Nonviral genetic transfer of Fas ligand induced significant growth suppression and apoptotic tumor cell death in prostate cancer in vivo

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  • An Erratum to this article was published on 08 September 2003


To accomplish efficient nonviral gene therapy against prostate cancer (PC), Epstein–Barr virus (EBV)-based plasmid vectors containing EBNA1 gene and oriP were employed and combined with a cationic polymer or cationic lipid. When EBV-plasmid/poly-amidoamine dendrimer complex was injected into PC-3-derived tumors established in severe combined immunodeficiency mice, a considerable expression of marker gene was obtained in the tumors, and the expression level was more than eight-fold higher than that achieved by conventional plasmid vector/dendrimer. Since most PC cells express the apoptotic signal molecule Fas (Apo-1/CD95) on their surface, Fas ligand (FasL) gene was transferred into PC cells to kill the tumor cells. In vitro transfection with pGEG.FasL (an EBV-plasmid with the FasL gene) significantly reduced the viability of PC cells, which subsequently underwent apoptosis. Intratumoral injections of pGEG.FasL into PC induced significant growth suppression of the xenograft tumors, in which typical characteristics of apoptosis were demonstrated by TUNEL staining and electron microscopic observations. When pGEG.FasL transfer was accompanied by systemic administrations of cisplatin, the tumors were inhibited even more remarkably, leading to prolonged survival of the animals. FasL gene transfection by means of EBV-based plasmid/cationic macromolecule complexes may provide a practical therapeutic strategy against PC.


Prostate cancer (PC) is the leading cause of death among males. Hormone ablation, radio- and chemo-therapies are widely conducted to treat advanced cases. In most instances, these therapies induce tumor regression for a few years, owing to the growth retardation and apoptosis of cancer cells. However, the tumors frequently relapse and become resistant to subsequent conventional anticancer treatments. Development of novel strategies against PC is desired.1,2 Phase I and II gene therapy trials against PC usually employ adenoviral and other viral vectors as gene delivery vehicles.3,4 In contrast, nonviral vectors are not frequently applied to gene therapy protocols against PC, because of their poor transduction efficiency in vivo. If this inefficiency is ameliorated, nonviral delivery systems may provide promising strategies for genetic treatment of PC, thanks to the lack of virus-related complications, low immunogenicity and stability.

We have previously reported that through employing the Epstein–Barr virus (EBV)-based plasmid vectors instead of conventional plasmid vectors, nonviral gene delivery is drastically improved in terms of transfection efficiency, as well as magnitude and longevity of expression.5 EBV-based plasmid vectors carry EBV nuclear antigen 1 (EBNA1) gene and oriP element derived from the viral genome. Binding to oriP in a sequence-specific manner, the EBNA1 facilitates nuclear transport, nuclear matrix binding and transcriptional up-regulation of the oriP-bearing plasmid. A variety of nonviral vehicles such as synthetic macromolecules,6,7,8,9,10,11 electroporation12,13,14 and particle-mediated procedure 15 have been successfully combined with the EBV system, while naked EBV plasmids are also quite useful in transferring therapeutic genes in vivo.16,17 Among the synthetic macromolecules, cationic polymers and cationic lipids conjugated with plasmid DNA (polyplex and lipoplex, respectively) are quite promising tools for cancer gene therapy.18,19 Compared with conventional polyplex and lipoplex systems, EBV-based plasmid vectors conjugated with cationic polymers and cationic lipids (EBV/polyplex and EBV/lipoplex, respectively) achieve quite successful genetic delivery, resulting in strong and persistent transgene expression in various tumor cells both in vitro6,7,8,9,10 and in vivo.8,10,11

Fas ligand (FasL) is a type 2 transmembrane glycoprotein belonging to the tumor necrosis factor superfamily.20 FasL leads to trimerization of Fas, the cell surface apoptotic signal molecule, and triggers sequential activation of cysteine proteases, subsequently inducing apoptosis in Fas-positive cells.21,22,23 Since most PC cells express membrane-bound Fas (mFas) on their surface, FasL gene transfer into PC cells may become a feasible therapeutic approach to this malignancy.24,25,26 One advantage of FasL gene transfer is that if all the cancer cells are not successfully transfected, the FasL-transfected cells, even if few in number, could induce apoptosis in neighboring cancer cells.

In the present study, we applied the EBV/polyplex and EBV/lipoplex systems to FasL gene transfer and analyzed the tumoricidal effects of the genetic treatment on PC.


