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Departments of
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Urology and
Internal Medicine, University of Iowa, Iowa City, IA 52242
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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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Given the tumor cell selectivity of TRAIL’s cytotoxicity from results obtained in vitro, recent studies have examined the safety and antitumor activity of soluble rTRAIL in vivo (20, 21, 22). TRAIL was found to be well tolerated when multiple doses were given to normal animals, and no observable histological or functional changes were observed in any of the tissues or organs examined. These results were dramatically different from those seen in animals given other apoptosis-inducing molecules, as injection of recombinant Fas ligand or anti-Fas mAb into animals rapidly induced massive hepatocyte degeneration, necrosis, and hemorrhage, and ultimately death (20, 23, 24). Moreover, multiple injections of soluble TRAIL into mice beginning the day after tumor implantation significantly suppressed the growth of the tumors, with many animals being tumor free (20, 21, 22). One potential drawback to these findings was that large amounts of soluble TRAIL were required to inhibit tumor formation. This may be due to the pharmacokinetic profile of soluble TRAIL that indicated that after i.v. injection the majority of the protein is cleared within 5 h (20). Increasing the in vivo t1/2 of soluble rTRAIL or developing an alternative means of delivery may increase the relative tumoricidal activity of TRAIL such that larger, more established tumors could be eradicated as efficiently as smaller tumors. The identification of alternate methods to deliver TRAIL to the tumor site, however, is also critical for the further development and testing of the antitumor activity of TRAIL in vivo. The results presented in this work describe the production of an adenoviral vector engineered to carry the gene for hTRAIL. Shortly after cell infection, TRAIL protein was detected, rapidly leading to the induction of apoptosis in TRAIL-sensitive tumor cells in vitro. Moreover, these results demonstrate the potential of using adenoviral-mediated delivery and local expression of TRAIL to destroy tumors in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The tripeptide caspase inhibitor, z-VAD-fmk, was obtained from Enzyme Systems Products (Livermore, CA). A stock solution of the inhibitor was prepared in DMSO and stored at 4°C. Brefeldin A (BFA) was purchased from Epicentre Technologies (Madison, WI), with a stock solution prepared in 100% EtOH and stored at -20°C. Abs against caspase-8 (provided by Dr. Marcus Peter, University of Chicago), poly(ADP-ribose) polymerase (PARP; PharMingen, San Diego, CA), and hTRAIL (PeproTech, Rocky Hill, NJ) were used for Western blotting according to manufacturer’s instruction. Soluble rhTRAIL was purchased from PeproTech and used at the indicated concentrations.
Cell lines
The human prostate carcinoma cell line (PC-3) was obtained from Dr. Michael Cohen (University of Iowa, Iowa City, IA). The human melanoma cell lines (WM 164 and WM 793) were obtained from Dr. Meenhard Herlyn (Wistar Institute, Philadelphia, PA). The human mammary adenocarcinoma cell line (MDA 231) was obtained from Dr. David Lynch (Immunex, Seattle, WA). The human bladder cancer cell line (RT-4) was obtained from Dr. Scott Crist (University of Iowa). PC-3, RT-4, WM 164, and WM 793 were cultured in DMEM supplemented with 10% FBS, penicillin, streptomycin, sodium pyruvate, nonessential amino acids, and HEPES (hereafter referred to as complete DMEM). MDA 231 was cultured in RPMI 1640 supplemented with 10% FBS, penicillin, streptomycin, sodium pyruvate, nonessential amino acids, and HEPES (hereafter referred to as complete RPMI). Normal human prostate epithelial cells (PrEC) were obtained from Clonetics (San Diego, CA) and cultured as directed.
Production of adenovirus encoding the hTRAIL gene
The cDNA for hTRAIL was obtained from Dr. Hideo Yagita (Juntendo University, Tokyo, Japan) (25). A replication-deficient adenovirus encoding the hTRAIL gene (Ad5-TRAIL) expressed from the CMV promotor was generated using standard methods by the University of Iowa Gene Transfer Vector Core (26). Briefly, the entire coding sequence of hTRAIL was cloned into the XhoI and NotI sites of pAd5CMVK-NpA. The resultant plasmid and adenovirus backbone sequences (Ad5) (27) that had the E1 (E1A and E1B) genes deleted were transfected into human embryonic kidney 293 cells, and viral particles were isolated and amplified for analysis of TRAIL expression. Recombinant adenoviruses encoding nuclear-targeted bacterial ß-galactosidase (Ad5-ßgal) or green fluorescent protein (Ad5-GFP) were used as virus controls. Recombinant adenoviruses were screened for replication competent virus by A549 plaque assay, and virus titer was determined by plaque assay on 293 cells. Purified viruses were stored in PBS with 3% sucrose and kept at -80°C until use.
Adenoviral infection
Cells were cultured in complete medium, and permitted to adhere for at least 6 h before adding adenovirus. Before infection, cells were washed with PBS, and then the vectors were added at the indicated number of PFU/cell in culture medium supplemented, as described above, but with only 2% FBS. After 4 h, cells were washed with PBS and incubated in complete medium for the remainder of the assay.
