Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice

Article metrics

Abstract

The application of adeno-associated virus (AAV) vectors to cancers is limited by their low transduction efficiency. Previously, we reported that γ-ray enhanced the second-strand synthesis, leading to the improvement of the transgene expression, and cytocidal effect of the herpes simplex virus type-1 thymidine kinase (HSVtk) and ganciclovir (GCV) system. In this study, we extended this in vitro findings to in vivo. First, the laryngeal cancer cell line (HEp-2) and HeLa were treated with AAVtk/GCV, the number of surviving cells was reduced as the concentration of GCV increased. Furthermore, the 4 Gy irradiation enhanced the killing effects of AAVtk/GCV by four-fold on HeLa cells and 15-fold on HEp-2 cells. Following the in vitro experiments, we evaluated the transgene expression and the antitumor activity of the AAV vectors in combination with γ-ray in nude mice inoculated with HEp-2 subcutaneously. The LacZ expression was observed in the xenografted tumors and significantly increased by γ-ray. The AAVtk/GCV system suppressed the tumors growth, and γ-ray augmented the antitumor activity by five-fold. These findings suggest that the combination of AAVtk/GCV system with radiotherapy is significantly effective in the treatment of cancers and may lead to reduction of the potential toxicity of both AAVtk/GCV and γ-ray.

Introduction

Current therapy for the head and neck cancer utilizes an aggressive multimodal approach with surgery, radiotherapy, and chemotherapy, depending on the tumor stage and patient characteristics. Despite advances in the therapeutic approaches, no substantial improvement in efficacy and survival has occurred over the past several decades. Conventional palliative treatments, such as chemotherapy, are often toxic and sometimes ineffective. In addition, no effective salvage therapy is available for whom standard treatment fails. Therefore, new treatments are needed both to improve survival in the long term and to obtain worthwhile, less toxic palliation in the short term.

Recent progress in gene therapy technologies has made it possible to develop novel therapeutic strategies for intractable cancers. Head and neck cancer is particularly well suited for gene transduction strategies. First, its location in the upper aerodigestive tract allows easy access for vector delivery and assessment of response. Second, targeting of gene transduction can be achieved by direct injection of the vector into the tumor. Finally, metastases mostly occur late in head and neck cancer progression, making local disease responsible for most of the morbidity and mortality.1,2 Therefore, any improvement in local disease control implies benefits for patients. At present, several virus vectors such as retroviral, adenoviral and adeno-associated virus (AAV) vectors3,4 have been utilized for the experiments of cancer gene therapy. AAV is a non-pathogenic virus with a single-stranded DNA genome.5,6 AAV vectors have emerged as a useful alternative to other vectors,7 and AAV have been evaluated in preclinical and clinical models for cystic fibrosis,8 Parkinson's disease9 and Hemophilia B.10 AAV vectors have a broad host range and can transduce head and neck cancer cells.11 However, an obstacle to these applications is a low transgene expression efficiency, mainly due to a limited second-strand synthesis.12,13 Recently, γ-ray irradiation has been reported to enhance the second-strand synthesis of the AAV vector genome and improve the transgene expression.14,15,16 In our previous study, we demonstrated that γ-rays enhance AAV-mediated transgene expression in maxillary sinus cancer cells in vitro.11 Thus, an AAV vector encoding suicide gene would kill target cells more efficiently when combined with γ-ray irradiation therapy. Although the herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system has shown to be effective for controlling tumor growth in animal models,17,18,19,20 this therapeutic approach alone sometimes fails to eradicate cancer cells, and tumors recur thereafter. Thus, alternative therapeutic modalities, such as combination therapy should be considered.21,22

In this study, we demonstrate effective suicide gene therapy using the AAV vector in combination with γ-ray irradiation, enhancing the antitumor activity both in vitro and in vivo.

Results

Comparison of the transduction efficiency of AAVLacZ between HeLa and HEp-2 cells

HeLa or HEp-2 cells were transduced with recombinant AAV with Escherichia coli LacZ expression cassette (AAVLacZ) at titers from 1×104 to 1×106 particles/cell (Figure 1a). The percentage of positive HEp-2 cells for 5-bromo-4-chloro-indonyl-β-D-galactopyranoside (X-gal) staining reached 72% when the cells were transduced with 1×106 particles/cell of AAVLacZ. The transduction efficiency of HEp-2 cells was almost as high as that of HeLa cells. Figure 1b shows the β-galactosidase expression level in HeLa and HEp-2 cells quantified by enzyme-linked immunosorbent assay (ELISA), when the cells were transduced with AAVLacZ at titers from 1×103 to 1×105 particles/cells. The amount of β-gal in both cells increased along with the increased concentration of AAVLacZ.

