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Experimental Therapeutics |
Departments of Medicine [X. L., M. Mara., J. Y., B. N., M. Marc.] and Molecular and Cellular Biology [M. Marc.], Baylor College of Medicine and Veterans Affairs Medical Center; Department of Thoracic and Cardiovascular Surgery, M. D. Anderson Cancer Center [J. A. R., S. K., B. F.]; and Department of Molecular and Cellular Biology, Texas Biotechnology Corporation [L. D.], Houston, Texas 77030
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In situ gene therapy represents an attractive alternative for the treatment of prostate cancer for several reasons. Primary prostate cancer is accessible by ultrasounds, and therapeutic genes can be directly inoculated into the neoplastic lesion. The prostate is relatively dispensable after the reproductive years so that, in contrast to cancers arising from organs regulating vital functions, specific promoters are unnecessary. In addition, disease progression or regression can be monitored using serial prostate-specific antigen measurements. Because prostate cancer is a relatively slow-growing disease, it is likely that treatment would include several inoculations of possibly more than one therapeutic gene. Another potential advantage of adenovirus-mediated gene therapy for prostate cancer is that intraprostatic gene delivery is associated with minimal side effects in patients treated to date (5) and minimal extraprostatic transduction of the therapeutic gene in experimental animals (6 , 7) . Thus, implementation of gene therapy for the primary lesion of prostate cancer is, at least in theory, a possible alternative to current treatment modalities. Nevertheless, reliable technology to reach metastatic lesions has not yet been developed.
Using a number of apoptotic substances, including lovastatin (8) , sodium phenyl acetate, and staurosporine (9 , 10) , we have identified at least two steps of the apoptotic pathway whose activation is necessary to achieve apoptosis of prostate cancer cell lines: (a) activation of the caspase pathway (9) ; and (b) release of apoptotic molecules such as cytochrome c from the mitochondria to the cytosol (10) . On the basis of these observations, we hypothesized that manipulation of these two steps of the apoptotic pathway by gene therapy may result in induction of therapeutic apoptosis of prostate cancer cells. Here we show that adenovirus-mediated overexpression of Bax induces therapeutic programmed cell by triggering the mitochondrial pathway of apoptosis in a variety of prostate cancer cells growing in vivo and in vitro.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
LNCaP (13)
, LNCaP-Bcl-2 (14)
, PC-3
(15)
, DU-145 (16)
, and TsuPr(1)
(17)
cells have been described previously
(10)
. LNCaP, LNCaP-Bcl-2, and TsuPr(1)
cells were grown in
RPMI 1640 supplemented with 10% FBS and 1% P&S (and 400 µg/ml G418
in LNCaP-Bcl-2). PC-3 cells were grown in F12 supplemented with 10%
FBS and 1% P&S. DU-145 cells were grown in DMEM + 10% FBS
and 1% P&S.
Adenoviral Constructs and Infection Protocol.
First-generation adenoviruses driven by the constitutively active Rous
sarcoma virus promoter and containing the cDNAs of caspase-3 and
caspase-7 (AvC3 and AvC7) have been described previously (9
, 18)
. The binary system for the overexpression of Bax consists of
two adenoviruses (Ad/PGK/GV16 and Ad/GT-Bax) that have also been
described previously (19)
. A previously described AvLac-Z
virus (9)
containing the Lac-Z cDNA was used as a control
for AvC3 and AvC7. A binary system overexpressing Lac-Z
(Ad/PGK/GV16 + Ad/GT-Lac-Z; Ref. 20
) was used as
a control in the experiments involving the binary system overexpressing
Bax.
