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Blood, 15 October 2002, Vol. 100, No. 8, pp. 3049-3050
CORRESPONDENCE
To the editor:
TRAIL-induced apoptosis of authentic myeloma cells does not
correlate with the procaspase-8/cFLIP ratio
We read with interest Mitsiades et al's recent
paper1 purporting to define the intracellular factors
regulating tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL) activity in myeloma cells. They demonstrated quite clearly the
crucial role for procaspase-8 activation in initiating TRAIL-induced
apoptosis and the clear correlation between the efficiency of
procaspase-8 activation and the degree of TRAIL-induced apoptosis.
Furthermore, they were able to demonstrate that maneuvers capable of
inhibiting antiapoptotic proteins or artificially elevating the levels
of intracellular procaspase-8 enhanced the apoptosis-inducing
capability of TRAIL. This preliminary data demonstrating the
"sensitization" of previously TRAIL-resistant myeloma cells with
novel agents are very interesting and, if it can be confirmed that
these strategies do not also sensitize nonmalignant cells, may well
provide a rationale for early-phase clinical trials. Of concern, however, is the claim that the degree of TRAIL resistance
of the cell lines studied was associated with a low procaspase-8/cFLIP
(FLICE inhibitory protein) ratio. No data are actually
presented to support this assertion. Scrutiny of the data that are
presented shows that there is no obvious correlation between the
physiologic levels of cFLIP and/or procaspase-8 and the degree of
TRAIL-induced apoptosis for the cell lines studied. This is clearly
exemplified by the "sensitive" cell lines MM1S and RPMI showing
quite different immunoblot findings, with high procaspase-8/low cFLIP
ratios and low procaspase-8/high cFLIP ratios, respectively. Our own
data from 5 authentic myeloma cell lines confirm the crucial
relationship between the efficiency of procaspase-8 activation and
TRAIL-induced apoptosis (Figure 1) and also show the lack of correlation between the physiologic procaspase-8/cFLIP levels and TRAIL-induced apoptosis (Figure 2).
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| Figure 1.
Immunoblot analysis of procaspase-8 activation in
TRAIL-treated myeloma cells.
The myeloma cells were incubated with 1 µg/mL leucine-zipper
TRAIL (Immunex, Seattle, WA) and harvested at 30 and 60 minutes
following incubation. The proportion of cells undergoing apoptosis,
shown by the percentage in parentheses at 60 minutes, was determined by
annexin-V staining. Cytosolic protein fractions (25 µg) were
separated on 4% to 16% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) gels, transferred to nitrocellulose, and
probed with a caspase-8-specific goat polyclonal antibody C-20
(Santa Cruz, Santa Cruz, CA). Proteins were visualized by enhanced
chemiluminescence (ECL). Membranes were stripped and reprobed with
monoclonal antibody to  -tubulin (Sigma, St Louis, MO). The
sensitive myeloma cell lines RPMI 8226, LP-1, and OPM-2 all show early
cleavage of procaspase-8 with generation of the intermediate p43/41
product (*) and the active P18 form. Resistant cell lines NCI H929 and
U266 demonstrate delayed procaspase-8 cleavage with minimal P18
generation.
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| Figure 2.
Determination of cFLIP and procaspase-8 levels in
untreated myeloma cells.
Untreated myeloma cells were lysed in whole cell lysate buffer and 80 µg protein separated on a 12% SDS-PAGE gel. Procaspase-8 was
detected as described above. Membranes were then stripped and reprobed
with the cFLIP monoclonal antibody G-11 (Santa Cruz). Tubulin detection
was used to confirm equal protein loading. The procaspase-8/cFLIP
ratios were calculated for each cell line densitometrically and do not
correlate with the amount of TRAIL-induced apoptosis (r = 0.7,
p = 0.19, Spearman rank correlation).