Figure 1
figure1

AAVLacZ expression in HeLa or HEp-2 cells. (a) Subconfluent HeLa (closed bar) or HEp-2 cells (hatched bar) were transduced with AAVLacZ at various doses ranging from 1×104 to 1×106 particles/cell. At 36 h post-transduction, the cells were fixed and stained with X-gal, and then positive cells were counted. (b) Subconfluent HeLa (closed bar) or HEp-2 cells (hatched bar) were transduced with 1×103 to 1×105 particles/cell of AAVLacZ. Thirty-six hours after transduction, the cells were lysed and β-gal was assayed by using the β-gal ELISA kit. Each bar represents the mean±s.d.

Effect of γ-ray irradiation on AAV-mediated transgene expression

γ-Rays have been shown to increase the transduction efficiency with AAV vectors, mainly by accelerating the rate of leading-strand synthesis of the AAV vector genome. According to our previous study,11 optimal transduction efficiency was obtained when the cells were transduced with 1×103 particles/cell of AAVLacZ immediately after irradiation. In Figure 2, γ-ray irradiation significantly increased LacZ expression in HeLa and HEp-2 cells in a dose-dependent manner. (one-way ANOVA: P<0.01).

Figure 2
figure2

Effect of γ-ray on AAV vector-mediated transgene expression. HeLa (closed square) or HEp-2 cells (open square) were transduced with 1×103 particles/cell of AAVLacZ immediately after γ-ray irradiation at doses ranging from 0 to 5 Gy. Thirty-six hours after transduction, the expression levels of LacZ were assayed by using the β-gal ELISA kit. Data were statistically analyzed by one-way ANOVA (*P<0.01). Each data point represents the mean±s.d.

γ-Rays enhance the second-strand synthesis of the AAV genome in HeLa and HEp-2 cells

To examine whether the second-strand synthesis of the AAV vector genome occurs more efficiently in γ-ray-irradiated cells, HeLa and HEp-2 cells were subjected to 0, 2, or 4 Gy of γ-ray irradiation, and then transduced with 1×104 particles/cell of AAVLacZ. Forty-eight hours after transduction, total DNA was isolated, treated with mung bean nuclease, and then loaded on 1% agarose gels. After transfer to nylon membranes, signals corresponding to the AAVLacZ genome were detected (Figure 3). Mung bean nuclease was used to digest the single-stranded DNA and to clearly visualize the double-stranded replicative form (RF) of the AAV vector genome. The RF was almost equal to 4.7 kb fragment derived from pAAVLacZ in size. At the dose of 4 Gy, in both HeLa and HEp-2 cells, the intensity of signal corresponding to the RF increased significantly, suggesting that the augmented transgene expression was associated with the conversion of the AAV vector genome to the double-stranded RF.

Figure 3
figure3

Second-strand synthesis of AAVLacZ genome after γ-ray irradiation in HeLa or HEp-2 cells. HeLa (a) or HEp-2 cells (b) were transduced with 1×104 particles/cell of AAVLacZ immediately after 0, 2, or 4 Gy of γ-ray irradiation. Two days later, total DNA was isolated in a low-salt condition. After mung bean nuclease treatment, the DNA samples were loaded on 1% agarose gels, transferred onto nylon membranes (Hybond N+, Amersham), and then hybridized with a radiolabeled CMV-specific probe. Signals were detected by using an imaging analyzer. Lane 1, a 4.7 kb fragment derived from pAAVLacZ; lane 2, mock transduced; lanes 3–5, AAVLacZ-transduced immediately after 0-, 2-, or 4-Gy irradiation respectively. RF: the double-stranded replicative form.

GCV treatment

Figure 4 shows the killing effect of various concentrations of GCV on HeLa and HEp-2 cells transduced with 1×105 particles/cell of recombinant AAV with HSVtk expression cassette (AAVtk) (closed bar). When the AAVtk-transduced cells were treated with 1 μg/ml of GCV, 75% of HeLa cells and 35% of HEp-2 cells were killed. As the concentration of GCV was increased, surviving cells were reduced and 98% of HeLa cells and 96% of HEp-2 cells were killed by exposure to 10 μg/ml of GCV, which was significantly higher than the killing rate in AAVLacZ-transduced cells (hatched bar) or mock-transduced cells (open bar) (two-way ANOVA: P<0.01).

