Sensitization of AIDS-Kaposi's Sarcoma Cells
to Apo-2 Ligand-Induced Apoptosis by
Actinomycin D
This information is current as
of June 15, 2017.
Shunsuke Mori, Kaoru Murakami-Mori, Shuji Nakamura,
Avi Ashkenazi and Benjamin Bonavida
J Immunol 1999; 162:5616-5623; ;
http://www.jimmunol.org/content/162/9/5616
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References
Sensitization of AIDS-Kaposi’s Sarcoma Cells to Apo-2
Ligand-Induced Apoptosis by Actinomycin D1
Shunsuke Mori,* Kaoru Murakami-Mori,* Shuji Nakamura,† Avi Ashkenazi,‡ and
Benjamin Bonavida2*
umor necrosis factor-a functions as a critical mediator of
immune responses and inflammatory reactions. Recent
advances in gene-cloning techniques showed evidence for
the existence of a TNF ligand family that consists of TNF-a, lymphotoxin (TNF-b), CD27 ligand (CD27L),3 4 –1BBL, CD30L,
CD40L, and Fas/Apo-1 ligand (1). The TNF ligand family members are all type II membrane glycoproteins with clear homology
to TNF-a within their C-terminal extracellular domains. As expected, these members share TNF-a-like activities such as cell
death induction. Recently, a novel TNF ligand family member,
named Apo-2 ligand (Apo-2L) (2) or TNF-related apoptosis-inducing ligand (TRAIL) (3), was cloned, using a consensus amino
acid sequence based on the most conserved region of this family.
Treatment with recombinant soluble Apo-2L (sApo-2L)/TRAIL,
or coculture with Apo-2L/TRAIL cDNA-transfected cells induced
rapidly cell death in transformed cell lines of diverse origin (2, 3).
T
*Department of Microbiology and Immunology, University of California School of
Medicine, Los Angeles, CA 90095; †Huntington Memorial Hospital, Pasadena, CA
91105; and ‡Molecular Oncology, Genentech, Inc., South San Francisco, CA 94080
Received for publication November 4, 1998. Accepted for publication February
12, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by a grant from the Boiron Research Foundation.
2
Address correspondence and reprint requests to Dr. Benjamin Bonavida, Department of Microbiology and Immunology, University of California School of Medicine,
10833 Le Conte Ave., Los Angeles, CA 90095-1747. E-mail address: [email protected]
3
Abbreviations used in this paper: CD27, CD27 ligand; Apo-2L, Apo-2 ligand;
TRAIL, TNF-related apoptosis-inducing ligand; sApo-2L, recombinant soluble Apo2L; DcR, decoy receptor; KS, Kaposi’s sarcoma; AIDS-KS, AIDS-associated KS; Act
D, actinomycin D; cFLIP, cellular FLICE (Fas-associated death domain protein-like
IL-1-converting enzyme) inhibitory proteins; FADD, Fas-associated death domain
protein; OM, oncostatin M.
Copyright © 1999 by The American Association of Immunologists
Two death domain-containing receptors, DR4 and DR5, have been
identified as Apo-2L/TRAIL receptors (4 –12). Unlike the Fas ligand, the expression of Apo-2L/TRAIL and its receptors is observed in various normal human tissues, suggesting that Apo-2L/
TRAIL must not be cytotoxic to most normal tissues in vivo (13).
It has also been reported that Apo-2L/TRAIL can bind a decoy
receptor 1 (DcR1; also called TRID, TRAIL-R3, or LIT) (5, 6, 11,
12, 14, 15) and DcR2 (also called TRAIL-R4 or TRUNDD) (16 –
18); however, these receptors cannot transduce the apoptotic signal
into the cells because of their lack of functional death domains.
Normal cells express high levels of DcR1 and DcR2, while malignant cells express only minimum amounts of these receptors
(13). Therefore, it is hypothesized that resistance of normal cells to
Apo-2L/TRAIL may be due to the expression of DcR1 and DcR2.
Kaposi’s sarcoma (KS) is the most common malignancy arising
in persons with HIV infection (AIDS-KS). The clinical course of
AIDS-KS is highly variable, ranging from a minimal disease presenting as an incidental finding, to a rapidly progressive or extensive disease resulting in significant morbidity and mortality (19).
Extracutaneous spread is common, involving most frequently the
oral cavity, the gastrointestinal tract, the lung, and the lymph
nodes. AIDS-KS is a highly vascular tumor consisting of proliferating spindle-shaped cells (KS cells), microvascular endothelial
cells, infiltrating mononuclear cells, and edema (19). Experimental
data using cultured KS cells indicate that KS cells contribute to the
development and progression of KS lesions by producing growthpromoting, inflammatory, and angiogenic cytokines (20 –24).
