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Departments of
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Immunobiology,
Protein Chemistry,
Biochemistry,
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Molecular Biology, and
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Hybridoma, Immunex Corporation, Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To further study the function of the death-inducing TRAIL receptors (TRAIL-R1 and -R2) alone or in combination with the receptors that do not induce death (TRAIL-R3 and -R4), mAbs were generated against each of the human TRAIL receptors and used to study a panel of TRAIL-sensitive and -resistant human melanoma cell lines characterized previously for mRNA expression of the four known TRAIL receptors 17 . These mAbs were evaluated in terms of their ability to induce apoptosis in the melanoma cell lines (when added in solution or when immobilized on culture plates), to block the binding of TRAIL to melanoma cells expressing TRAIL-R1 and/or -R2, and to inhibit the death of TRAIL-sensitive target cells upon exposure to TRAIL. These mAbs also allowed us to test whether resistance to the cytotoxic effects of TRAIL is influenced by the expression of the putative "decoy receptors", TRAIL-R3 and/or TRAIL-R4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human melanoma cell lines (WM 9, 35, 98-1, 164, 793, 1341-D, and 3211) were provided by Dr. M. Herlyn (Wistar Institute, Philadelphia, PA) and cultured in DMEM supplemented with 10% FBS, penicillin, streptomycin, and glutamine.
Generation of anti-TRAIL receptor mAb
BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were immunized with a purified fusion protein consisting of the extracellular domain of human TRAIL-R1, -R2, -R3, or -R4 coupled to the constant region of human IgG1 (huTRAIL-R:Fc) in Titermax (Cytrx Corporation, Norcross, GA). Mice were boosted three times, and spleen cells were fused with the murine myeloma NS1 in the presence of 50% polyethylene glycol in PBS followed by culture in DMEM/HAT and DMEM/HT selective media. Supernatants from positive wells were tested for the ability to bind the appropriate TRAIL receptor in an ELISA (cell-based ELISA using CV1 cells transfected with TRAIL receptor cDNA) and for reactivity to huTRAIL-R:Fc in Western blots. Hybridomas that produced Abs that bound to huTRAIL-R:Fc but not human IgG1 were cloned by three rounds of limiting dilution. mAb isotypes were determined to be IgG1 (M270, M272, M412, M413, M431, and M445), IgG2a (M271, M273), and IgG2b (M411); all mAbs were purified by protein A affinity chromatography.
Leucine zipper-human TRAIL (LZ-huTRAIL)
The LZ-huTRAIL expression plasmid 12 and the production and purification of LZ-huTRAIL have been described elsewhere 28 .
Analysis of LZ-huTRAIL binding to melanoma cells
The surface expression of TRAIL receptor(s) was determined by flow cytometric analysis by measuring the binding of LZ-huTRAIL 17 . Briefly, cells were incubated with 10 µg/ml LZ-huTRAIL in 3% BSA in PBS (PBSA) for 30 min on ice. The cells were washed in PBS, followed by the addition of a biotinylated anti-leucine zipper mAb (M15; 10 µg/ml in 3% PBSA) for 30 min on ice. After incubation, cells were washed in PBS and then incubated for 30 min on ice with phycoerythrin-conjugated streptavidin (SA) (diluted 1/200 in 3% PBSA; Sigma, St. Louis, MO). In some cases, cells were preincubated with the TRAIL receptor-specific mAb (10 µg/ml for 30 min on ice) to determine which mAb could inhibit LZ-huTRAIL binding. Cells were analyzed on a FACScan (Becton Dickinson, San Jose, CA).
Inhibition of LZ-huTRAIL binding to TRAIL-R1:Fc
To determine whether any of the anti-TRAIL-R1 mAb could block the binding of LZ-huTRAIL to TRAIL-R1, a modified ELISA was used. We coated 96-well ELISA plates (Corning, Corning, NY) with 1 µg/ml goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) for 4 h at 4°C (all subsequent steps were also performed at 4°C), followed by the addition of TRAIL-R1:Fc (1 µg/ml in 5% nonfat dry milk (NFDM) in PBS-Tween 20 (0.05% v/v)) overnight. Wells were washed with PBS-Tween and subsequently blocked with 5% NFDM in PBS-Tween. After 1 h, the milk was removed and the anti-TRAIL-R1 mAbs were added (10 µg/ml starting concentration diluted in 5% NFDM in PBS-Tween) for 1 h. Wells were washed, followed by the addition of LZ-huTRAIL (1 µg/ml diluted in 5% NFDM in PBS-Tween) for 1 h. After washing, biotinylated anti-leucine zipper mAb (M15; 1 µg/ml diluted in 5% NFDM in PBS-Tween) was added for 1 h. Following the washing of the wells, horseradish peroxidase (HRP)-SA (diluted 1/500 in 5% NFDM in PBS-Tween; Zymed, San Francisco, CA) was added for 1 h. Wells were washed, and colorimetric substrate (TMP microwell peroxidase substrate system; Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added. Upon color development, H2SO4 was added to stop the reaction, and the plate was analyzed on an ELISA plate reader at OD450. The percent inhibition of LZ-huTRAIL binding was determined using the following equation: 100 x (1 - [experimental group OD - background OD]/[untreated group O.D. - background OD]).