Reported gene assays for measuring adenoviral infection
Analysis of adenoviral infection efficiency and transferred gene expression was performed using Ad5-GFP and Ad5-ßgal, respectively. Cells infected with Ad5-GFP were analyzed by flow cytometry on a FACScan (Becton Dickinson, San Jose, CA) at various time points after infection to determine infection efficiency. Cells infected with Ad5-ßgal were assayed for ß-galactosidase activity with the Galacto-Light Plus chemiluminescent reporter gene assay system (Tropix, Bedford, MA) to determine the level of transferred gene expression.
In vitro killing of human tumor cell lines with adenoviral vectors
Tumor cell sensitivity to Ad5-ßgal, Ad5-GFP, or Ad5-TRAIL was assayed using the following procedure. Cells were added to 96-well plates (2 x 104 cells/well) in complete medium, and then allowed to adhere for at least 6 h before infection with the various adenoviral vectors, as described above. As a positive control, soluble rhTRAIL was added to the target cells at the indicated concentrations. In some experiments, z-VAD-fmk (20 µM) TRAIL-R2:Fc (20 µg/ml; Immunex), Fas:Fc (20 µg/ml; PharMingen), or brefeldin A (5 µg/ml) was added to the medium during and after infection for the remainder of the assay. Cell death was determined after 20 h by crystal violet staining, as described (28). Results are presented as percent cell death: (1 - (OD cells treated per OD cells not treated)) x 100. For analysis of apoptosis, tumor cell targets were incubated as described above and apoptotic cell death was measured by flow cytometry using FITC-conjugated annexin V (R&D Systems, Minneapolis, MN) and propidium iodide (Sigma, St. Louis, MO), as described (29, 30).
Western blotting
Cells were lysed in PBS containing 1% Nonidet P-40, 0.35 mg/ml PMSF, 9.5 µg/ml leupeptin, and 13.7 µg/ml pepstatin A. The lysed cells were centrifuged at 14,000 x g to remove cellular debris, and protein concentrations of the lysates were determined by the colorimetric bicinchoninic acid analysis (Pierce, Rockford, IL). Equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membrane (Novex, San Diego, CA), and blocked with 5% nonfat dry milk in PBS Tween-20 (0.05% v/v) overnight. The membrane was incubated with the anti-caspase-8, anti-PARP, or anti-hTRAIL mAb (diluted according to manufacturer’s instructions) for 1 h. After washing, the membrane was incubated with an anti-mouse or anti-rabbit HRP Ab (diluted 1/1000; Amersham, Arlington Heights, IL) for 1 h. Following several washes, the blots were developed by chemiluminescence according to the manufacturer’s protocol (Renaissance chemiluminescence reagent; DuPont NEN, Boston, MA).
Flow cytometry
Surface expression of TRAIL was determined by measuring the binding of the anti-hTRAIL mAb M181 (mouse IgG1; Immunex). Briefly, cells were incubated with 10 µg/ml M181 or MOPC-21 (nonspecific mouse IgG1 isotype control; Sigma) in 3% BSA in PBS (PBSA) for 30 min on ice. Following three washes with PBS, cells were incubated with a PE-conjugated, Fc-specific goat anti-mouse F(ab')2 (Jackson ImmunoResearch, West Grove, PA) for 30 min on ice. Finally, after three washes in PBS, the cells were analyzed on a FACScan (Becton Dickinson).
Cell-mediated killing of human tumor cells
WM 164 cells were infected with 1000 PFU/cell Ad5-TRAIL for 4 h as above, incubated in complete medium for 12 h, washed, and resuspended in complete medium. PC-3 tumor cells were labeled with 100 µCi of 51Cr for 1 h at 37°C, washed three times, and resuspended in complete medium. To determine TRAIL-induced death, 51Cr-labeled tumor cells (104/well) were incubated with varying numbers of Ad5-TRAIL/WM 164 effector cells for 8 h. As a positive control, soluble TRAIL was added to the target cells at the indicated concentrations. In some cultures, TRAIL-R2:Fc or Fas:Fc (20 µg/ml) was added to the Ad5-TRAIL/WM 164 effector cells 15 min before adding tumor cell targets. Assays were performed in round-bottom 96-well plates, and the percent specific lysis was calculated as: 100 x (experimental cpm - spontaneous cpm)/(total cpm - spontaneous cpm). Spontaneous and total release were determined in the presence of either medium alone or 1% Nonidet P-40, respectively. The presence of TRAIL-R2:Fc or Fas:Fc during the assay had no effect on the level of spontaneous release of 51Cr by the target cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cDNA encoding full-length hTRAIL was inserted into the E1
region of a replication-deficient Ad5 construct under the control of
the CMV immediate early promoter (Fig. 1
A). This plasmid was
transfected into human embryonic kidney 293 cells to propagate the
virus. Ad5-TRAIL-infected 293 or uninfected 293 cells were lysed, and
the cellular proteins were separated by nonreducing SDS-PAGE to assay
for TRAIL expression by Western blotting. Amino acid sequence analysis
of the TRAIL cDNA predicts a weight of 32.5 kDa for TRAIL monomers
(9, 10). As demonstrated in Fig. 1B, prominent
bands migrating at 32–35 kDa and 55–58 kDa are clearly evident, which
correspond to TRAIL monomers and dimers, respectively. In contrast, no
corresponding bands were present in the uninfected 293 cell lysate. Ab
specificity was demonstrated by using rTRAIL comprised of the entire
extracellular domain of 169 aa, which yields a monomer of 19.6 kDa.
