Mediated Herpes Simplex Virus Thymidine Research Articles

Adenovirus-mediated herpes simplex virus thymidine kinase gene therapy combined with ganciclovir induces hepatoma cell apoptosis.

The present study aimed to examine the apoptotic effects of adenovirus (ADV)-mediated herpes simplex virus thymidine kinase (ADV-TK) combined with ganciclovir (GCV) in tissues obtained from patients with hepatocellular carcinoma in order to provide a theoretical basis for the development of this gene therapy program. Apoptosis detection was conducted using the terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling assay and the apoptosis index was compared between the experimental; and control groups. Furthermore, the protein expression levels of caspase-3, B-cell lymphoma-2 (Bcl-2), Bcl-2-assoicated protein X (Bax) and nuclear factor (NF)-κB were examined in pathological specimens using immunohistochemical staining. The Bax/Bcl-2 ratio and the release of cytochrome c were examined using western blot analysis. Results indicated that combined ADV-TK and GCV treatment significantly increased the number of apoptotic cells compared with the control group (P<0.05). Immunohistological analysis revealed that ADV-TK and GCV treatment significantly increased the number of caspase-3-positive cells, reduced the Bax/Bcl-2 ratio and NF-κB expression levels and promoted the release of cytochrome c compared with the control group (P<0.01). In conclusion, the present results suggest that combined ADV-TK and GCV treatment exerts its effect through the apoptotic signaling pathway.

P08.42 Long-term treatment with Valganciclovir improves lentiviral suicide gene therapy for glioblastoma

Previously we have shown that lentiviral vector mediated herpes simplex virus thymidine kinase (HSV-Tk) / ganciclovir (GCV) therapy is a very promising therapeutic option for GBM treatment. However, after long-term remission, tumor recurrence was observed in treated animals and interestingly a fraction of recurrent glioma cells expressed HSV-Tk. We hypothesized that short-term GCV treatment, which is used in clinical trials for suicide gene therapy of GBM, fails to eliminate a fraction of glioma cells which are slow-proliferating; thus, a longer period of prodrug administration might provide a better outcome. Here, we compared long-term treatment with the prodrug valganciclovir (valGCV), which is tailored for oral administration, to short-term treatment with GCV within the context of suicide gene therapy for experimental GBM. After orthotopic implantation of patient derived GBM spheroids and visible tumor growth on MRI, we injected lentiviral vectors expressing TK.007, a highly active mutant of HSV-tk. Animals were treated with either short-term GCV (3 weeks) or long-term valGCV (3 months). Although both treatment groups showed remission and subsequent tumor recurrence, animals in the valGCV treatment group survived significantly longer and had a longer recurrence-free time period. When analyzing the recurrent tumors, we observed that these were more invasive and less angiogenic compared to primary tumors in the control groups. Furthermore, recurrent tumors showed upregulation of tyrosine kinase receptor expression, in particular EGFR. These results open up the possibility for a combination treatment of lentiviral suicide gene therapy with tyrosine kinase receptor inhibitors in the future.

Bifidobacterium infantis-mediated HSV-TK/GCV suicide gene therapy induces both extrinsic and intrinsic apoptosis in a rat model of bladder cancer

Bladder cancer is the most common cancer in the urological tract. Here, we investigated the molecular pathways involved in the apoptosis of rodent bladder cancer, which was treated with Bifidobacterium infantis (BI)-mediated herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) suicide gene therapy. We engineered the BI-TK system, which consists of BI with the recombinant plasmid PGEX-1λT carrying HSV-TK (that is, TK) gene. Tumor-bearing rats were randomly divided into three groups and tail vein injected with normal saline (group A), BI/PGEX-1λT (group B) or BI-TK (group C), followed by intraperitoneal injection of GCV. The BI-TK/GCV system (group C) was shown to inhibit tumor growth and caused the apoptosis of tumor cells as assessed by in situ terminal transferase dUTP nick-end labeling assay. While varied extents of apoptosis were detected in tumor cells from all groups, tumor cells treated with BI-TK/GCV system (group C) exhibited the highest level of apoptosis (P < 0.05), consistent with our previous studies. Furthermore, we found that the expression levels of Fas, FasL, Cyt-C and caspase-9 in tumor tissues derived from group C were significantly higher than the other two groups (P < 0.001). Therefore, our results have demonstrated that the BI-TK/GCV therapy system exhibits a sustainable antitumor growth activity in the rodent model of bladder cancer. Mechanistically, both extrinsic and intrinsic apoptosis pathways are involved in the BI-TK/GCV antitumor functionality.

