Development of a Conditionally Replicating Pseudorabies Virus for HER-2/neu-overexpressing Bladder Cancer Therapy
Authors: Ai-Li Shiau, Yu-Ping Lin, Gia-Shing Shieh, Chih-Hau Su, Wen-Luan Wu, Yuh-Shyan Tsai, Chih-Wei Cheng, Ming-Derg Lai, Chao-Liang Wu
Journal: Molecular Therapy (2006)

Overexpression of the HER-2/neu oncogene, a frequent molecular event in a variety of cancers including bladder cancer, is associated with tumor progression and poor prognosis. Therapeutic strategies to targeting HER-2/neu-overexpressing cancer cells have shown promise. Pseudorabies virus (PrV), a herpesvirus of swine, may be exploited as an oncolytic agent for human cancer. Herein, we generated a conditionally replicating glycoprotein E-defective PrV mutant carrying glycoprotein D and herpes simplex virus type 1 thymidine kinase genes, which are essential for viral entry and replication, under the transcriptional control of the HER-2/neu promoter. The recombinant PrV, designated YP2, selectively replicated in and lysed HER-2/neu-overexpressing human bladder, mouse bladder, and hamster oral cancer cells in vitro. Notably, YP2 retarded MBT-2 bladder tumor growth in mice by more than 50% and more than half of the mice survived for over 50 days, whereas all the control mice survived less than 30 days. Taken together, our results suggest that YP2 may have therapeutic potential for the treatment of invasive bladder cancer. Furthermore, because HER-2/neu is overexpressed in a broad spectrum of cancers, this conditionally replicating PrV may be broadly applicable. Overexpression of the HER-2/neu oncogene, a frequent molecular event in a variety of cancers including bladder cancer, is associated with tumor progression and poor prognosis. Therapeutic strategies to targeting HER-2/neu-overexpressing cancer cells have shown promise. Pseudorabies virus (PrV), a herpesvirus of swine, may be exploited as an oncolytic agent for human cancer. Herein, we generated a conditionally replicating glycoprotein E-defective PrV mutant carrying glycoprotein D and herpes simplex virus type 1 thymidine kinase genes, which are essential for viral entry and replication, under the transcriptional control of the HER-2/neu promoter. The recombinant PrV, designated YP2, selectively replicated in and lysed HER-2/neu-overexpressing human bladder, mouse bladder, and hamster oral cancer cells in vitro. Notably, YP2 retarded MBT-2 bladder tumor growth in mice by more than 50% and more than half of the mice survived for over 50 days, whereas all the control mice survived less than 30 days. Taken together, our results suggest that YP2 may have therapeutic potential for the treatment of invasive bladder cancer. Furthermore, because HER-2/neu is overexpressed in a broad spectrum of cancers, this conditionally replicating PrV may be broadly applicable. IntroductionConventional therapies for transitional cell carcinoma of the bladder often result in poor prognosis, indicating the need for novel therapeutic strategies. HER-2/neu is a cell surface proto-oncogene that is often overexpressed in various cancers. Higher frequency of HER-2/neu overexpression has been observed in muscle-invasive bladder cancer, which correlates with enhanced metastasis and a poor clinical outcome.1Jimenez RE et al.Her-2/neu overexpression in muscle-invasive urothelial carcinoma of the bladder: prognostic significance and comparative analysis in primary and metastatic tumors.Clin Cancer Res. 2001; 7: 2440-2447PubMed Google Scholar, 2Kruger S et al.HER2 overexpression in muscle-invasive urothelial carcinoma of the bladder: prognostic implications.Int J Cancer. 2002; 102: 514-518Crossref PubMed Scopus (157) Google Scholar, 3Gandour-Edwards R et al.Does HER2/neu expression provide prognostic information in patients with advanced urothelial carcinoma?.Cancer. 2002; 95: 1009-1015Crossref PubMed Scopus (122) Google Scholar, 4Kruger S et al.Overexpression of c-erbB-2 oncoprotein in muscle-invasive bladder carcinoma: relationship with gene amplification, clinicopathological parameters and prognostic outcome.Int J Oncol. 