Higher order forms of the rTRAIL migrating at approximately 40 kDa
(dimer) and 60 kDa (trimer) were also present. The differences in
monomer and dimer sizes reflect the fact that one is only the
extracellular domain (rTRAIL) while the other is full length
(Ad-TRAIL-derived). The lack of detectable higher order multimers in
the 293/Ad-TRAIL lysate may be due to the method of cellular lysis and
only analyzing the solubilized proteins not associated with the cell
membrane. It is possible that full-length TRAIL trimers require support
from the cell membrane to remain in this form, which would be absent
from the soluble component of the cell lysate containing mostly
monomeric and dimeric forms. Thus, these results demonstrate that the
adenoviral-mediated gene transfer of hTRAIL results in transgene
expression in human cells.
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One of the advantages of using an adenoviral vector lies in the
ability to infect epithelial cell populations. Group C adenovirus, such
as Ad5, requires the interaction between the viral fiber capsid protein
to the coxsackievirus and adenovirus receptor, or CAR, and the viral
penton base binding to certain integrins (e.g.,
vß3 and
vß5) for entry into
the cell by receptor-mediated endocytosis (31, 32, 33).
Therefore, it was critical to determine whether a panel of human tumor
cell lines (MDA 231, mammary adenocarcinoma; PC-3, prostate carcinoma;
RT-4, bladder papilloma; WM 164, melanoma; and WM 793, melanoma) would
be receptive to adenoviral infection before examining the effects of
Ad5-TRAIL infection. The tumor cells were infected with either an
adenovirus carrying the enhanced green fluorescent protein gene
(Ad5-GFP) or the ß-galactosidase gene (Ad5-ßgal) for 4 h, and
then analyzed 20 h later. When infected with 1000 PFU/cell
Ad5-GFP, all of the tumor cell lines demonstrated a high percentage of
infectivity, ranging from 84.7 to 99.1%, as measured by flow cytometry
(Fig. 2A). In addition, normal
PrEC were also found to be highly susceptible to Ad5-GFP infection
(95.3%). Infection with Ad5-ßgal revealed that all of the cell types
produced protein from the transferred gene in a PFU/cell-dependent
manner; however, there were greater differences in ß-galactosidase
activity between the different cell types than seen when examining GFP
production (Fig. 2B). Thus, these results indicate that
adenoviral-mediated transfer of the ß-galactosidase and GFP reporter
genes into the cells of interest resulted in efficient gene
transcription and translation into protein, suggesting that infection
with Ad5-TRAIL should produce TRAIL protein in a similar percentage of
cells.
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With the demonstration that the human tumor cell line panel was
adequately infected with adenovirus, subsequent experiments were
performed to examine the consequences of Ad5-TRAIL infection. The tumor
cells were infected with either Ad5-ßgal or Ad5-TRAIL for 4 h,
and then cultured for an additional 20 h before determining the
amount of cell death. As indicated in Fig. 3A, minimal cell death of PC-3
cells was observed upon infection with Ad5-ßgal. In contrast, a
significant increase in cell death was seen with Ad5-TRAIL infection.
Moreover, the level of cell death induced by Ad5-TRAIL infection was
comparable with that of soluble TRAIL-induced death. This cytotoxic
activity was seen with other TRAIL-sensitive tumor cell targets, but
not with the TRAIL-resistant melanoma cell line WM 164 or normal PrEC
(Table I). Analysis of TRAIL protein
production by Western blot revealed detectable levels in PC-3 cell
lysates by 1 h postinfection, with levels increasing over the
entire time course (Fig. 3B). In contrast, lysates from
uninfected PC-3 cells or PC-3 cells examined 20 h after Ad5-ßgal
infection had no detectable TRAIL protein present. Thus, these results
demonstrate that tumor cells infected with Ad5-TRAIL produce TRAIL
protein that, presumably, leads to their death.