Although a number of modalities have been used for 15 yr, cure
or long term complete remission from KS is unlikely with the
currently available therapeutic modalities (25). We have reported
that AIDS-KS cells are resistant to Fas-mediated apoptosis (26). In
addition, AIDS-KS cells are resistant to chemotherapeutic drugs
(25). Several lines of evidence suggest that KS cells may acquire
0022-1767/99/$02.00
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Kaposi’s sarcoma (KS) is the most frequent malignancy associated with HIV infection (AIDS-KS), a complication that leads to
high mortality and morbidity. AIDS-KS cells are resistant to killing by chemotherapeutic drugs/NK cells and Fas-induced apoptosis, suggesting that the acquisition of antiapoptotic characteristics by AIDS-KS cells may contribute to their prolonged survival.
Apo-2 ligand (Apo-2L)/TNF-related apoptosis-inducing ligand, a new member of the TNF family, has been identified as an
apoptosis-inducing molecule. In this study we examined the sensitivity of 10 different AIDS-KS isolates to Apo-2L-mediated
cytotoxicity. AIDS-KS cells were relatively resistant to Apo-2L; however, Apo-2L and actinomycin D (Act D) used in combination
synergistically potentiated the induction of cell death in nine of the 10 isolates. Apo-2L induced apoptosis in >80% of AIDS-KS
cells pretreated with Act D. The caspase inhibitors, zIETD-fmk and zDEVD-fmk, inhibited apoptosis in AIDS-KS by sApo-2L,
suggesting that caspase 3-like and caspase 8 or 10 activities are essential for Apo-2L-mediated apoptosis. Act D treatment of
AIDS-KS cells markedly and selectively down-regulated Bcl-xL expression, while the expressions of decoy receptors 1 and 2, Bax,
cellular FLICE (Fas-associated death domain protein-like IL-1-converting enzyme) inhibitory protein, FADD (Fas-associated
death domain protein), procaspase 8, and p53 were not affected. These findings suggest the possible involvement of Bcl-xL in Act
D-induced sensitization of AIDS-KS cells to Apo-2L-mediated apoptosis. Furthermore, Act D did not sensitize PBMC or fibroblast
cells to Apo-2L. Thus, Apo-2L and Act D used in combination may be of therapeutic value in the treatment of AIDS-KS. The
Journal of Immunology, 1999, 162: 5616 –5623.
The Journal of Immunology
resistance to several apoptotic stimuli through the expression of
antiapoptotic molecules such as Bcl-2 (27) and Bcl-xL (28). The
resistance of KS cells to apoptosis may hamper the development of
therapeutic agents for the treatment of AIDS-KS. In this study we
present evidence that actinomycin D (Act D) sensitizes AIDS-KS
cells to sApo-2L-mediated apoptosis. In addition, we address the
possible roles of apoptosis-related molecules in Act D-induced
sensitization of AIDS-KS cells to sApo-2L.
Materials and Methods
Cells and reagent
Cytotoxicity assay
AIDS-KS cells or human foreskin fibroblast cells were seeded in 24-well
culture plates and cultured for 18 –24 h at 37°C. Each well was washed
twice with medium and incubated in the presence or the absence of Act D,
with or without sApo-2L or CH-11. Eighteen hours later, the number of
adherent cells was counted. The medium was removed, and the plates were
washed with Hanks’ solution (Life Technologies). After detaching the cells
with trypsin/EDTA (Life Technologies), the number of cells was counted
using a particle counter (Coulter, Hialeah, FL). Cell viability was also
evaluated using a cell proliferation kit-XTT assay (Boehringer Mannheim,
Indianapolis, IN). The cell viability determined by counting the adherent
cells correlated with that determined by the XTT assay. The data are represented as the mean 6 SD (n 5 3). Cytotoxicity for PBMC was determined using a cell proliferation kit-XTT assay according to the manufacturer’s instructions. Cell viability (%) 5 100 3 (OD450 – 650 obtained from
sApo-2L-treated cells/OD450 – 650 obtained from untreated cells).
Morphology
KS-10B cells (2 3 104 cells) were cultured for 18 h in RPMI 1640 supplemented with 10% FBS in a 24-well culture plate. The sApo-2L
(500 ng/ml) and Act D (10 ng/ml) were added to the cultures and then
incubated for 18 h. The cells were observed under a phase-contrast microscope (Olympus Optical, Tokyo, Japan).