790 SYSTEMIC T-CELL ACTIVATION FOLLOWING NEOADJUVANT IN SITU GENE THERAPY IN HIGH-RISK PROSTATE CANCER PATIENTS

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2012790 SYSTEMIC T-CELL ACTIVATION FOLLOWING NEOADJUVANT IN SITU GENE THERAPY IN HIGH-RISK PROSTATE CANCER PATIENTS Takefumi Satoh, Makoto Kubo, Ken-ichi Tabata, Shinji Kurosaka, Kazumasa Matsumoto, Tetsuo Fujita, Fumiya Obata, Yasutomo Nasu, Hiromi Kumon, Dov Kadmon, Malcolm K. Brenner, Timothy C. Thompson, and Shiro Baba Takefumi SatohTakefumi Satoh Kanagawa, Japan More articles by this author , Makoto KuboMakoto Kubo Kanagawa, Japan More articles by this author , Ken-ichi TabataKen-ichi Tabata Kanagawa, Japan More articles by this author , Shinji KurosakaShinji Kurosaka Kanagawa, Japan More articles by this author , Kazumasa MatsumotoKazumasa Matsumoto Kanagawa, Japan More articles by this author , Tetsuo FujitaTetsuo Fujita Kanagawa, Japan More articles by this author , Fumiya ObataFumiya Obata Kanagawa, Japan More articles by this author , Yasutomo NasuYasutomo Nasu Okayama, Japan More articles by this author , Hiromi KumonHiromi Kumon Okayama, Japan More articles by this author , Dov KadmonDov Kadmon Houston, TX More articles by this author , Malcolm K. BrennerMalcolm K. Brenner Houston, TX More articles by this author , Timothy C. ThompsonTimothy C. Thompson Houston, TX More articles by this author , and Shiro BabaShiro Baba Kanagawa, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.878AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Neoadjuvant in situ cytotoxic gene therapy can potentially trigger a systemic immune response, which could impact occult micro-metastatic disease. We are currently conducting adenoviral vector mediated Herpes Simplex Virus-thymidine kinese (HSV-tk) gene plus ganciclovir (GCV) therapy as neoadjuvant intraprostatic injection for localized high-risk prostate cancer. This study evaluates the systemic T-cell response following gene therapy. METHODS We enrolled 5 men with clinically localized prostate cancer but high risk for recurrence (Kattan nomogram score > 115) in this Phase I–II trial. Intraprostatic viral injections (two) were followed by 2 weeks of GCV and prostatectomy 4 weeks later. Peripheral blood lymphocytes were analyzed by fluorescent antibody cell sorting (FACS). Samples were taken at the time of HSV-tk virus injection and at selected intervals afterwards. 100 μl of heparin-treated blood was incubated with dual color labeled antibody pairs: CD45/CD14, CD3/CD19, CD3/CD8, CD3/CD4, CD8/HLA-DR, CD4/HLA-DR, CD3/HLA-DR, CD3/CD56+CD16 and CD4/CD25. Forward and side scatter was set to distinguish lymphocyte, macrophage, and granulocyte populations with an EPICS XL flow cytometer. RESULTS A reduction in serum PSA was observed immediately after vector injection and GCV therapy in all patients. The mean reduction was 31.1% and ranged from 24.8 to 38.9%. The pretreatment mean percentage of CD8+ T cells positive for the HLA-DR marker of activation was 10.6%. For day 2, day 7, day 14, day 16 and day 56 post treatment, the mean percent of CD8+ DR+ T cells increased by 14.0%, 12.3%, 19.7%, 25.4% and 14.9%, which were statistically significant (day 14; p = 0.0431, day 16; p = 0.0431). CONCLUSIONS We present evidence of systemic T-cell responses following HSV-tk + GCV gene therapy under clinical trial condition. There was an increase in activated CD8+ T cells in the peripheral blood following vector injection suggesting the potential for activation of components of cell-mediated immune response in this neoadjuvant setting. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e323 Peer Review Report Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takefumi Satoh Kanagawa, Japan More articles by this author Makoto Kubo Kanagawa, Japan More articles by this author Ken-ichi Tabata Kanagawa, Japan More articles by this author Shinji Kurosaka Kanagawa, Japan More articles by this author Kazumasa Matsumoto Kanagawa, Japan More articles by this author Tetsuo Fujita Kanagawa, Japan More articles by this author Fumiya Obata Kanagawa, Japan More articles by this author Yasutomo Nasu Okayama, Japan More articles by this author Hiromi Kumon Okayama, Japan More articles by this author Dov Kadmon Houston, TX More articles by this author Malcolm K. Brenner Houston, TX More articles by this author Timothy C. Thompson Houston, TX More articles by this author Shiro Baba Kanagawa, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Ultrasound-targeted microbubble destruction mediated herpes simplex virus-thymidine kinase gene treats hepatoma in mice

