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Advances in Brief |
Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery [A. P., B. F., R. Y., S. K., J. A. R., S. G. S.] and Departments of Surgical Oncology [K. K. H] and Molecular Pathology [T. J. M.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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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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Our initial attempts to develop an adenoviral vector containing the Bak gene were complicated by high levels of apoptosis induced in the packaging 293 cell line. This problem was overcome by our development of a binary adenoviral vector system containing two adenoviral vectors. One of the two adenoviral vectors, Ad/GT-Bak, contains a Bak gene under the control of the GT promoter and the GAL4/GV16 fusion protein. This Ad/GT-Bak vector can be grown to large quantities in 293 cells without toxicity to the packaging cell line. Bak gene expression can then be induced in target tissues by coadministration of the Ad/GT-Bak vector with the second adenoviral vector in our system, Ad/GV16 (which produces the GAL4/GV16 fusion protein).
Using this novel system, we have found that overexpression of the Bak gene induces high levels of apoptosis in p53 wild-type, mutant, and null tumor cell lines, regardless of the level of the antiapoptotic members of the Bcl-2 family (i.e., Bcl-2 and Bcl-XL). This effect was seen in vitro and in vivo and was accompanied by marked tumor regression of lung cancer cells in a nu/nu s.c. tumor model. Interestingly, despite high levels of transduction, Bak overexpression did not result in apoptosis in a caspase 3-deficient cell line (MCF-7). Furthermore, the addition of the caspase inhibitor z-DEVD-fmk abrogated Bak-mediated tumor killing in tumor cell lines that were previously Bak sensitive. Together, these results suggest that adenoviral-mediated overexpression of the proapoptotic Bak gene is indeed a novel strategy for treating tumors, although the mechanism appears to be caspase dependent.
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Materials and Methods |
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Adenovirus Production.
Construction of the Ad/GT-LacZ and Ad/GV16 vectors was reported
previously (2)
. Ad/CMV-GFP (described hereafter as Ad/GFP)
was obtained from Dr. T. J. Liu (Department of Head and
Neck Surgery, University of Texas M. D. Anderson Cancer Center,
Houston, TX). The Ad/GT-Bak vector was constructed by placing Bak cDNA
(obtained from T. J. M.) downstream of the GAL4/TATA promoter (GT) to
generate the shuttle plasmid pAd/GT-Bak. This plasmid was cotransfected
into 293 cells along with a 35-kb ClaI fragment purified
from human adenoviral type 5 to generate the Ad/GT-Bak vector. The
transduction efficiencies of adenoviral vectors in various cancer cell
lines were determined by infecting cells with Ad/GT-LacZ and then
determining the titers needed to transduce at least 80% of the cells.
Flow Cytometry Analysis.
Apoptotic cell death was examined in terms of changes in cell
morphology and flow cytometric analysis of cells for propidium iodide
exclusion and by TUNEL assay 24–48 h after transduction. Specimens
were analyzed in an EPICS Profile II flow cytometer (Coulter Corp.,
Hialeah, FL).
Western Blot Analysis.
Total cell lysates were prepared by lysing plated cell monolayers with
SDS-PAGE sample buffer. The proteins were transferred to Hybond
enhanced chemiluminescence membranes (Amersham Corp., Arlington
Heights, IL) and incubated with the primary and secondary antibodies
according to the Amersham enhanced chemiluminescence protocol.
In Vivo Tumor Growth and Ad/Bak Treatment.
H1299 and A549 cells (5 x 106
cells/0.2 ml) were injected s.c. into the flanks of female, athymic,
5–6-week-old, nu/nu mice. Once tumors grew to approximately
5 mm x 5 mm, they were then injected directly with
Ad/GT-Bak + Ad/GV16, control vector Ad/GT-Bak + Ad/GFP, control vector Ad/GT-LacZ + Ad/GV16, or PBS. Each
injection was given through a single pass of a 31-gauge hypodermic
needle. A second virus injection was given 3 days after the first
injection. A third virus injection was given 3 days after the second
injection. Tumor volume was calculated by assuming a spherical shape
and using the formula volume = (a x b2)/2, where a and
b are the maximum and minimum diameters, respectively.