DNA fragmentation assays
KS-10B cells were cultured in RPMI 1640 supplemented with 10% FBS in
75-cm2 culture flask. The sApo-2L (250 ng/ml) and Act D (10 ng/ml) were
added to cultures, then incubated for 18 h. Detached and attached cells (;1
million cells) were collected, washed three times with PBS, and then suspended in 100 ml of ice-cold 70% ethanol. After incubation for 20 min at
room temperature, the cells were washed, suspended in staining buffer
(PBS/10 mg/ml propidium iodide/50 mg/ml RNase A), incubated for 30
min at room temperature, and subjected to flow cytometric analysis using
an EPICS XL flow cytometer (Coulter).
RT-PCR
Confluent AIDS-KS cells were cultured for 18 h in the presence or the
absence of Act D (10 ng/ml). Total RNA was prepared using TRIzol (Life
Technologies). First-strand cDNA was synthesized in 60 ml of reaction
mixture containing 6 mg of total RNA, using a cDNA Preamplification Kit
(Life Technologies). The cDNA synthesis reaction mixtures were subjected
to PCR amplification (100 ml) under the following conditions for DcR1,
DcR2, cellular FLICE (Fas-associated death domain protein-like IL-1-converting enzyme) inhibitory protein (cFLIP), and b-actin: 30 cycles at 94°C
for 1 min, at 55°C for 1 min, and at 72°C for 1 min. PCR was performed
using the following upstream and downstream primers. DcR1 (upstream),
59-CAG TGT AAA GAA GGC ACC TTC CGG-39; DcR1 (downstream),
59-GCA GGA GTC CCT GGG CTG GTG-39; DcR2 (upstream), 59-CAC
TAC CTT ATC ATC ATA GTG GTT TT-39; DcR2 (downstream), 59GAA GGA CAT GAA CGC CGC CGG AAA AG-39; cFLIP (upstream),
59-GAC CCT TGT GAG CTT CCC TAG TC-39; cFLIP (downstream),
59-GAG CAG GTG GGT CTC CAC AGC-39; and b-actin (upstream),
59-ATC TGGCAC CAC ACC TTC TAC AAT GAG CTG CG-39; b-actin
(downstream), 59-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT
GC-39. Ten microliters of PCR reaction were subjected to agarose gel
electrophoresis. Densitometric analysis was performed using Scan Analysis (Biosoft, London, U.K.). The intensity of the PCR products generated
from 5 ml of Act D-untreated cell-derived template was set as 1.0.
Western blot analysis
Confluent AIDS-KS cells were incubated for 18 h in the presence or the
absence of Act D (10 ng/ml). The cells were lysed at 4°C in RIPA buffer
(50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate,
150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 mg/ml of aprotinin, 1 mg/ml
leupeptin, 1 mg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF). The cell
lysates (5–30 mg) were electrophoresed on 10 –20 or 12% SDS-PAGE
(Novex, San Diego, CA) and subjected to Western blot analysis. The transfer of proteins from gels onto Hybond nitrocellulose membranes (Amersham, Arlington Heights, IL) was electrophoretically conducted in a transblotting cell (Bio-Rad, Hercules, CA). The membranes were blocked by
immersing for 1 h at room temperature in 5% nonfat skim milk/PBS and
then incubated with the respective Ab for 18 h at room temperature. Mouse
anti-Bcl-x and FADD (Fas-associated death domain protein) mAbs were
purchased from Transduction Laboratory (Lexington, KY). Mouse antiBax mAb and rabbit anti-p53 polyclonal Ab were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Mouse anti-procaspase 8 mAb was
obtained from PharMingen (San Diego, CA). Rabbit anti-cFLIP polyclonal
Ab was obtained from Upstate Biotechnology. After washing in PBS/0.1%
Tween-20, the membranes were incubated for 2 h with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG Ab (New England Biolabs,
Beverly, MA). After washing in PBS/0.1% Tween-20, the membranes
were developed with a Phototope Western blot detection kit (New England
Biolabs).
Results
Sensitization of AIDS-KS cells to sApo-2L-induced cell death
We determined the cytotoxic effect of sApo-2L on 10 kinds of
AIDS-KS cells obtained from tissues of different patients with HIV
infection. AIDS-KS cells were incubated with 500 ng/ml of sApo2L. As shown in Table I, KS-7, KS-11, and KS-22 were moderately sensitive to 500 ng/ml of sApo-2L, and the other KS cells
were resistant. Protein synthesis inhibitors or chemotherapeutic
drugs are known to markedly augment Fas- or TNF-a-mediated
cytotoxicity for some types of cells (31). We have also reported
that the combination of an agonistic anti-Fas mAb, CH-11, and a
subtoxic concentration of Act D (10 ng/ml) induced significant
levels of cell death in AIDS-KS cell populations (Fig. 1A) (26).
