From the Department of Biomedical Sciences, University of California, Riverside, California 92521
Received for publication, February 27, 2003 , and in revised form, April 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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The Bcl-2 family of apoptotic regulators is characterized by the presence
of Bcl-2 homology
(BH)1 domains and is
subdivided into three groups comprising the antiapoptotic members Bcl-2 and
Bcl-XL, the proapoptotic Bax-like proteins including Bak and Bax,
and BH-3 domain-only proteins including Bid, Bik, Bim, and others. The BH-3
domain is a motif of 16 residues forming an amphipathic helix required
for dimerization among Bcl-2 family members and for death induction.
Dimerization between Bcl-2 family members requires the BH-3 domain of one
partner and a groove formed among BH-1, BH-2, and BH-3 domain helices of the
other partner (4), although the
role of dimerization in the regulation of mitochondrial membrane permeability
and cytochrome c release remains incompletely resolved
(5). Recent studies have
revealed that BH-3-only domain proteins require at least one Bax-like partner
to induce cell death (6,
7). Bid is one such
proapoptotic protein that induces a conformational change in Bax or Bak
resulting in a channel-forming complex in mitochondrial membranes
(811).
Bid is recognized as an intracellular link connecting the death receptor
pathway and mitochondrial death machinery through caspase-8 activation
(5). Full-length Bid, normally
present in cytosol, is cleaved by activated caspase-8 to form tBid
(carboxyl-terminal region of Bid), which translocates to the mitochondria
causing cytochrome c release. Although tBid is a potent inducer of
apoptosis, recent studies have shown that under some conditions full-length
Bid can induce cytochrome c release in the absence of proteolytic
cleavage (12).
Because Bid processing can link the extrinsic and intrinsic cell death pathways and amplify death receptor signaling, we have proposed Bid overexpression as a potential therapy for the management of cancer. In the present report, we show that a Tet-OffTM adenoviral vector expressing Bid (Ad-Bid) can result in high levels of Bid expression, tBid cleavage, and apoptosis in human NSCLC lines. The proapoptotic effects of Ad-Bid and tBid processing were viral dose-dependent and enhanced by chemotherapeutic agents such as cisplatin or Fas death receptor engagement. Ad-Bid could restore chemosensitivity to cisplatin in the absence of wild-type p53, suggesting Ad-Bid (alone or in combination with chemotherapy or death receptor engagement) as a potential therapy against cancers lacking functional p53.
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EXPERIMENTAL PROCEDURES |
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AntibodiesAntibodies against human caspase-8, full-length Bid, DFF45, Bak, and Bax were purchased from Pharmingen. Antibodies against human cytochrome c and cytochrome c oxidase subunit 4 were purchased from Clontech. Anti-actin was purchased from Sigma. Secondary horseradish peroxidase-conjugated goat anti-rabbit antibody was obtained from Jackson Immunoresearch Laboratories (West Grove, PA). Antibody against tBid (p15) was purchased from BIOSOURCE International, Inc. (Camarillo, CA).
Adenovirus ProductionRecombinant Tet-OffTM adenovirus
containing human Bid was constructed according to the manufacturer's protocol
(Adeno-XTM Tet-OffTM expression system, Clontech). Human Bid cDNA
was kindly provided by Dr. Gerhard Wagner (Harvard Medical School, Boston,
MA). The construction and characterization of a recombinant adenovirus
expressing human p53 (Ad-p53) and Escherichia coli
-galactosidase (Ad-LacZ) were described previously
(13). Viruses were propagated
in 293 packaging cells; tetracycline was present in the culture medium for
Ad-Bid. Viral titer was determined by plaque assay. Cells were plated in
6-well plates at 1 x 106 cells/well 1 day prior to virus
infection. Unless otherwise indicated, cells were infected with adenovirus at
a total m.o.i. of 5 plaque-forming units/cell. For Bid-infected cells, the
ratio of the two vectors (Ad-Bid and Ad-Tet-OffTM) was 1:1.
Anti-Fas and Drug TreatmentAnti-Fas (IPO-4, Kamiya, Seattle, WA) and cisplatin (Sigma) were used at a concentration of 1 and 2.5 µg/ml, respectively. For combination treatments, cells were plated with 5-m.o.i. Ad-Bid or Ad-LacZ for 24 h before adding anti-Fas or cisplatin and were cultured for an additional 24 h before proliferation was determined using the CellTiter96TM proliferation assay (Promega, Madison, WI).