Western blotting
TRAIL receptor/OPG blot. Melanoma cells were lysed in PBS containing 1% Nonidet P-40, 0.35 mg/ml PMSF, 9.5 µg/ml leupeptin, and 13.7 µg/ml pepstatin A. The lysates were centrifuged at 14,000 x g to remove cellular debris, and the protein concentrations of the extracts were determined by colorimetric bicinchoninic acid analysis (Pierce, Rockford, IL). Equal amounts of protein were separated by SDS-PAGE, transferred to a nitrocellulose membrane (Novex, San Diego, CA), and blocked with 5% NFDM in PBS-Tween 20 overnight at 4°C. The membrane was incubated with TRAIL receptor mAb (1 µg/ml in 5% NFDM in PBS-Tween) or OPG polyclonal Ab (diluted 1/1000 in 5% NFDM in PBS-Tween; provided by Dr. E. Clark, University of Washington, Seattle, WA) for 1 h. After washing, the membrane was incubated for 1 h with an anti-mouse HRP Ab (Amersham, Arlington Heights, IL). Following several washes, the blots were developed by chemiluminescence according to the manufacturer’s protocol (Renaissance chemiluminescence reagent, DuPont-New England Nuclear, Boston, MA).
Poly(ADP-ribose) polymerase (PARP) blot. Melanoma cells were incubated with 10 µg/ml TRAIL-R1 and/or -R2 mAb for 30 min, followed by the addition of 100 ng/ml LZ-huTRAIL. Cells were lysed after 4 h, and protein concentrations were determined, electrophoresed, and transferred to a nitrocellulose membrane as outlined above. The membrane was incubated with a rabbit anti-PARP Ab (Research Diagnostics, Flanders, NJ) for 1 h. After washing, the membrane was incubated for 1 h with an anti-rabbit HRP Ab (Amersham). Following several washes, the blots were developed by chemiluminescence as described above.
Cytotoxicity assays
Tumor cell sensitivity to TRAIL or TRAIL-R1/-R2 mAb was assayed by incubating the cells in 96-well plates with the indicated amount of purified LZ-huTRAIL, TRAIL receptor-specific mAb, or both in solution or immobilized to the culture plate. In some experiments, mAb to TRAIL-R1 and/or -R2, carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk), carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk) (20 µM; Enzyme Systems Products, Livermore, CA), or actinomycin D (Act D) (30 ng/ml) was added to the culture medium before incubation with LZ-huTRAIL or mAb. Cell death was determined by chromium release 5 or crystal violet staining 18 after 8 or 24 h, respectively, as described previously. Results are presented as the percentage of cell death using the following equations: 100 x ([experimental group cpm - background cpm]/]maximum cpm - background cpm]) for the chromium release experiments or 100 x (1 - [experimental group OD]/[untreated group OD]) for the crystal violet experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments analyzing the TRAIL receptor family have been limited
by the lack of receptor-specific Abs. Thus, mAbs were generated that
could serve as tools to measure protein expression and dissect
individual receptor function. Soluble fusion proteins consisting of the
extracellular portion of each human TRAIL receptor coupled to the Fc
domain of human IgG1 (huTRAIL-R:Fc) were used to immunize mice and
generate mAbs that were reactive with the specific huTRAIL-R:Fc but not
human IgG1 by ELISA (data not shown) or Western blotting (Fig. 1
A). In addition, the
anti-TRAIL receptor mAb only reacted with the appropriate
CV1/EBV-encoded nuclear Ag cells transfected with cDNAs for each
of the four TRAIL receptors in a slide binding assay (data not shown),
further demonstrating receptor specificity and no cross-reactivity with
the other three TRAIL receptors.