Therefore, we examined the combined effect of sApo-2L and Act
D treatment on AIDS-KS cell viability. Combination treatment of
sApo-2L with Act D efficiently induced cell death in KS isolates,
except in KS-8 (Table I). We further analyzed the effects of
sApo-2L and Act D (10 ng/ml) on KS-10B and KS-11. AIDS-KS
cells were incubated with various concentrations of sApo-2L in the
presence or the absence of Act D (10 ng/ml) (Fig. 1B). In the
presence of Act D, sApo-2L induced cell death in KS-10B and
KS-11 in a concentration-dependent manner.
To determine whether the cytotoxic effect of Act D and sApo-2L
on KS cells was the result of a synergistic or an additive effect,
KS-9 cells were treated with various concentrations of sApo-2L or
Act D (Fig. 1C). Based on the methods described by Berenbaum
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AIDS-KS cells were developed from the pleural effusion of lung KS (KS-6,
KS-7, KS-9, KS-11, KS-21, KS-22, KS-23), lung KS (KS-3, KS-8), and
oral mucosa KS (KS-10B) of HIV-infected patients. KS-21, KS-22, and
KS-23 cells were developed at the Institute of Molecular Medicine, Huntington Memorial Hospital (Pasadena, CA), by using previously described
methods (29). KS-3, KS-6, KS-7, KS-9, KS-10B, and KS-11 were developed in the Laboratory of Tumor Cell Biology, National Cancer Institute,
National Institutes of Health (Bethesda, MD). AIDS-KS cells were maintained in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS and conditioned medium from human oncostatin
M (OM)-expressing Chinese hamster ovary cells to a final OM concentration of 15 ng/ml (30). During the study KS-6, KS-8, and KS-11 cells died,
and these cells were not available for all tests performed in the other KS
cell cultures. Human foreskin fibroblast cells were maintained in EMEM
supplemented with 10% FBS (29). Human PBMC were purified using Ficoll-Hypaque and cultured in RPMI 1640 supplemented with 10% FBS.
Act D was purchased from Sigma (St. Louis, MO). The sApo-2L was
prepared as previously described (6). Caspase inhibitors, zVAD-fmk and
zDEVD-fmk, were purchased from Calbiochem-Novabiochem (San Diego,
CA). The zIETD-fmk was obtained from MBL (Nagoya, Japan). The agonistic anti-Fas mAb (CH-11) was purchased from Upstate Biotechnology
(Lake Placid, NY).
5617
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APOPTOSIS INDUCTION IN AIDS-KS CELLS BY Apo-2L/TRAIL
Table I. Synergistic cytotoxicity of combination treatment with Act D
and sApo-2L on AIDS-KS isolates a
Cell Viability (%)
AIDS-KS
Cell Isolates
Origin
Act D
(10 ng/ml)
sApo-2L
(500 ng/ml)
Act D/sApo-2L
(10/500 ng/ml)
KS-3
KS-6
KS-7
KS-8
KS-9
KS-10B
KS-11
KS-21
KS-22
KS-23
L
PE
PE
L
PE
MU
PE
PE
PE
PE
93.0
89.9
83.5
85.7
83.6
79.5
83.4
74.5
88.7
66.1
86.6
76.4
67.9
85.2
80.0
90.2
59.2
79.5
54.4
84.4
64.9 (80.0)b
43.3 (66.3)b
27.4 (51.4)b
70.7 (74.9)b
18.1 (63.6)b
21.3 (69.7)b
8.2 (31.6)b
31.9 (54.0)b
16.7 (43.1)b
30.4 (50.5)b
(32), we confirmed that the cytotoxic effect of combination of Act
D and sApo-2L on KS-9 was the result of synergy. Next, we examined the kinetics of cell death induction by sApo-2L. When
KS-10B and KS-11 cells were pretreated for 18 h with Act D (10
ng/ml), most sApo-2L-treated cells displayed cell blebbing within
2 h, and almost all cells were detached within 6 h (Fig. 1D). These
data indicate that Act D sensitizes AIDS-KS cells to sApo-2L. The
effect of Act D and sApo-2L on KS-3 was less pronounced compared with those on the other KS isolates (Fig. 2A). However,
efficient cell death induction was obtained by increasing the concentration of Act D (Fig. 2B) or by pretreatment of KS-3 with Act
D (Fig. 2C).