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There are 4 known surface receptors for TRAIL, 2 of which (TRAIL-R1 and
TRAIL-R2) appear to be capable of inducing apoptosis upon binding of
TRAIL.2 Our understanding of TRAIL-TRAIL receptor interactions beyond this is limited. But the earlier belief that the 2 "decoy" TRAIL receptors (TRAIL-R3 and TRAIL-R4) lacking intracellular death domains somehow protect cells from TRAIL has been
shown by our group and others to be incorrect.3-5 For
TRAIL to be exploited maximally as an antitumor agent, how it works must be more thoroughly understood, particularly the exact roles of the
known TRAIL receptors. Acceptance of the perhaps premature assertion
that myeloma-cell TRAIL sensitivity is regulated by physiologic levels
of procaspase-8 and/or cFLIP will do little to improve this lack of understanding.
Andrew Spencer, Sung-Lin Yeh, Karly Koutrevelis, and Cindy Baulch-Brown
Correspondence: Andrew Spencer, BMT Programme, The Alfred
Hospital, Commercial Road, Prahran, Victoria, Australia; e-mail:
aspencer{at}netspace.net.au
References
1.
Mitsiades N, Mitsiades CS, Poulaki V, Anderson KC, Treon SP.
Intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human multiple myeloma cells.
Blood.
2002;99:2162-2171[Abstract/Free Full Text].
2.
Gura T.
How TRAIL kills cancer cells, but not normal cells [news; comment].
Science.
1997;277:768[Free Full Text].
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Griffith TS, Chin WA, Jackson GC, Lynch DH, Kubin MZ.
Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells.
J Immunol.
1998;161:2833-2840[Abstract/Free Full Text].
4.
Keane MM, Ettenberg SA, Nau MM, Russell EK, Lipkowitz S.
Chemotherapy augments TRAIL-induced apoptosis in breast cell lines.
Cancer Res.
1999;59:734-741[Abstract/Free Full Text].
5.
Lincz LF, Yeh TX, Spencer A.
TRAIL-induced eradication of primary tumour cells from multiple myeloma bone marrows is not related to TRAIL receptor expression or prior chemotherapy.
Leukemia.
2001;15:1650-1657[CrossRef][Medline]
[Order article via Infotrieve].
Response:
Resistance to tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL) is modulated by FLICE-inhibitory protein (FLIP) and
procaspase-8 in multiple myeloma cells
At the center of the concerns raised by Dr Spencer et al is the
role of FLICE-inhibitory protein (FLIP) and procaspase-8 in mediating
resistance to tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL, Apo2 ligand)-mediated apoptosis in myeloma cells. In our
studies, TRAIL resistance was associated with underexpression of
procaspase-8 and overexpression of FLIP. In support of these conclusions were experiments that demonstrated the following: (1)
TRAIL-resistant myeloma cell lines tended to exhibit low levels of
procaspase-8 and high levels of FLIP, as well as other antiapoptotic molecules such as cellular inhibitors of apoptosis protein-2 (cIAP-2); (2) forced overexpression of procaspase-8 in TRAIL-resistant myeloma cells increased TRAIL-sensitivity; (3) antisense oligonucleotides against FLIP increased TRAIL-sensitivity of TRAIL-resistant myeloma cells; and (4) pharmacologic modulators of FLIP expression (eg, protein
kinase C [PKC] inhibitors, cycloheximide, etc) that
down-regulated the expression of FLIP also led to increased
TRAIL-sensitivity of TRAIL-resistant myeloma cells. Similar findings
have been published by others who have studied TRAIL-mediated apoptotic
pathways in other tumor cell models, and have akin to us shown
that underexpression of procaspase-8 and/or overexpression of FLIP
(including high caspase-8 homologue FLIP [cFLIP]/procaspase-8 levels)
are associated with resistance to TRAIL.1-3 We have
attempted to dissect out differences in our respective study approaches
that may have led to discordant results. While Spencer et
al's letter does not provide the methodology for the included