ObjectiveThe purpose of the study was to explore the anti-tumor effect of ultrasound -targeted microbubble destruction mediated herpes simplex virus thymidine kinase (HSV-TK) suicide gene system on mice hepatoma.MethodsForty mice were randomly divided into four groups after the models of subcutaneous transplantation tumors were estabilished: (1) PBS; (2) HSV-TK (3) HSV-TK+ ultrasound (HSV-TK+US); (4) HSV-TK+ultrasound+microbubbles (HSV-TK+US+MB). The TK protein expression in liver cancer was detected by western-blot. Applying TUNEL staining detected tumor cell apoptosis. At last, the inhibition rates and survival time of the animals were compared among all groups.ResultsThe TK protein expression of HSV-TK+MB+US group in tumor-bearing mice tissues were significantly higher than those in other groups. The tumor inhibitory effect of ultrasound-targeted microbubble destruction mediated HSV-TK on mice transplantable tumor was significantly higher than those in other groups (p < 0.05), and can significantly improve the survival time of tumor-bearing mice.ConclusionUltrasound-targeted microbubble destruction can effectively transfect HSV-TK gene into target tissues and play a significant inhibition effect on tumors, which provides a new strategy for gene therapy in liver cancer.

Inhibitory effects of HSV-tk/GCV system mediated by recombinant adeno-associated virus 2 on rabbit lens epithelial cells

To study the inhibitory effects of recombinant adeno-associated virus 2 (rAAV2)-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system on the rabbit lens epithelial cells (N/N1003A) in vitro and to investigate the mechanism of cell death. After N/N1003A cells had been transfected with rAAV2-EGFP, expression of enhanced green fluorescent protein (EGFP) were observed by inverted fluorescent microscope and the transfection efficiency was detected by flow cytometry. N/N1003A cells were infected by recombinant virus rAAV2/HSV-tk as the treated group, and the uninfected N/N1003A cells were used as the controls. The dose- and time-dependent efficiency and bystander effect of HSV-tk/GCV system on the cells were studied by MTT assay. Apoptosis and necrosis were observed by phase contrast microscope, electron microscope and Hoechst33258 stain. Apoptotic cell rate and cell cycle were detected by flow cytometry. rAAV2 vector encoding EGFP gene could be transfected into N/N1003A cells stably and efficiently. The effects of GCV on these two groups were dose-dependent (F = 13.076. 239, P < 0.001). The difference of percentages of survival cells between the study group and the control group at various doses of GCV was statistically significant (F = 53,47.119, P < 0.001). The 50% of the inhibitory concentration (IC50) of GCV in the study group was 2 mg/L and was 524 mg/L in the control group. The killing efficiency of GCV increased with the prolongation of time and showed significant bystander effect. Cell apoptosis and necrosis were observed in N/N1003A-tk cells transfected by GCV, and the percentage of apoptotic cells was significantly higher than that of the control group (t = 3.83, P < 0.01). The percentages of N/N1003A-tk cells in the S phase of the cell cycle was significantly higher than that of the control group (t = 3.55, P < 0.01). Whereas the percentages of the G0/ G1 phase in GCV treated cells was significantly lower than that of the control group ( t = 4.29, P < 0.01). GCV can kill efficiently the N/N1003A cells infected by recombinant virus rAAV2/HSV-tk, and there is strong bystander effect. Recombinant adeno-associated virus-mediated HSV-tk/GCV suicide gene system may provide an effective approach for the treatment of lens posterior capsular opacification.