The sApo-2L/Act D-induced AIDS-KS cell death occurs via
apoptosis
To characterize the Apo-2L-induced cell death in AIDS-KS cells,
we examined the morphological changes following sApo-2L treatment of these cells (Fig. 3A). When KS-10B cells were treated for
18 h with sApo-2L (500 ng/ml) plus Act D (10 ng/ml), most cells
were detached from the culture dishes and formed extensive blebbing on the cell surface, a characteristic of apoptosis. In contrast,
the effect of either agent used alone was extremely weak. We evaluated apoptosis by the propidium iodide staining method. As
shown in Fig. 3B, flow cytometric analysis revealed that the combined treatment of sApo-2L (250 ng/ml) and Act D (10 ng/ml)
induced DNA fragmentation in 55.1% of the KS cell population,
while only small numbers of sApo-2L-treated or Act D-treated KS
cells underwent apoptosis. The number of apoptotic cells shown in
Fig. 3B correlated well with the viability assessed by counting the
number of adherent cells shown in Fig. 1B. These findings demonstrate that the combination treatment with sApo-2L and Act D
induces KS cell death by apoptosis.
Act D failed to sensitize fibroblast cells and PBMC to sApo-2L
To examine whether the combination treatment with Act D and
sApo-2L also kills normal cells, cytotoxic assays were performed
with foreskin fibroblast cells and PBMC. As shown in Fig. 4, foreskin fibroblast cells and PBMC were resistant to sApo-2L. Act D
inhibited the growth of fibroblast cells and reduced the viability of
PBMC; however, the combination of Act D and sApo-2L failed to
induce synergistic cell death in both cell types. These data show
that Act D cannot sensitize fibroblast cells and PBMC to sApo-2L.
FIGURE 1. Synergistic effects of Act D and CH-11 on viability of KS
cells (A) on sApo-2L (B). AIDS-KS cells (1–2 3 104 cells) were seeded in
24-well culture plates. After preculture for 18 h, the medium was changed,
and the cells were cultured for 18 h with or without Act D (10 ng/ml) and
various concentrations of CH-11 or sApo-2L. The adherent cells were
counted using a Coulter particle counter. The data represent the mean 6
SD (n 5 3). Open bars, without Act D; solid bars, with Act D (10 ng/ml).
C, Synergistic effect of Act D and sApo-2L on viability of KS-9. The data
represent the mean 6 SD (n 5 3). Open bars, without Act D; hatched bars,
Act D (5 ng/ml); dotted bars, Act D (10 ng/ml); solid bars, Act D (20
ng/ml). The combination effect of sApo-2L and Act D meets the criteria of
synergism as described by Berenbaum (32). The concentrations of Act D
and sApo-2L necessary to induce 50% killing in KS-9 cells are .20 and
1000 ng/ml, respectively. One combination for 50% killing consisted of 10
ng/ml Act D and 125 ng/ml sApo-2L. According to Berenbaum, the interaction index was calculated as follows: 10/20 1 125/1000 5 0.625 , 1.0,
which means synergism. The other combination consisted of 5 ng/ml Act
D and 250 ng/ml sApo-2L. In this case, the interaction index was 5/20 1
250/1000 5 0.5. Thus, the interaction index of sApo-2L and Act D clearly
meets the criteria of synergism, thereby indicating that Act D and sApo-2L,
synergistically induced cell death in KS-9. D, Kinetics of sApo-2L-induced
cell death. AIDS-KS cells were cultured for 18 h in the absence or the
presence of Act D (10 ng/ml). The sApo-2L was added in culture, and the
number of cells was counted during the indicated incubation periods. The
viability of Act D-treated cells in the presence (M) or the absence (E) of
sApo-2L (250 ng/ml) and the viability of non-Act D-treated cells in the
presence (f) or the absence (F) of sApo-2L (250 ng/ml) are shown.
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a
AIDS-KS cells were isolated from KS in pleural effusion (PE), lung KS (L), and
oral mucosal KS (MU). Viability was determined as follows: 100 3 (numbers of
sApo-2L and/or Act D-treated adherent cells)/(untreated adherent cells). The data
represent mean 6 SD (n 5 3). The values in parentheses represent the calculated
additive effects of treatment with Act D alone and sApo-2L alone.
b
Synergy was calculated according to Berenbaum (32).
The Journal of Immunology
5619
The sApo-2L-induced apoptosis in AIDS-KS cells is caspase
dependent
Caspases play essential roles in many types of apoptosis (33).