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Although crystal violet staining of the tumor cells infected with
the adenoviral vectors or treated with rTRAIL as presented in Fig. 3
indicates the amount of cell death, it does not discriminate between
apoptotic and necrotic cell death. Previous reports have demonstrated
that TRAIL-induced cell death occurs through an apoptotic mechanism
characterized by the activation of a cascade of intracellular
proteases, or caspases, and the cleavage of numerous intracellular
proteins (9, 10, 14, 34, 35, 36). To confirm that the tumor
cell death following Ad5-TRAIL infection was mediated through an
apoptotic mechanism, caspase activation and cellular protein cleavage
were examined. Thus, PC-3 cells were infected with Ad5-TRAIL for 4
h, cell lysates were prepared at various times after infection, and the
cellular proteins were separated by SDS-PAGE for Western blot analysis
of caspase-8 activation and PARP cleavage. Activation of caspase-8
occurred within 2 h after infection, while PARP cleavage took
place by 4 h after infection (Fig. 4A). By 20 h after
infection, the levels of the active p18 subunit of caspase-8 and 85-kDa
fragment of PARP had dropped below the level of detection, due to
extensive apoptotic destruction. To further demonstrate the importance
of caspase activation in the death of Ad5-TRAIL-infected cells, the
caspase inhibitor z-VAD-fmk (carbobenzyloxy-Val-Ala-Asp (OMe)
fluoromethyl ketone) was added to the culture medium throughout the
assay. z-VAD-fmk completely inhibited PC-3 cell death, whereas equal
concentrations of the peptide vehicle (DMSO) did not (Fig. 4B).
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C), providing further evidence
that the death of the Ad5-TRAIL-infected tumor cells was through an
apoptotic mechanism. Morphological changes, such as membrane blebbing
and the release of apoptotic bodies, were also observed in cells
infected with Ad5-TRAIL using light microscopy (data not shown). Ad5-TRAIL-induced death can be inhibited with BFA, but not TRAIL receptor:Fc
The results obtained to date demonstrated that Ad5-TRAIL infection
leads to TRAIL protein production and the subsequent induction of
apoptotic cell death. However, it was important to also demonstrate the
cell death to be a TRAIL-dependent phenomenon with the expression of
TRAIL on the surface of the infected cells. Thus, the TRAIL-resistant
(both soluble TRAIL and Ad5-TRAIL) human melanoma WM164 was infected
with Ad5-ßgal or Ad5-TRAIL as in previous experiments, and then
analyzed for TRAIL expression by flow cytometry after 8 h.
TRAIL-resistant WM 164 cells were used in this study because the cell
death that occurred in the TRAIL-sensitive PC-3 cells following
Ad5-TRAIL infection made them difficult to analyze accurately as
nonspecific staining increased as the cells became apoptotic. Whereas
no TRAIL was detectable on uninfected or Ad5-ßgal-infected WM 164
cells, those cells infected with Ad5-TRAIL did express TRAIL on the
cell surface (Fig. 5A).
Interestingly, treatment of the Ad5-TRAIL-infected WM 164 cells with
BFA resulted in the inhibition of TRAIL expression at the cell surface.
BFA blocks the anterograde migration of proteins through the Golgi
complex, and thus prevents their expression on the cell surface. The
BFA treatment did not, however, inhibit the production of TRAIL
protein, as both treated and untreated Ad5-TRAIL-infected WM 164
cell lysates contained TRAIL protein as determined by Western blotting
(Fig. 5B).
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A) at concentrations that
completely inhibited cell death induced by soluble TRAIL (Fig. 6B). It was reasoned that since the PC-3 cells are adherent,
nonpolarized cells TRAIL could be expressed on surfaces that were
inaccessible to TRAIL-R2:Fc, but still able to engage the TRAIL-R1 or
TRAIL-R2 expressed there. Seeing that BFA could inhibit the expression
of TRAIL (Fig. 5A), the same experiment was tried, but in
the presence of BFA or its vehicle EtOH. In this setting, the addition
of BFA was able to block the cell death resulting from Ad5-TRAIL
infection, whereas EtOH did not (Fig. 6C). This inhibition
by BFA did not interfere with the signaling mechanism of TRAIL-R1/R2 as
BFA-treated PC-3 cells were as sensitive to soluble TRAIL-induced death
as those cultured without BFA (data not shown). Further support for
TRAIL-mediated killing following Ad5-TRAIL infection was obtained by
using Ad5-TRAIL-infected PrEC or WM 164 cells to induce apoptosis in
PC-3 target cells. PrEC or WM 164 was infected with 1000 PFU/cell
Ad5-TRAIL for 4 h, and then 12 h later incubated with
51Cr-labeled PC-3 cells at various E:T cell
ratios. Whereas uninfected PrEC or WM 164 demonstrated no cytolytic
activity against the PC-3 target cells, the Ad5-TRAIL-infected cells
displayed comparable activity to soluble TRAIL (Fig. 6D).