1106. Results of a Phase I Clinical Trial of Adenoviral-Interferon-Beta Gene Therapy for Malignant Mesothelioma and Malignant Pleural Effusions

Our group is investigating intrapleural (IPl) interferon-beta (IFN- β) gene transfer for malignant pleural mesothelioma (MPM) and malignant pleural effusions (MPE). We previously conducted trials involving adenoviral (Ad)-mediated herpes simplex virus thymidine kinase gene (Ad.HSVtk) delivery and ganciclovir (GCV) in 34 MPM patients. These studies demonstrated dose-related gene transfer and anti-Ad immune responses. Two of 3 surviving patients have minimal residual disease 7.5 years after Ad.HSVtk/GCV.

43. Long-Term T Cell Activation Following Combined In Situ Gene Therapy and Intensity-Modulated Radiotherapy Compared to Gene Therapy as Mono-Therapy in Prostate Cancer Patients

Introduction: A program combining in situ gene therapy and intensity-modulated radiation therapy (IMRT) was implemented for the treatment of prostate cancer because the complementary mechanisms of cytotoxicity may have an enhanced efficacy. The in situ gene therapy comprised of adenoviral vector mediated Herpes Simplex Virus-thymidine kinase (AdHSVtk) + valacyclovir (VCV). We are currently conducting clinical trials using this approach. This study explores long-term immune responses following combined radio-gene therapy compared to gene therapy as mono-therapy.

923. Combining In Situ Gene Therapy with Radiotherapy and Hormonal Therapy in the Treatment of Stage D1 Prostate Cancer

Introduction: Clinical trial of combination adenoviral vector mediated Herpes Simplex Virus-thymidine kinase (HSV-tk) + valacyclovir (VCV) in situ gene therapy with radiotherapy (RT) and hormonal therapy was conducted for Stage D1 prostate cancer. We now report the safety, efficacy, and immune responses using this approach.

Enhanced systemic T-cell activation after in situ gene therapy with radiotherapy in prostate cancer patients

Purpose In situ cytotoxic gene therapy can potentially trigger a systemic immune response, which could impact occult metastatic disease. We are currently conducting three clinical trials using in situ adenoviral vector mediated herpes simplex virus–thymidine kinase (HSV- tk) gene delivery followed by the HSV- tk prodrug ganciclovir (GCV) or valacyclovir (VCV). This study evaluates the systemic T-cell response after gene therapy in each trial. Methods and materials The study protocol included three separate clinical trials in the Baylor Prostate Cancer SPORE Program: Trial A gene therapy in prostate cancer patients failing radiotherapy (36 patients), Trial B neoadjuvant gene therapy in pre–radical prostatectomy patients (22 patients), and Trial C gene therapy in combination with radiotherapy for prostate cancer (27 patients). Heparinized blood was collected at the time of vector injection and at selected intervals afterward. A complete blood count was performed, and peripheral blood lymphocytes were analyzed by fluorescent antibody cell sorting after labeling with dual color–labeled antibody pairs. Results The pretreatment mean percentage of activated CD8+ T cells (DR+CD8+ T cells) was 12.23%, 16.72%, and 14.09% (Trials A, B, and C, respectively). Two weeks posttreatment, this increased to 22.87%, 26.15%, and 39.04% (Trials A, B, and C, respectively), and these increases were statistically significant ( p = 0.0188, p = 0.0010, p < 0.0001, respectively). The increase of DR+CD8+ T cells was significantly larger in Trial C than in Trial A ( p = 0.0044) or Trial B ( p = 0.0288). Total CD8+ T cells significantly increased at 2 weeks posttreatment in Trial B and C ( p = 0.0013, p = 0.0004, respectively). Interestingly, only in Trial C were significant increases in activated CD4+ T cells seen at 2 weeks ( p = 0.0035). Conclusions This is the first report of systemic T-cell responses after HSV- tk+GCV/VCV gene therapy under three clinical trial conditions. There was an increase in activated CD8+ T cells in the peripheral blood after vector injection, suggesting the potential for activation of components of cell-mediated immune response in all trial conditions. The addition of radiotherapy to in situ gene therapy seems to further increase the total CD8+ T cells and activated CD4+ T cells.