Two days before each experiment 1 x 105 cells were seeded in a six-well plate. On the day of the infection, one of the six wells was trypsinized, and the cells were counted. This information was used to infect each cell line at the desired MOIs. Infections were in 5% CO2 incubators at 37% for 1 h using 500 µl of infection medium (the same medium used for each cell line + 2% FBS and 1% P&S) on a rocker. Pilot experiments with AvLac-Z determined the optimal MOI for each cell line. In these experiments (data not shown), all cells became Lac-Z-positive without manifesting toxicity for up to 7 days after infection at MOIs between 10: 1 and 100:1. Thus, a MOI of 100:1 was used in the experiments shown in this report. Similar results were obtained using the control binary system Ad/PGK/GV16 + Ad/GT-Lac-Z (not shown). The experiments shown here used a MOI of 100:1 of the binary system, in which one-third of the viral particles were Ad/PGK/GV16 and two-thirds were Ad/GT-Lac-Z or Ad/GT-Bax.
In vivo experiments were carried on with PC-3 cells. s.c.
tumors of PC-3 cells were obtained by s.c. inoculation of 5 x 105
cells mixed in 20% Matrigel (diluted
in normal saline to a volume of 100 µl). Tumors were inoculated in
the right and left flanks of 12 animals (total tumors,
n = 24). Twenty days after inoculation,
tumors reached a diameter of 
0.4 mm with a 100% penetrance. At this
point, tumors were injected with 1 x 1010 pfu of a mixture containing one-third
Ad/PGK/GV16 + two-thirds Ad/GT-Lac-Z (the control group,
n = 12) or one-third Ad/PGK/GV16 + two-thirds Ad/GT-Bax (the therapeutic group, n = 12) every 5 days for a total of three inoculations. Tumor size
was determined with a caliper at a 5-day interval, and the size was
determined using the formula
(m1)2 x
m2 x 0.5236, where
m1 is the shorter axis, and
m2 the longer axis (21)
. Ten
days after the last treatment, mice were sacrificed, and the tumors
were weighed. A complete autopsy of the mice was done to rule out
macroscopic side effects. Statistical analysis was done using a
two-tailed paired Student’s t test and ANOVA.
Analysis of the Apoptotic Pathway.
The apoptotic pathway was analyzed as described previously. Briefly, 3
days after infection with the AvC7 and AvC3 viruses and 24 h after
infection with the binary system overexpressing Bax, cells were
harvested and analyzed as described previously for caspase-7,
caspase-3, or Bax overexpression, pro-caspase-9, pro-caspase-3, and
pro-caspase-7 processing, cytochrome c redistribution, DFF
cleavage, DEVDase activity, and TUNEL (8, 9, 10)
.
Western Analysis and Subcellular Fractionation.
Western analysis was performed as described previously
(8, 9, 10)
. In each experiment, the same number of µg of
cell lysate was loaded, as specified in each case. In some experiments,
ß-actin was simultaneously immunodetected, to verify loading of
similar amounts of cell lysates. Subcellular fractionation was
performed using serial centrifugation steps as described by Gross
et al. (22
, 23)
with some modifications.
Briefly, cells were washed twice in ice-cold PBS, resuspended in five
volumes of extraction buffer [containing 220 mM
mannitol, 68 mM sucrose, 50
mM PIPES-KOH (pH 7.4), 50
mM KCl, 5 mM EGTA, 2
mM MgCl2, 1
mM DTT, and protease inhibitors (Sigma; added at
1;100 dilution)] and kept on ice for 15 min. Cells were then spun at
400 x g for 10 min at 4°C to separate out
nuclei and unbroken cells. This supernatant was centrifuged at
10,000 x g for 10 min at 4°C to collect
the heavy membrane, mitochondria-enriched pellet. The new supernatant
was then spun at 100,000 x g for 30 min at
4°C to separate the light membrane endoplasmic reticulum-enriched
pellet (not used in these experiments) from the supernatant (containing
the cytosol). Pilot experiments demonstrated the ability of this
technique to yield subcellular fractions enriched with mitochondria or
cytosol. For instance, cytochrome c was recovered uniquely
from the mitochondrial fraction in cells not undergoing apoptosis and
from both the cytosolic and mitochondrial fractions when cells were
undergoing apoptosis. In contrast, the proteins voltage-dependent anion
channel (located on the outer mitochondrial membrane) was recovered
uniquely from the mitochondrial fraction, regardless of whether the
cells were undergoing apoptosis (not
shown).5
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and other cell lines (not shown) was time dependent. Despite
substantial pro-caspase-3 overexpression (Fig. 2)
, we were unable to detect DEVDase activity (not shown), TUNEL (not
shown), or cleavage of apoptotic substrates such as DFF (Fig. 2)
in any
of the cell lines tested. Thus, adenovirus-mediated pro-caspase-3
overexpression was not followed by autocatalytic activation of the
overexpressed protein and did not induce therapeutic apoptosis of
prostate cancer cell lines.