Caspase inhibitors block Apo-2L/TRAIL-induced apoptosis (34,
35). We tested the effects of caspase inhibitors, zVAD-fmk,
zDEVD-fmk, and zIETD-fmk, on sApo-2L-induced apoptosis of
AIDS-KS cells (Fig. 5). In vitro, zVAD-fmk is a general caspase
inhibitor, and zDEVD-fmk and zIETD-fmk inhibit caspase 3-like
and caspase 8 and 10 activities, respectively. AIDS-KS cells were
pretreated for 18 h with Act D, then the cells were further incubated with each caspase inhibitor for 1 h. The viability was determined after 5-h stimulation with sApo-2L. All three caspase inhibitors significantly inhibited sApo-2L-induced apoptosis in Act
D-treated KS cells. These data indicate that caspase 3-like and
caspase 8 or 10 activities are critical in sApo-2L-induced apoptosis
in AIDS-KS cells.
Molecular mechanism of Act D-induced sensitization of AIDSKS cells to sApo-2L
Act D is an inhibitor of transcription. To investigate the mechanism of sensitization of KS cells to sApo-2L by Act D, we examined the effect of Act D on the expression of both the decoy receptors for sApo-L, DcR1 and DcR2 and cFLIP (36, 37), a cellular
homologue of viral FLIP (38), using RT-PCR (Fig. 6). We performed PCR from 5 and 0.5 ml of RT reaction mixture. As
shown in Fig. 6, no significant down-regulation of these mol-
FIGURE 3. Apo-2L-induced cell death was mediated through apoptosis. KS-10B cells were treated for 18 h with Act D (10 ng/ml), sApo-2L
(500 ng/ml), and Act D (10 ng/ml)/sApo-2L (500 ng/ml). A, Morphological
changes were observed under phase contrast microscopy. B, DNA fragmentation assay. Cells were treated for 15 min with 70% ethanol. After
washing with PBS, the cells were suspended in PBS including propidium
iodide (10 mg/ml) and RNase A (50 mg/ml), and then subjected to flow
cytometry using an EPICS XL. Cells (2 3 104) were analyzed. The numbers
of DNA fragmented cells are indicated at the right corner of each panel.
ecules was observed in Act D-treated KS cells. Further, the
levels of cFLIP protein in AIDS-KS cells were not changed
following Act D treatment (Fig. 7).
Members of the Bcl-2 family regulate apoptosis induced by various stimuli (39 – 41). Bcl-2 and Bcl-xL function as antiapoptotic
molecules, while Bax and Bcl-xS function as proapoptotic molecules. The p53 regulates the expression of these Bcl-2 family molecules (39 – 41). As shown in Fig. 7, high levels of p53 proteins
were expressed in Act D-untreated cells, and Act D treatment did
not affect the expression of p53. Bax proteins are abundantly expressed, and Act D treatment did not affect the expression of Bax
proteins. Bcl-xL and Bcl-xS are generated from a single gene by
alternative splicing (42). Anti-Bcl-x mAb recognized only a molecule with a Mr of 30 kDa, indicating that AIDS-KS cells preferentially expressed the Bcl-xL protein. Act D treatment markedly
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FIGURE 2. Synergistic effect of Act D and sApo-2L on the viability of
KS-3 as a function of increasing concentrations of sApo-2L in the presence
of Act D (10 ng/ml; A), increasing the concentration of Act D with
sApo-2L (250 ng/ml; B), or pretreatment with Act D (C). KS-3 cells were
pretreated for 18 h with or without Act D (10 ng/ml) and incubated for 6 h
with sApo-2L. The data represent the mean 6 SD (n 5 3). A and C, Open
bars, without Act D; solid bars, with Act D plus sApo-2L (250 ng/ml). B,
Open bars, without sApo-2L; solid bars, with sApo-2L (250 ng/ml).
5620
APOPTOSIS INDUCTION IN AIDS-KS CELLS BY Apo-2L/TRAIL
FIGURE 4. Effects of sApo-2L and Act D on viability of PBMC and
foreskin fibroblast cells. PBMC (1 3 105 cells/100 ml) were incubated for
18 h in a 96-well culture well with or without Act D in the presence or the
absence of sApo-2L (500 ng/ml). Viability was determined by the XTT
assay. Foreskin fibroblast cells were seeded in a 24-well culture plate and
incubated for 18 h. Each well was washed twice with medium and incubated in the presence or the absence of Act D, with or without sApo-2L.
Eighteen hours later, the number of adherent cells was counted using a
Coulter particle counter. The data represent the mean 6 SD (n 5 3).
reduced the level of the Bcl-xL protein. The expressions of bcl-2
mRNA and Bcl-2 protein in AIDS-KS cells were low compared
with their expression in the Daudi B lymphoma (26, 28).