This activity was TRAIL specific, as inclusion of soluble TRAIL-R2:Fc,
but not Fas:Fc, to the Ad5-TRAIL-infected WM 164 cells blocked target
cell lysis (Fig. 6E). Similar results were also obtained
with PrEC (data not shown). Collectively, the results presented in
Figs. 5 and 6 demonstrate that the apoptotic death following Ad5-TRAIL
infection results from TRAIL expression on the cell surface, where it
binds to either TRAIL-R1 or TRAIL-R2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The identification of TRAIL several years ago generated a great deal of
interest, when it was determined that it appeared to have the ability
to induce apoptosis in a variety of tumor cell lines, but not in normal
cells in vitro. Moreover, it was observed that TRAIL mRNA is
constitutively expressed in a wide variety of cells and tissues. These
were unusual characteristics for a death-inducing molecule in the TNF
family, as the expression of TNF, lymphotoxin-, and Fas ligand is
tightly regulated since they can have toxic effects on normal tissues.
The tumor-specific activity of TRAIL was extended in vivo with the
observation that treatment of SCID and nude mice bearing human tumors
with soluble TRAIL significantly inhibited tumor outgrowth without any
observable toxic side effects to the host (20, 21, 22). This
inhibition of tumor outgrowth, though, required high amounts of TRAIL
given over several days shortly after tumor implantation.
Pharmacokinetic analysis revealed that soluble TRAIL given to mice i.v.
displayed an elimination t1/2 of just
under 5 h (20). Given that many normal tissues
express mRNA for at least one of the four TRAIL receptors, this
suggests that almost all the tissues in the body have the potential to
bind and sequester soluble TRAIL and, thus, prevent it from reaching
the tumor.
An alternative approach would be to administer TRAIL locally, where it would exist at a greater concentration and have a better chance of significantly inducing tumor cell death. Such localized, intratumoral injections of soluble TRAIL would, however, be limited in that only a relatively small volume could be administered, suggesting that a potentially suboptimal amount of TRAIL protein would be used. In contrast, adenovirus can be produced at high titers, such that small volumes would contain high numbers of infectious adenoviral particles carrying the hTRAIL gene. The use of the CMV promoter to drive the transcription of the transferred hTRAIL gene serves as an additional mechanism to further increase the local concentration of TRAIL protein, because it may not be transcriptionally regulated in the same manner as the TRAIL promoter. Only when the process of apoptosis has disrupted cellular functions sufficiently to affect protein production in the Ad5-TRAIL-infected tumor cell will the generation of TRAIL stop.
The concept of gene therapy has recently developed into a viable method of treating malignant transformation and cancer progression. Whereas some therapies have focused on replacing the absent critical functional genes in the target cells to restore a normal phenotype, other approaches have been based on introducing genes that encode immunostimulatory molecules to activate the immune system against the tumor. Many of these studies have employed the use of replication-deficient adenoviral vectors derived from Ad5 to transfer the gene of interest into the target tumor cells. For example, adenoviral vectors encoding CD80, IFN-ß, IL-2, IL-7, and IL-12 have all demonstrated the ability to stimulate antitumor responses after administration (41, 42, 43, 44, 45, 46). The combination of adenovirus-mediated delivery of the herpes simplex virus thymidine kinase gene and ganciclovir therapy has proved efficacious in treating prostate cancer (47, 48). Also, adenoviral vectors expressing Fas ligand have been tested in the treatment of prostate cancer models, experimental glioma, and renal carcinoma (49, 50, 51). Even with these promising observations, immunogenicity remains a potential problem with adenoviral-based vectors. Abs present in the patient may quickly neutralize the adenovirus before it can deliver its genetic load, as there is widespread immunity to a variety of adenovirus serotypes in humans. Recent results from a phase I clinical trial, however, reported the safety of using adenoviral vectors as a gene delivery vehicle in humans and demonstrated successful transgene expression even in the presence of preexisting immunity to adenovirus (43). Intratumoral administration of Ad5-TRAIL may provide the virus with an appropriate environment for infection of the tumor and surrounding tissue, which will lead to gene expression and sufficient TRAIL protein production to induce tumor cell death. Such therapeutic studies with Ad5-TRAIL are in progress.
It was surprising that there were differences in tumor cell death
following Ad5-TRAIL infection as compared with soluble TRAIL-induced
death. The relative activity of Ad5-TRAIL is dependent upon its ability
to infect a target cell. Adenovirus infection requires the expression
of CAR (coxsackievirus and adenovirus receptor) and the expression of
certain integrins, such as
vß3 and
vß5
(31, 32, 33). All of the tumor cell lines and the normal PrEC
were highly susceptible to adenovirus infection, suggesting that each
cell type differentially regulated the translation of TRAIL mRNA into
protein. Additional regulation may result in the transport of any TRAIL
that is produced from the cytoplasm to the cell surface at different
rates. The surface expression of TRAIL is required for apoptosis
induction, as the inhibition of protein transport by BFA inhibited cell
death, but not the production of TRAIL protein, following Ad5-TRAIL
infection.