Figure 4
figure4

Survival of HeLa and HEp-2 cells upon AAVtk/GCV. HeLa (a) or HEp-2 cells (b) were mock transduced (open bar), transduced with 1×105 particles/cell of AAVLacZ (hatched bar), or AAVtk (closed bar). Twenty-four hours after transduction, the cells were exposed to different concentrations of GCV. After a 7-day incubation in the presence of GCV, surviving cells were counted. Data were analyzed by two-way ANOVA. Asterisks mean that the data obtained for AAVtk transduction were significantly different from those with or without transduction of AAVLacZ (P<0.01). Each bar represents the mean±s.d. (n=3).

Enhanced cytocidal effect of the AAVtk/GCV system by γ-ray irradiation

To facilitate comparison of the therapeutic outcome, the killing effect of GCV (3 μg/ml) on HeLa or HEp-2 cells transduced with various doses of AAVtk with or without γ-ray irradiation was evaluated (Figure 5). When HeLa and HEp-2 cells were transduced with 3×104 particles/cell of AAVtk without irradiation, 48% of the HeLa cells and 40% of the HEp-2 cells were killed by the exposure to the GCV. As the dose of AAVtk was increased, the number of surviving cells were reduced, and 98% of the HeLa cells and 95% of the HEp-2 cells were killed when transduced with 3×105 particles/cell of AAVtk, which was significantly higher than the killing rate in the case of AAVLacZ-transduced cells as expected. To investigate whether γ-ray enhances the killing effect of AAVtk/GCV, HeLa and HEp-2 cells were irradiated with 2 or 4 Gy of γ-ray immediately before the transduction with various doses of AAVtk, and then cultured in 3 μg/ml of GCV. When the HeLa cells were transduced with 3×104 particles/cell of AAVtk, 70% of the 2 Gy irradiated cells and 85% of the 4 Gy irradiated cells were killed by the addition of GCV. When the HeLa cells were transduced at 3×105 particles/cell, γ-ray irradiation enhanced the killing effects of AAVtk/GCV system by four-fold. γ-Ray irradiation also enhanced the killing effects on HEp-2 cells by 15-fold. The enhancement by γ-ray irradiation was calculated from the ratio of 4 Gy irradiated survival rate to non-irradiated survival rate. These results show that γ-ray irradiation enhances the killing effects of AAVtk/GCV system significantly (two-way ANOVA: P<0.01).

Figure 5
figure5

Enhancement of the cytocidal effect of AAVtk by γ-ray irradiation. HeLa (a) or HEp-2 cells (b) were transduced with various doses of AAVLacZ or AAVtk with or without γ-ray irradiation. Twenty-four hours after transduction, the cells were treated with 3 μg/ml of GCV. After a 7-day incubation in the presence of GCV, surviving cells were counted. Closed squares: AAVLacZ-transduced cells; closed circles: AAVLacZ-transduced cells with 4 Gy irradiation; closed triangles: AAVtk-transduced cells; open squares: AAVtk-transduced cells with 2 Gy irradiation; open circles: AAVtk-transduced cells with 4 Gy irradiation. Asterisks mean that the AAVtk-transduced and irradiated cells were significantly different from the AAVtk-transduced and non-irradiated cells (P<0.01). Each data point represents the mean±s.d.

γ-Ray enhances the transgene expression in vivo

To assess the enhancement of transgene expression by γ-ray irradiation in vivo, the AAVLacZ-transduced tumors were stained with X-gal (Figure 6). Compared with non-irradiated control, the tumors irradiated at 4 Gy showed increased number of X-gal-positive cells. To quantify the amount of β-galactosidase, we homogenized the non-irradiated or 4 Gy irradiated tumors and measured with the β-gal ELISA kit. As shown in Figure 7, the amount of β-galactosidase in 4 Gy irradiated tumors was 2.5 times larger than that in non-irradiated tumors (one-way ANOVA: P<0.01).

Figure 6
figure6

The effect of γ-ray irradiation on AAV-mediated transgene expression in subcutaneous tumors in BALB/c mice. The tumors were established by HEp-2 cells into the flanks of the mice xenografts. (a) Transduction with 5×1011 particles/tumor of AAVLacZ without γ-ray irradiation. (b) Transduction with AAVLacZ immediately after 4 Gy irradiation. Three days after AAVLacZ injection, the tumors were excised from mice and frozen in OTC embedding compound on a liquid-nitrogen bath. Cryostat sections were made and fixed with 0.05% glutaraldehyde in PBS. Histochemical staining for β-galactosidase activity was performed in the sections as described in Materials and methods.

