caspase 8 and -3 activation in Jurkat (data not shown) and MOLT-4 cells, leading to proteolytic cleavage of cellular proteins such as poly(ADP-ribose)polymerase (1) . Treatment of Jurkat cells with purified Apo2L also induced caspase 8 and -3 cleavage and activation, similar to previous reports (28 , 29) , indicating that radiation may mediate its apoptotic effects by engaging the caspase cascade not only through increased levels of the DR5 receptor, but also by increasing the levels of Apo2L. Bid activation indicates the requirement for caspase amplification in the Apo2L/DR5 apoptotic pathway by a mechanism reported to take place through translocation of activated Bid to mitochondria, facilitating cytochrome c release and activation of caspase 9 and then caspase 3 (20 , 22 , 30) .
The observations that radiation can induce Apo2L and that low doses of radiation can cooperate synergistically with Apo2L in enhancing cell death may have implications for clinical therapy. This is reminiscent of the synergistic or additive cell killing between TNF and radiation reported in several solid tumor cell lines (31) . Gene therapy approaches have been proposed based on radiation-responsive promoters driving TNF expression (32) , and TNF expression has been shown to sensitize certain radiation-resistant tumors (33) . In addition, there are essential differences between the Apo2L-DR4 or DR5 pathway and those using TNF and FasL, which further indicate that Apo2L might be a safer agent. Thus, at least in some systems, it has been shown that Apo2L kills tumor cells and not normal cells because of the presence on the latter of DcRs (3 , 5) . More recently, the observations of a lack of cytotoxicity of Apo2L toward normal cells and tissues have been extended successfully to mice (34) and nonhuman primates (35) . Several chemotherapeutioc agents have been also shown to have a synergistic activity with Apo2L (35 , 36) , indicating that combination therapies using Apo2L are likely to be widely applicable.
The present investigations enhance our understanding of the mechanism of radiation and Apo2L-induced apoptosis. The observed synergy between Apo2L and low, therapeutic doses of radiation can form the basis for developing strategies for pharmacological intervention, with potential for clinical application. In particular, the specificity of Apo2L cytotoxicity for tumor cells and its systemic distribution reaching metastases and the surgical precision with which ionizing radiation can now be delivered in the clinic could constitute a very attractive combination for enhanced clinical response of tumors resistant to radiation therapy.
Note Added in Proof
After this work was completed, it was reported that radiation can
sensitize breast carcinoma cells both in vitro and
in vivo to Apo2L-induced apoptosis. This synergistic
effect was p53-dependent and was suggested to be the result of
radiation-induced up-regulation of DR5 (37)
. However,
Apo2L is not induced following irradiation of these breast carcinoma
cells,5
indicating that the synergy between radiation and Apo2L might work
through different mechanisms in different cell types.
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ACKNOWLEDGMENTS |
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constructs, X. Wang (U. T.
Southwestern) for the Bid antibody, and Amy Raber (Cleveland Clinic
Flow Cytometry Core) for expert assistance. The Becton Dickinson FACS
Vantage Cell Sorter was purchased through a generous gift from the Keck
Foundation. We thank Drs. C. Bevins, Q. Chen, R. M. Macklis, S.
Mazumder, G. Chen, and F. Dong for helpful suggestions on the
manuscript. |
FOOTNOTES |
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1 This work was supported by research grants from
NIH (CA81504 and CA82858). 
2 To whom requests for reprints should be
addressed, at Department of Cancer Biology, NB40, Lerner Research
Institute, Cleveland Clinic Foundation, Cleveland, OH 44195. Phone:
(216) 444-9970; Fax: (216) 445-6269; E-mail: almasaa{at}ccf.org
3 The abbreviations used are: TNF, tumor necrosis
factor; Apo2L, Apo2 ligand; ApoP, Apo2L promoter; DR, death receptor;
Fas, CD95/Fas/Apo1; FasL, Fas ligand; DcR, decoy receptor; PBMC,
peripheral blood mononuclear cell; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; RT-PCR, reverse transcription-PCR; pNA, p-nitroanilide;
IETD, acetyl-Ile-Glu-Thr-Asp-pNA; DEVD, acetyl-Asp-Glu-Val-Asp-pNA; Gy,
gray; FACS, fluorescence-activated cell sorter; TRAIL, TNF-related
apoptosis-inducing ligand.
4 Q. Chen, B. Gong, A. Zhou, E. Hsi, E. Hsi, M.
Hussein, and A. Almasan. Induction of Apo2L and modulation of
Bcl-2-related proteins regulate type I interferon-induced apoptosis in
multiple myeloma, submitted for publication.
5 B. Gong and A. Almasan, unpublished
observations.
Received 12/10/99. Accepted 8/ 9/00.