Whereas surface expression of TRAIL is essential for the observed tumoricidal activity of Ad5-TRAIL, the sensitivity of the cell to TRAIL-induced apoptosis is also an important component of this phenomenon. This was best demonstrated by the fact that the melanoma cell line WM 164 and the normal PrEC, which were resistant to soluble TRAIL-mediated apoptosis, were also resistant to effects of Ad5-TRAIL infection. The identification of two TRAIL receptors that are capable of initiating the apoptotic machinery (TRAIL-R1 and TRAIL-R2) and two that are not (TRAIL-R3 and TRAIL-R4) led to the initial hypothesis that the expression of TRAIL-R3 and/or TRAIL-R4 conferred resistance to TRAIL-induced death (11, 12, 18, 19). However, this hypothesis was formulated by examining TRAIL receptor mRNA expression in several normal tissues and tumor cell lines, and from experiments in which TRAIL-R3 or TRAIL-R4 were overexpressed in transfected cells. Most of the tumor cell lines used in this study express TRAIL-R3 and/or TRAIL-R4, yet were sensitive to TRAIL (soluble or Ad5-TRAIL-derived)-mediated death (36, 52), so it is unlikely that expression of either TRAIL-R3 or TRAIL-R4 plays a role in determining their resistance to TRAIL. One possible explanation for the resistance of the WM 164 cells to Ad5-TRAIL may come from a component of the cell death machinery called FLIP (Fas-associated death domain-like IL-1-converting enzyme inhibitory protein). FLIP is believed to inhibit the death receptor signaling machinery at its most proximal point by preventing the interaction of caspase-8 and/or Fas-associated death domain protein to the death domains of cross-linked death receptors, and thus inhibit any downstream apoptotic signaling events (53). Intracellular levels of FLIP are high in the TRAIL-resistant melanoma cell line WM 164 (36), and high FLIP levels have also been shown to correlate with resistance to TRAIL-mediated apoptosis in primary vs transformed keratinocytes (54). While FLIP may have a protective function in the WM 164 tumor cell line, it is likely to be one of several intracellular proteins that cooperate with other molecules (both intracellular and at the cell surface) to regulate TRAIL-induced death in tumor cell lines.
The participation of TRAIL-R3 and TRAIL-R4 in regulating TRAIL sensitivity may be greater, however, in normal cells/tissues or primary tumors than in established tumor cell lines. The treatment of normal human umbilical vein or microvascular endothelial cells with phospholipase C (to strip the GPI-linked TRAIL-R3 from the surface) and cycloheximide (to prevent the reexpression of any TRAIL-R3) sensitized these cells to TRAIL (13). This would suggest that TRAIL-R3 is a key regulator of the sensitivity of normal cells to TRAIL-induced death, but the addition of cycloheximide may inhibit the production of some other protein (perhaps FLIP) critical for TRAIL resistance. Furthermore, it is not known how much TRAIL-R3 or TRAIL-R4 is needed to inhibit the formation of a competent TRAIL-R1 or TRAIL-R2 signaling complex. RT-PCR analysis of the normal PrEC detected mRNA species for all four TRAIL receptors (T.S.G., unpublished observation), making it possible for TRAIL-R3 and/or TRAIL-R4 to enter into the TRAIL-ligated receptor complex on the PrEC, and thus making it incapable of initiating apoptosis. As with the tumor cell lines, it is difficult to determine at this time what is the exact mechanism that regulates TRAIL sensitivity and resistance in normal cells and tissues. Additional studies will be required to determine whether regulation is determined at the surface (i.e., TRAIL-R3, TRAIL-R4 expression), within the cell (i.e., FLIP, Bcl-2 family member), or both.
The observed suicide-like death of the Ad5-TRAIL-infected tumor cells
is not the only mechanism by which tumor cells may die with this kind
of gene transfer therapy in vivo. The intralesional injection of
Ad5-TRAIL would likely result in the infection of both cancerous and
normal cells surrounding the injection site. While the normal PrEC
tested in the report were not killed when infected with Ad5-TRAIL, they
still produced TRAIL protein from the transferred gene, as evidenced by
the fact that they could then be used to kill PC-3 cells in a
TRAIL-dependent manner (Fig. 6, D and E). This
suggests that it would not be imperative for Ad5-TRAIL to infect the
tumor cell, as infection in either the tumor itself or the surrounding
normal tissue would lead to the localized production of TRAIL. In
addition, the apoptotic death resulting from Ad5-TRAIL infection may
help initiate a T cell-mediated immune response against any remaining
tumor cells. Recent reports have demonstrated that immature dendritic
cells can engulf apoptotic bodies and present Ags derived from these
cell fragments in an MHC class I-restricted fashion upon maturation,
resulting in the CTL activity (55, 56). Likewise, the
combination of Ad5-TRAIL with an immunostimulatory cytokine (i.e.,
IL-12, IFN-
) may result in the initiation of a tumor-specific immune
response against any remaining tumor cells. Conversely, one potential
disadvantage to using the Ad5-TRAIL gene therapy would be the chance of
inducing TRAIL expression on a normally TRAIL-resistant tumor cell,
giving it another means to evade or repel cell-mediated attempts of
tumor rejection. Indeed, mammary adenocarcinoma cells engineered to
express TRAIL demonstrated enhanced growth in vivo compared with the
parental tumor cells (57). This situation is similar to
that reported for a number of Fas ligand-expressing tumors (8, 58, 59, 60). Careful evaluation of the primary tumor will be
necessary before Ad5-TRAIL injection to minimize this possibility.