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and other cell lines
(not shown) with kinetics similar to pro-caspase-3. Overexpressed
pro-caspase-7 was catalytically activated (Fig. 3)
, induced DFF cleavage (Fig. 2)
, and led to TUNEL in LNCaP and
LNCaP-Bcl-2 but not in PC-3, TsuPr(1)
, and DU-145 cells (Fig. 3)
.
Further analysis showed that activation of the apoptotic pathway was
accompanied by cleavage of the proapoptotic molecule Bid (Fig. 4B)
and translocation of cytochrome c from the
mitochondria to the cytoplasm (Fig. 4C)
. In the cell lines
where caspase-7 did not undergo autocatalytic activation or in all of
the cell lines infected with the control virus AvLac-Z, Bid was
uncleaved and cytochrome c was not translocated (Fig. 4
,
A, B, and D). Fig. 2
shows that
adenovirus-mediated pro-caspase-7 overexpression was associated with
the presence of smaller bands when the various cell lines were analyzed
by Western analysis using the anti-caspase-7 antibody. These bands were
recognized by an antibody, the epitope of which is between amino acids
4 and 125 of pro-caspase-7. Thus, it is likely that they represent
either NH2-terminal proteolytic products of the
full-length protein that are recognized by the
NH2-terminal antibody. In alternative, they may
represent truncated proteins initiated at the level of one of the
internal methionines localized before residue 125. In either case, the
smaller bands cannot be the active subunits of active caspase-7,
because these are of COOH-terminal derivation and would not be
recognized by the antibody.
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Overexpression of Bax Induces Apoptosis of Prostate Cancer Cell
Lines.
Because we were unable to engage the apoptotic apparatus by
overexpressing the distal caspases, we turned our attention to more
proximal molecules. Specifically, we looked into the reported ability
of pro-apoptotic Bcl-2 family members, such as Bax, to trigger
apoptosis by functionally inactivating the mitochondria and forcing the
release of cytochrome c (22)
. Bax was
overexpressed with a system using two adenoviruses (19)
.
The first (Ad/PGK/GV16) produces a powerful transcription factor, the
GAL4-VP16 fusion protein under the control of the constitutively active
PGK promoter. The second (Ad/GT-Bax) produces Bax under the control of
a GAL/TATA minipromoter. Thus, the constitutively produced GAL4-VP16
binds the GAL/TATA minipromoter and drives transcription of the Bax
cDNA. Using this system, Bax was dramatically overexpressed in each of
the cell lines used within 24 h (as illustrated for PC-3 cells in
Fig. 5A
). No Bax overexpression was present in cells infected with
the Ad/PGK/GV16 + Ad/GT-Lac-Z binary system (the control for
the Bax overexpression system; Fig. 5A
). Within 24 h
after infection, Bax overexpression was followed by redistribution of
cytochrome c to the cytoplasm (Fig. 5, G and H)
. This, in turn, was followed by activation of caspase-9,
caspase-3, and caspase-7, induction of DEVDase activity, cleavage of
the apoptotic substrate DFF, induction of TUNEL, and DNA laddering.