Figure 7
figure7

γ-Ray increased the expression level of β-galactosidase. The tumors were established by HEp-2 cells into the flanks of the mice xenografts. The tumors were injected with 5×1011 particles of AAVLacZ immediately after γ-ray irradiation at the dose of 0 or 4 Gy. Three days after AAVLacZ injection, the tumors were excised, homogenized, and assayed for the amount of β-galactosidase. Data were analyzed by one-way ANOVA. Asterisk means that the β-galactosidase expression level in 4 Gy irradiated tumors was significantly different from that in non-irradiated tumors (P<0.01). Each bar represents the mean±s.d. (n=3).

Suppressive effects of combination therapy upon tumor growth in vivo

To examine the killing effect of this system in vivo, tumor nodules were established in the flanks of BALB/c nude mice by subcutaneous injection of the HEp-2 cells. Once tumors were established, those animals were irradiated at 4 Gy and then directly injected with 1×1012 particles of AAVLacZ or AAVtk in the tumors. Administration of GCV (50 mg/kg) or phosphate-buffered saline (PBS) intraperitoneally twice a day was started at 24 h after the vector injection and continued for 2 weeks. The tumors were measured every 3 days.

Five treatment groups of 4 or 5 animals each were established: Group 1 is the AAVLacZ-transduced animals with PBS administration (n=4). Group 2 is the AAVtk-transduced animals with GCV administration (n=5). Group 3 is the AAVLacZ-transduced animals with 4 Gy irradiation and GCV administration (n=5). Group 4 is the AAVtk-transduced animals with 4 Gy irradiation and PBS administration (n=4). Group 5 is the AAVtk-transduced animals with 4 Gy irradiation and GCV administration.

The representative growth curve of the tumors after treatment is shown in Figure 8. The AAVtk/GCV system suppressed the growth rate of xenografted tumors by half, and γ-ray irradiation augmented the antitumor activity five-fold as assessed by the relative tumor volume to the controls. The combined approach with AAVtk/GCV system and γ-ray irradiation suppressed the tumor growth for 30 days. These data were analyzed by two-way ANOVA (P<0.01).

Figure 8
figure8

In vivo tumor growth inhibition with AAVtk/GCV treatment and γ-ray irradiation. The tumors derived from HEp-2 cells were established in the flanks of BALB/c nude mice by subcutaneous injection of the cell lines. Once established (a diameter of 4–5 mm), the tumors were irradiated at 4 Gy and then directly injected with 1×1012 particles of AAVLacZ or AAVtk. Administration of GCV (50 mg/kg) or PBS intraperitoneally twice a day was started 24 h after the virus injection and continued for 2 weeks. The tumors were measured every 3 days with calipers in two perpendicular diameters. Closed squares: AAVLacZ-injected animals (n=4); open squares: AAVtk-injected animals with GCV administration (n=5); closed triangles: AAVLacZ-injected animals with 4 Gy irradiation and GCV administration (n=5); closed circles: AAVtk-injected animals with 4 Gy irradiation and PBS administration (n=4); open circles: AAVtk-injected animals with 4 Gy irradiation and GCV administration. The relative tumor volume (T/T0) was calculated as the ratio of tumor volumes at any time after the treatment (T) to that before the treatment (T0). Data were analyzed by two-way ANOVA (P<0.01). Each data point represents the mean±s.d.

Discussion

In this study, we demonstrated that γ-ray irradiation enhanced AAV vector-mediated transgene expression and cytocidal effect of AAVtk/GCV on target cells. These findings were also shown in animal experiments, ie γ-ray irradiation enhanced transgene expression and killing effect of the AAVtk/GCV upon the grafted tumors.

There is a possibility that the killing effect was contributed by HSVtk/GCV and γ-ray irradiation without AAV vectors. However, we made several experiments about the second-strand synthesis utilizing AAVLacZ and demonstrated that the enhanced killing effect can be explained by the higher conversion efficiency of AAV vector genome to double-stranded form. Although several studies have been reported on the higher conversion efficiency, the mechanism by which a genetic stress enhances the second-strand synthesis is not fully understood.12,13,14,15,16 Qing et al23 reported that dephosphorylation of the single-stranded D sequence-binding protein facilitated second-strand synthesis of the AAV vector genome. Sanlioglu et al24 reported that the enhancement of AAV vector transduction by UV and adenovirus E4orf6 correlated with induction of two distinct molecular conversion pathways, and UV led to increased abundance of circular AAV vector genome. Some studies have shown that DNA repair synthesis is required for efficient transduction rather than replicative cellular DNA synthesis.13,25 The machinery of cellular DNA repair synthesis may play an important role in converting the single-stranded AAV vector genome to a double-stranded form, which is activated by γ-ray irradiation.