2002; 21: 981-987PubMed Google Scholar Overexpression of HER-2/neu enhances invasion, angiogenesis, and increased survival of cancer cells, leading to increased cancer metastases.5Yu D Hung MC Overexpression of ErbB2 in cancer and ErbB2-targeting strategies.Oncogene. 2000; 19: 6115-6121Crossref PubMed Scopus (335) Google Scholar Furthermore, overexpression of HER-2/neu confers on cancer cells increased resistance to various cancer therapies, resulting in poor response to cancer treatment. HER-2/neu-targeted strategies have shown promise as potential therapies for HER-2/neu-overexpressing cancer cells.5Yu D Hung MC Overexpression of ErbB2 in cancer and ErbB2-targeting strategies.Oncogene. 2000; 19: 6115-6121Crossref PubMed Scopus (335) Google Scholar These cells are believed to contain transcription factors that strongly activate the HER-2/neu promoter, and suicide gene therapy has been developed that exploits their transcriptional profile.6Harris JD Gutierrez AA Hurst HC Sikora K Lemoine NR Gene therapy for cancer using tumour-specific prodrug activation.Gene Ther. 1994; 1: 170-175PubMed Google ScholarPseudorabies virus (PrV), a neurotropic herpesvirus, has a wide host range, including several mammalian species, but is non-pathogenic for humans.7Enquist LW Husak PJ Banfield BW Smith GA Infection and spread of alphaherpesviruses in the nervous system.Adv Virus Res. 1998; 51: 237-347Crossref PubMed Google Scholar Systemic and clinical manifestations are dependent on virus strain–specific virulence and age of the animals, ranging from severe neurological signs, leading to death, to mild respiratory symptoms with elevated temperature and loss of appetite, resulting in decreased weight gain. Glycoproteins D (gD), E (gE), and I (gI) are important for interactions between virions and host cells.8Hampl H Ben Porat T Ehrlicher L Habermehl KO Kaplan AS Characterization of the envelope proteins of pseudorabies virus.J Virol. 1984; 52: 583-590PubMed Google Scholar, 9Wathen MW Wathen LM Isolation, characterization, and physical mapping of a pseudorabies virus mutant containing antigenically altered gp50.J Virol. 1984; 51: 57-62PubMed Google Scholar The gD protein is essential for viral penetration, but not required for direct cell-to-cell spread.10Rauh I Mettenleiter TC Pseudorabies virus glycoproteins gII and gp50 are essential for virus penetration.J Virol. 1991; 65: 5348-5356PubMed Google Scholar, 11Peeters B Pol J Gielkens A Moormann R Envelope glycoprotein gp50 of pseudorabies virus is essential for virus entry but is not required for viral spread in mice.J Virol. 1993; 67: 170-177PubMed Google Scholar, 12Mulder W Pol J Kimman T Kok G Priem J Peeters B Glycoprotein D-negative pseudorabies virus can spread transneuronally via direct neuron-to-neuron transmission in its natural host, the pig, but not after additional inactivation of gE or gI.J Virol. 1996; 70: 2191-2200PubMed Google Scholar The gE protein is dispensable for viral replication but is required for transneuronal spread of PrV. Deletion of gE reduces virulence,13Mettenleiter TC Zsak L Kaplan AS Ben-Porat T Lomniczi B Role of a structural glycoprotein of pseudorabies in virus virulence.J Virol. 1987; 61: 4030-4032PubMed Google Scholar because gE-deleted PrV has a reduced capability to infect second- and third-order neurons of the olfactory and trigeminal routes in the nervous system.14Mulder WA et al.Glycoprotein gE-negative pseudorabies virus has a reduced capability to infect second- and third-order neurons of the olfactory and trigeminal routes in the porcine central nervous system.J Gen Virol. 1994; 75: 3095-3106Crossref PubMed Scopus (69) Google Scholar, 15Babic N Klupp B Brack A Mettenleiter TC Ugolini G Flamand A Deletion of glycoprotein gE reduces the propagation of pseudorabies virus in the nervous system of mice after intranasal inoculation.Virology. 1996; 219: 279-284Crossref PubMed Scopus (71) Google Scholar The complex formed by gE and gI is one of the prime determinants for neuroinvasion and neurovirulence. PrV-encoded thymidine kinase (TK) supports viral DNA replication and plays an important role in virulence and neuroinvasiveness.16McGregor S Easterday BC Kaplan AS Ben Porat T Vaccination of swine with thymidine kinase-deficient mutants of pseudorabies virus.Am J Vet Res. 1985; 46: 1494-1497PubMed Google Scholar, 17Ferrari M et al.A comparative study of pseudorabies virus (PRV) strains with defects in thymidine kinase and glycoprotein genes.J Comp Pathol. 