The N-terminus of the ICP10 gene of type 2 herpes simplex virus (HSV-2) encodes a serine/threonine protein kinase (PK) domain that facilitates HSV-2 replication by activating the Ras/MEK/MAPK mitogenic pathway and suppressing apoptosis. We recently demonstrated that deletion of this oncogenic PK domain converts it to a potent oncolytic agent. This mutant, which we have designated FusOn-H2, preferentially replicates in and thus lyses tumor cells in which the Ras signaling pathway is constitutively activated. Here we show that FusOn-H2 exerts strong ability in inducing apoptosis in different lines of human tumor cells and in esophageal tumors growing in mice. The apoptotic effect produced by FusOn-H2 was not restricted to infected cells but extended to uninfected bystander cells, thereby increasing the lethality of the virus. These results add a novel killing mechanism to those previously assigned to FusOn-H2, rendering it an attractive candidate for clinical trials.
The current generation of oncolytic herpes simplex viruses was constructed exclusively from type 1 virus (HSV-1) and usually has deletions in key genes that allow the virus to replicate preferentially in dividing cells, such as tumor cells (see Varghese and Rabkin1 for a review). Genes that have been deleted in these constructs encode thymidine kinase,2 the large subunit of ribonucleotide reductase3, 4 and uracyl N-glycosylase,5 to name only a few. The principal function of these viral genes is to generate adequate nucleotide pools for efficient viral DNA replication in less permissive cellular environments, such as that of quiescent neurons. Hence, where nucleotide pools are abundant, as in tumor cells, viruses mutated in these genes can preferentially replicate in and destroy the cells. Other oncolytic HSVs have been derived from viruses with mutations in the γ34.5 gene, which functions as a virulence factor by markedly enhancing the viral burst size of infected cells through suppression of the shutoff of host protein synthesis.6, 7, 8, 9 Such mutant HSVs are highly attenuated for neurovirulence but retain the ability to replicate in dividing cells, albeit at a reduced level compared to wild-type HSV.10, 11
We recently constructed a unique oncolytic virus, based on the HSV-2 backbone, that we designated as FusOn-H2.12 To generate FusOn-H2, we deleted the serine/threonine protein kinase (PK) domain of the ICP10 gene, the homolog of the ICP6 gene in HSV-1. This domain can activate the Ras/MEK/MAPK mitogenic pathway by binding to and phosphorylating the GTPase-activating protein Ras-GAP and is therefore required for efficient HSV-2 replication.13, 14 Deletion of the PK domain from the viral genome impairs virus growth in normal cells and in non-transformed cells with inactive Ras signaling,12, 15 but does not substantially affect virus replication in tumor cells,12 where Ras signaling is frequently activated by mutations in the Ras gene itself or by alterations in upstream or downstream signaling components.16 Preliminary studies indicate that FusOn-H2 kills tumor cells by at least two mechanisms: direct cytolysis and syncytia formation.12, 17
The induction of apoptosis in tumor cells is an attractive antitumor strategy that, in principle, could be implemented with oncolytic HSVs.18 However, since premature apoptosis represents an important mechanism of host cell defense against viral infections,19 HSV and several other viruses have developed mechanisms to prevent the early apoptotic death of infected cells. Immediately upon infection, HSV expresses several antiapoptotic proteins to counteract the induction of apoptotic genes in the host cell (see Goodkin, Morton and Blaho20 for a review). Since the PK domain encoded by the ICP10 gene of HSV-2 is considered to have antiapoptotic activity,21 we reasoned that its deletion in the FusOn-H2 oncolytic virus might enable the virus to kill tumor cells by inducing apoptosis, thus adding to its repertoire of antitumor mechanisms. To investigate this possibility, we assessed the killing activity of FusOn-H2 against a panel of human tumor cell lines, both in vitro and in vivo, using semi-quantitative assays of chromatin condensation and DNA laddering. The results demonstrate strong apoptotic activity by FusOn-H2 in different lines of human tumor cells and suggest that this activity may extend to uninfected bystander cells.
FusOn-H2 induces apoptosis in human tumor cells of different tissue origins
The antiapoptotic activities of certain gene products block apoptosis in HSV-2-infected cells unless viral protein synthesis is blocked with translation inhibitors such as cycloheximide.22, 23 In principle, deletion of the PK domain of the viral ICP10 gene, which reportedly has antiapoptotic function,21 should render cells susceptible to apoptosis induced by HSV-2 or its derivatives.
To test whether the PK domain deletion in FusOn-H2 permits the induction of apoptotic death in tumor cells, we infected a panel of human tumor cell lines of different tissue origins (EC9706, human esophagus cancer; SKOV3, human ovarian cancer; SW403 and SW480, human colon cancer), using the virus at a multiplicity of infection (MOI) of 10 (Figure 1). Baco-1, an oncolytic virus derived from HSV-1, was included as a control. The cells were seeded into six-well plates and infected with the viruses on the following day. Twenty-four hours post infection, the cells were stained with Hoechst dye and observed for intense, compact fluorescent nuclear staining, which is indicative of chromatin condensation, a characteristic cellular change associated with apoptosis. At an MOI of 10 PFU/cell, approximately 80% tumor cells showed FusOn-H2-induced apoptosis as judged from their intense fluorescent staining pattern. By contrast, uninfected tumor cells were essentially devoid of intense Hoechst staining in the nucleus, as were tumor cells infected with either the parental wt186 virus or Baco-1.