Several studies reported that AAV vectors were useful for the treatment of cancers in model experiments. Kunke et al3 showed that expressing the antisense of human papillomavirus early gene effectively killed the tumors derived from cervical cancer cells. Suicide gene therapy, in particular, HSVtk-expressing AAV vectors, was reported in the application to several kinds of cancers. The AAV vectors expressing HSVtk and interleukin 2 effectively killed glioma cells implanted into brains of nude mice.26 The expression of HSVtk driven by a liver-specific promoter via AAV vectors in tumors experimentally produced by implantation of hepatocellular carcinoma cells successfully retarded the tumor progression.27 We previously demonstrated the enhancement of the cytocidal effect of AAVtk/GCV system by γ-ray in vitro.11 The AAVtk/GCV system effectively killed the cancer cells depending on the concentration of GCV. Moreover, when the cancer cells were irradiated prior to transduction with AAVtk, they were killed more efficiently in a dose-dependent manner.

To extend these in vitro findings to an animal model, in the present study, we investigated the enhancement of AAV-mediated transgene expression by γ-ray irradiation in nude mice. To confirm whether γ-ray enhances the transgene expression, the xenografted tumors were injected with AAVLacZ. γ-Ray irradiation increased the number of X-gal-positive cells and the amount of β-galactosidase as expected in the tumors in vivo. Furthermore, significantly higher growth inhibition of the xenografted tumors was observed in the 4 Gy irradiation plus AAVtk/GCV group compared with the AAVtk/GCV system alone, the 4 Gy irradiation plus AAVLacZ/GCV, or the AAVtk/PBS. The combination therapy using other vectors and irradiation has been reported,21,22 but the augmentation of the second-strand synthesis by γ-ray irradiation is unique to AAV vectors.

Apoptosis induced by γ-ray irradiation enhances the bystander effects, which may allow nearby untransduced cells to take up the apoptotic vesicles containing phosphorylated toxic GCV metabolites.21 Thus, the combination therapy of AAV-mediated suicide gene therapy with radiotherapy or other genotoxic stress such as chemotherapy seems to be valuable for the treatment of cancers.

Recently, the HSVtk mutants with improved GCV-mediated killing and bystander effect have been developed.28,29,30 Since GCV has side effects such as pancytopenia31 and acute renal failure,32 the concentration of GCV should be kept as low as possible. In this study, the concentration of GCV was higher than the standard experiments, this concentration was chosen because the difference in the killing effects was most prominent among the groups, and the animals well tolerated throughout the study.

Our model would be another alternative to improve AAV-mediated suicide gene therapy of cancer. Although several studies were reported on combining radiotherapy and viral vector mediated gene therapy,22,33 our therapeutic model made it with lower dose of irradiation. AAV-mediated suicide gene therapy and γ-ray irradiation may provide a more effective and safer alternative for the treatment of head and neck cancer.

Materials and methods

Cell lines

To compare the transgene expression between head and neck cancer cells and the cells used in the standard experiments, we used HeLa cells other than the head and neck cancer cells. HeLa cells and a human laryngeal carcinoma cell line, HEp-2 cells (a gift from the Cell Resource Center for Biomedical Research, Tohoku University), were cultured in DMEM/F12 (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml of penicillin and 100 μg/ml of streptomycin (Irvine Scientific, Santa Ana, CA, USA) at 37°C in 5% CO2.

Plasmids

The plasmid pAAVLacZ contains the cytomegalovirus (CMV) promoter, human growth hormone first intron, E. coli LacZ gene, and SV40 early polyadenylation sequence between two inverted terminal repeats. A 1.8 kb DNA fragment encoding the HSVtk gene was obtained by double digestion with Hinc II and Pvu II of plasmid M234 (a gift from Dr Y Mishina, Yokohama City University, Japan) and subcloned into the pAAVLacZ in the place of the LacZ gene (pAAVtk). pW1909 is an AAV helper plasmid harboring rep/cap sequences, in which the p5 promoter was moved to downstream of the poly A signal to enhance AAV vector production.35 An adenovirus helper plasmid plAd5 contains the adenovirus early genes: E2a, E4, and VA.36

AAV vector production

AAV vectors were produced based on the plasmid transfection.36 Briefly, subconfluent 293 cells were cotransfected with AAV vector plasmid, pW1909, and adenovirus helper plasmid by a calcium phosphate precipitation method. Recombinant AAV was harvested by three cycles of freeze/thaw. The vector solution was then purified twice on a CsCl gradient as described previously.36 The vector titer was determined by a quantitative dot blot hybridization of DNase-treated stocks.