Western Blot AnalysisCell lines plated 24 h previously in 6-well plates were washed once with ice-cold PBS containing 5 mM EDTA and 1 mM sodium orthovanadate and were harvested by scraping into each well 0.2 ml of ice-cold lysis buffer (1% Triton X-100, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol (v/v), 2 mM EDTA, 1 mM sodium orthovanadate (v/v), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 mg/ml leupeptin). Cell lysates were clarified by centrifugation (5 min at 16,000 x g at 4 °C), and the protein concentration was determined using the DC protein assay (Bio-Rad). For cytochrome c detection in the cytosol, mitochondrial and cytosolic fractions were separated using the ApoAlert® cell fractionation kit (Clontech). Equal amounts of protein were transferred electrophoretically to a Hybond polyvinylidene difluoride transfer membrane (Amersham Biosciences) and incubated with primary and secondary antibodies according to the Supersignal® West Pico chemiluminescence protocol (Pierce).
Flow Cytometric Analysis of Mitochondrial Membrane Potential after Ad-Bid or Anti-Fas TreatmentMitochondrial membrane potential was measured using DiOC6 (3, 3'-dihexyloxacarbocyanine iodide; final concentration 100 nM, excitation wavelength 488 nm, emission 529 nm) as described (14). Cells were plated in 6-well plates at a density of 1 x 106 cells/well for 24 h before infection with Ad-Bid and Ad-Tet-OffTM at a m.o.i. of 5 plaque-forming units/cell. As a control, cells also were treated with anti-Fas and cycloheximide at a concentration of 1 and 10 µg/ml, respectively. Sixteen h after viral infection or anti-Fas treatment, adherent and floating cells were harvested, washed once in PBS, and incubated with DiOC6 for 20 min at 37 °C. Following incubation, cells were washed with ice-cold PBS, resuspended in 500 µl of PBS on ice, and immediately analyzed with a FACScan (fluorescence-activated cell sorter scan; BD Biosciences). The percentage of cells showing decreased DiOC6 fluorescence represents cells with decreased mitochondrial membrane potential.
Flow Cytometric Analysis of ApoptosisCells were plated in 24-well plates at a density of 2 x 105 cells/well 1 day prior to infection at a m.o.i. of 5 plaque-forming units/cell. After 48 h, adherent cells and floating cells were harvested, washed once with PBS, and fixed in 70% (v/v) ethanol overnight at 4 °C. Fixed cells were resuspended in PBS containing 0.2% Triton X-100 and 1 mg/ml RNase for 5 min and then stained with propidium iodide at 50 µg/ml to determine subdiploid DNA content using a FACScan (fluorescence-activated cell sorter scan). Doublets, cell debris, and fixation artifacts were gated out, and sub-G0/G1 DNA content was determined using Cell Quest software, version 3.3.
Inhibition and Measurement of Caspase ActivityCaspase-8- and -9-like activity was determined using ApoAlert® caspase colorimetric assay kits (Clontech). Caspase-3-like activity was determined using the ApoTarget® caspase/CPP32 colorimetric assay kit (BIOSOURCE International, Inc.). The -fold increase in protease activity was determined by comparing the levels of caspase activity in treated cells with those in untreated cells or control cells (Ad-LacZ). As a positive control for caspase activity, Jurkat cells were treated with recombinant human soluble SUPERFasTM ligand (50 ng/ml) purchased from Alexis Corporation (San Diego, CA). For caspase inhibition, H460 cells were preincubated with 50 mM zVAD-fmk (BioVision, Inc., Mountain View, CA) for 30 min prior to infection. Cells were cultured for 24 h after infection before being harvested for Western blot analysis of Bid cleavage.
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RESULTS |
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Mitochondria-associated Changes in NSCLC Induced by Ad-BidActivation of effector caspase-3 and -9 suggested that Ad-Bid was inducing mitochondrial membrane changes and cytochrome c release like that observed with Bax and Bak overexpression (15, 16). Because BH-3 domain-only proteins such as Bid require at least one Bax-like partner to induce apoptosis (8), we examined Bak and Bax expression in the three lung cancer cell lines by Western blotting. Bax was expressed equivalently in all three cell lines (Fig. 3A), whereas Bak expression was considerably more variable with A549 cells expressing low levels as reported (16).
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To determine whether Ad-Bid infection corresponded with changes in mitochondrial permeability, cell lines were treated with the fluorescent dye DiOC6 to target mitochondrial events. As shown in Fig. 3B, Ad-Bid caused a significant reduction in DiOC6 fluorescence, indicating mitochondrial depolarization. The changes elicited by Ad-Bid treatment were more striking than those observed with anti-Fas. These events were accompanied by a sharp increase in cytosolic cytochrome c detectable by Western blotting (Fig. 3C) and seen only in Ad-Bid-treated cells and not in either mock-infected or tetracycline-treated Ad-Bid-infected cells. The mitochondria-associated protein Cox4 is shown as a control. Experiments were repeated three times with similar findings.