Fig. 5
shows this sequence of events in PC-3 cells, compared with cells
treated with the control binary system Ad/PGK/GV16 + Ad/GT-Lac-Z. A similar sequence of events was also observed in the
other cell lines, with the only exception of DU-145 cells, where no
activation of caspase-7 was observed. Nevertheless, in all cell lines
Bax overexpression resulted in induction of DEVDase activity and TUNEL
(Fig. 6)
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, three intratumoral injections of Ad/PGK/GV16 + Ad/GT-Bax
caused a highly significant (P < 0.0006)
25% regression of PC-3 tumors that was only evident after 15 days of
exposure to the Bax virus and 5 days after the last injection. This
represented a 4-fold reduction in tumor volume compared with those
treated with the control virus combination of Ad/PGK/GV16 + Ad/GT-Lac-Z (P < 0.00005). No macroscopic
damage was evident in the area surrounding the s.c. tumor, the liver,
lungs, heart, and kidneys of the animals in both groups.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The molecular mechanisms for the inability of overexpressed pro-caspase-3 to undergo activation are unclear. A previous report demonstrated that ectopic overexpression of pro-caspase-3 is followed by apoptosis of the host cells only if one uses a chimeric caspase-3 molecule in which the order of the various subunits is rearranged (27) . Thus, it is conceivable that the native pro-caspase-3 molecule is not a good substrate for autoactivation after overexpression. It is also possible that overexpressed pro-caspase-3 is sequestered into a compartment that precludes autoactivation by, for example, acidification-dependent release of the safety catch.6
The ability of pro-caspase-7 to undergo cleavage in some cell lines but not others suggests a cell-specific mechanism that confers protection from apoptosis. Antiapoptotic molecules may prevent caspase-7 activation. For example, we showed by Western analysis that the caspase inhibitor XIAP (28) is expressed to a higher degree in DU-145 cells compared with LNCaP cells (not shown). Thus, it is possible that XIAP, other members of the IAP family (29) , or the differential expression of IAP inhibitors such as Smac/Diablo (30 , 31) may play a role in preventing activation of overexpressed caspases in prostate cancer cell lines. Interestingly, in LNCaP and LNCaP-Bcl-2 cells, active caspase-7 activated upstream, proximal components of the mitochondrial pathway by, for example, cleaving the proapoptotic Bcl-2 family member Bid. In addition, caspase-7 induced apoptosis through direct cleavage of downstream, distal apoptotic substrates such as DFF.
Bax overexpression caused cytochrome c release, activation of the caspase pathway, and apoptosis of every prostate cancer cell line. It is possible that cytoplasmic release of the mitochondrial protein Smac (30 , 31) contributed to the apoptotic effect of overexpressed Bax. These observations imply that manipulation of Bax expression has broad application to the induction of therapeutic apoptosis. Bax overexpression resulted in apoptotic death also of cell lines such as PC-3 and DU-145, which are resistant to some forms of chemically induced apoptosis (10) . Furthermore, Bax overexpression had the ability to bypass the antiapoptotic effect of Bcl-2, which is stably overexpressed in LNCaP-Bcl-2 cells (14) , and Bcl-XL, which is naturally overexpressed in PC-3 cells.5 Furthermore, Bax overexpression induced massive apoptosis in DU-145 cells, where the Bax protein is not expressed because of a nonsense mutation of the gene (32) .
The apparent general ability of Bax overexpression to engage the apoptotic machinery and induce cell death in vitro suggested that Bax overexpression may be effective in vivo in models of prostate cancer. Indeed, three inoculations of the binary system overexpressing Bax caused regression of s.c. PC-3 tumors and remarkable inhibition of tumor size compared with the continued growth of tumors injected with the control virus. Because Bax overexpression induced apoptosis in PC-3 cells, which are particularly resistant to apoptosis (10 , 33 , 34) , these results support the likely success of this strategy in treating all of the genotypically and phenotypically diverse cell types in both organ-confined and metastatic prostate cancer. Additional support of the idea that Bax can act as a global inducer of therapeutic apoptosis comes also from the experience of other investigators, who have used different cell lines and experimental models with results similar to ours (19 , 20 , 35) . Furthermore, it is likely that treatment optimization may cause complete tumor regression. For example, the dose chosen in the current experiments was dictated by technical considerations such that we used the largest volume that could be inoculated in these relatively small tumors without causing a major spread to either the surrounding tissues or the skin of the animals.