Efficient gene transfer into PC cell lines by means of the EBV/polyplex and EBV/lipoplex

EBV-based and conventional plasmid vectors with Escherichia coli β-galactosidase (β-gal) marker gene (Figure 1) were coupled with cationic polymer (poly amidoamine (PAMAM) dendrimer) (SuperFect™, Qiagen, Hilden, Germany) or cationic lipid (Effectene™, Qiagen) at various molar ratios, and the resultant DNA/synthetic vector complexes were transfected into the human PC cell lines, PC-3 and LNCaP.24,25,26 Comparing the two delivery vehicles, the PC-3 cells were more effectively transfected by PAMAM dendrimer than by Effectene, whereas Effectene was superior to PAMAM dendrimer as far as LNCaP cells were tested (data not shown). In the following experiments PC-3 and LNCaP cells were transfected by PAMAM dendrimer and Effectene, respectively.

Figure 1

Plasmids used in this study. Maps of pGEG.FasL, pGEG.β, pGEG.luc, pGEG.4, pG.β and pG.luc are schematically illustrated. prom: promoter; polyA: SV40 polyA additional signal; β-gal: E. coli β-galactosidase gene; luc: firefly luciferase gene; FasL: human Fas ligand gene.

Figure 2a shows the kinetics of β-gal activities. The enzyme activities in pGEG.β-transfected cells were 22.3-fold (PC-3 on day 4) and 13.7-fold (LNCaP on day 5) stronger than those in pG.β-transfected cells. As shown in Figure 3, approximately 38% of PC-3 cells stained with X-gal on day 4 when the maximum β-gal activity was obtained through the pGEG.β/dendrimer transfection. In the meanwhile, virtually 100% of LNCaP cells given pGEG.β/Effectene were strongly positive in a dense blue color at their optimum time point (day 5). A PC-3 cell may express a larger amount of β-gal than an LNCaP cell (Figures 2a and 3). In contrast, only 5% (PC-3) and 12% (LNCaP) of pG.β-transfected cells stained with X-gal.

Figure 2

Successful transfection in vitro and in vivo into PC. (a) PC-3 and LNCaP cells were transfected with pGEG.β (solid bars) or pG.β (open bars) as described in the Materials and methods. Cells were harvested on the indicated days and β-gal assay was performed. β-gal activity is standardized with the amount of protein, and means ±sd of hexaplicate samples are shown. (b) pGEG.luc (solid bar) or pG.luc (open bar) was combined with PAMAM dendrimer and injected into established PC-3 tumors three times every other day. As a control, some tumors were given injections of PBS (shaded bar). Seven days after the first transfection, tumors were excised and luciferase activity was measured. The luciferase activities are standardized with the amount of protein, and means ±sd of hexaplicate samples are shown. (c) Cells were transfected with the indicated plasmids, and 4 (PC-3) or 5 (LNCaP) days later, sFasL in the supernatant was measured by ELISA. As a control, the supernatant of untransfected cells was also examined. Data are presented as the means ±sd of triplicate samples. *P<0.05.

Figure 3

Strong marker gene expression in PC cells transfected with EBV-based plasmid vector in vitro. PC-3 and LNCaP cells were transfected with the indicated plasmids as in Figure 2. The cells were fixed and stained with X-gal on day 4 (PC-3) or 5 (LNCaP) post-transfection (original magnification is ×400).

We also investigated the transfection efficiency of the EBV/polyplex in vivo. The plasmids with firefly luciferase marker gene (Figure 1) were coupled with cationic polymer, and complexes were injected via a micro-infusion pump to PC-3-derived tumors established in severe combined immunodeficiency (SCID) mice. As shown in Figure 2b, pGEG.luc elicited approximately 8.6-fold higher marker gene expression than did pG.luc. Luciferase activity was not demonstrated in control tumors injected with PBS. Insofar as we tested, injections of naked EBV plasmid failed to induce significant marker gene expression in the tumors (data not shown). Injections of the synthetic vectors did not induce any inflammatory response in the PC tumors.

PC cells transfected with FasL gene underwent apoptosis in vitro and in vivo

Plasmid vectors encoding FasL were transfected into PC cells in vitro, and the transgene product in the culture supernatant was evaluated by ELISA analysis. PC cells transfected with pGEG.FasL secreted a considerable level of soluble FasL (sFasL), while the FasL production of untransfected cells was below the detection level (PC-3) or very faint (LNCaP) (Figure 2c).

The cytotoxic effect of FasL gene transfer was then assessed using tetrazolium salt-based assay. As shown in Figure 4, pGEG.FasL showed remarkable cytotoxicity against PC-3 and LNCaP cells (P<0.05, compared with pGEG.β-transfectants). Viability of PC cells was partially affected by pGEG.β. This is ascribed to the suppression of proliferation, rather than to cell death, of transfectants (data not shown).