DNA Fragmentation and Death in Ad-Bid-infected NSCLCTo
document that mitochondrial membrane changes were accompanied by the induction
of cell death, lung cancer cell lines were examined for sub-G1 DNA
content using propidium iodide staining and flow cytometry 48 h after Ad-Bid
infection. Infection with the Ad-LacZ control resulted in little, if any, DNA
fragmentation (range 0.87.8%), whereas Ad-Bid infection resulted in a
marked increase in sub-G1 DNA content (range 11.080.0%) in
all three cell lines (Fig.
4A). For the sake of comparison, DNA fragmentation also
is shown for Ad-p53, a therapy currently in clinical trials for lung cancer
and head and neck cancer
(1719).
Ad-Bid was consistently more effective than Ad-p53 in the induction of DNA
fragmentation and cell death (Fig.
4) at comparable multiplicities of infection. Such effects were
most striking in H460 and A549 cells, where Ad-p53 resulted in 20% loss
of cell viability 48 h following infection with 5 m.o.i. of virus, whereas
Ad-Bid at the same viral dose and time resulted in
90% cell death. In
H358 cells, which are relatively resistant to Ad-p53-induced apoptosis
(
10% loss of cell viability), Ad-Bid induced >50% cell death. Similar
findings were observed in three independent experiments.
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Enhanced Apoptosis in NSCLC Infected with Ad-Bid Treated with Cisplatin
or Anti-FasThe goal of preclinical studies is to maximize tumor
cell killing, particularly in more resistant tumors such as the p53-null tumor
H358. Because Bid can be processed by caspase-8 activation, we reasoned that
enhanced killing might be observed with Ad-Bid in combination with activators
of caspase-8. To this end, we examined the combined effects of Ad-Bid and
either cisplatin or anti-Fas on tumor cell killing. Both anti-Fas and
cisplatin treatment activated caspase-8 in H460 and H358 cells. In H460 cells,
anti-Fas induced an approximately 5-fold increase in caspase-8 activity,
whereas cisplatin induced a 45-fold increase
(Fig. 5A). In H358
cells, anti-Fas and cisplatin treatment resulted in a more modest activation
of caspase-8 with a 1.52-fold activation by anti-Fas and a
23-fold activation by cisplatin. As a positive control, caspase-8
activation is shown for both cell lines following treatment with soluble Fas
ligand (Fig. 5A).
These experiments were repeated twice with similar findings. H358 and H460
cell lines express low levels of Fas and are moderately sensitive to anti-Fas
killing (20,
21).
Fig. 5B shows the loss
of cellular viability in Ad-LacZ-infected H460 and H358 treated with anti-Fas
antibody; in both cases, anti-Fas treatment resulted in 40% cell killing.
When Bid was overexpressed, however, both lung cancer cell lines showed a
marked enhancement of Fas sensitivity. In the case of H460, Ad-Bid treatment
resulted in the death of
90% of the cells (consistent with
Fig. 4), and the addition of
anti-Fas further diminished viable cell counts to <5% of the starting
population. In the case of the more resistant cancer cell line H358, Bid
overexpression resulted in the death of
50% of the cells (consistent with
Fig. 4), and the addition of
anti-Fas diminished viability to <5% of the starting population. Likewise,
when Bid was overexpressed, both cell lines show a marked sensitivity to
cisplatin. Ad-LacZ-transduced H460 cells showed
60% cell death after
treatment with cisplatin, whereas the Ad-Bid and cisplatin combination induced
>95% cell death (Fig.
5C). In the H358 lung cancer cells, Ad-LacZ treatment in
combination with cisplatin induced
35% cell death, whereas the Ad-Bid and
cisplatin combination increased killing to
90%. These experiments were
repeated four times with similar findings. As expected based upon the loss of
cellular viability, Ad-Bid in combination with anti-Fas or cisplatin also
increased DFF45 cleavage (Fig.
6A) and tBid processing
(Fig. 6B) in both cell
lines (n = 2). Taken together, these data suggest that agents capable
of activating caspase processing of Bid may enhance Ad-Bid-induced tumor cell
killing in an additive or superadditive manner.
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DISCUSSION |
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Our studies suggest that Bid overexpression in NSCLC cell lines results in potent apoptosis as measured by changes in mitochondrial transmembrane potential, cytochrome c release, DNA fragmentation, and loss of cell viability (Fig. 3, B and C, and Fig. 4). Cell death induced by Ad-Bid is p53-independent in that Bid effectively kills both p53 wild-type (A549, H460) and p53-null (H358) cells, suggesting that Bid, like other Bcl-2 family members, functions downstream of p53. Ad-Bid can induce apoptosis in NSCLC cells expressing high levels of antiapoptotic Bcl-2 family members (16, 23), suggesting that Bid can overcome Bcl-2 inhibition of the Bak and Bax conformational changes required for the induction of cell death (2428). Because Bid can induce apoptosis in partnership with either Bak or Bax, Ad-Bid also may be effective in tumors containing mutations in one of these proteins (29, 30).