The development of alternate or adjuvant forms of cancer therapy is crucial, due to the increasing rates of many cancers throughout the world. For example, prostate cancer is one of the most prevalent cancers among U.S. males, with annual death rates currently estimated at over 40,000 (61). Current treatment for localized prostate cancer is limited to surgery or radiation therapy, whereas androgen ablation is generally accepted as the best method for treating metastatic prostate cancer. Unfortunately, a significant number of patients with advanced prostate cancer fail to demonstrate any initial positive response to androgen ablation therapy. Moreover, prostatic cells often lose their dependency on androgen during cancer progression, and androgen ablation becomes ineffective, leading to tumor progression and death within 3 yr. The development of an adenovirus expressing the gene for hTRAIL is the first of what will likely be numerous gene therapy vectors to treat cancer in the future.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas S. Griffith, Department of Urology, 428 MRC, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242-1089.
3 Abbreviations used in this paper: TRAIL, TNF-related apoptosis-inducing ligand; Ad5, adenovirus 5; BFA, brefeldin A; EtOH, ethanol; FLIP, Fas-associated death domain-like IL-1-converting enzyme inhibitory protein; GFP, green fluorescent protein; hTRAIL, human TRAIL; PARP, poly(ADP-ribose) polymerase; PrEC, prostate epithelial cells; Ad5-ßgal, recombinant adenovirus encoding nuclear-targeted bacterial ß-galactosidase; Ad5-GFP, recombinant adenovirus encoding nuclear-targeted green fluorescent protein.
Received for publication March 7, 2000. Accepted for publication June 13, 2000.
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  L. S. Sasportas, R. Kasmieh, H. Wakimoto, S. Hingtgen, J. A. J. M. van de Water, G. Mohapatra, J. L. Figueiredo, R. L. Martuza, R. Weissleder, and K. Shah Assessment of therapeutic efficacy and fate of engineered human mesenchymal stem cells for cancer therapy PNAS, March 24, 2009; 106(12): 4822 - 4827. [Abstract] [Full Text] [PDF]
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 S. Hingtgen, X. Ren, E. Terwilliger, M. Classon, R. Weissleder, and K. Shah Targeting multiple pathways in gliomas with stem cell and viral delivered S-TRAIL and Temozolomide Mol. Cancer Ther., November 1, 2008; 7(11): 3575 - 3585. [Abstract] [Full Text] [PDF]
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 R. L. VanOosten and T. S. Griffith Activation of Tumor-Specific CD8+ T Cells after Intratumoral Ad5-TRAIL/CpG Oligodeoxynucleotide Combination Therapy Cancer Res., December 15, 2007; 67(24): 11980 - 11990. [Abstract] [Full Text] [PDF]
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M. F. Corsten, R. Miranda, R. Kasmieh, A. M. Krichevsky, R. Weissleder, and K. Shah MicroRNA-21 Knockdown Disrupts Glioma Growth In vivo and Displays Synergistic Cytotoxicity with Neural Precursor Cell Delivered S-TRAIL in Human Gliomas Cancer Res., October 1, 2007; 67(19): 8994 - 9000. [Abstract] [Full Text] [PDF]
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C. Lin, W. Wei, J. Zhang, S. Liu, Y. Liu, and D. Zheng Formyl peptide receptor-like 1 mediated endogenous TRAIL gene expression with tumoricidal activity Mol. Cancer Ther., October 1, 2007; 6(10): 2618 - 2625. [Abstract] [Full Text] [PDF]
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 E. Terzioglu, A. Bisgin, A. D. Sanlioglu, M. Ulker, V. Yazisiz, S. Tuzuner, and S. Sanlioglu Concurrent gene therapy strategies effectively destroy synoviocytes of patients with rheumatoid arthritis Rheumatology, May 1, 2007; 46(5): 783 - 789. [Abstract] [Full Text] [PDF]
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 C. Carlo-Stella, C. Lavazza, A. Locatelli, L. Vigano, A. M. Gianni, and L. Gianni Targeting TRAIL Agonistic Receptors for Cancer Therapy Clin. Cancer Res., April 15, 2007; 13(8): 2313 - 2317. [Abstract] [Full Text] [PDF]
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 K. Miller-Jensen, K. A. Janes, Y.-L. Wong, L. G. Griffith, and D. A. Lauffenburger Adenoviral vector saturates Akt pro-survival signaling and blocks insulin-mediated rescue of tumor-necrosis-factor-induced apoptosis J. Cell Sci., September 15, 2006; 119(18): 3788 - 3798. [Abstract] [Full Text] [PDF]