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B); this panel had
been characterized previously for its expression of TRAIL receptor
mRNA 17 . The results of this screening identified several pairs of
TRAIL-sensitive and -resistant cell lines as having the same TRAIL
receptor expression pattern. For example, WM 35 (sensitive) and WM 3211
(resistant) only expressed TRAIL-R2. TRAIL-R2 and -R3 were expressed by
WM 793 (sensitive) and WM 164 (resistant), whereas WM 98-1
(sensitive) and WM 1341-D (resistant) expressed both TRAIL-R1 and
TRAIL-R2. Lastly, the TRAIL-sensitive line WM 9 was found to express
all four TRAIL receptors. The seven melanoma cell lines were also
analyzed for OPG expression, and none were found to express this
protein (data not shown). The doublet seen in the blot with the
anti-TRAIL-R2 mAb (M413) is consistent with previous observations
predicting two variants of TRAIL-R2 resulting from an 11-aa deletion at
the amino terminus 12 . These results on TRAIL receptor protein
expression are concordant with the RT-PCR results examining TRAIL
receptor mRNA expression 17 and extend the earlier observed lack of
correlation between decoy TRAIL receptor (TRAIL-R3 and -R4) mRNA
expression and TRAIL resistance. Anti-TRAIL-R1 and -R2 mAb-induced apoptosis
The results of previous studies using Fas-specific mAbs have
demonstrated that although they may act as antagonists when added in
soluble form, these mAbs can act as potent agonists when appropriately
cross-linked 19 . Thus, it was of interest to determine whether the
anti-TRAIL-R1 and -R2 mAbs acted in a similar fashion. None of the
anti-TRAIL-R1 and only one of the anti-TRAIL-R2 mAbs (M412)
tested were able to induce lysis of TRAIL-sensitive melanoma cells when
added to cultures in solution in an 8-h 51Cr release assay,
and the lysis induced by M412 was weak (Fig. 2). Moreover, the TRAIL-resistant
melanoma lines (WM 164, 1341-D, and 3211) were also resistant to
soluble anti-TRAIL-R1 and -R2 mAb-induced lysis. Interestingly, all
of the anti-TRAIL-R2 and two of the TRAIL-R1 mAbs that failed to or
minimally induced lysis of TRAIL-sensitive melanoma cells when added in
solution displayed increased lytic ability when immobilized to the
culture plate (Fig. 3). Again, this lysis
was only seen with the TRAIL-sensitive melanoma lines and not with the
TRAIL-resistant lines. Thus, these results demonstrate that
melanoma cell sensitivity to anti-TRAIL receptor mAb is comparable
with that of TRAIL, and further suggest that the nonsignaling TRAIL-R3
and -R4 do not play a major role in resistance to TRAIL-induced
apoptosis.
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Three methods were employed to determine whether the
anti-TRAIL-R1 and -R2 mAbs were able to block LZ-huTRAIL binding to
the appropriate receptors and prevent TRAIL-induced apoptosis. Using
the TRAIL-sensitive melanoma cell lines shown to express only TRAIL-R2
(WM 35), TRAIL-R1 and -R2 (WM 98-1), or TRAIL-R2 and -R3 (WM 793), the
anti-TRAIL-R2 mAbs were first assessed for their ability to block
the binding of LZ-huTRAIL as measured by flow cytometry. Cells were
incubated with the anti-TRAIL-R2 mAb, control mAb, or no mAb for 20
min, followed by incubation with LZ-huTRAIL. Secondary reagents were
then added to measure the level of LZ-huTRAIL binding to the cells.
M413 mAb was found to completely inhibit LZ-huTRAIL binding to WM 35
(Fig. 4A). However, as
expected M413 could not fully inhibit LZ-huTRAIL binding to either WM
98-1 or WM 793 cells, which express additional TRAIL receptors. The
anti-TRAIL-R2 mAb, M412, was unable to inhibit LZ-huTRAIL binding
as seen with M413.
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B). In addition, M270 could also inhibit LZ-huTRAIL
binding, but only at the highest Ab concentration. To confirm that this
assay accurately measured the inhibition of LZ-huTRAIL binding, a
similar experiment was performed using TRAIL-R2:Fc and the
anti-TRAIL-R2 mAbs M412 and M413 that are shown in Fig. 4A. Indeed, only M413 inhibited LZ-huTRAIL binding to
TRAIL-R2:Fc, whereas M412 did not (Fig. 4C). Collectively,
these results demonstrate that two of the anti-TRAIL-R1 mAbs and
one of the anti-TRAIL-R2 mAbs are capable of inhibiting the binding
of LZ-huTRAIL to their respective receptors.
Next we tested the capability of the M271 and M413 mAbs to act as
antagonists by protecting these TRAIL-sensitive melanoma cell lines
from TRAIL-induced death. The data demonstrate that the addition of
M413 significantly decreased the amount of death seen following
LZ-huTRAIL addition to WM 35 and WM 793 target cells, both of which
express TRAIL-R2 but not TRAIL-R1 (Fig. 5A). Whereas the preincubation
of M412 was unable to prevent the TRAIL-induced death of the melanoma
cells, there was an increase in cell death upon incubation with M412
and LZ-huTRAIL. This increase probably resulted from the cytotoxic
activity of both M412 and LZ-huTRAIL, because M412 was the only
anti-TRAIL-R2 mAb that demonstrated this activity when in solution
(Fig. 2). The fact that no such inhibition was observed with WM 98-1
target cells suggests that death in this case was mediated via TRAIL-R1
signaling. Based on the results from Fig. 4B demonstrating
that the anti-TRAIL-R1 mAb M271 could inhibit LZ-huTRAIL binding to
TRAIL-R1:Fc, we tested whether the coincubation of M271 and M413 could
inhibit the TRAIL-induced death of WM 98-1. Whereas no inhibition of
death was seen with the individual mAb, there was significant
inhibition when both mAbs were added before LZ-huTRAIL (Fig. 5B).