Figure 4

PC cells transfected with pGEG.FasL decreased in viability. PC-3 or LNCaP cells were transfected with the indicated plasmids as in Figure 2. Nontransfected cells were prepared as controls (shaded bars). Cytotoxicity was measured 4 (PC-3) or 5 (LNCaP) days after the transfection by the tetrazolium salt reduction assay. The results represent the means ±sd of triplicate samples. *P<0.05.

PC-3 and LNCaP cells transfected with pGEG.FasL were analyzed by APO Percentage™ (Biocolor Ltd, Belfast, Northern Ireland). With this system, each apoptotic cell in the culture can be identified under an inverted microscope even at a low-power magnification; an apoptotic cell stains red, while a viable or necrotic cell remains unlabelled or stains pale pink.27,28 Considerable proportions of PC-3 and LNCaP cells showed typical signs of apoptosis following pGEG.FasL transfer (Figure 5a). In contrast, few pGEG.β-transfectants stained red.

Figure 5

FasL gene transfer induced apoptosis in PC cells. (a) PC-3 (upper panels) and LNCaP (lower panels) cells were transfected with the indicated plasmids combined with PAMAM dendrimer (PC-3) or Effectene (LNCaP). On day 4 (PC-3) or 5 (LNCaP) post-transfection, cells were stained with APOPercentage™ as described in Materials and methods (original magnification is ×400). (b, c) PC-3 tumors established in SCID mice were injected with the indicated plasmids combined with PAMAM dendrimer. On day 7, tumors were stained with TUNEL method (b, original magnification is ×600) or observed under an electron microscopy (c, original magnification is ×3000).

To assess whether the FasL vector also induces apoptotic death of PC in vivo, pGEG.β or pGEG.FasL was delivered into PC-3-derived tumors via the PAMAM dendrimer. TUNEL staining revealed that the pGEG.FasL/dendrimer-injected tumors were abundant in apoptotic nuclei with double-strand DNA breaks, while apoptosis was not evident in the PC-3 tumors treated with pGEG.β (Figure 5b). Electron microscopic observation consistently demonstrated that pGEG.FasL-transfected tumor cells underwent apoptosis with typical characteristics, including extensive chromatin condensation, nuclear fragmentation and plasma membrane blebbing (Figure 5c). In sharp contrast, the pGEG.β-transfected tumor cells remained intact and normal in morphology.

In vivo transduction with pGEG.FasL elicited significant suppression of PC-derived tumor growth

We then tested whether FasL gene transfer is capable of inducing therapeutic antitumor effects against PC in vivo. When PC-3 and LNCaP tumors were repetitively injected with pGEG.FasL, the growth rate of PC-3 tumors was significantly reduced, while the LNCaP tumors were drastically suppressed (Figure 6). The results indicate that EBV-plasmid/cationic macromolecule complexes are useful tools that enable successful transduction in vivo of FasL gene into PC leading to significant tumor remission.

Figure 6

In vivo therapeutic effect of the FasL gene transfection. pGEG.FasL (□) or pGEG.4 () was combined with PAMAM dendrimer (PC-3) or Effectene (LNCaP) and injected into the PC tumors established in SCID mice (day 0). The injections were repeated on days 0, 2 and 4 (arrows). Control tumors were treated with PBS (). Means ±sd are plotted (n=6 in each group). *P<0.05.

Combination therapy of pGEG.FasL transfer and cisplatin (CDDP) administration

To obtain a more remarkable therapeutic outcome, we combined FasL gene transfer with an anticancer agent, CDDP. In in vitro experiments, addition of CDDP for 24 h augmented the tumoricidal effect of the pGEG.FasL transfection in a dose-dependent manner (Figure 7a). The CDDP treatment for this short period did not influence PC-3 viability per se, while LNCaP cells were partially killed by the treatment. As above (Figure 4, left panel), PC-3 cells were moderately reduced in viability after treatment with pGEG.4/dendrimer, which suppressed proliferation of the cancerous cells without inducing apoptosis (Figure 7a, left panel). The efficacy of the combination therapy was also confirmed by in vivo experiments; intratumoral pGEG.FasL transfection in combination with systemic administration of CDDP strongly hampered the growth of PC-3-derived tumors, significantly extending the longevity of the tumor-bearing animals (Figure 7b). The antitumor effect of the combination therapy was more evident than either gene therapy alone or chemotherapy alone.