experiments nor list the antibodies used to determine procaspase-8 and
FLIP protein expression, there are concerns raised by the
interpretation of these data in view of the cell lines and methodology
previously used by these investigators. One key concern is the
suboptimal level of TRAIL-induced tumor cell death noted for the RPMI
8226/S myeloma cell line in their previous study,4 as well
as outright resistance to TRAIL for both U266 and NCI H929 myeloma
cells per their letter. But in deference to their own studies, Spencer
and colleagues acknowledge that NCI H929 myeloma cells are
TRAIL-sensitive in their Leukemia article,4
while describing these cells as TRAIL-resistant in their letter. One
likely explanation for this discrepancy is that Spencer et al measure
TRAIL-induced apoptosis by annexin V only after one hour of TRAIL
treatment. This likely is a very early time point to determine the full
extent of TRAIL-induced apoptosis, because as shown in our previous
studies,5 myeloma cells become annexin V-positive after 4 to 6 hours of TRAIL treatment. This likely explains why in their letter
Spencer et al have incorrectly classified the NCI H929 and U266 cell
lines as TRAIL-resistant. In our hands, as well as those of many other
investigators, all of the above cell lines have exhibited marked
sensitivity to TRAIL,6-9 including the NCI H929 cell line,
which exhibited an inhibitory concentration 50% (IC50) of
below 100 ng/mL and showed TRAIL sensitivity similar to the
TRAIL-sensitive LP-1 and OPM-2 myeloma cells reported by Spencer et al
in Figure 1 of their letter. One possible explanation for these
discordant findings is the technique used to determine induction of
apoptosis by TRAIL. The authors curiously report in Leukemia
that they first undertook to remove dead cells following TRAIL
treatment by density gradient centrifugation over Lymphoprep (Nycomed
Pharma, Oslo, Norway) before establishing the percentage of
cells that underwent apoptosis, thus potentially underestimating cell
death. The rationale for taking this step remains unclear and hampers
the valid interpretation of these studies since appropriately establishing TRAIL sensitivity is a prerequisite to discerning intracellular regulatory pathways. It does remain unclear, as suggested by Spencer et al, whether
the use of Epstein-Barr virus (EBV) genome containing cell lines is a
confounding factor in these and other studies since modulation of both
caspase-8 and FLIP have been reported as a potential mechanism of EBV
tumorigenesis in Burkitt lymphoma.10 But a certain subset
(or subsets) of multiple myeloma (MM) may be EBV-related in
view of reports that have demonstrated EBV genomic presence in freshly
obtained MM patient cells.10-13 Interestingly, this subset
may include those patients with CD20+ disease since EBV
nuclear antigen 3C (EBNA-3C) modulates the PU.1 transcription
factor14 that is normally down-regulated in plasma cells
and is responsible, along with the PU.1 interacting partner (Pip), for
CD20 expression (reviewed in Treon et al15). This point is
especially worth mentioning since the cell lines in our study that were
moderately or highly TRAIL resistant were CD20+ and have
been reported to be EBV+ as well.5
Nicholas Mitsiades, Constantine Mitsiades, Kenneth C. Anderson, and Steven P. Treon
Correspondence: Steven P. Treon, Department of Medical Oncology,
Dana Farber Cancer Institute and Harvard Medical School, 44 Binney St,
LG102, Boston, MA 02115; e-mail: steven_treon{at}dfci.harvard.edu
References
1.
Kim Y, Suh N, Sporn M, Reed JC.
An inducible pathway for degradation of FLIP protein sensitizes tumor cells to TRAIL induced apoptosis.
J Biol Chem.
2002;277:22320-22329[Abstract/Free Full Text].
2.
Harper N, Farrow SN, Kaptein A, Cohen GM, MacFarlane M.
Modulation of tumor necrosis factor apoptosis-inducing ligand induced NF-kB activation by inhibition of apical caspases.
J Biol Chem.