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 J.-M. Yun, M.-H. Kweon, H. Kwon, J.-K. Hwang, and H. Mukhtar Induction of apoptosis and cell cycle arrest by a chalcone panduratin A isolated from Kaempferia pandurata in androgen-independent human prostate cancer cells PC3 and DU145 Carcinogenesis, July 1, 2006; 27(7): 1454 - 1464. [Abstract] [Full Text] [PDF]
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J. K. Earel Jr., R. L. VanOosten, and T. S. Griffith Histone Deacetylase Inhibitors Modulate the Sensitivity of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Resistant Bladder Tumor Cells Cancer Res., January 1, 2006; 66(1): 499 - 507. [Abstract] [Full Text] [PDF]
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 T. J. Kemp, A. T. Ludwig, J. K. Earel, J. M. Moore, R. L. VanOosten, B. Moses, K. Leidal, W. M. Nauseef, and T. S. Griffith Neutrophil stimulation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) results in the release of functional soluble TRAIL/Apo-2L Blood, November 15, 2005; 106(10): 3474 - 3482. [Abstract] [Full Text] [PDF]
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X. Zhang, R. M. Cheung, R. Komaki, B. Fang, and J. Y. Chang Radiotherapy Sensitization by Tumor-Specific TRAIL Gene Targeting Improves Survival of Mice Bearing Human Non-Small Cell Lung Cancer Clin. Cancer Res., September 15, 2005; 11(18): 6657 - 6668. [Abstract] [Full Text] [PDF]
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J. Shi, D. Zheng, Y. Liu, M. H. Sham, P. Tam, F. Farzaneh, and R. Xu Overexpression of Soluble TRAIL Induces Apoptosis in Human Lung Adenocarcinoma and Inhibits Growth of Tumor Xenografts in Nude Mice Cancer Res., March 1, 2005; 65(5): 1687 - 1692. [Abstract] [Full Text] [PDF]
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A. T. Ludwig, J. M. Moore, Y. Luo, X. Chen, N. A. Saltsgaver, M. A. O'Donnell, and T. S. Griffith Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand: A Novel Mechanism for Bacillus Calmette-Guerin-Induced Antitumor Activity Cancer Res., May 15, 2004; 64(10): 3386 - 3390. [Abstract] [Full Text] [PDF]
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D. Jacob, J. Davis, H. Zhu, L. Zhang, F. Teraishi, S. Wu, F. C. Marini III, and B. Fang Suppressing Orthotopic Pancreatic Tumor Growth with a Fiber-Modified Adenovector Expressing the TRAIL Gene from the Human Telomerase Reverse Transcriptase Promoter Clin. Cancer Res., May 15, 2004; 10(10): 3535 - 3541. [Abstract] [Full Text] [PDF]
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S. Armeanu, U. M. Lauer, I. Smirnow, M. Schenk, T. S. Weiss, M. Gregor, and M. Bitzer Adenoviral Gene Transfer of Tumor Necrosis Factor-related Apoptosis-Inducing Ligand Overcomes an Impaired Response of Hepatoma Cells but Causes Severe Apoptosis in Primary Human Hepatocytes Cancer Res., May 15, 2003; 63(10): 2369 - 2372. [Abstract] [Full Text] [PDF]
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 A. F. Taghiyev, N. V. Guseva, H. Harada, C. M. Knudson, O. W. Rokhlin, and M. B. Cohen Overexpression of BAD Potentiates Sensitivity to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Treatment in the Prostatic Carcinoma Cell Line LNCaP Mol. Cancer Res., May 1, 2003; 1(7): 500 - 507. [Abstract] [Full Text] [PDF]
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M. Ehtesham, P. Kabos, M. A. R. Gutierrez, N. H. C. Chung, T. S. Griffith, K. L. Black, and J. S. Yu Induction of Glioblastoma Apoptosis Using Neural Stem Cell-mediated Delivery of Tumor Necrosis Factor-related Apoptosis-inducing Ligand Cancer Res., December 15, 2002; 62(24): 7170 - 7174. [Abstract] [Full Text] [PDF]
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 Q. Wang, X. Wang, A. Hernandez, M. R. Hellmich, Z. Gatalica, and B. M. Evers Regulation of TRAIL Expression by the Phosphatidylinositol 3-Kinase/Akt/GSK-3 Pathway in Human Colon Cancer Cells J. Biol. Chem., September 20, 2002; 277(39): 36602 - 36610. [Abstract] [Full Text] [PDF]
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T. Lin, J. Gu, L. Zhang, X. Huang, L. C. Stephens, S. A. Curley, and B. Fang Targeted Expression of Green Fluorescent Protein/Tumor Necrosis Factor-related Apoptosis-inducing Ligand Fusion Protein from Human Telomerase Reverse Transcriptase Promoter Elicits Antitumor Activity without Toxic Effects on Primary Human Hepatocytes Cancer Res., July 1, 2002; 62(13): 3620 - 3625. [Abstract] [Full Text] [PDF]
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