Figure 7

Combination therapy of FasL gene transfer and CDDP. (a) PC-3 and LNCaP were transfected with the indicated plasmid as in Figure 2, and 3 (PC-3) or 4 (LNCaP) days later, CDDP was added to the culture at the indicated concentrations. Twenty-four hours later, cytotoxicity was measured as in Figure 4. Bars represent mean ±sd of triplicate samples. (b) Mice bearing PC-3 tumors were given intratumoral injections of pGEG.FasL/PAMAM dendrimer (triangles and squares) or PBS (circles and diamonds) on days 0, 2, 4, 7, 9 and 11 (closed arrows). Intraperitoneal administrations of CDDP (9.0 mg/kg body weight) (diamonds and squares) or PBS (circles and triangles) were performed on days 1 and 8 (open arrows). Volumes of tumors (left panel) and survival curves of mice (right panel) are shown. Bars in the left panel indicate the means ±sd *P<0.05. P-values in the right panel are: P=0.042 between pGEG.FasL+CDDP and Control (PBS), P=0.047 between pGEG.FasL+CDDP and CDDP alone, and P=0.019 between pGEG.FasL+CDDP and pGEG.FasL alone (n=6 in each group).


The major impediment of nonviral vectors is that transfection efficiency has been relatively poor compared to viral vectors.18,19 Although PC-3 and LNCaP are commonly used as experimental models for PC, there have been no reports documenting nonviral systems that worked on these PC cells as effectively as our systems.29 In the present study, we employed PAMAM dendrimer and Effectene as synthetic nonviral vectors. The former is a spherical polymer rich in positive charges,30 while the latter is a nonliposomal cationic lipid.31 As far as we tested, these two reagents yielded the highest transfection efficacies among commonly used synthetic macromolecules. The transfection efficiencies are not obstructed by serum (data not shown). It is also noteworthy that PAMAM dendrimer and Effectene are less toxic to cells than are many other gene delivery vehicles. They are stable and hardly oxidized compared to other cationic liposomes. The EBV/polyplex and EBV/lipoplex systems are quite effective in transferring genes into PC cells not only in vitro but also in vivo.

Some earlier reports suggested that Fas-mediated apoptosis could be a target for therapeutic intervention in PC.24,25,26 Although agonistic anti-Fas antibodies (eg CH-11) also kill Fas-positive tumor cells,25,26 they could cause severe hepatotoxicity, which may restrict clinical application of the antibodies.32,33 If FasL gene transduction provides the transgene product exclusively on the cell surface of tumors, systemic adverse effects like hepatotoxicity may be avoided. Reportedly, LNCaP cells are relatively resistant to anti-Fas antibody treatment.25 FasL gene transfer may be more effective in inducing apoptosis in PC cells than do anti-Fas antibodies.

Membrane-bound FasL (mFasL) is converted into sFasL through enzymatic cleavage by matrix metalloproteinase-like enzymes.34,35 Although sFasL has very weak apoptosis-inducing activity,36,37 several groups have engineered noncleavable mutant or chimeric FasL molecules to evade the toxicity of sFasL possibly converted from mFasL.38,39 As shown by the ELISA analysis, PC cells transfected with pGEG.FasL/PAMAM dendrimer secreted significant concentrations of sFasL. By our hands, the sFasL was unable to induce apoptosis in Fas-sensitive T leukemia cell lines, Jurkat and CEM, while mFasL on the pGEG.FasL transfectants successfully induced apoptosis in these leukemic cells (data not shown). Nevertheless, noncleavable FasL may be preferable to wild type. This may be particularly important in case FasL vectors equipped with a tumor-specific promoter (eg prostate-specific antigen (PSA) promoter) will be systemically administered to patients to eradicate metastatic tumors.

FasL gene transfer may induce not only Fas-mediated apoptosis but also immune responses against tumors.40,41 The antitumor immunity may be of great value for the treatment of metastatic PC. In the present study, we aimed at estimating the former effect in immunocompromised hosts, which did not show obvious inflammatory reaction in the tumors (data not shown). Arai et al reported that FasL gene transfer into Fas-negative colon carcinoma in syngenic hosts unexpectedly resulted in marked tumor regression.40 Drozdzik et al reported that fibroblasts engineered to express FasL did not exert toxic effects on Fas-negative cell lines in vitro, whereas the fibroblasts abrogated their tumorigenicity in syngenic mice.41 Immune cells elicited by the FasL may have contributed to the antitumor effects. At present, however, it remains controversial whether FasL mediates antitumor immunity.

We have also demonstrated the effectiveness in vivo of combination therapy comprising FasL gene transfer and systemic administration of a representative anticancer agent, CDDP (Figure 7). The CDDP is one of the most commonly used chemotherapeutic agents in the treatment of advanced PC. However, the overall response rate induced by CDDP alone is insufficient. As previously reported, certain anticancer agents including CDDP augment Fas-mediated apoptosis in tumor cells, affecting the Fas-mediated intracellular signaling pathways. If tumor cells are exposed to a subtoxic dose of anticancer agent for a short period, the drug sensitizes the cancer cells to FasL-mediated cytotoxicity.42,43 Furthermore, treatment of PC cells with both CDDP and anti-Fas antibody resulted in a strong cytotoxic effect and apoptosis.43 Combination therapy of FasL gene transfer and CDDP may be useful against PC, particularly those that are relatively resistant to either chemotherapy alone or FasL genetic transfer alone. This approach may also be helpful in minimizing side effects, because the dosages of CDDP and FasL gene may be reduced.