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). Thus,
these results demonstrate that the addition of M413 mAb in solution can
block LZ-huTRAIL binding to TRAIL-R2 and inhibit TRAIL-induced death in
cells expressing TRAIL-R2 in the absence but not the presence of
TRAIL-R1. The results also show that LZ-huTRAIL binding to TRAIL-R1 can
be blocked by two of the anti-TRAIL-R1 mAbs (M271 and M273), and
that the addition of both M271 and M413 can inhibit the TRAIL-induced
death of cells expressing both TRAIL-R1 and -R2.
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Previous studies examining TRAIL-induced apoptosis revealed the
activation of several caspases and determined that TRAIL-resistant
cells could be converted to TRAIL-sensitive upon the addition of Act D
17 . To determine whether these events also occurred during
anti-TRAIL-R1 and -R2 mAb-induced death, the sensitive melanoma
line WM 98-1 was incubated with immobilized LZ-huTRAIL, M271, or M413
mAb along with the caspase inhibitors zVAD or zIETD 20, 21, 22, 23 . The death
induced by each reagent was inhibited by the addition of zVAD or zIETD
(Fig. 7). Next, the TRAIL-resistant lines
WM 164 and WM 1341-D were incubated with immobilized LZ-huTRAIL, M413,
or M271 in the presence or absence of Act D. Significant cell death was
induced only in the presence of Act D, whereas minimal death occurred
in its absence (Fig. 8). Similar results
were also seen when other anti-TRAIL-R1 and -R2 mAbs were used with
Act D (data not shown). These results indicate that mAb cross-linking
of the death-inducing TRAIL receptors does not always lead to apoptotic
cell death, and that the mAb-induced death results from the activation
of the signaling cascade seen during TRAIL-induced apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using mAbs against the four TRAIL receptors, we were able to survey a panel of human melanoma cell lines for TRAIL receptor protein expression; this panel had been characterized previously for TRAIL receptor mRNA by RT-PCR 17 . We found the expression of the TRAIL receptor proteins to be completely concordant with the mRNA expression, such that no correlation between the expression of the presumed decoy TRAIL receptor proteins and resistance or sensitivity to TRAIL was observed. Although a lower amount of TRAIL-R3 and -R4 protein was detected in the cells expressing these receptors as compared with TRAIL-R1 and -R2, it is difficult to determine whether this low protein level prevents these TRAIL receptors from conferring resistance to TRAIL-mediated apoptosis. This uncertainty is largely due to the fact that it is not currently known how the different TRAIL receptors interact with one another on the cell surface, nor is it known how much TRAIL-R3 or -R4 must be present on the surface of normal (i.e., untransfected) cells to disrupt the formation of TRAIL-R1 or -R2 signaling complexes to provide protection from the cytotoxic effects of TRAIL, as initially predicted in overexpression experiments 10, 11, 13, 14, 15 . Although TRAIL-R3 or -R4 may act as a decoy when overexpressed subsequent to transfection, the results presented here suggest that they do not appear to be protective under physiological conditions. The mAbs used in this paper will permit further study of these issues.
In addition to analyzing the expression of the different TRAIL receptors, the production of agonistic mAbs that were reactive specifically against the death-inducing receptors TRAIL-R1 and -R2 made it possible to bypass the proposed protective nature of TRAIL-R3 and -R4, thereby examining cell sensitivity solely through TRAIL-R1 or -R2 cross-linking. The decoy receptor hypothesis proposed that the inclusion of these protective receptors into a trimerized receptor complex bound by TRAIL would prevent death-inducing signaling complex formation and thus confer resistance to TRAIL-induced apoptosis 10, 11, 15 . The testing of this decoy receptor hypothesis with anti-TRAIL-R1 or -R2 mAb demonstrated that mAb-induced apoptosis only occurred in the melanoma cell lines that were also sensitive to the natural ligand TRAIL, regardless of the presence or absence of TRAIL-R3 and/or -R4. Moreover, the TRAIL-resistant cells only became sensitive to the anti-TRAIL-R1 and -R2 mAbs when cultured with Act D, further supporting the idea that intracellular regulation of TRAIL-induced apoptosis plays a greater protective role in TRAIL sensitivity or resistance. We have recently found a correlation between high levels of the antiapoptotic protein FADD-like IL-1ß-converting enzyme-inhibitory protein 24 in these human melanoma cell lines and resistance to TRAIL-induced apoptosis 17 . Although these data suggest a role for FADD-like IL-1ß-converting enzyme-inhibitory protein in the susceptibility of tumor cells to TRAIL, the potential contribution of other intracellular molecules in regulating TRAIL receptor signaling and apoptosis cannot be excluded.