2000; 123: 152-163Crossref PubMed Scopus (21) Google Scholar Moreover, it is required for production of infectious virions in non-dividing cells.18Mulder WA Priem J Pol JM Kimman TG Role of viral proteins and concanavalin A in in vitro replication of pseudorabies virus in porcine peripheral blood mononuclear cells.J Gen Virol. 1995; 76: 1433-1442Crossref PubMed Scopus (14) Google Scholar Genetically modified herpes simplex virus (HSV) mutants are effective oncolytic viruses targeting rapidly dividing cells in animal models,19Martuza RL Malick A Markert JM Ruffner KL Coen DM Experimental therapy of human glioma by means of a genetically engineered virus mutant.Science. 1991; 252: 854-856Crossref PubMed Scopus (767) Google Scholar, 20Kooby DA et al.Oncolytic viral therapy for human colorectal cancer and liver metastases using a multi-mutated herpes simplex virus type-1 (G207).FASEB J. 1999; 13: 1325-1334Crossref PubMed Scopus (151) Google Scholar and have completed Phase I clinical trials in treating recurrent high-grade glioma.21Rampling R et al.Toxicity evaluation of replication-competent herpes simplex virus (ICP 34.5 null mutant 1716) in patients with recurrent malignant glioma.Gene Ther. 2000; 7: 859-866Crossref PubMed Scopus (515) Google Scholar, 22Markert JM et al.Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial.Gene Ther. 2000; 7: 867-874Crossref PubMed Scopus (809) Google Scholar PrV can infect and destroy both mouse and human tumors in vivo without signs of disease, suggesting its potential use as an oncolytic agent for human tumors.23Boldogkoi Z Bratincsak A Fodor I Evaluation of pseudorabies virus as a gene transfer vector and an oncolytic agent for human tumor cells.Anticancer Res. 2002; 22: 2153-2159PubMed Google Scholar Given that deletion of virulence genes of PrV is necessary for safety concerns, but also renders PrV less able to replicate and thereby less cytolytic or oncolytic, in this study we generated YP2 virus, a gE/TK-defective PrV mutant carrying both gD and HSV-1 TK genes under the transcriptional control of the HER-2/neu promoter. Replication and spread of YP2 would be expected to be restricted due to lack of gD and TK expression in normal cells or cancer cells expressing low or negligible levels of HER-2/neu. The fact that gD is essential but not required for cell-to-cell spread raises the question of the safety of YP2. Nevertheless, it has been shown that gD-negative virions released from non-complementing cells are not infectious and unable to be transmitted from inoculated animals to other animals, or even to other species.10Rauh I Mettenleiter TC Pseudorabies virus glycoproteins gII and gp50 are essential for virus penetration.J Virol. 1991; 65: 5348-5356PubMed Google Scholar, 11Peeters B Pol J Gielkens A Moormann R Envelope glycoprotein gp50 of pseudorabies virus is essential for virus entry but is not required for viral spread in mice.J Virol. 1993; 67: 170-177PubMed Google Scholar Furthermore, although gD-negative PrV can spread transneuronally via direct neuron-to-neuron transmission, the tansneuronal spread is abrogated after additional inactivation of gE.12Mulder W Pol J Kimman T Kok G Priem J Peeters B Glycoprotein D-negative pseudorabies virus can spread transneuronally via direct neuron-to-neuron transmission in its natural host, the pig, but not after additional inactivation of gE or gI.J Virol. 