Results for six to seven animals from each treatment group were
averaged and expressed as the mean ± SD.
To evaluate apoptosis, tumors were removed 12 h after the third injection of virus in vivo, fixed in 10% formalin, embedded in paraffin blocks, and then assessed by histological analysis and TUNEL assays. For TUNEL assay, sections were dewaxed, rehydrated (55°C for 15 min), washed in xylene, and then rehydrated through a graded series of ethanol and redistilled water. Tissue sections were then incubated with proteinase K, permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate, and labeled with the TUNEL reaction mixture.
Statistical Analysis.
ANOVA and a two-tailed Student’s t test were used for
statistical analysis when appropriate. Significance was set at
P < 0.05.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. \
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demonstrates, Ad/GT-Bak induced high
levels of apoptosis in vitro in all cell lines except MCF-7
as early as 24 h after transduction. The mock-infected (PBS) or
virus control-infected (Ad/GFP and Ad/GT-LacZ) cells demonstrated no
apoptosis (1–3%), despite being infected with similar viral titers.
The Ad/GT-Bak-infected cells, however, demonstrated 40–60% apoptosis
24–48 h after infection (Fig. 1C)
. This apoptosis was
confirmed by characteristic morphological changes, PARP cleavage, and
caspase activation (Fig. 1B)
. PARP and caspase 3, which are
useful as indices of apoptosis, were only cleaved in cells that had
been transduced with Ad/GT-Bak + Ad/GV16. MCF-7 cells were
the only cells that failed to undergo apoptosis, despite adequate Bak
transduction (Fig. 1, B and C)
. Interestingly,
the levels of antiapoptotic Bcl-2 family members did not appear to be
greater in the MCF-7 cells than in the Bak-sensitive cell lines (Fig. 1A)
, suggesting that these proteins were not the cause of
the resistance in MCF-7 cells. As Fig. 1B
demonstrates,
however, MCF-7 cells differed from the other cell lines in
lacking a functional caspase 3. Additionally, no PARP cleavage was
noted in MCF-7 cells after Ad/GT-Bak infection, possibly because of the
caspase 3 deficiency.
To test whether Ad/GT-Bak-induced apoptosis and PARP cleavage were
caspase dependent, we infected H1299 tumor cells with Ad/GT-Bak in the
presence or absence of the caspase blocker z-DEVD-fmk. As Fig. 2A
shows, Bak-induced apoptosis was completely abrogated by
caspase inhibition. Additionally, PARP cleavage was blocked by the
addition of z-DEVD-fmk, despite Bak overexpression (Fig. 2B)
. These results suggest that: (a) MCF-7 cells
may be resistant to Ad/GT-Bak killing because they are caspase
deficient; and (b) Ad/GT-Bak-mediated tumor killing is
caspase dependent.
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). In addition, these observations held true in both p53-null (H1299)
and p53 wild-type (A549; data not shown) cells, suggesting that the
mechanism was p53 independent.
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, demonstrated more of the cellular debris and
morphological changes associated with apoptosis (Fig. 4B)
,
and demonstrated increased TUNEL staining (Fig. 4C)
. In
contrast, treatment with Ad/GT-LacZ or Ad/GFP yielded no histologically
detectable apoptotic responses. Additionally, intratumoral injection of
Ad/GT-Bak caused no significant systemic toxicity. Together, these
results demonstrated the potential antitumor effect of Ad/GT-Bak when
directly injected into tumors.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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To our knowledge, ours is the first report of an antitumoral effect of
adenovirally mediated overexpression of the proapoptotic Bak gene. Bak
is located on chromosome 6 and shares homology with the entire Bcl-2
family, including its antiapoptotic and proapoptotic members
(7)
. In fact, the family members interact through highly
conserved areas of Bcl-2 homology (BH1, BH2, and BH3) that allow
hetero- and homodimerization and the consequent close regulation of
apoptosis. In the case of Bax, this scheme has been well worked out in
a rheostat model in which excess proapoptotic Bax suppresses Bcl-2 and
induces apoptosis via cytochrome c (8)
. Less is
known about Bak, but there is evidence that Bak functions in a similar
manner (7
, 9
, 10)
. If true, such a mechanism of action
would favor the use of adenoviral vectors because high levels of
proapoptotic Bak would presumably be able to overwhelm the levels of
antiapoptotic Bcl-2 members, inducing the cell to undergo
programmed cell death. Our study suggests that this may indeed be the
case because we found that overexpression of Bak was able to induce
apoptosis in cancer cells even in the presence of high levels of
antiapoptotic Bcl-2 family members (Fig. 1A)
.