To our knowledge, the mAbs used in this study have also led to the first demonstration that naturally expressed TRAIL-R1 can signal for cell death. Previous studies with TRAIL-R1 relied on overexpression of the receptor on transfected cells to demonstrate the apoptosis-inducing nature of this TRAIL receptor 9 . The anti-TRAIL-R1 mAbs are able to directly induce apoptotic cell death in TRAIL-R1-expressing, TRAIL-sensitive melanoma cells. Because none of the cell lines available express TRAIL-R1 alone, we could not determine whether any of the TRAIL-R1 mAbs are capable of blocking LZ-huTRAIL binding to the cells and inhibiting cell death as seen with M413. However, the use of an ELISA-based assay with TRAIL-R1:Fc found that two of the anti-TRAIL-R1 mAbs could significantly inhibit LZ-huTRAIL binding in this setting, suggesting that the mAbs against TRAIL-R1 could inhibit the TRAIL-induced apoptosis of cells that only express TRAIL-R1 or be combined with M413 to prevent the death of cells expressing TRAIL-R1 and -R2. This finding proved to be correct when examining the ability of the anti-TRAIL-R1 mAb M271 or anti-TRAIL-R2 mAb M413 to inhibit the TRAIL-induced death of WM 98-1 (TRAIL-R1+ and -R2+) melanoma cells when added individually or together. Protection was only seen when both blocking mAbs were added before adding LZ-huTRAIL. This protection was also seen when the other blocking anti-TRAIL-R1 mAbs were combined with M413 (data not shown).
Recent findings have demonstrated that another receptor, OPG, is capable of binding TRAIL 16 . OPG is a secreted TNF receptor-related protein that can block osteoclastogenesis in vitro and increase bone density and act as a protective agent against ovariectomy-associated bone loss in vivo when overexpressed 25 . Thus, soluble OPG may function as a competitive inhibitor of its cognate TNF family ligand, preventing the ligand from binding to its cellular receptor. In the search for a ligand for OPG, it was determined that OPG binds TRAIL and can inhibit the TRAIL-induced apoptosis of Jurkat cells, even though the binding affinity of OPG is slightly weaker than the affinities of the TRAIL receptors for TRAIL 16 . Further in vitro studies with OPG have found that TRAIL can inhibit the osteoclastogenic ability of OPG 16 . These two observations suggest that OPG and TRAIL may function to inhibit one another. The release of OPG from a cell may also serve as a natural inhibitor of TRAIL-induced death, similar to the protection conferred by soluble Fas 26, 27 . It is unlikely that OPG is involved in determining TRAIL resistance in human melanoma cells, because none of the seven human melanoma cell lines used in this study were found to express OPG protein as determined by Western blot analysis (data not shown). However, OPG may play a role in preventing TRAIL-induced apoptosis in normal tissues or other tumor cell types. Additional studies will need to be performed to address these possibilities.
The existence of four TRAIL receptors, two with the ability to signal for apoptosis (TRAIL-R1 and -R2) and two without (TRAIL-R3 and -R4), as well as the soluble TRAIL-binding protein OPG demonstrates the biological complexity of this receptor-ligand system. The results presented here show that TRAIL-R1 or -R2 ligation does not always lead to death and suggest that TRAIL-induced apoptosis is not regulated simply through either the competitive binding of TRAIL by TRAIL-R3 or -R4 or by the transduction of protective signals mediated by either of these receptors. It seems likely that multiple factors function together to provide resistance against the cytotoxic effects of TRAIL, the most important of which appears to be intracellular regulation of caspase activation 17 . Although many questions still remain unanswered regarding the biological function of each of these TRAIL receptors and the signals generated upon their ligation, the TRAIL receptor-specific mAbs described here will serve as valuable tools in the future study of these receptors.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: TRAIL, TNF-related apoptosis-inducing ligand; OPG, osteoprotegerin; PBSA, 3% BSA in PBS; PARP, poly(ADP-ribose) polymerase; HRP, horseradish peroxidase; SA, streptavidin; NFDM, nonfat dry milk; Act D, actinomycin D; LZ-huTRAIL, leucine zipper-human TRAIL.