Transduction of HeLa or HEp-2 cells with AAV vectors

One day before transduction, 1×105 cells were plated onto 3.5 cm dishes in triplicate. The cells were transduced with different amounts of AAVLacZ.

Assay for β-galactosidase activity

Thirty-six hours after transduction with AAVLacZ, the cells were fixed by 0.05% glutaraldehyde in PBS and stained with X-gal (Takara, Tokyo, Japan) in PBS containing 5 mM K3[Fe(CN)6], 5 mM K4[Fe(CN)6], and 1 mM MgCl2.37 For each dish, 500 cells were counted by light microscopy and the percentage of blue stained cells was determined. An average of three wells was determined for each variable. In addition, the amount of β-galactosidase was quantified by using the β-gal ELISA kit (Boehringer-Mannheim, Hilden, Germany).

The enhancement of transgene expression by γ-ray irradiation

To examine whether γ-ray enhances the transgene expression in HeLa and HEp-2 cells, the cells were plated at a density of 1×105 cells/well in six-well culture plates 24 h prior to irradiation at doses of 0–5 Gy. Irradiation was done using GAMMACELL-40 (Atomic Energy of Canada, Ottawa, Canada) at a dose rate of 0.83 Gy/min. The cells were then transduced with 1×103 particles/cell of AAVLacZ immediately after irradiation. Thirty-six hours after transduction, we measured the amount of β-galactosidase with the β-gal ELISA.

Analysis of the second-strand synthesis of the vector genome

HeLa and HEp-2 cells grown in 10 cm dishes (1×106/dish) were transduced with 1×104 particles/cell of AAVLacZ immediately after irradiation. Two days later, total DNA was isolated under a low-salt condition (10 mM Tris–HCl [pH 7.5], 5mM EDTA, and 0.5% sodium dodecyl sulfate) to prevent annealing of the AAVLacZ genomes.38 Forty micrograms of genomic DNA digested with 80 units of mung bean nuclease (Takara, Tokyo, Japan) was resolved on 1% agarose gels, transferred to nylon membranes (Hybond N+; Amersham, Buckinghamshire, UK) and then hybridized with CMV promoter-specific probe radiolabeled with random primer labeling kit (Amersham, Buckinghamshire, UK) in 50% formamide, 6× SSC, 0.5% SDS, 5× Denhardt's solution, and 100 μg/ml of denatured salmon sperm DNA at 42°C overnight. The membranes were washed, and then analyzed by using an image analyzer (BAS-1500, Fuji, Tokyo, Japan).

GCV treatment

The cells were plated and transduced with AAVtk at the dose of 1×105 particles/cell. Twenty-four hours after transduction with AAV vectors, culture media were replaced by fresh media containing various concentrations of GCV ranging from 0 to 10 μg/ml. After a 7-day incubation in the presence of GCV, surviving cells were counted. The survival rate was calculated from the ratio of the number of surviving cells to the number of cells not treated with GCV. To evaluate the synergistic effect of the AAVtk/GCV system and γ-ray irradiation, the cells were irradiated at doses of 2 or 4 Gy just before AAV vector transduction.

Animal experiments

Female BALB/c nude mice (between 4 and 5 weeks of age) were purchased from CLEA Japan, Inc. (Tokyo, Japan) and maintained in the specific pathogen-free animal facility at Jichi Medical School. The HEp-2 cells (2×106/mouse) with 25% Matrigel (Collaborative Research, Bedford, MA, USA) were injected subcutaneously into the flanks of the mice. The tumors were irradiated by the GAMMACELL-40.

Assay for β-galactosidase in tumors

To examine whether γ-rays enhance the transgene expression in vivo, 5×1011 particles of AAVLacZ were injected into the tumors in a diameter of 4–5 mm immediately after 4 Gy irradiation. Three days after AAVLacZ was injected, the tumors were excised from mice and frozen in OTC embedding compound on a liquid-nitrogen bath. Cryostat sections (20 μm thick) were made with a Cryotome CR-502 (Nakagawa, Tokyo, Japan) and were fixed with 0.05% glutaraldehyde in PBS. Histochemical staining for β-galactosidase activity was performed in the sections, as described above and counterstained with nuclear fast red. Furthermore, to quantify the amount of β-galactosidase in the non-irradiated or 4 Gy irradiated tumor, these samples were homogenized for 10 s in a tissue homogenizer. The homogenates were centrifuged twice and aliquots of the supernatants were prepared for the assay with β-gal ELISA kit (Boehringer-Mannheim). Four mice were used in each treatment group.