Since the prostate is a capsulated organ and is not essential to the patient's life, injections of vectors into localized PC are safe and tolerable.44,45,46 Unlike many other malignancies, PC can be targeted with specific regulatory elements such as PSA or prostate-specific membrane antigen (PSMA) promoters.39,46 In this regard, PC is quite suited for FasL gene therapy, and the present system may provide a novel therapeutic regimen against PC.

Materials and methods

Plasmid vectors

pGEG.4 is an EBV-based plasmid vector that contains EBV oriP, EBNA1 gene (derived from p220.247) driven by a CAG promoter (a strong, hybrid promoter composed of cytomegalovirus immediate early enhancer and chicken beta-actin promoter),48 and another CAG promoter followed by an SV40 polyA additional signal.17 pGEG.FasL was constructed by inserting the XhoI-BamHI 1.1 kb fragment containing the full-length human Fas ligand cDNA and a polyA signal from pME18S.hFasL (a generous gift from Dr Yonehara) into the pGEG.4 that was deprived of the SV40 polyA additional signal. pGEG.β and pGEG.luc contain expression units for β-gal and firefly luciferase gene, respectively, under the control of the CAG promoter.14,17 pG.β and pG.luc are non-EBV conventional plasmid vectors with CAG-β-gal and CAG-luciferase, respectively.17 All plasmids were purified using Qiagen Maxiprep Endo-free kits (Qiagen).

In vitro gene transfer

PC-3 and LNCaP were maintained in RPMI1640 medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% (PC-3) or 6% (LNCaP) fetal bovine serum (FBS). PC-3 cells were seeded into six-well plates at a concentration of 1.0×105 in 2 ml of complete medium per well. After 24 h of cultivation at 37°C in 5% CO2/95% humidified air (standard condition), 2 μg of plasmid DNA was dissolved in 100 μl of RPMI1640 medium without FBS and antibiotics, followed by addition of 20 μg of PAMAM dendrimer according to the manufacturer's protocol. To transfect LNCaP, cells were seeded into six-well plates (2.0×105 in 2 ml of complete medium per well). Twenty-four hours later, 0.4 μg of plasmid DNA was combined with 6 μg of Effectene™, and the generated lipoplex was added to the cells according to the manufacturer's protocol. The cells were cultured under the standard condition until analysis.

X-gal staining of cultured cells

Cells were fixed with 1% glutaraldehyde/PBS for 10 min.After washing three times with PBS, cells were incubated at 37°C in X-gal staining solution (0.005% (v/v) 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal), 1 mM MgCl2, 150 mM NaCl, 3 mM K4(Fe(CN)6), 3 mM K3(Fe(CN)6), 60 mM Na2HPO4 and 0.1% Triton X-100). Twenty-four hours later, the staining solution was replaced by 1 mM Na2-EDTA/PBS to terminate the enzyme reaction.

β-gal assay

Cells were scraped from plates by trypsinization, washed twice with PBS and resuspended in 50 μl of Tris-HCl (pH 7.8). After freezing and thawing twice, the lysate was centrifuged at 14,000 g for 5 min β-gal activity in the supernatant was assayed with a β-gal assay kit (Invitrogen, San Diego, CA, USA). The optical density (OD) was measured at 420 nm, and the activity was calculated as follows: β-gal units=(380×OD420)/30/mg protein, where 380 is a conversion factor and 30 is the incubation time in minutes. The protein concentration of the supernatant was assessed according to the method of Bradford.49

ELISA analysis

Four (PC-3) or 5 (LNCaP) days after the transfection, culture supernatant was replaced by 2 ml of fresh complete medium. Twenty-four hours later, the culture medium was harvested and sFasL concentration was measured using an sFasL ELISA kit (MBL, Nagoya, Japan).

Cell viability analysis

Cell viability was evaluated by colorimetric method using tetrazolium salt.50 Briefly, triplicate aliquots of cells were transfected as described above. Following cultivation under the standard condition, culture supernatant was replaced by 1000 μl of fresh complete medium containing 0.5 μmol of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8, Nacalai Tesque, Kyoto, Japan). After incubation for an additional 1 h, the culture supernatant was harvested and OD at 450 nm was measured. Cytotoxicity was calculated as follows: cytotoxicity (%)=(1−(absorbance of experimental wells/average absorbance of control wells))×100.