Limitations of currently available treatments have driven interest to develop new experimental therapies for prostate cancer. Attempts have been made to transduce the prostate with various therapeutic genes, including HSV-TK (5 , 36, 37, 38, 39) , p53 (40 , 41) , p16 (41 , 42) , p21 (41 , 43) , IL-12 (44) , and C-CAM (45) using a variety of vectors and prostate specific (7 , 46) or constitutively active viral promoters. Most of the gene therapy approaches for prostate cancer that have been reported consist in the inoculation of adenoviruses containing the HSV-TK construct (47) . The use of genes such as Bax represents an evolution from HSV-TK, because the former is cytotoxic without requiring exposure to substances like gancyclovir. On the basis of the results presented in this report, we think that the main issue with the use of apoptotic genes is not whether they will cause cell death but rather how to optimally target them to specific tissues and tumors. Although we did not identify any side effects attributable to the extravasation of the Bax virus to tissue other than the s.c. tumors, it is predictable that death genes such as Bax will induce a suicidal response in every tissue in which they are concentrated. This is especially true if they are driven, as in our studies, by a constitutively active promoter. Previous literature suggesting that treatment of patients with the intraprostatic delivery of adenoviruses containing HSV-TK is not associated with significant side effects (5 , 39) . Similarly, intraprostatic inoculation of adenoviruses in dogs is not associated with significant extraprostatic spread of the virus of interest (6 , 7) . Nevertheless, we think that prostate-specific promoters should be developed to target Bax overexpression uniquely to prostatic epithelium. Thus, one of the key challenges for this field is to identify prostate specific promoters that restrict expression of the therapeutic gene to prostatic epithelium. Promoters specific to prostate epithelial cells have been used successfully to create transgenic models of prostate cancer (48, 49, 50) and to direct gene expression to prostate cancer cells (7 , 46 , 51) . The questions that remain to be asked is whether these promoters are powerful enough to sufficiently overexpress the protein of interest to obtain an apoptotic effect. In conclusion, although Bax overexpression is a powerful way to induce apoptosis in experimental models of prostate cancer, further work is necessary before this approach can be used for the treatment of human disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Veterans Affairs Merit Review
Program, the Department of Defense Prostate Cancer Research Program,
the Baylor Specialized Program of Research Excellence on Prostate
Cancer, and the Chao Foundation (to M. M.). 
2 These authors share first authorship.
3 To whom requests for reprints should be
addressed, at Department of Medicine, Baylor College of Medicine and
Veterans Affairs Medical Center, 2002 Holcombe Boulevard, Houston, TX
77030. Phone: (713) 794-7945; Fax: (713) 794-7714; E-mail: marcelli{at}bcm.tmc.edu
4 The abbreviations used are: DFF, DNA
fragmentation factor; TUNEL, terminal deoxynucleotidyl
transferase-mediated nick end labeling; P&S, penicillin and
streptomycin; PGK, phosphoglycerokinase; FBS, fetal bovine serum; MOI,
multiplicity of infection.
5 X-Y. Li, M. Marani, R. Mannucci, B. Kinsey, F.
Andriani, I. Nicoletti, L. Denner, and M. Marcelli. Overexpression of
BCL-XL underlies the molecular basis for resistance to
staurosporine-induced apoptosis on PC-3 cells, submitted for
publication.
6 Roy and Nicholson, personal
communication.
Received 8/ 4/00. Accepted 11/ 1/00.
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