Received for publication August 6, 1998. Accepted for publication November 16, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity 7:813.[Medline]
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P. Horak, D. Pils, A. Kaider, A. Pinter, K. Elandt, C. Sax, C. C. Zielinski, R. Horvat, R. Zeillinger, A. Reinthaller, et al. Perturbation of the Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Cascade in Ovarian Cancer: Overexpression of FLIPL and Deregulation of the Functional Receptors DR4 and DR5 Clin. Cancer Res., December 15, 2005; 11(24): 8585 - 8591. [Abstract] [Full Text] [PDF]
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T. Shiraishi, T. Yoshida, S. Nakata, M. Horinaka, M. Wakada, Y. Mizutani, T. Miki, and T. Sakai Tunicamycin Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Human Prostate Cancer Cells Cancer Res., July 15, 2005; 65(14): 6364 - 6370. [Abstract] [Full Text] [PDF]
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E. Ishikawa, M. Nakazawa, M. Yoshinari, and M. Minami Role of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand in Immune Response to Influenza Virus Infection in Mice J. Virol., June 15, 2005; 79(12): 7658 - 7663. [Abstract] [Full Text] [PDF]
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K. Motoki, E. Mori, A. Matsumoto, M. Thomas, T. Tomura, R. Humphreys, V. Albert, M. Muto, H. Yoshida, M. Aoki, et al. Enhanced Apoptosis and Tumor Regression Induced by a Direct Agonist Antibody to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor 2 Clin. Cancer Res., April 15, 2005; 11(8): 3126 - 3135. [Abstract] [Full Text] [PDF]
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H. Cui, T. Li, and H.-F. Ding Linking of N-Myc to Death Receptor Machinery in Neuroblastoma Cells J. Biol. Chem., March 11, 2005; 280(10): 9474 - 9481. [Abstract] [Full Text] [PDF]
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X. Y. Zhang, X. D. Zhang, J. M. Borrow, T. Nguyen, and P. Hersey Translational Control of Tumor Necrosis Factor-related Apoptosis-inducing Ligand Death Receptor Expression in Melanoma Cells J. Biol. Chem., March 12, 2004; 279(11): 10606 - 10614. [Abstract] [Full Text] [PDF]
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N. Harper, M. A. Hughes, S. N. Farrow, G. M. Cohen, and M. MacFarlane Protein Kinase C Modulates Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis by Targeting the Apical Events of Death Receptor Signaling J. Biol. Chem., November 7, 2003; 278(45): 44338 - 44347. [Abstract] [Full Text] [PDF]
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 A. Younes and M. E. Kadin Emerging Applications of the Tumor Necrosis Factor Family of Ligands and Receptors in Cancer Therapy J. Clin. Oncol., September 15, 2003; 21(18): 3526 - 3534. [Abstract] [Full Text] [PDF]
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X. Chen, K. Kandasamy, and R. K. Srivastava Differential Roles of RelA (p65) and c-Rel Subunits of Nuclear Factor {kappa}B in Tumor Necrosis Factor-related Apoptosis-inducing Ligand Signaling Cancer Res., March 1, 2003; 63(5): 1059 - 1066. [Abstract] [Full Text] [PDF]
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P. Schneider, D. Olson, A. Tardivel, B. Browning, A. Lugovskoy, D. Gong, M. Dobles, S. Hertig, K. Hofmann, H. Van Vlijmen, et al. Identification of a New Murine Tumor Necrosis Factor Receptor Locus That Contains Two Novel Murine Receptors for Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) J. Biol. Chem., February 7, 2003; 278(7): 5444 - 5454. [Abstract] [Full Text] [PDF]
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T. M. LaVallee, X. H. Zhan, M. S. Johnson, C. J. Herbstritt, G. Swartz, M. S. Williams, W. A. Hembrough, S. J. Green, and V. S. Pribluda 2-Methoxyestradiol Up-Regulates Death Receptor 5 and Induces Apoptosis through Activation of the Extrinsic Pathway Cancer Res., January 15, 2003; 63(2): 468 - 475. [Abstract] [Full Text] [PDF]
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 S. A. Renshaw, J. S. Parmar, V. Singleton, S. J. Rowe, D. H. Dockrell, S. K. Dower, C. D. Bingle, E. R. Chilvers, and M. K. B. Whyte Acceleration of Human Neutrophil Apoptosis by TRAIL J. Immunol., January 15, 2003; 170(2): 1027 - 1033. [Abstract] [Full Text] [PDF]
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 H. Higuchi, S. F. Bronk, M. Taniai, A. Canbay, and G. J. Gores Cholestasis Increases Tumor Necrosis Factor-Related Apoptotis-Inducing Ligand (TRAIL)-R2/DR5 Expression and Sensitizes the Liver to TRAIL-Mediated Cytotoxicity J. Pharmacol. Exp. Ther., November 1, 2002; 303(2): 461 - 467. [Abstract] [Full Text] [PDF]