NSCLC cell lines infected with Ad-Bid express large quantities of Bid protein (Fig. 1A). When Bid was overexpressed, both full-length Bid (24 kDa) and tBid (15 kDa) were detectable by Western blotting (Fig. 1A). Detection of tBid was dependent on the viral dose used for infection (Fig. 1B) with tBid observed at viral doses of 5 m.o.i. or greater. Interestingly, Ad-Bid infection consistently induced both caspase-9- and caspase-3-like activity in NSCLC cells at levels 45-fold above that observed with mock-infected cells (Fig. 2A). In NSCLC cell lines infected with Ad-LacZ or Ad-p53 (another proapoptotic gene), caspase-9 and -3 activities were elevated in the range of 23-fold, respectively. Thus, Bid overexpression elevated downstream caspase activities above the level observed with adenoviral infection alone (Ad-LacZ) or adenoviral expression of another apoptotic gene (Ad-p53). The mechanism underlying elevated caspase activities in Ad-Bid-infected cells is unresolved but likely reflects tBid-induced amplification of the mitochondrial pathway of apoptosis (cytochrome c release and activation of caspase-9). Interestingly, although tBid represented only a small fraction of the total Bid protein following Ad-Bid infection (Fig. 1), the addition of zVAD-fmk blocked the generation of both tBid and cell death (Fig. 2, B and C), suggesting that endogenous caspase-mediated processing of Bid was required for its biological effects.
Because Bid is recognized as an intracellular link connecting death
receptors and the mitochondrial apoptotic pathway through caspase-8 activation
(4,
5), Ad-Bid-induced apoptosis
should be enhanced by agents that increase caspase-8 such as cisplatin and
anti-Fas. As shown in Fig.
5A, both agents activated caspase-8 activity in NSCLC
cell lines and potently enhanced cell killing by Ad-Bid
(Fig. 5, B and
C). As expected with increased caspase-8 activity, tBid
and DFF45 cleavage also were elevated in the combination Ad-Bid treatments
(Fig. 6). Of particular note is
the restoration of chemosensitivity to cisplatin in p53-null H358 after Ad-Bid
treatment. These cells normally are resistant to cisplatin killing, showing
about 35% cell death when treated with the control virus Ad-LacZ and cisplatin
(2.5 µg/ml). Ad-Bid alone induced 50% cell death, whereas the
combination of Ad-Bid and cisplatin resulted in >90% loss of cell
viability. These studies suggest that Ad-Bid can induce chemosensitization in
the absence of functional p53 and that Ad-Bid in combination with cisplatin or
Fas receptor engagement might offer a strategy to maximize the apoptotic
signal in malignant cells, including those lacking p53 function.
In summary, we have shown that adenovirally mediated overexpression of Bid leads to the rapid and potent activation of apoptosis in malignant NSCLC cell lines in a p53-independent manner and in the presence of high levels of antiapoptotic Bcl-2 family members. Caspase-dependent processing of tBid occurs in Ad-Bid-infected cells and can be enhanced further through caspase-8 (cisplatin, Fas engagement), leading to additive or superadditive cell killing. These results suggest that gene transfer with Bid alone or Bid in combination with either chemotherapy or death receptor engagement may be a useful strategy for the treatment of malignant disease. Because Bid killing is non-selective (i.e. not specific to malignant cells), the development of tissue- and tumor-specific promoter systems will be required to ensure specificity and limit death in normal tissues. Additional studies currently are underway to develop Ad-Bid therapeutic vectors that can be used for preclinical evaluation.
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FOOTNOTES |
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To whom correspondence should be addressed: University of California, Dept. of
Biomedical Sciences, 1274 Webber Hall, Riverside, CA 92521. Tel.:
909-787-2583; Fax: 909-787-2438; E-mail:
laurie.owenschaub{at}ucr.edu.
1 The abbreviations used are: BH, Bcl-2 homology; tBid, truncated Bid; Ad,
adenovirus; Ad-Bid, adenoviral vector expressing Bid; Tet, tetracycline;
NSCLC, non-small cell lung cancer(s); m.o.i., multiplicity of infection; PBS,
phosphate-buffered saline; DiOC6, 3,
3'-dihexyloxacarbocyanine iodide; DFF, DNA fragmentation factor; fmk,
fluoromethyl ketone; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fmk.
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ACKNOWLEDGMENTS |
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REFERENCES |
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