Adenovirus-mediated herpes simplex virus thymidine kinase gene therapy in BT4C rat glioma model

Adenovirus (Adv)-mediated herpes simplex virus thymidine kinase (adv/tk) gene therapy combined with ganciclovir (GCV) medication is a promising approach for the treatment of malignant glioma. However, optimal administration and the effect of possible adjuvant treatments have not been fully examined. In the present study, we examined the efficacy of adv/tk/GCV gene therapy in a syngeneic BT4C rat malignant glioma model, either as a single administration or given as three injections during three consecutive days. The effect of combined adv-mediated macrophage colony-stimulating factor (MCSF) and adv/tk gene transfer was also studied. BT4C malignant glioma cells were injected into the right corpus callosum of BDIX rats (n=112). Before gene therapy, the presence of tumors was verified by MRI. The rats were divided into eight groups as follows: group I (n=20) received a single adv/tk gene transfer (total dose 4x10(8) pfu) and GCV treatment for 14 days; group II (n=5) received the same gene transfer without GCV; group III (n=28) received three adv/tk injections (total dose 4x10(8) pfu) on three consecutive days and GCV for 14 days; group IV (n=5) received three similar adv/tk injections without GCV medication; group V (n=13) received three adv/MCSF injections (total dose 2x10(8) pfu) on three consecutive days and GCV medication; group VI (n=12) received three adv/tk and adv/MCSF (total dose 6x10(8) pfu) injections on three consecutive days followed by GCV medication; and group VII (n=12) the same treatment without GCV. Group VIII (n=17) consisted of wild-type BT4C malignant glioma tumors without any treatment. Treatment effect and tissue responses were characterized by general histology, immunohistochemistry, MRI, and survival of the study groups. The best treatment effect and survival was found in rats treated with adv/tk gene transfer once a day for three consecutive days (P<.05). No improvement of the treatment effect was seen after the combined adv/tk and adv/MCSF gene transfer compared with the repeated adv/tk gene transfer. The results show that 20% of the rats can be cured (survival >6 months) after optimized adv/tk gene therapy. It is concluded that repeated intratumoral administration of adv/tk is a promising approach for the treatment of malignant glioma tumors in vivo.

Systemic T-cell activation following in situ gene therapy in prostate cancer patients

Purpose/Objective: Neoadjuvant in situ cytotoxic gene therapy can potentially trigger a systemic immune response, which could impact occult micro-metastatic disease. We are currently conducting three clinical trials using in situ adenoviral vector mediated Herpes Simplex Virus-thymidine kinese (HSV-tk) gene plus ganciclovir (GCV) therapy. This study evaluates the systemic T-cell response following gene therapy in each trial.

Adenovirus-mediated suicide-gene therapy in an orthotopic murine bladder tumor model.

Patients with high-grade transitional-cell carcinoma (TCC) of the bladder frequently experience recurrence and progress and have a low response rate to chemotherapy in metastatic TCC. In this study, we evaluated the feasibility and long-term efficacy of suicide-gene therapy using adenovirus (Ad)-mediated herpes simplex virus thymidine kinase gene (HSV-TK) and prodrug ganciclovir (GCV) as a potential therapeutic approach in murine-orthotopic models of TCC. A replication defective adenoviral vectors containing toxic HSV-TK gene under the transcriptional control of RSV (Rous sarcoma virus) promoter (Ad-RSV-TK) was used. Orthotopic bladder TCC was established with 1 x 106 murine (MBT-2) TCC cells in syngenic C3H/He female mice. Intratumoral injection of Ad-RSV-TK in combination with GCV (20 mg/kg body weight/day i.p. b.i.d. x 7 days) was administered in vivo for the determination of treatment efficacy and long-term host survival in separate controlled experiments. In vivo experiments demonstrated greater than three-fold reductions in MBT-2 tumor growth for the animals treated with Ad-RSV-TK (5 x 108 plaque forming units (pfu)/GCV therapy (P < 0.01)). Central tumor necrosis and apoptosis were revealed by histomorphology and immunohistochemistry compared with other control animals (non-treated, GCV alone, Ad-RSV-TK alone). Direct intratumoral injection with Ad-RSV-TK/GCV also resulted in significantly improved survival over the control groups in separated experiment (log-rank test, P < 0.05). Suicide-gene therapy using Ad-RSV-TK/GCV provides an effective therapy in an experimental murine orthotopic bladder cancer by significantly inhibiting tumor growth and improving long-term host survival.

Enhanced antitumor effect and reduced vector dissemination with fiber-modified adenovirus vectors expressing herpes simplex virus thymidine kinase.