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 M. Matysiak, A. Jurewicz, D. Jaskolski, and K. Selmaj TRAIL induces death of human oligodendrocytes isolated from adult brain Brain, November 1, 2002; 125(11): 2469 - 2480. [Abstract] [Full Text] [PDF]
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T. S. Griffith, J. M. Fialkov, D. L. Scott, T. Azuhata, R. D. Williams, N. R. Wall, D. C. Altieri, and A. D. Sandler Induction and Regulation of Tumor Necrosis Factor-related Apoptosis-inducing Ligand/Apo-2 Ligand-mediated Apoptosis in Renal Cell Carcinoma Cancer Res., June 1, 2002; 62(11): 3093 - 3099. [Abstract] [Full Text] [PDF]
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J. J. Lum, A. A. Pilon, J. Sanchez-Dardon, B. N. Phenix, J. E. Kim, J. Mihowich, K. Jamison, N. Hawley-Foss, D. H. Lynch, and A. D. Badley Induction of Cell Death in Human Immunodeficiency Virus-Infected Macrophages and Resting Memory CD4 T Cells by TRAIL/Apo2L J. Virol., November 15, 2001; 75(22): 11128 - 11136. [Abstract] [Full Text] [PDF]
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 D. Y. Zang, R. G. Goodwin, M. R. Loken, E. Bryant, and H. J. Deeg Expression of tumor necrosis factor-related apoptosis-inducing ligand, Apo2L, and its receptors in myelodysplastic syndrome: effects on in vitro hemopoiesis Blood, November 15, 2001; 98(10): 3058 - 3065. [Abstract] [Full Text] [PDF]
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P. Secchiero, P. Mirandola, D. Zella, C. Celeghini, A. Gonelli, M. Vitale, S. Capitani, and G. Zauli Human herpesvirus 7 induces the functional up-regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) coupled to TRAIL-R1 down-modulation in CD4+ T cells Blood, October 15, 2001; 98(8): 2474 - 2481. [Abstract] [Full Text] [PDF]
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A. E. Tollefson, K. Toth, K. Doronin, M. Kuppuswamy, O. A. Doronina, D. L. Lichtenstein, T. W. Hermiston, C. A. Smith, and W. S. M. Wold Inhibition of TRAIL-Induced Apoptosis and Forced Internalization of TRAIL Receptor 1 by Adenovirus Proteins J. Virol., October 1, 2001; 75(19): 8875 - 8887. [Abstract] [Full Text] [PDF]
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X. D. Zhang, X. Y. Zhang, C. P. Gray, T. Nguyen, and P. Hersey Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis of Human Melanoma Is Regulated by Smac/DIABLO Release from Mitochondria Cancer Res., October 1, 2001; 61(19): 7339 - 7348. [Abstract] [Full Text] [PDF]
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C. S. Mitsiades, S. P. Treon, N. Mitsiades, Y. Shima, P. Richardson, R. Schlossman, T. Hideshima, and K. C. Anderson TRAIL/Apo2L ligand selectively induces apoptosis and overcomes drug resistance in multiple myeloma: therapeutic applications Blood, August 1, 2001; 98(3): 795 - 804. [Abstract] [Full Text] [PDF]
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A. V. Franco, X. D. Zhang, E. Van Berkel, J. E. Sanders, X. Y. Zhang, W. D. Thomas, T. Nguyen, and P. Hersey The Role of NF-{{kappa}}B in TNF-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis of Melanoma Cells J. Immunol., May 1, 2001; 166(9): 5337 - 5345. [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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A. Chuntharapai, K. Dodge, K. Grimmer, K. Schroeder, S. A. Marsters, H. Koeppen, A. Ashkenazi, and K. J. Kim Isotype-Dependent Inhibition of Tumor Growth In Vivo by Monoclonal Antibodies to Death Receptor 4 J. Immunol., April 15, 2001; 166(8): 4891 - 4898. [Abstract] [Full Text] [PDF]
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R. D. Pettersen, G. Bernard, M. K. Olafsen, M. Pourtein, and S. O. Lie CD99 Signals Caspase-Independent T Cell Death J. Immunol., April 15, 2001; 166(8): 4931 - 4942. [Abstract] [Full Text] [PDF]
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 E. H. Alexander, J. L. Bento, F. M. Hughes Jr., I. Marriott, M. C. Hudson, and K. L. Bost Staphylococcus aureus and Salmonella enterica Serovar Dublin Induce Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Expression by Normal Mouse and Human Osteoblasts Infect. Immun., March 1, 2001; 69(3): 1581 - 1586. [Abstract] [Full Text] [PDF]