1996; 70: 2191-2200PubMed Google Scholar By contrast, YP2 replicates and lyses cancer cells overexpressing HER-2/neu owing to gD and TK expression through the transcriptional activation of the HER-2/neu promoter. In this study, we show that YP2 selectively replicated in and lysed HER-2/neu-overexpressing bladder cancer cells, and retarded tumor growth and prolonged survival in mice bearing syngeneic bladder tumors. Our results suggest that this HER-2/neu promoter-driven oncolytic PrV may have therapeutic potential for treating bladder cancer and possibly other tumors overexpressing HER-2/neu.ResultsConstruction and characterization of HER-2/neu promoter–driven oncolytic PrVThe strategy for constructing YP2, a gE/TK-defective PrV mutant carrying both gD and HSV-1 TK genes driven by the HER-2/neu promoter, is depicted in Figure 1a. YP2 was primarily characterized by PCR to amplify the gD and HSV-TK genes. As shown in Figure 1b, regions corresponding to the gD and HSV-TK genes of 262 and 1,131 bp in size, respectively, were detected in YP2 DNA, whereas only the HSV-TK gene was amplified in CW1 DNA by PCR. The PCR products were verified by automated DNA sequencing (data not shown). To confirm the genomic structure of YP2, virion DNA was cleaved with BamHI, electrophoresed, transferred to a nylon membrane, and hybridized to a radiolabeled gD-specific probe. In TNL, the gD gene resides within the 7.4-kb BamHI fragment of the PrV genome.24Pritchett RF Bush CE Chang TJ Wang JT Zee YC Comparison of the genomes of pseudorabies (Aujeszky's disease) virus strains by restriction endonuclease analysis.Am J Vet Res. 1984; 45: 2486-2489PubMed Google Scholar CW1 was derived from TNL, in which the gD gene was replaced by the HSV-TK gene.25Shiau AL Liu CW Wang SY Tsai CY Wu C A simple selection system for construction of recombinant gD-negative pseudorabies virus as a vaccine vector.Vaccine. 2002; 20: 1186-1195Crossref PubMed Scopus (9) Google Scholar The HSV-TK expression cassette in CW1 was replaced with the HER-2/neu promoter-gD-IRES-HSV-TK fragment to generate YP2. Southern hybridization with the gD probe yielded signals only with DNA derived from TNL and YP2 (Figure 1c). Whereas a single band corresponding to approximately 7.4 kb was observed with TNL with a 2-h exposure time, no signal was detected with YP2 (data not shown). The content of the BamHI-digested DNA from TNL was much higher than that from YP2 or CW1, as evidenced by ethidium bromide staining (data not shown). To increase the sensitivity, we prolonged the exposure time. With a longer exposure time (20 h), YP2 gave a single band corresponding to approximately 10.5 kb, whereas a strong 7.4-kb band was noticed with TNL (Figure 1c). As expected, no signal was detected with CW1 even when the blot was overexposed. Taken together, the data from PCR and Southern hybridization analyses confirm the genomic structure of YP2.Detection of HER-2/neu protein expression and promoter activity in bladder and oral cancer cellsFirst, we examined the expression levels of HER-2/neu protein in a panel of cancer cell lines and mouse NIH3T3 fibroblasts by immunoblot analysis (Figure 2a). HER-2/neu was highly expressed in TSGH-8301 (human bladder cancer), MBT-2 (murine bladder cancer), and HCDB-1 (hamster oral cancer) cells, with the highest levels in TSGH-8301 cells. The lowest levels of HER-2/neu expression were found in NIH3T3 and TCC-SUP (human bladder cancer) cells. Furthermore, to compare the transcriptional activity of the HER-2/neu promoter among these cells, we made use of a luciferase reporter assay. As shown in Figure 2b, the cytomegalovirus (CMV) early promoter was active in NIH3T3, MBT-2, and HCDB-1 cells, with highest activity found in NIH3T3 cells. However, HER-2/neu promoter activity was detected only in MBT-2 and HCDB-1 cells, and not in NIH3T3 cells. Thus, the ratio of HER-2/neu to CMV promoter activity was higher in MBT-2 and HCDB-1 cells than in NIH3T3 cells (Figure 2c). In human bladder cancer cells, the same ratio in TSGH-8301 cells was greater than that in J82 and TCC-SUP cells (Figure 2d). Notably, the human HER-2/neu promoter activity was weaker in non-human cancer cells than in human cancer cells, compared with the CMV promoter (Figure 2c and d). Taken together, these results indicate that TSGH-8301 cells possessed significantly higher HER-2/neu promoter activity and expressed more HER-2/neu proteins than J82 and TCC-SUP cells. In non-human cells, MBT-2 and HCDB-1 cancer cells exhibited much higher HER-2/neu promoter activity than NIH3T3 fibroblasts.Figure 2Expression of HER-2/neu proteins and transcriptional activities of HER-2/neu and CMV promoters in various cells. (a) Expression of HER-2/neu proteins in various cancer cells and NIH3T3 fibroblasts examined by