There are at least two hurdles confronting the use of the adenovirus (Ad)-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system for the treatment of cancer. One is inefficient Ad vector-mediated gene transfer into tumor cells lacking the primary receptor, i.e., the coxsackievirus and adenovirus receptor (CAR). The other is hepatotoxicity due to unwanted vector spread into the liver, even when Ad vectors are injected intratumorally. Herein, we present an attractive strategy for overcoming such limitations based on use of a fiber-modified Ad vector containing an RGD peptide motif in the fiber knob. HSVtk-expressing Ad vectors containing mutant fiber (AdRGD-tk) or wild-type fiber (Ad-tk) were injected intratumorally into CAR-negative B16 melanoma cells inoculated into mice, after which GCV was injected intraperitoneally for 10 days. AdRGD-tk showed approximately 25 times more antitumor activity than Ad-tk. Histopathological studies suggested that liver damage in mice injected with AdRGD-tk was significantly lower than that in mice injected with Ad-tk. Intratumoral administration of luciferase-expressing Ad vectors containing the mutant fiber (AdRGD-L2) resulted in nearly 40 times more luciferase production in the tumor, but 8 times less production in the liver than the conventional Ad vectors (Ad-L2). These results indicate that combination of fiber-modified vectors and a HSVtk/GCV system is a potentially useful and safe approach for the treatment of tumors lacking CAR expression, and that fiber-modified vectors could be of great utility for gene therapy and gene transfer experiments.

Adenovirus-mediated suicide-gene therapy using the herpes simplex virus thymidine kinase gene in cell and animal models of human prostate cancer: changes in tumour cell proliferative activity.

To determine the feasibility and efficacy of suicide-gene therapy using adenovirus (Ad)-mediated herpes simplex virus thymidine kinase (HSV-TK) and the prodrug acyclovir, and to evaluate changes in the biological phenotype for tumour cell proliferative activity after suicide-gene therapy in animal models of human prostate cancer. Using a replication-defective adenoviral vector (cytomegalovirus, CMV) containing the beta-galactosidase gene (Ad-CMV-beta-gal) as a control and Ad-CMV-TK as the therapeutic vector under the transcriptional control of the CMV promoter, transduction efficiency was assessed in vitro by infecting LNCaP and PC-3 androgen-dependent and independent human prostate cancer cells with Ad-CMV-beta-gal, and using X-gal staining. The TK activity in prostate cancer cells infected with Ad-CMV-TK was determined by measuring TK-mediated [3H]-gancyclovir phosphorylation. The sensitivity of LNCaP and PC-3 cells to Ad-CMV-TK in vitro was determined after infection with the therapeutic vector with or without acyclovir. The inhibition of PC-3 tumour growth in vivo induced by the Ad-CMV-TK/acyclovir suicide-gene system was assessed in separate and controlled experiments using human prostate cancer mouse models. Ki-67 proliferative antigen and proliferating cell nuclear antigen (PCNA), both useful proliferative indices, were evaluated using immunohistochemical staining (MIB-1 monoclonal antibody and monoclonal anti-PCNA antibody) in formalin-fixed, paraffin-embedded tissues from gene therapy-treated and control animals. The mean TK activity was significantly higher in LNCaP and PC-3 cells infected with Ad-CMV-TK than in cells infected with Ad-CMV-beta-gal, used as a control (P < 0.05). The growth of human prostate cancer cells with Ad-CMV-TK was significantly inhibited by adding acyclovir in vitro (P < 0.05). In the in vivo experiments using the PC-3 human prostate cancer mouse model, tumour volume and growth was lower in mice treated with Ad-CMV-TK/acyclovir than in those treated with Ad-CMV-TK only, acyclovir only or untreated (controls) (P < 0.05). Histochemical staining of tumour tissues showed that Ad-CMV-TK/acyclovir destroyed PC-3 tumours through tumour cell death and apoptosis, with local lymphatic infiltration. The mean PCNA labelling index in prostate cancer cells of mice treated with Ad-CMV-TK/acyclovir was significantly lower than that in untreated controls (P < 0.05, Mann-Whitney U-test). The Ki-67 labelling index in prostate cancer cells of mice treated with Ad-CMV-TK/acyclovir was also lower than that in untreated controls (P < 0.05, Student's t-test). Adenovirus-mediated suicide-gene therapy using the HSV-TK gene decreased the proliferative activity of PC-3 human prostatic cancer cells in vivo. Adenovirus-mediated suicide-gene therapy using an HSV-TK/acyclovir system provided effective therapy in an experimental human prostate cancer mouse model, by significantly inhibiting tumour growth and decreasing the proliferative activity of human prostate cancer cells. Such therapy could be developed as a novel method for treating patients with androgen-independent prostate cancer.