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REFERENCES |
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B and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity, 7: 813-820, 1997.[Medline]
mRNA after cellular exposure to ionizing radiation. Proc. Natl. Acad. Sci. USA, 86: 10104-10107, 1989. sensitizes prostate cancer cells to -irradiation-induced apoptosis. Cancer Res., 59: 1606-1614, 1999.This article has been cited by other articles:
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  C. MOSER, S. A. LANG, and O. STOELTZING Heat-shock Protein 90 (Hsp90) as a Molecular Target for Therapy of Gastrointestinal Cancer Anticancer Res, June 1, 2009; 29(6): 2031 - 2042. [Abstract] [Full Text] [PDF]
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 F. Chiarini, F. Fala, P. L. Tazzari, F. Ricci, A. Astolfi, A. Pession, P. Pagliaro, J. A. McCubrey, and A. M. Martelli Dual Inhibition of Class IA Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin as a New Therapeutic Option for T-Cell Acute Lymphoblastic Leukemia Cancer Res., April 15, 2009; 69(8): 3520 - 3528. [Abstract] [Full Text] [PDF]
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M. M. Shareef, N. Cui, R. Burikhanov, S. Gupta, S. Satishkumar, S. Shajahan, M. Mohiuddin, V. M. Rangnekar, and M. M. Ahmed Role of Tumor Necrosis Factor-{alpha} and TRAIL in High-Dose Radiation Induced Bystander Signaling in Lung Adenocarcinoma Cancer Res., December 15, 2007; 67(24): 11811 - 11820. [Abstract] [Full Text] [PDF]
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V. N. Ivanov, H. Zhou, and T. K. Hei Sequential Treatment by Ionizing Radiation and Sodium Arsenite Dramatically Accelerates TRAIL-Mediated Apoptosis of Human Melanoma Cells Cancer Res., June 1, 2007; 67(11): 5397 - 5407. [Abstract] [Full Text] [PDF]
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 Z. Tang, J. A. Bauer, B. Morrison, and D. J. Lindner Nitrosylcobalamin Promotes Cell Death via S Nitrosylation of Apo2L/TRAIL Receptor DR4 Mol. Cell. Biol., August 1, 2006; 26(15): 5588 - 5594. [Abstract] [Full Text] [PDF]
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 X. Zhang, R. M. Cheung, R. Komaki, B. Fang, and J. Y. Chang Radiotherapy Sensitization by Tumor-Specific TRAIL Gene Targeting Improves Survival of Mice Bearing Human Non-Small Cell Lung Cancer Clin. Cancer Res., September 15, 2005; 11(18): 6657 - 6668. [Abstract] [Full Text] [PDF]
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 C. Crowder, O. Dahle, R. E. Davis, O. S. Gabrielsen, and S. Rudikoff PML mediates IFN-{alpha}-induced apoptosis in myeloma by regulating TRAIL induction Blood, February 1, 2005; 105(3): 1280 - 1287. [Abstract] [Full Text] [PDF]
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M. S. Merchant, X. Yang, F. Melchionda, M. Romero, R. Klein, C. J. Thiele, M. Tsokos, H. U. Kontny, and C. L. Mackall Interferon {gamma} Enhances the Effectiveness of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Agonists in a Xenograft Model of Ewing's Sarcoma Cancer Res., November 15, 2004; 64(22): 8349 - 8356. [Abstract] [Full Text] [PDF]
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X. Zhang, T.-G. Jin, H. Yang, W. C. DeWolf, R. Khosravi-Far, and A. F. Olumi Persistent c-FLIP(L) Expression Is Necessary and Sufficient to Maintain Resistance to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Mediated Apoptosis in Prostate Cancer Cancer Res., October 1, 2004; 64(19): 7086 - 7091. [Abstract] [Full Text] [PDF]
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A. W. Tolcher, J. Kuhn, G. Schwartz, A. Patnaik, L. A. Hammond, I. Thompson, H. Fingert, D. Bushnell, S. Malik, J. Kreisberg, et al. A Phase I Pharmacokinetic and Biological Correlative Study of Oblimersen Sodium (Genasense, G3139), an Antisense Oligonucleotide to the Bcl-2 mRNA, and of Docetaxel in Patients with Hormone-Refractory Prostate Cancer Clin. Cancer Res., August 1, 2004; 10(15): 5048 - 5057. [Abstract] [Full Text] [PDF]
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 A. I Kassis In vivo validation of the bystander effect Human and Experimental Toxicology, February 1, 2004; 23(2): 71 - 73. [Abstract] [PDF]
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 M. Chawla-Sarkar, J. A. Bauer, J. A. Lupica, B. H. Morrison, Z. Tang, R. K. Oates, A. Almasan, J. A. DiDonato, E. C. Borden, and D. J. Lindner Suppression of NF-{kappa}B Survival Signaling by Nitrosylcobalamin Sensitizes Neoplasms to the Anti-tumor Effects of Apo2L/TRAIL J. Biol. Chem., October 10, 2003; 278(41): 39461 - 39469. [Abstract] [Full Text] [PDF]
