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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
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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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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-,
lymphotoxin (TNF-ß), 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- within their C-terminal extracellular
domains. As expected, these members share TNF--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). Two death
domain-containing receptors, DR4 and DR5, have been identified as
Apo-2L/TRAIL receptors (4, 5, 6, 7, 8, 9, 10, 11, 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, 17, 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 growth-promoting, inflammatory, and angiogenic cytokines (20, 21, 22, 23, 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 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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 ± SD (n = 3). Cytotoxicity for PBMC was determined using a cell proliferation kit-XTT assay according to the manufacturer’s instructions. Cell viability (%) = 100 x (OD450–650 obtained from sApo-2L-treated cells/OD450–650 obtained from untreated cells).
Morphology
KS-10B cells (2 x 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 µl of ice-cold 70%
ethanol. After incubation for 20 min at room temperature, the cells
were washed, suspended in staining buffer (PBS/10 µg/ml propidium
iodide/50 µg/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 µl of reaction mixture containing 6 µg of total RNA, using a cDNA Preamplification Kit (Life Technologies). The cDNA synthesis reaction mixtures were subjected to PCR amplification (100 µl) under the following conditions for DcR1, DcR2, cellular FLICE (Fas-associated death domain protein-like IL-1-converting enzyme) inhibitory protein (cFLIP), and ß-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), 5'-CAG TGT AAA GAA GGC ACC TTC CGG-3'; DcR1 (downstream), 5'-GCA GGA GTC CCT GGG CTG GTG-3'; DcR2 (upstream), 5'-CAC TAC CTT ATC ATC ATA GTG GTT TT-3'; DcR2 (downstream), 5'-GAA GGA CAT GAA CGC CGC CGG AAA AG-3'; cFLIP (upstream), 5'-GAC CCT TGT GAG CTT CCC TAG TC-3'; cFLIP (downstream), 5'-GAG CAG GTG GGT CTC CAC AGC-3'; and ß-actin (upstream), 5'-ATC TGGCAC CAC ACC TTC TAC AAT GAG CTG CG-3'; ß-actin (downstream), 5'-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3'. 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 µl 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 µg/ml of aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF). The cell lysates (5–30 µg) 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 anti-Bax 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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 sApo-2L. 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--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.
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C). Based on the methods described by Berenbaum (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).
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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.
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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.
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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.
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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 µl of RT reaction mixture. As
shown in Fig. 6, no significant down-regulation of these molecules 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).
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, 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 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-1ß, and TNF- (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 IL-6/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 Apo-2L/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 essential 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, 5, 6, 10, 34). Yeh et al. (53) showed that DR4-induced apoptosis is not inhibited in fibroblast cells derived from FADD-deficient 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, 40, 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, 57, 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 mRNA in AIDS-KS cells was very low (26). Similar findings were reported by Foreman (28). In contrast, the expressions of cFLIP mRNA and Bax, p53, FADD, and procaspase 8 proteins were not affected by Act D treatment. Marked down-regulation of DcR1 and DcR2 mRNA was not observed in AIDS-KS cells by Act D. Griffith et al. (50) reported that there was no correlation between the resistance of cells (>60 different tumors) to TRAIL and the expression of DcR1 and DcR2. Thus, it seems unlikely that DcR1 and DcR2 expression are responsible for resistance of AIDS-KS cells to sApo-2L. Scaffidi et al. (58) demonstrated that Fas signals are transduced exclusively through the mitochondria-dependent pathway in Jurkat and CEM cells, although most Fas-sensitive cells are killed through the mitochondria-independent pathway. Thus, it is possible that Apo-2L-induced signal may pass exclusively through the mitochondria in AIDS-KS cells. Down-regulation of Bcl-xL may be associated with sensitization of AIDS-KS cells by Act D.
Recently, it has been reported that the sensitivity of several melanoma
cells to Apo-2L/TRAIL is increased by cyclohexamide or Act D treatment
(50, 59). However, the underlying mechanism of sensitization remains
unknown. Chemotherapeutic drugs up-regulated the level of DR5 mRNA in a
p53-dependent manner (8). Up-regulation of DR5 was also reported by
TNF- in p53-independent manner (60). Up-regulation of DR5 may lead
to an increase in sensitivity to sApo-2L.
In summary, our data show that Act D sensitizes AIDS-KS cells to sApo-2L-mediated apoptosis. Sensitization was achieved with subtoxic concentrations of Act D. Furthermore, we show that normal fibroblasts and PBMC are not sensitized by Act D to sApo-2L killing. Therefore, our findings in vitro showing synergistic cytotoxic activity of sApo-2L in combination with Act D support their use in vivo in the therapy of drug-resistant AIDS-KS.
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Acknowledgments |
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Footnotes |
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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:
3 Abbreviations used in this paper: CD27, CD27 ligand; Apo-2L, Apo-2 ligand; TRAIL, TNF-related apoptosis-inducing ligand; sApo-2L, recombinant soluble Apo-2L; 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.
Received for publication November 4, 1998. Accepted for publication February 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B pathway. Immunity 7:821.[Medline]
B. Immunity 7:831.[Medline]
B and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity 7:813.[Medline]
) is a potent growth factor for AIDS-associated Kaposi’s sarcoma (KS) cells: the soluble form of gp130 is antagonistic for sIL-6R-induced AIDS-KS cell growth. Int. Immunol. 8:595.. Cancer Res. 58:159.This article has been cited by other articles:
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  L. Galligan, D. B. Longley, M. McEwan, T. R. Wilson, K. McLaughlin, and P. G. Johnston Chemotherapy and TRAIL-mediated colon cancer cell death: the roles of p53, TRAIL receptors, and c-FLIP Mol. Cancer Ther., December 1, 2005; 4(12): 2026 - 2036. [Abstract] [Full Text] [PDF]
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 A. R. Jazirehi, C.-P. Ng, X.-H. Gan, G. Schiller, and B. Bonavida 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., December 1, 2001; 7(12): 3874 - 3883. [Abstract] [Full Text] [PDF]
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 H. Thakkar, X. Chen, F. Tyan, S. Gim, H. Robinson, C. Lee, S. K. Pandey, C. Nwokorie, N. Onwudiwe, and R. K. Srivastava Pro-survival Function of Akt/Protein Kinase B in Prostate Cancer Cells. RELATIONSHIP WITH TRAIL RESISTANCE J. Biol. Chem., October 12, 2001; 276(42): 38361 - 38369. [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. Nesterov, X. Lu, M. Johnson, G. J. Miller, Y. Ivashchenko, and A. S. Kraft Elevated Akt Activity Protects the Prostate Cancer Cell Line LNCaP from TRAIL-induced Apoptosis J. Biol. Chem., March 30, 2001; 276(14): 10767 - 10774. [Abstract] [Full Text] [PDF]
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T. F. Burns and W. S. El-Deiry Identification of Inhibitors of TRAIL-induced Death (ITIDs) in the TRAIL-sensitive Colon Carcinoma Cell Line SW480 Using a Genetic Approach J. Biol. Chem., October 5, 2001; 276(41): 37879 - 37886. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or the absence (
) of sApo-2L (250 ng/ml) and the
viability of non-Act D-treated cells in the presence (
) or the
absence (•) of sApo-2L (250 ng/ml) are shown.