Apoptosis detection in vitro

Apoptosis was detected using the APOPercentage™ kit.27,28 Briefly, cells were transfected with FasL gene as above. Four (PC-3) or 5 (LNCaP) days later, culture supernatant was removed from the wells and 1000 μl of complete medium containing 50 μl of APOPercentage Dye was added to the cells. Sixty min later, the supernatant was drained from the wells and after washing twice with PBS, cells were observed under an inverted microscope.

In vivo transfection into PC tumors

All the in vivo experiments were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine. To establish PC tumors, 6×106 (PC-3) or 107 (LNCaP) cells were mixed with 50 μl of Matrigel™ (Becton-Dickinson, NJ, USA) and subcutaneously inoculated into the flanks of 8 to 9-week-old SCID mice (CLEA, Osaka, Japan). Seven (PC-3) or 10 (LNCaP) days later, the tumors developed to sizes larger than 60 mm3 (day 0). A total of 10 μg of plasmid DNA was conjugated with 90 μg of PAMAM dendrimer and injected into the PC-3 tumors. To transfect the LNCaP tumors, 100 μl of lipoplex containing 5 μg of plasmid DNA and 25 μg of Effectene was injected. The injections were performed on days 0, 2 and 4 at a velocity of 20 μl/min using a micro-infusion pump (Termo, Tokyo, Japan) as described.51 Luciferase activity was examined on day 7 as described previously.14 To assess growth rate of tumors, the diameters of PC tumors were scaled with a digital scalper and their volumes were calculated as follows: tumor volume=a×b2/2 (mm3), where a is the long diameter and b the short diameter.

Apoptosis detection in vivo

For TUNEL staining, PC-3 tumors were fixed in 4% paraformaldehyde at 4°C for 24 h, embedded in OCT compound, and frozen at −80°C. Serial 10 μm sections were made, and TUNEL staining was performed using the in situ Apoptosis Detection Kit (Takara, Kyoto, Japan) according to the supplier's protocol. For electron microscopic observation, tumors were fixed in 2% glutaraldehyde at 4°C for 2 h, followed by washing with 0.1 M phosphate buffer and immersion in 1% osmium tetroxide for 2 h. Following dehydration in graded series of ethanol, the specimen was embedded in Spurr resin, before staining of the sections with uranyl acetate and lead citrate. Observation was made with a Hitachi 7000 electron microscope set at 75 kV.

Combination therapy in vitro and in vivo by pGEG.FasL and CDDP

PC-3 and LNCaP cells were transfected with polyplex or lipoplex as above. After cultivation for 2 (PC-3) or 4 (LNCaP) days, CDDP was added at various concentrations, and 24 h later, cell viability was measured by the tetrazolium salt reduction assay. In vivo combination therapy was performed as follows: polyplex was injected into subcutaneous PC-3 tumors three times a week for 2 weeks. Some groups of mice were also given intraperitoneal injections of CDDP (9.0 mg/kg body weight) on days 0 and 7.

Statistical evaluation

For the β-gal assays, cytotoxicity assays and comparison of tumor volumes, Fisher's protected least significant difference (Fisher's PLSD) test was used. For Kaplan–Meier analysis, survival differences between groups were evaluated using Logrank test. A P-value of 0.05 or less was considered significant.


  1. 1

    Morris MJ, Scher HI . Novel strategies and therapeutics for the treatment of prostate carcinoma. Cancer 2000; 89: 1329–1348.

  2. 2

    Oh WK, Kantoff PW . Treatment of locally advanced prostate cancer: is chemotherapy the next step? J Clin Oncol 1999; 17: 3664–3675.

  3. 3

    Harrington KJ et al. Gene therapy for prostate cancer: current status and future prospects. J Urol 2001; 166: 1220–1233.

  4. 4

    Shalev M et al. Gene therapy for prostate cancer. Br J Urol 2001; 57: 8–16.

  5. 5

    Mazda O . Application of Epstein–Barr virus and its genetic elements to gene therapy. In: Cid-Arregui A, Garcia-Carranca A (eds). Viral Vectors: Basic Science and Gene Therapy. Eaton Publishing: Massachusetts, 2000, pp 325–337.

  6. 6

    Satoh E et al. Efficient gene transduction by Epstein–Barr virus (EBV)-based vectors coupled with cationic liposome and HVJ-liposome. Biochem Biophys Res Commun 1997; 238: 795–799.

  7. 7

    Harada Y et al. Highly efficient suicide gene expression in hepatocellular carcinoma cells by Epstein–Barr virus-based plasmid vectors combined with polyamidoamine dendrimer. Cancer Gene Ther 2000; 7: 27–36.