2001;276:34743-34752[Abstract/Free Full Text].
3.
Grotzer MA, Eggert A, Zuzak TJ, et al.
Resistance to TRAIL-induced apoptosis in primitive neuroectodermal brain tumor cells correlates with a loss of caspase 8 expression.
Oncogene.
2000;19:4604-4610[CrossRef][Medline]
[Order article via Infotrieve].
4.
Lincz LF, Yeh TX, Spencer A.
TRAIL induced eradication of primary tumour cells from multiple myeloma patient bone marrows is not related to TRAIL receptor expression or prior chemotherapy.
Leukemia.
2001;15:1650-1657[CrossRef][Medline]
[Order article via Infotrieve].
5.
Mitsiades N, Mitsiades CS, Poulaki V, Anderson KC, Treon SP.
Intracellular regulation of tumor necrosis factor-related apoptosis inducing ligand-induced apoptosis in human myeloma cells.
Blood.
2002;99:2162-2171[Abstract/Free Full Text].
6.
Mitsiades C, Treon SP, Mitsiades N, et al.
TRAIL/Apo2L ligand selectively induces apoptosis and overcomes drug resistance in multiple myeloma: therapeutic applications.
Blood.
2001;98:795-804[Abstract/Free Full Text].
7.
Jazirehi AR, Ng CP, Gan XH, Schiller G, Bonavida B.
Adriamycin sensitizes the adriamycin resistant 8226/Dox40 human multiple myeloma cells to Apo2L/tumor necrosis factor related apoptosis-inducing ligand-mediated (TRAIL) apoptosis.
Clin Cancer Res.
2001;7:3874-3883[Abstract/Free Full Text].
8.
Chen Q, Gong B, Ahmed ASM, et al.
Apo2L/TRAIL and Bcl-2 related proteins regulate type I interferon-induced apoptosis in multiple myeloma.
Blood.
2001;98:2183-2192[Abstract/Free Full Text].
9.
Gazitt Y.
TRAIL is a potent inducer of apoptosis in myeloma cells derived from multiple myeloma patients and is not cytotoxic to hematopoietic stem cells.
Leukemia.
1999;13:1817-1824[CrossRef][Medline]
[Order article via Infotrieve].
10.
Tepper CG, Seldin MF.
Modulation of caspase-8 and FLICE-inhibitory protein expression as a potential mechanism of Epstein-Barr virus tumorigenesis in Burkitt's lymphoma.
J Immunol.
2001;167:5404-5411[Abstract/Free Full Text].
11.
Dominici M, Luppi M, Campioni D, et al.
PCR with degenerate primers for highly conserved DNA polymerase gene of the herpesvirus family shows neither human herpesvirus 8 nor a related variant in bone marrow stromal cells from multiple myeloma patients.
Int J Cancer.
2000;86:76-82[CrossRef][Medline]
[Order article via Infotrieve].
12.
Voelkerding KV, Sandhaus LM, Kim HC, et al.
Plasma cell malignancy in the acquired immune deficiency syndrome: association with Epstein Barr virus.
Am J Clin Path.
1989;92:222-228[Medline]
[Order article via Infotrieve].
13.
Chandburn A, Cesarman E, Knowles DM.
Molecular pathology of post-transplantation lymphoproliferative disorders.
Semin Diagn Pathol.
1997;14:15-26[Medline]
[Order article via Infotrieve].
14.
Lin J, Johannsen E, Robertson E, Kieff E.
Epstein-Barr virus nuclear antigen 3 C putative repression domain mediates coactivation of the LMP1 promoter with EBNA-2.
J Virol.
2002;76:232-242[Abstract/Free Full Text].
15.
Treon SP, Belch AR, Kelliher A, et al.
CD20-directed serotherapy in multiple myeloma: biological considerations and therapeutic applications.
J Immunother.
2002;25:72-81[Medline]
[Order article via Infotrieve].
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