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 K.-Y. Jen and V. G. Cheung Transcriptional Response of Lymphoblastoid Cells to Ionizing Radiation Genome Res., September 1, 2003; 13(9): 2092 - 2100. [Abstract] [Full Text] [PDF]
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S. Ray and A. Almasan Apoptosis Induction in Prostate Cancer Cells and Xenografts by Combined Treatment with Apo2 Ligand/Tumor Necrosis Factor-related Apoptosis-inducing Ligand and CPT-11 Cancer Res., August 1, 2003; 63(15): 4713 - 4723. [Abstract] [Full Text] [PDF]
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J. H. Kim, M. Ajaz, A. Lokshin, and Y. J. Lee Role of Antiapoptotic Proteins in Tumor Necrosis Factor-related Apoptosis-inducing Ligand and Cisplatin-augmented Apoptosis Clin. Cancer Res., August 1, 2003; 9(8): 3134 - 3141. [Abstract] [Full Text] [PDF]
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 S. Ghaffari, Z. Jagani, C. Kitidis, H. F. Lodish, and R. Khosravi-Far Cytokines and BCR-ABL mediate suppression of TRAIL-induced apoptosis through inhibition of forkhead FOXO3a transcription factor PNAS, May 27, 2003; 100(11): 6523 - 6528. [Abstract] [Full Text] [PDF]
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T. Naka, K. Sugamura, B. L. Hylander, M. B. Widmer, Y. M. Rustum, and E. A. Repasky Effects of Tumor Necrosis Factor-related Apoptosis-inducing Ligand Alone and in Combination with Chemotherapeutic Agents on Patients' Colon Tumors Grown in SCID Mice Cancer Res., October 15, 2002; 62(20): 5800 - 5806. [Abstract] [Full Text] [PDF]
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L. Y. Xue, N. J. Butler, G. M. Makrigiorgos, S. J. Adelstein, and A. I. Kassis From the Cover: Bystander effect produced by radiolabeled tumor cells invivo PNAS, October 15, 2002; 99(21): 13765 - 13770. [Abstract] [Full Text] [PDF]
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P. Secchiero, A. Gonelli, P. Mirandola, E. Melloni, L. Zamai, C. Celeghini, D. Milani, and G. Zauli Tumor necrosis factor-related apoptosis-inducing ligand induces monocytic maturation of leukemic and normal myeloid precursors through a caspase-dependent pathway Blood, September 18, 2002; 100(7): 2421 - 2429. [Abstract] [Full Text] [PDF]
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S. Mazumder, B. Gong, Q. Chen, J. A. Drazba, J. C. Buchsbaum, and A. Almasan Proteolytic Cleavage of Cyclin E Leads to Inactivation of Associated Kinase Activity and Amplification of Apoptosis in Hematopoietic Cells Mol. Cell. Biol., April 1, 2002; 22(7): 2398 - 2409. [Abstract] [Full Text] [PDF]
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 A. L. Greenway, D. A. McPhee, K. Allen, R. Johnstone, G. Holloway, J. Mills, A. Azad, S. Sankovich, and P. Lambert Human Immunodeficiency Virus Type 1 Nef Binds to Tumor Suppressor p53 and Protects Cells against p53-Mediated Apoptosis J. Virol., February 22, 2002; 76(6): 2692 - 2702. [Abstract] [Full Text] [PDF]
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A. Sreekumar, M. K. Nyati, S. Varambally, T. R. Barrette, D. Ghosh, T. S. Lawrence, and A. M. Chinnaiyan Profiling of Cancer Cells Using Protein Microarrays: Discovery of Novel Radiation-regulated Proteins Cancer Res., October 1, 2001; 61(20): 7585 - 7593. [Abstract] [Full Text] [PDF]
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Q. Chen, B. Gong, A. S. Mahmoud-Ahmed, A. Zhou, E. D. Hsi, M. Hussein, and A. Almasan Apo2L/TRAIL and Bcl-2-related proteins regulate type I interferon-induced apoptosis in multiple myeloma Blood, October 1, 2001; 98(7): 2183 - 2192. [Abstract] [Full Text] [PDF]
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P. Secchiero, A. Gonelli, C. Celeghini, P. Mirandola, L. Guidotti, G. Visani, S. Capitani, and G. Zauli Activation of the nitric oxide synthase pathway represents a key component of tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity on hematologic malignancies Blood, October 1, 2001; 98(7): 2220 - 2228. [Abstract] [Full Text] [PDF]
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M. Chawla-Sarkar, D. W. Leaman, and E. C. Borden Preferential Induction of Apoptosis by Interferon (IFN)-{beta} Compared with IFN-{{alpha}}2: Correlation with TRAIL/Apo2L Induction in Melanoma Cell Lines Clin. Cancer Res., June 1, 2001; 7(6): 1821 - 1831. [Abstract] [Full Text] [PDF]
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R. Di Pietro, P. Secchiero, R. Rana, D. Gibellini, G. Visani, K. Bemis, L. Zamai, S. Miscia, and G. Zauli Ionizing radiation sensitizes erythroleukemic cells but not normal erythroblasts to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by selective up-regulation of TRAIL-R1 Blood, May 1, 2001; 97(9): 2596 - 2603. [Abstract] [Full Text] [PDF]
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