  8. 8

    Maruyama-Tabata H et al. Effective suicide gene therapy in vivo by EBV-based plasmid vectors coupled with polyamidoamine dendrimer. Gene Ther 2000; 7: 53–60.

  9. 9

    Tanaka S et al. Targeted killing of carcinoembryonic antigen (CEA)-producing cholangiocarcinoma cells by PAMAM dendrimer-mediated transfer of an Epstein–Barr virus (EBV)-based plasmid vector carrying the CEA-promoter. Cancer Gene Ther 2000; 7: 1241–1249.

  10. 10

    Iwai M et al. Polyethylenimine-mediated suicide gene transfer induces a therapeutic effect for hepatocellular carcinoma in vivo by using an Epstein–Barr virus-based plasmid vector. Biochem Biophys Res Commun 2002; 291: 48–54.

  11. 11

    Asada H et al. Significant antitumor effects obtained by autologous tumor cell vaccine engineered to secrete Interleukin (IL)-12 and IL-18 by means of the EBV/lipoplex. Mol Ther 2002; 5: 609–616.

  12. 12

    Mazda O, Satoh E, Yasutomi K, Imanishi J . Extremely efficient gene transfection by Epstein–Barr virus vectors into lympho-hematopoietic cell lines. J Immunol Methods 1997; 204: 143–151.

  13. 13

    Satoh E et al. Successful transfer of adenosine deaminase (ADA) gene in vitro into human peripheral blood CD34+ cells by transfecting Epstein–Barr virus (EBV)-based episomal vectors. FEBS Lett 1998; 441: 39–42.

  14. 14

    Kishida T et al. In vivo electroporation-mediated transfer of Interleukin-12 and Interleukin-18 genes induces significant anti-tumor effects against melanoma in mice. Gene Ther 2001; 8: 1234–1240.

  15. 15

    Nishizaki K et al. In vivo gene gun-mediated transduction into rat heart with Epstein–Barr virus-based episomal vectors. Ann Thorac Surg 2000; 70: 1332–1337.

  16. 16

    Tomiyasu K et al. Direct intra-cardiomuscular transfer of B2-adrenergic receptor gene augments cardiac output in cardiomyopathic hamsters. Gene Ther 2000; 7: 2087–2093.

  17. 17

    Cui FD et al. Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein–Barr virus (EBV)-based plasmid vectors. Gene Ther 2001; 8: 1508–1513.

  18. 18

    Schatzlein AG . Nonviral vectors in cancer gene therapy: principles and progress. Anticancer Drugs 2001; 12: 275–304.

  19. 19

    Han S, Mahato RI, Sung YK, Kim SW . Development of biomaterials for gene therapy. Mol Ther 2000; 2: 302–317.

  20. 20

    Suda T, Takahashi T, Golstein P, Nagata S . Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 1993; 75: 1169–1178.

  21. 21

    Yonehara S, Ishii A, Yonehara M . A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J Exp Med 1989; 169: 1747–1756.

  22. 22

    Schneider P et al. Characterization of Fas (Apo-1, CD95)–Fas ligand interaction. J Biol Chem 1997; 272: 18827–18833.

  23. 23

    Owen-Schaub L et al. Fas and Fas ligand interactions in malignant disease. Int J Oncol 2000; 17: 5–12.

  24. 24

    Hedlund TE et al. Adenovirus-mediated expression of Fas ligand induces apoptosis of human prostate cancer cells. Cell Death Differ 1999; 6: 175–182.

  25. 25

    Hedlund TE, Duke RC, Schleicher MS, Miller GJ . Fas-mediated apoptosis in seven human prostate cancer cell lines: correlation with tumor stage. Prostate 1998; 36: 92–101.

  26. 26

    Takeuchi T et al. Modulation of growth and apoptosis response in PC-3 and LNCAP prostate-cancer cell lines by Fas. Int J Cancer 1996; 67: 709–714.

  27. 27

    Kisseleva MV, Cao L, Majerus PW . Phosphoinositide-specific inositol polyphosphate 5-phosphatase IV inhibits Akt/PKB phosphorylation and leads to apoptotic cell death. J Biol Chem 2001; 277: 6266–6272.

  28. 28

    Joyce DE et al. Gene expression profile of antithrombotic protein C defines new mechanisms modulating inflammation and apoptosis. J Biol Chem 2001; 276: 11199–11203.

  29. 29

    Yoshimura I, Suzuki S, Tadakuma T, Hayakawa M . Suicide gene therapy on LNCaP human prostate cancer cells. Int J Urol 2001; 8: S5–S8.

  30. 30

    Tang MX, Szoka FC . The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther 1997; 4: 823–832.