More recently, several reports have indicated that FADD is essential for Apo-2L/TRAIL-induced apoptosis (7, 9, 12). Following
stimulation of Fas, the death-inducing signaling complex, Fas/
FADD/pro-caspase 8 complex, is formed, leading to the activation
of a caspase cascade and the induction of apoptosis (13, 43). Since
we have shown their involvement of the caspase 8-like activity in
sApo-2L-induced apoptosis in AIDS-KS cells (Fig. 5), we examined the expression of FADD and procaspase 8 (Fig. 7). The levels
of FADD and procaspase 8 proteins were unaffected by Act D treatment. Together, these findings indicate that Act D preferentially
down-regulated the expression of anti-apoptotic Bcl-xL protein.
Discussion
FIGURE 5. Effects of caspase inhibitors on sApo-2L-mediated cell
death in AIDS-KS cells. KS-10B cells were incubated for 18 h with Act D
(10 ng/ml). Before stimulation, KS 10B cells were treated for 1 h with each
of the caspase inhibitors, and the cells were incubated for 6 h with sApo-2L
(500 ng/ml). The number of Act D-treated cells was set as 100%. The data
represent the mean (n 5 2). Vertical bar, sApo-2L (500 ng/ml); dotted bar,
zVAD-fmk plus sApo-2L; solid bar, zDEVD-fmk plus sApo-2L; hatched
bar, zIETD-fmk plus sApo-2L.
Currently, various chemotherapeutic treatments are implemented
for patients with rapidly progressive, extensive, or symptomatic
KS; however, it has been difficult to control symptoms caused by
KS (25). Most patients with AIDS-KS present with severe immune
dysregulation caused by HIV infection, and therefore, there is a
limitation to the use of high dosages of chemotherapeutic drugs
(25). In this study we have obtained evidence in vitro that the
Apo-2L-Act D combination treatment can reduce the minimum
effective dosage of Act D. Act D and sApo-2L efficiently induced
cell death in nine of 10 AIDS-KS isolates. Further, following pretreatment with Act D, sApo-2L killed AIDS-KS cells more efficiently. Transcripts of Apo-2L/TRAIL and its receptors, DR4 and
DR5, were detected in many types of normal tissues, suggesting
that these tissues may be resistant to Apo-2L/TRAIL (13). In this
study we found that Act D cannot sensitize PBMC and fibroblast
cells to sApo-2L. Consequently, sApo-2L and subtoxic Act D may
prove to be beneficial biological therapeutic agents in the absence
of undesirable drug-mediated side effects on AIDS-KS patients.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 6. Effects of Act D treatment on the expression of DcR1,
DcR2, and cFLIP mRNA. AIDS-KS cells were incubated for 18 h with or
without Act D (10 ng/ml), and RNA was purified. First-strand cDNA was
synthesized using reverse transcriptase. PCR was performed using 5 and
0.5 ml of RT reaction. Each PCR product was electrophoresed on 3%
agarose gel for KS-9, KS-10B, and KS-11. For KS-10B, PCR for b-actin
was performed from 1 and 0.1 ml. The numbers below the picture indicate
the relative expression of each mRNA. The intensity of PCR was described
in Materials and Methods
The Journal of Immunology
5621
FIGURE 7. Effects of Act D on the expression of p53, FADD, procaspase 8, Bax, cFLIP, and Bcl-xL proteins. KS-9, KS-10B, and KS-11 cells were
cultured for 18 h in the presence or the absence of Act D (10 ng/ml). Cell lysates were subjected to Western blot analysis.
sential for induction of apoptosis in AIDS-KS cells by sApo-2L.
The caspase cascade in apoptosis may be activated by both a mitochondria-dependent and a mitochondria-independent pathway.
In the mitochondria-independent pathway, the caspase cascade is
activated by death-inducing signaling complex, which consists of
death receptors, apoptotic adaptor molecules, and initiator types of
caspases, such as caspase 2 and 8. TRADD (TNFR-associated
death domain protein), FADD, RIP (receptor interacting protein),
and RAIDD (RIP-associated ICH-1/Ced-3 homologous death domain protein are known as apoptotic adaptor molecules (13, 43, 51,
52). In the mitochondrion-dependent pathway, the initiator
caspase, caspase 9, is activated by Apaf-1 and cytochrome c,
which is released from mitochondria following apoptotic stimuli
(39, 40). It is as yet unknown which pathway is involved in the
Apo-2L-induced apoptosis. Ectopic expression of dominant negative FADD did not inhibit apoptosis by Apo-2L, indicating that a
FADD-independent pathway is linked to activation of a caspase
cascade (4 – 6, 10, 34). Yeh et al. (53) showed that DR4-induced
apoptosis is not inhibited in fibroblast cells derived from FADDdeficient mice, while TNF receptor type I-, Fas-, and DR3-induced
apoptosis was inhibited. However, other laboratories demonstrated
that the dominant negative FADD inhibits DR4- or DR-5-induced
apoptosis (7, 9, 12, 54). Pan et al. (5) claimed no association of
DR4 or DR5 with FADD, TRADD, and caspase 8, while others
presented the association of DR4 or DR5 with TRADD, FADD,
TRAF (TNFR-associated factor), and RIP (9, 12). Further, Pan et
al. (5) showed that DR4 and DR5 are associated with FLICE2
(caspase 10b). Our findings showing inhibition of sApo-2L-induced apoptosis in AIDS-KS cells by zIETD-fmk may suggest the
involvement of caspase 10.