In vivo gene therapy

To examine the tumor growth inhibition, 1×1012 particles of AAVtk or AAVLacZ as control were injected into the tumors immediately after 4 Gy irradiation. Administration of GCV (50 mg/kg) or PBS intraperitoneally twice a day was started 24 h after the virus infection and continued for 2 weeks. The tumors were measured every 3 days with calipers in two perpendicular diameters. The tumor volume was calculated as 0.5×L×W2, where L is the length (mm) and W is the width (mm).39 The tumors reaching a diameter of 4–5 mm were used as the starting point for the study of tumor growth or regression.

References

  1. 1

    Merino OR, Lindberg RD, Fletcher GH . An analysis of distant metastases from squamous cell carcinoma of the upper respiratory and digestive tracts. Cancer 1977; 40: 145–151.

  2. 2

    Clayman GL et al. In vivo molecular therapy with p53 adenovirus for microscopic residual head and neck squamous carcinoma. Cancer Res 1995; 55: 1–6.

  3. 3

    Kunke D et al. Preclinical study on gene therapy of cervical carcinoma using adeno-associated virus vectors. Cancer Gene Ther 2000; 7: 766–777.

  4. 4

    Su H, Lu R, Ding R, Kan YW . Adeno-associated viral-mediated gene transfer to hepatoma: thymidine kinase/interleukin 2 is more effective in tumor killing in non-ganciclovir (GCV)-treated than in GCV-treated animals. Mol Ther 2000; 1: 509–515.

  5. 5

    Berns KI, Rose JA . Evidence for a single-stranded adenovirus-associated virus genome: isolation and separation of complementary single strands. J Virol 1970; 5: 693–699.

  6. 6

    Blacklow NR et al. A seroepidemiologic study of adenovirus-associated virus infection in infants and children. Am J Epidemiol 1971; 94: 359–366.

  7. 7

    Muzyczka N . Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol 1992; 158: 97–129.

  8. 8

    Wagner JA et al. Efficient and persistent gene transfer of AAV-CFTR in maxillary sinus. Lancet 1998; 351: 1702–1703.

  9. 9

    Muramatsu S et al. Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes. Hum Gene Ther 2002; 13: 345–354.

  10. 10

    Kay MA et al. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 2000; 24: 257–261.

  11. 11

    Kanazawa T et al. Gamma-rays enhance rAAV-mediated transgene expression and cytocidal effect of AAV-HSVtk/ganciclovir on cancer cells. Cancer Gene Ther 2001; 8: 99–106.

  12. 12

    Ferrari FK, Samulski T, Shenk T, Samulski RJ . Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J Virol 1996; 70: 3227–3234.

  13. 13

    Fisher KJ et al. Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis. J Virol 1996; 70: 520–532.

  14. 14

    Alexander IE, Russell DW, Miller AD . DNA-damaging agents greatly increase the transduction of nondividing cells by adeno-associated virus vectors. J Virol 1994; 68: 8282–8287.

  15. 15

    Alexander IE, Russell DW, Spence AM, Miller AD . Effects of gamma irradiation on the transduction of dividing and nondividing cells in brain and muscle of rats by adeno-associated virus vectors. Hum Gene Ther 1996; 7: 841–850.

  16. 16

    Peng D et al. Transduction of hepatocellular carcinoma (HCC) using recombinant adeno-associated virus (rAAV): in vitro and in vivo effects of genotoxic agents. J Hepatol 2000; 32: 975–985.

  17. 17

    Trask TW et al. Phase I study of adenoviral delivery of the HSV-tk gene and ganciclovir administration in patients with current malignant brain tumors. Mol Ther 2000; 1: 195–203.

  18. 18

    Hasenburg A et al. Thymidine kinase gene therapy with concomitant topotecan chemotherapy for recurrent ovarian cancer. Cancer Gene Ther 2000; 7: 839–844.

  19. 19

    Sutton MA et al. In vivo adenovirus-mediated suicide gene therapy of orthotopic bladder cancer. Mol Ther 2000; 2: 211–217.

  20. 20

    Makinen K et al. Evaluation of herpes simplex thymidine kinase mediated gene therapy in experimental pancreatic cancer. J Gene Med 2000; 2: 361–367.

  21. 21

    Kawashita Y et al. Regression of hepatocellular carcinoma in vitro and in vivo by radiosensitizing suicide gene therapy under the inducible and spatial control of radiation. Hum Gene Ther 1999; 10: 1509–1519.

  22. 22

    Rogulski KR et al. Double suicide gene therapy augments the antitumor activity of a replication-competent lytic adenovirus through enhanced cytotoxicity and radiosensitization. Hum Gene Ther 2000; 11: 67–76.