immunoblot analysis. Expression of β-actin served as the quantitative control. (b) HER-2/neu and CMV promoter activities were assessed by the luciferase reporter assay. Cells were transfected with pGL-HER-2-Luc or pGL-CMV-Luc and the transfection efficiency was standardized to the cotransfected plasmid pTCY-LacZ expressing β-gal driven by the β-actin promoter. Relative promoter activity is expressed as a ratio of relative luciferase activity driven by the HER-2/neu promoter divided by that driven by the CMV promoter in (c) MBT-2, HCDB-1, and NIH3T3 cells, and (d) TSGH-8301, J82, and TCC-SUP cells. Each value represents the mean±SD (n=3; P<0.05 for J82 vs TSGH-8301 and P<0.01 for TCC-SUP vs TSGH-8301 by Student's t-test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)YP2-induced cytopathic effect in bladder and oral cancer cellsWe then examined YP2-induced cytopathic effect (CPE) in various cell lines by light microscopy and crystal violet staining. Both YP2 and TNL (a wild-type virulent PrV strain) caused CPE in MBT-2 cells that overexpress HER-2/neu, whereas CW1 (a gD/gE-negative PrV), as expected, did not induce CPE (Figure 3a). Figure 3b shows that TNL dose-dependently lysed all the cell lines examined at 3 days post-infection, whereas YP2 exerted differential cytolytic effects on these cells. YP2 caused CPE in a dose-dependent manner in HER-2/neu-overexpressing TSGH-8301 and MBT-2 cells. Notably, neither non-HER-2/neu-overexpressing cancer cells (TCC-SUP and J82 cells) nor NIH3T3 fibroblasts showed CPE when infected with YP2. Nevertheless, TCC-SUP cells were as susceptible as TSGH-8301 cells to TNL infection, whereas J82 cells were more resistant to TNL-induced cytolysis. Interestingly, CPE in HCDB-1 cells infected with YP2 appeared more pronounced than that observed in cells infected with TNL. In a separate experiment, cell viability was also measured by trypan blue exclusion 4 days after infection with YP2, TNL, or CW1 at 2 × 104 TCID50. The viability of YP2- or CW1-infected NIH3T3 cells did not differ significantly from that of the mock-infected counterpart, whereas viable cells in TNL-infected cells were scarcely detected (Figure 3c). By contrast, the viability of MBT-2 cells decreased dramatically after YP2 or TNL infection, but remained unchanged after CW1 infection when compared with mock-infected cells (Figure 3d). Collectively, YP2-induced CPE was evident in HER-2/neu-overexpressing cells, including human bladder, mouse bladder, and hamster oral cancer cells. Nevertheless, CPE was observed neither in NIH3T3 fibroblasts nor in bladder cancer cells with lower or negligible levels of HER-2/neu expression, such as TCC-SUP and J82 cells.Figure 3CPE in various cell lines after infection with YP2, TNL, or CW1. (a) CPE in MBT-2 cells infected with YP2 or TNL, but not CW1. MBT-2 cells were infected with 2 × 105 TCID50 of YP2, TNL, or CW1 and monitored for CPE by photomicrographic examination at 3 days post-infection (original magnification × 200). (b) CPE in MBT-2, NIH3T3, TSGH-8301, TCC-SUP, J82, and HCDB-1 cells after infection with TNL or YP2. Cells were infected with varying doses of TNL or YP2, and monitored for CPE by crystal violet staining at 3 days post-infection. Cell viability in (c) NIH3T3 and (d) MBT-2 cells after infection with 2 × 104 TCID50 of TNL, CW1, or YP2. The viable cell numbers were determined by Trypan blue exclusion at 4 days post-infection. Each value represents the mean±SD (n=4; ***P<0.001 compared with mock-infected cells by Student's t-test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)As the safety issue would be of obvious concerns if PrV is used for cancer therapy, we injected 8-week-old C3H/HeN mice with YP2, CW1, or TNL via the intraperitoneal route and determined their 50% lethal dose (LD50). The LD50 of TNL was 4.04 × 103 TCID50, whereas LD50 was >5 × 106 TCID50 and >2 × 107 TCID50 for CW1 and YP2, respectively, as all the doses of YP2 and CW1 used for injection did not cause any death in the mice. Therefore, YP2 has been attenuated at least to a similar degree of virulence to CW1. The fact that the monoclonal antibody Herceptin developed as a targeted therapy for HER-2/neu-overexpressing cancers may induce cardiotoxicity in some patients prompted us to examine whether HER-2/neu promoter-driven YP2 had a cardiotoxic effect.26Slamon DJ et al.Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.N Engl J Med. 2001; 344: 783-792Crossref PubMed Scopus (9256) Google Scholar C3H/HeN mice that have been injected with YP2 (2 × 107 TCID50) via the cardiac route remained healthy and exhibited no gross lesions throughout the experimental period of 3 weeks (data not shown). Collectively, these results suggest that YP2 was attenuated and safe when injected into mice as compared with wild-type PrV.Correlation between the levels of YP2-induced CPE and HER-2/neu promoter activity in MBT-2 cellsBecause YP2 replication was dependent on the production of gD and TK, which was under the transcriptional control of the HER-2/neu promoter, we investigated whether a difference of the HER-2/neu promoter activity in MBT-2 cells influenced the cytolytic effect of YP2. As adenovirus E1A can repress the HER-2/neu promoter activity,27Yu D Suen TC Yan DH Chang LS Hung MC Transcriptional repression of the neu protooncogene by the adenovirus 5 E1A gene products.Proc Natl Acad Sci USA. 1990; 87: 4499-4503Crossref PubMed Scopus (137) Google Scholar we transfected MBT-2 cells with pTCY-E1A, an E1A expression vector under the transcriptional control of the β-actin promoter, and examined YP2-induced CPE. Figure 4a shows that E1A dose-dependently suppressed the HER-2/neu promoter activity as determined by the luciferase reporter assay. Accordingly, the degree of YP2-induced CPE decreased as the transfected E1A DNA concentration increased (Figure 4b). It has been reported that treatment of 12-O-tetradecanoylphorbol 13-acetate (TPA) in conjunction with retinoid acid synergistically enhances the transcriptional activity of the HER-2/neu promoter.28Hudson LG Ertl AP Gill GN Structure and inducible regulation of the human c-erb B2/neu promoter.J Biol Chem. 1990; 265: 4389-4393PubMed Google Scholar MBT-2 cells exerted higher HER-2/neu promoter activity when treated with TPA and retinoid acid (Figure 4c), which rendered MBT-2 cells more susceptible to YP2-induced cytolytic effects (Figure 4d). Taking this together, we demonstrate that the cytolytic effect of YP2 was positively correlated with the strength of the HER-2/neu promoter activity in cancer cells. Furthermore, these results are consistent with the idea that the replication and cytolytic effect of YP2 were controlled by the HER-2/neu promoter in HER-2/neu-overexpressing cancer cells.Figure 4Modulation of HER-2/neu promoter activity affected YP2-induced cytolysis in MBT-2 cells. MBT-2 cells were transfected with pTCY-E1A (1.2, 0.8, 0.4, and 0 μg) and pGL-HER-2-Luc (0.5 μg). The total amount of plasmid DNA for transfection was kept constant by the addition of pTCY-LacZ. (a) Cell lysates were harvested 30 h after transfection and their luciferase activities were determined. (b) Alternatively, 24 h after transfection, cells were infected with various doses of YP2 and monitored for CPE by crystal violet staining at 3 days post-infection. (c) MBT-2 cells were transfected with pGL-HER-2-Luc (0.5 μg) and pTCY-LacZ (0.2 μg). After 24 h, cells were treated with TPA (10 nM) and retinoic acid (10 μM) for 6 h and their luciferase activities were determined. (d) Alternatively, MBT-2 cells were infected with various doses of YP2. After 5 h, cells were washed, fed with complete medium containing TPA and retinoic acid, and cultured for an additional 18 h. Cells were then provided fresh medium and CPE was monitored by crystal violet staining 3 days later. Each value represents the mean±SEM (n=3; ***P<0.001 by Student's t-test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Antitumor effects of YP2 on immunocompetent mice bearing MBT-2 tumorsWe next exploited a syngeneic MBT-2 bladder tumor model in immunocompetent C3H/HeN mice, which is more relevant to clinical settings, to investigate antitumor effects of YP2. Figure 5a shows that YP2 treatment significantly retarded MBT-2 bladder tumor growth by more than 50% compared with CW1 or phosphate-buffered saline (PBS) treatment (P<0.0001). Increased