Several lines of evidence show that Bcl-2 and Bcl-xL function as
antiapoptotic molecules mainly at the level of the mitochondria
(39 – 41). It was reported that Bcl-xL binds Apaf-1 and inhibits
activation of caspase 9 (55). Overexpression of Bcl-2 and Bcl-xL
inhibits apoptosis or necrosis induced by a variety of stimuli (56 –
58). Further, Bcl-2 is strongly stained in spindle cells of advanced
AIDS-KS (27). Foreman et al. (28) reported that high levels of
Bcl-x are detected in AIDS-KS lesions, and cultured AIDS-KS
cells preferentially express Bcl-xL. These studies suggest that high
levels of Bcl-2 and Bcl-xL may lead to prolonged survival of
AIDS-KS cells. In this study we show that AIDS-KS spindle cells
preferentially express Bcl-xL, which is markedly reduced by Act D
treatment. Previously, we have reported that the level of bcl-2
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Several lines of evidence show that the AIDS-KS lesion is, at
least in its early stages, a reversible hyperplasia initiated by a complex cascade of cytokines and growth factors (20, 21, 44). Experimental data corroborate that KS cells, central elements in KS lesion, proliferate rapidly in response to various external stimuli,
such as OM (30, 45), IL-6/soluble IL-6R complexes (46), IL-1b,
and TNF-a (20, 47). Recently, we have reported that endogenous
basic fibroblast growth factor is essential for the proliferation of
AIDS-KS cells induced by external cytokines (23). We have also
reported that dexamethasone synergistically enhances OM- or IL6/soluble IL-6R-mediated proliferation of AIDS-KS cells (47), a
finding that may explain the clinical observations that corticosteroid therapy is associated with the development of new KS (48)
and the deterioration of preexisting KS (49). KS lesions are rapidly
aggravated in response to environmental factors such as opportunistic infection and glucocorticoid use for immune suppression
therapy (21, 44). Large amounts of basic fibroblast growth factor
and vascular endothelial growth factor produced from KS cells
(24) may induce severe angiogenesis with bleeding and edema,
resulting in increasing mortality. Therefore, agents that induce
rapid death of AIDS-KS cells may be more effective compounds
for the treatment of AIDS-KS. Herein, we show that the combination treatment of sApo-2L with Act D induced a more rapid
cytotoxic response to AIDS-KS cells than Act D used alone. For
instance, sApo-2L-induced cell blebbing became detectable within
2 h in almost 100% of KS cells pretreated with Act D. These
findings suggest that the combined therapy of sApo-2L and Act D
is anticipated to improve the efficiency of KS treatment. In addition to Act D, we have also tested the effect of chemotherapeutic
drugs, such as adriamycin, cisplatinum, and etoposide. Combination treatment of any of these drugs with sApo-2L synergistically
induced cell death, although the sensitization effects were much
less pronounced than those achieved with Act D (data not shown).
It has been proposed that induction of apoptosis by Apo-2L/
TRAIL requires caspase activation. Crm A, a viral caspase inhibitor, and YVAD-CHO, a tetrapeptide ICE inhibitor, inhibit Apo2L/TRAIL-induced apoptosis (34, 35). In the present study we
show that zVAD-fmk, zDEVD-fmk, and zIETD-fmk markedly inhibited sApo-2L-induced cell death in KS. The caspase 8 inhibitor,
but not the caspase 3 inhibitor, blocked sApo-2L-induced apoptosis in melanoma (50). However, in our studies, there was
significant inhibitory effect by both caspase inhibitors. These data
indicate that caspase 3-like and caspase 8 or 10 activities are es-
5622
APOPTOSIS INDUCTION IN AIDS-KS CELLS BY Apo-2L/TRAIL
Acknowledgments
We thank Dr. W. B. Corey (Huntington Memorial Hospital, Pasadena, CA)
for critical support and Dr. A. Jewett for useful discussions. We also thank
Samantha Nguyen for preparing the manuscript.
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