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N. Mitsiades, V. Poulaki, C. Mitsiades, and M. Tsokos Ewing's Sarcoma Family Tumors Are Sensitive to Tumor Necrosis Factor-related Apoptosis-inducing Ligand and Express Death Receptor 4 and Death Receptor 5 Cancer Res., March 1, 2001; 61(6): 2704 - 2712. [Abstract] [Full Text]
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H. Matsuzaki, B. M. Schmied, A. Ulrich, J. Standop, M. B. Schneider, S. K. Batra, K. S. Picha, and P. M. Pour Combination of Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) and Actinomycin D Induces Apoptosis Even in TRAIL-resistant Human Pancreatic Cancer Cells Clin. Cancer Res., February 1, 2001; 7(2): 407 - 414. [Abstract] [Full Text]
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W. D. Thomas, X. D. Zhang, A. V. Franco, T. Nguyen, and P. Hersey TNF-Related Apoptosis-Inducing Ligand-Induced Apoptosis of Melanoma Is Associated with Changes in Mitochondrial Membrane Potential and Perinuclear Clustering of Mitochondria J. Immunol., November 15, 2000; 165(10): 5612 - 5620. [Abstract] [Full Text] [PDF]
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L. Zamai, P. Secchiero, S. Pierpaoli, A. Bassini, S. Papa, E. S. Alnemri, L. Guidotti, M. Vitale, and G. Zauli TNF-related apoptosis-inducing ligand (TRAIL) as a negative regulator of normal human erythropoiesis Blood, June 15, 2000; 95(12): 3716 - 3724. [Abstract] [Full Text] [PDF]
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H. Walczak, A. Bouchon, H. Stahl, and P. H. Krammer Tumor Necrosis Factor-related Apoptosis-inducing Ligand Retains Its Apoptosis-inducing Capacity on Bcl-2- or Bcl-xL-overexpressing Chemotherapy-resistant Tumor Cells Cancer Res., June 1, 2000; 60(11): 3051 - 3057. [Abstract] [Full Text] [PDF]
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M. Giovarelli, P. Musiani, G. Garotta, R. Ebner, E. Di Carlo, Y. Kim, P. Cappello, L. Rigamonti, P. Bernabei, F. Novelli, et al. A ""Stealth Effect"": Adenocarcinoma Cells Engineered to Express TRAIL Elude Tumor-Specific and Allogeneic T Cell Reactions J. Immunol., November 1, 1999; 163(9): 4886 - 4893. [Abstract] [Full Text] [PDF]
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 N. A. Fanger, C. R. Maliszewski, K. Schooley, and T. S. Griffith Human Dendritic Cells Mediate Cellular Apoptosis via Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (Trail) J. Exp. Med., October 18, 1999; 190(8): 1155 - 1164. [Abstract] [Full Text] [PDF]
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L. M. Sedger, D. M. Shows, R. A. Blanton, J. J. Peschon, R. G. Goodwin, D. Cosman, and S. R. Wiley IFN-{gamma} Mediates a Novel Antiviral Activity Through Dynamic Modulation of TRAIL and TRAIL Receptor Expression J. Immunol., July 15, 1999; 163(2): 920 - 926. [Abstract] [Full Text] [PDF]
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X. D. Zhang, A. Franco, K. Myers, C. Gray, T. Nguyen, and P. Hersey Relation of TNF-related Apoptosis-inducing Ligand (TRAIL) Receptor and FLICE-inhibitory Protein Expression to TRAIL-induced Apoptosis of Melanoma Cancer Res., June 1, 1999; 59(11): 2747 - 2753. [Abstract] [Full Text] [PDF]
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C. A. Benedict, P. S. Norris, T. I. Prigozy, J.-L. Bodmer, J. A. Mahr, C. T. Garnett, F. Martinon, J. Tschopp, L. R. Gooding, and C. F. Ware Three Adenovirus E3 Proteins Cooperate to Evade Apoptosis by Tumor Necrosis Factor-related Apoptosis-inducing Ligand Receptor-1 and -2 J. Biol. Chem., January 26, 2001; 276(5): 3270 - 3278. [Abstract] [Full Text] [PDF]
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A. Truneh, S. Sharma, C. Silverman, S. Khandekar, M. P. Reddy, K. C. Deen, M. M. Mclaughlin, S. M. Srinivasula, G. P. Livi, L. A. Marshall, et al. Temperature-sensitive Differential Affinity of TRAIL for Its Receptors. DR5 IS THE HIGHEST AFFINITY RECEPTOR J. Biol. Chem., July 21, 2000; 275(30): 23319 - 23325. [Abstract] [Full Text] [PDF]
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6 h. M412 (10 µg/ml), M413 (10 µg/ml), W6/32 (10
µg/ml), or medium was added to each well 30 min before adding
LZ-huTRAIL. B, LZ-huTRAIL-induced death of WM 98-1
(TRAIL-R1+, -R2+) following treatment with
soluble M413 (10 µg/ml), M271 (10 µg/ml), or M413 and M271 (10
µg/ml each). Protection against LZ-huTRAIL-induced death was only
provided by pretreating with both M413 and M271. Each value represents
the mean of three wells. For clarity, SE bars were omitted from the
graph but were <10% for all data points. Experiments were performed
three times with each cell line.