The one cell line that was resistant to Ad/GT-Bak killing was MCF-7,
although high levels of Bak protein were induced in such cells (Fig. 1, A–C)
. The reason for this seeming contradiction may be that
the MCF-7 cell line lacks a functional caspase 3 as a result of a
genomic mutation that introduces a premature stop codon into the
caspase 3 mRNA (11)
. In the present case, the fact that
the caspase inhibitor z-DEVD-fmk completely abrogated Ad/GT-Bak induced
apoptosis in cell lines that were previously Bak sensitive supports the
hypothesis that Ad/GT-Bak-mediated tumor killing is caspase dependent
(Fig. 2, A and B)
. Unlike Bax, which has been
reported to have caspase-dependent and -independent pathways of
activation, our studies suggest that Bak-induced apoptosis is
predominantly caspase dependent (12)
. In addition, our
studies suggest that this process is p53 independent because
Bak-induced apoptosis occurred equally well in p53 wild-type, null, or
mutant cells. This observation may be explained by the fact that the
Bcl-2 family members function downstream from p53 in the apoptotic
cascade. Indeed, we have observed that Ad-p53 appears to induce
apoptosis in part by up-regulation of Bak and Bax and subsequently
inducing apoptosis, perhaps by caspase activation (13)
.
These observations are important because if Bak functions downstream
from p53, then the clinical use of the Bak vector may be independent of
p53. If so, then treatment of p53 mutant or -resistant cancer cells
with adenoviral Bak might be possible
In summary, we have shown for the first time that adenoviral-mediated overexpression of Bak by Ad/GT-Bak leads to rapid apoptosis of cancer cells in vitro and sustained tumor regression in vivo. This antitumor strategy does not appear to be dependent on p53 status and appears to work even in the presence of high levels of antiapoptotic Bcl-2 family members. The mechanism of action of Bak-mediated apoptosis, however, appears to be caspase dependent. In short, this study suggests that adenoviral overexpression of Bak and other proapoptotic Bcl-2 family members, despite the dependence on caspase, is a novel strategy for cancer gene therapy and is worthy of further exploration.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the National Cancer
Institute and the NIH (Grant P01 CA78778-01A1; to J. A. R.), by
Specialized Program of Research Excellence in Lung Cancer Grant
P50-CA70907, by gifts to the Division of Surgery and Anesthesiology
from Tenneco and Exxon for the Core Laboratory Facility, by The
University of Texas M. D. Anderson Cancer Center Support Core Grant
CA16672, and by a sponsored research agreement with Introgen
Therapeutics, Inc. 
2 To whom requests for reprints should be
addressed, at Department of Thoracic and Cardiovascular Surgery, The
University of Texas M. D. Anderson Cancer Center, 1515 Holcombe
Boulevard, Box 109, Houston, TX 77030. Phone: (713) 745-4530; Fax:
(713) 794-4901; E-mail: sswisher{at}mdanderson.org
3 The abbreviations used are: PARP, poly(ADP
ribose) polymerase; TUNEL, terminal deoxynucleotidyl
transferase-mediated nick end labeling; GFP, green fluorescence
protein.
Received 9/28/99. Accepted 12/17/99.
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