  31. 31

    Kang SH, Zirbes EL, Kole R . Delivery of antisense oligonucleotides and plasmid DNA with various carrier agents. Antisense Nucleic Acid Drug Dev 1999; 9: 497–505.

  32. 32

    Galle PR et al. Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J Exp Med 1995; 182: 1223–1230.

  33. 33

    Fujino M et al. Controlled Fas ligand gene expression by Cre/loxP-mediated switching system: high levels of FasL expression result in lethal hepatitis. Transplant Proc 1999; 31: 2695–2696.

  34. 34

    Tanaka M et al. Fas ligand in human serum. Nat Med 1996; 2: 317–322.

  35. 35

    Kayagaki N et al. Metalloproteinase-mediated release of human Fas ligand. J Exp Med 1995; 182: 1777–1783.

  36. 36

    Schneider P et al. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J Exp Med 1998; 187: 1205–1213.

  37. 37

    Tanaka M, Itai T, Adachi M, Nagata S . Downregulation of Fas ligand by shedding. Nat Med 1998; 4: 31–36.

  38. 38

    Aoki K et al. Restricted expression of an adenoviral vector encoding Fas ligand (CD95L) enhances safety for cancer gene therapy. Mol Ther 2000; 1: 555–565.

  39. 39

    Rubinchik S et al. A complex adenovirus vector that delivers FASL-GFP with combined prostate-specific and tetracycline-regulated expression. Mol Ther 2001; 4: 416–426.

  40. 40

    Arai H, Gordon D, Nabel EG, Nabel GJ . Gene transfer of Fas ligand induces tumor regression in vivo. Proc Natl Acad Sci 1997; 94: 13 862–13 867.

  41. 41

    Drozdzik M et al. Antitumor effect of allogenic fibroblasts engineered to express Fas ligand (FasL). Gene Ther 1998; 5: 1622–1630.

  42. 42

    Frost P, Ng CP, Belldegrun A, Bonavida B . Immunosensitization of prostate carcinoma cell lines for lymphocytes (CTL, TIL, LAK)-mediated apoptosis via the Fas–Fas-ligand pathway of cytotoxicity. Cell Immunol 1997; 180: 70–83.

  43. 43

    Uslu R et al. Chemosensitization of human prostate carcinoma cell lines to anti-fas-mediated cytotoxicity and apoptosis. Clin Cancer Res 1997; 3: 963–972.

  44. 44

    Belldegrun A et al. Interleukin 2 gene therapy for prostate cancer: phase I clinical trial and basic biology. Hum Gene Ther 2001; 12: 883–892.

  45. 45

    Teh BS et al. Phase I/II trial evaluating combined radiotherapy and in situ gene therapy with or without hormonal therapy in the treatment of prostate cancer – a preliminary report. Int J Radiat Oncol Biol Phys 2001; 51: 605–613.

  46. 46

    Chen Y et al. CV706, a prostate cancer-specific adenovirus variant, in combination with radiotherapy produces synergistic antitumor efficacy without increasing toxicity. Cancer Res 2001; 61: 5453–5460.

  47. 47

    Yates JL, Warren N, Sugden B . Stable replication of plasmids derived from Epstein–Barr virus in various mammalian cells. Nature (London) 1985; 313: 812–815.

  48. 48

    Niwa H, Yamamura K, Miyazaki J . Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 1991; 108: 193–199.

  49. 49

    Bradford MM . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976; 72: 248–254.

  50. 50

    Matsuoka M, Wispriyono B, Igisu HA . Increased cytotoxicity of cadmium in fibroblasts lacking c-fos. Biochem Pharmacol 2000; 59: 1573–1576.

  51. 51

    Coll JL et al. In vivo delivery to tumors of DNA complexed with linear polyethylenimine. Hum Gene Ther 1999; 10:1659–1666.

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We would like to thank Dr Shin Yonehara (Institute for Virus Research, Kyoto University) for kindly providing us with the human Fas ligand gene and Dr Jun-ichi Miyazaki (Department of Nutrition and Physiological Chemistry, Osaka University Medical School) for providing the CAG promoter. This research was supported by a Grant-in-Aid from the Japanese Ministry of Education, Science and Culture (No. 13470339).

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Correspondence to O Mazda.

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Nakanishi, H., Mazda, O., Satoh, E. et al. Nonviral genetic transfer of Fas ligand induced significant growth suppression and apoptotic tumor cell death in prostate cancer in vivo. Gene Ther 10, 434–442 (2003) doi:10.1038/

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  • prostate cancer
  • EBV-based plasmid vector
  • episomal vector
  • Fas ligand
  • EBV/polyplex
  • EBV/lipoplex

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