  23. 23

    Qing K et al. Adeno-associated virus type 2-mediated gene transfer: correlation of tyrosine phosphorylation of the cellular single-stranded D sequence-binding protein with transgene expression in human cells in vitro and murine tissues in vivo. J Virol 1998; 72: 1593–1599.

  24. 24

    Sanlioglu S, Duan D, Engelhardt JF . Two independent molecular pathways for recombinant adeno-associated virus genome conversion occur after UV-C and E4orf6 augmentation of transduction. Hum Gene Ther 1999; 10: 591–602.

  25. 25

    Russell DW, Alexander IE, Miller AD . DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors. Proc Natl Acad Sci USA 1995; 92: 5719–5723.

  26. 26

    Okada H et al. Gene therapy against an experimental glioma using adeno-associated virus vectors. Gene Ther 1996; 3: 957–964.

  27. 27

    Su H, Lu R, Chang JC, Tissue-specific expression of herpes simplex virus thymidine kinase gene delivered by adeno-associated virus inhibits the growth of human hepatocellular carcinoma in athymic mice. Proc Natl Acad Sci USA 1997; 94: 13 891–13 896.

  28. 28

    Qiao J, Black ME, Caruso M . Enhanced ganciclovir killing and bystander effect of human tumor cells transduced with a retroviral vector carrying a herpes simplex virus thymidine kinase gene mutant. Hum Gene Ther 2000; 11: 1569–1576.

  29. 29

    Howard BD et al. Transduction of human pancreatic tumor cells with vesicular stomatitis virus G-pseudotyped retroviral vectors containing a herpes simplex virus thymidine kinase mutant gene enhances bystander effects and sensitivity to ganciclovir. Cancer Gene Ther 2000; 7: 927–938.

  30. 30

    Valerie K et al. Improved radiosensitization of rat glioma cells with adenovirus-expressed mutant herpes simplex virus-thymidine kinase in combination with acyclovir. Cancer Gene Ther 2000; 7: 879–884.

  31. 31

    Buhles WC et al. Ganciclovir treatment of life- or sight-threatening cytomegalovirus infection: experience in 314 immunocompromised patients. Rev Infect Dis 1988; 10: S495–S506.

  32. 32

    Snydman DR . Ganciclovir therapy for cytomegalovirus disease associated with renal transplants. Rev Infect Dis 1988; 10: S554–S562.

  33. 33

    Hanna NN et al. Virally directed cytosine deaminase/5-fluorocytosine gene therapy enhances radiation response in human cancer xenografts. Cancer Res 1997; 57: 4205–4209.

  34. 34

    Wilkie NM et al. Hybrid plasmids containing an active thymidine kinase gene of Herpes simplex virus 1. Nucleic Acids Res 1979; 7: 859–877.

  35. 35

    Ogasawara Y et al. Efficient production of adeno-associated virus vectors using split-type helper plasmids. Jpn J Cancer Res 1999; 90: 476–483.

  36. 36

    Matsushita T et al. Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Ther 1998; 5: 938–945.

  37. 37

    Cepko C . Preparation of a specific retrovirus producer cell line. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds). Current Protocols in Molecular Biology. John Wiley & Sons: New York, 1995, pp 9.10.1–9.10.13.

  38. 38

    Ozawa K, Kurtzman G, Young N . Replication of the B19 parvovirus in human bone marrow cell cultures. Science 1986; 233: 883–886.

  39. 39

    Kung AL et al. Suppression of tumor growth through disruption of hypoxia-inducible transcription. Nat Med 2000; 6: 1335–1340.

Download references

Acknowledgements

We thank Dr M Nakazawa (Department of Radiology, Jichi Medical School) for technical advice and helpful discussion. We also thank Avigen Inc., for providing the plasmids, pAAVLacZ, pW1909 and plAd, and the Cell Resource Center for Biomedical Research, Tohoku University for providing the HEp-2 cells.

This work was supported in part by grants from the Ministry of Health, Labor and Welfare of Japan, Grants-in-Aid for Science Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, CREST (Core Research for Evolutional Science and Technology), and Special Coordination Funds for promoting Science and Technology of the Science and Technology Agency of Japanese Government.

Author information

Correspondence to K Ozawa.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kanazawa, T., Mizukami, H., Okada, T. et al. Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice. Gene Ther 10, 51–58 (2003) doi:10.1038/sj.gt.3301837

Download citation

Keywords

  • adeno-associated virus vector
  • herpes simplex thymidine kinase
  • irradiation
  • animal experiments
  • head and neck neoplasms

Further reading