survival was also observed in YP2-treated mice in comparison with CW1-treated (P=0.0028) or PBS-treated (P=0.0022) mice (Figure 5b). Immunofluorescent staining with swine antiserum against PrV demonstrated that PrV proteins were detected within MBT-2 tumors after intratumoral injection with either YP2 or TNL, whereas only background expression was observed with the tumor of PBS-treated animals (Figure 5c). Furthermore, hematoxylin–eosin (H&E) staining revealed that the intratumoral infiltrate of mononuclear cells was more pronounced in the tumors injected with YP2 or TNL than those injected with PBS (Figure 5c). In the TSGH-8301 tumor xenograft model in non-obesediabetic/severe combined immunodeficient mice, YP2 only marginally reduced tumor volume in tumor-bearing mice (data not shown). Nevertheless, mice treated with YP2 developed more necrosis areas in the tumors than those treated with CW1 or PBS (data not shown).Figure 5Antitumor effects of YP2 on immunocompetent mice bearing bladder tumors. Groups of eight or nine C3H/HeN mice that had been inoculated subcutaneously with 2 × 106 MBT-2 cells at day 0 were treated intratumorally with 2 × 104 TCID50 of YP2 or CW1, or with PBS daily from days 7 to 13. (a) The mean tumor volume is shown and the data are presented as mean±SD (P<0.0001 for YP2 Vs CW1 or PBS by Student's t-test). (b) Kaplan–Meier survival curves at day 50 are shown (P=0.0028 for YP2 vs CW1 and P=0.0022 for YP2 vs PBS by the log-rank test). (c) Detection of PrV proteins within MBT-2 tumors in vivo. C3H/HeN mice bearing MBT-2 tumors were injected intratumorally with 2 × 106 TCID50 of YP2 or TNL, or PBS for 3 consecutive days. Immunofluorescent staining of cryostat sections of tumors with swine anti-PrV antiserum and fluorescein-conjugated goat anti-swine IgG was performed at 24 h for TNL- or PBS-treated animals and at 72 h for YP2-treated animals after last virus injection. The stained sections were examined under fluorescence and bright-field microscopes. Immunofluorescent images (left panel) and bright-field images (middle panel) in the same field are displayed (original magnification × 640). Formalin-fixed, paraffin-embedded sections were examined by H&E stain (right panel, original magnification × 400).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionIn this study, we exploited an engineered PrV as an oncolytic agent for treating bladder cancer overexpressing HER-2/neu. Whereas PrV could infect human glioblastoma cells and induce cytolytic effects in vitro, it only modestly infected human glioblastoma xenografts in vivo, with relatively little spread within the tumor.29Wollmann G Tattersall P van den Pol AN Targeting human glioblastoma cells: comparison of nine viruses with oncolytic potential.J Virol. 2005; 79: 6005-6022Crossref PubMed Scopus (100) Google Scholar Moreover, PrV seems to grow less well in human cells than in swine or rodent cells.23Boldogkoi Z Bratincsak A Fodor I Evaluation of pseudorabies virus as a gene transfer vector and an oncolytic agent for human tumor cells.Anticancer Res. 2002; 22: 2153-2159PubMed Google Scholar In this study, we also show that mouse bladder and hamster oral cancer cells were more susceptible to cytolysis induced by wild-type PrV than human bladder cancer cells. Furthermore, human bladder cancer cells exhibited differential susceptibility to PrV-induced cytolytic effects. Based on a simple system for generating recombinant PrV vectors carrying foreign genes,25Shiau AL Liu CW Wang SY Tsai CY Wu C A simple selection system for construction of recombinant gD-negative pseudorabies virus as a vaccine vector.Vaccine. 2002; 20: 1186-1195Crossref PubMed Scopus (9) Google Scholar, 30Shiau AL Liu PS Wu CL Novel strategy for generation and titration of recombinant adeno-associated virus vectors.J Virol. 2005; 79: 193-201Crossref PubMed Scopus (20) Google Scholar we constructed a conditionally replicating gE-deleted PrV mutant. We reasoned that transcriptional retargeting of gD and TK genes might provide a means to achieve selective virulence for tumors while retaining attenuated virulence for normal cells. We generated YP2 virus by reintroducing gD and HSV-TK genes linked by