To further evaluate the ability of FusOn-H2 to induce apoptosis in tumor cells, we analyzed the genomic DNA content from infected cells for the appearance of 200 base pair laddering, another characteristic indicative of apoptosis. Three tumor cell lines, all used in the previous experiment, were infected with viruses at 10 PFU/cell or were mock infected. At 24 h post infection, cells were harvested and their genomic DNA extracted and separated on a 1% agarose gel (Figure 2). DNA laddering was observed in cells infected with FusOn-H2, but not in those infected with wt186 or Baco-1, confirming the results of the chromatin condensation assay. Thus, FusOn-H2 with its characteristic deletion of the PK domain in the ICP10 gene readily induces apoptosis in human tumor cells.
Induction of apoptosis in bystander cells
An exceptionally high level of apoptosis was observed in EC9706 cells after their infection with FusON-H2, prompting us to ask whether all of the apoptotic cells had been infected by the virus. As FusOn-H2 carries the gene that encodes green fluorescent protein (GFP) inserted in its genome,12 its presence in cells can be conveniently determined by detecting the expression of this marker gene. We therefore infected EC9706 cells with FusOn-H2 and then stained them with Hoechst dye (Figure 3). Both GFP expression and Hoechst dye staining were apparent under different wavelengths of fluorescent light. Interestingly, an appreciable number of cells that had not been infected by FusOn-H2, based on visual inspection for the lack of GFP expression, had Hoechst dye staining patterns indicative of apoptosis. On average, there was 1 FusOn-H2-infected cell for every 2.6 cells that showed chromatin condensation (Table 1). This suggests that the induction of apoptosis in these cells may not only be a direct consequence of virus infection, but may also represent a substantial bystander effect induced by FusOn-H2-infected cells.
FusOn-H2-induced apoptosis accelerates tumor cell death
As shown in Figure 4, there was an obvious difference in time to a cytopathic effect (CPE) between tumor cells infected with FusOn-H2 and Baco-1. Cells infected with FusOn-H2 at a dose of 1 PFU/cell showed a complete CPE within 24 h that included cell aggregation, rounding and then detachment from each other, whereas those infected with Baco-1 at the same dose appeared morphologically normal at 24 h and did not show any signs of CPE until 72 h post infection (data not shown). Although we previously described the ability of FusOn-H2 to induce syncytia formation by tumor cells,12 this cell membrane fusion was not obvious except with FusOn-H2-infected SKOV3 cells, possibly because the relatively high virus dose used in this experiment led to a cell infection rate of nearly 100%. The fact that FusOn-H2 could induce apoptosis when syncytia formation was not obvious indicates that the fusogenic phenotype of this virus did not directly contribute to its ability to induce apoptosis. The report of a similarly constructed PK mutant that could induce apoptosis despite lacking a fusogenic phenotype supports this notion.21 Regardless of the mechanism, these results demonstrate that FusOn-H2 induces the apoptotic death of tumor cells immediately after virus infection, while the oncolytic effect of virus replication, exemplified by Baco-1 infection, is delayed for at least 72 h.
FusOn-H2-induced apoptosis is an important antitumor mechanism of the virus in vivo
We next evaluated the antitumor activity of FusOn-H2 in vivo against tumor xenografts established from one of the tumor cell lines used in previous experiments. Baco-1 was included in this experiment to permit a direct comparison of the therapeutic effect of these two viruses. Tumor xenografts were established on the right flank of nude mice by subcutaneous injection of 5 × 106 freshly harvested EC9706 cells. When the tumor diameter reached approximately 5 mm, mice received a single intratumoral injection of either FusOn-H2 or Baco-1 at a dose of 3 × 106 PFU, with phosphate-buffered saline (PBS) injection serving as a control. The tumors were measured regularly for 6 weeks, and the tumor growth ratio was determined by dividing the tumor volume before therapy by the volume measured at different time points after therapy (Figure 5). Therapeutic administration of FusOn-H2 essentially stopped all tumor growth within 1 week, followed by progressive tumor shrinkage. By the end of the experiment, the average tumor size was only about half that before the instigation of virotherapy, and more than half the mice were completely tumor free. By contrast, Baco-1 injection did not produce any discernible therapeutic effects until day 21. Even then, the tumor shrinkage was transient, with renewed tumor growth apparent by day 35. Overall, the therapeutic effect of FusOn-H2 was consistently and significantly stronger than that of Baco-1 (P<0.05). These results indicate that the apoptotic death and accompanying bystander effect induced by FusOn-H2 in esophageal cancer cells is likely a major antitumor mechanism in vivo.
To confirm that apoptotic cell death was indeed induced by FusOn-H2 within the tumor mass, we collected tumor samples 3 days after intratumoral injection of oncolytic viruses and stained them for Terminal transferase dUTP nick end labeling (Tunel) assay. Representative fields from two tumor samples of each virus treatment group are shown in Figure 6. The results show widespread apoptosis in tumors treated with FusOn-H2, but only weak TUNEL staining in those treated with Baco-1. These findings corroborate earlier experiments, indicating a dominant role for virus-induced apoptotic death in the antitumor activity of FusOn-H2.
A hallmark of tumor cells is their resistance to apoptosis. Consequently, several strategies have been devised to overcome this block for therapeutic purposes.24 Here we demonstrate that a new oncolytic virus, FusOn-H2, constructed from HSV-2 by deleting the PK domain of the ICP10 gene, has a potent ability to induce the apoptotic death of tumor cells. Moreover, this property is coupled with an apoptotic effect on surrounding tumor cells not infected by the virus, although the mechanism remains unclear. Recently, it was demonstrated that mda-7/IL-24, a secreted cytokine of the IL-10 family, could induce tumor-specific apoptosis in transduced as well as surrounding tumor cells.25 Thus, upregulation of mda-7/IL-24 or a similar cytokine may be one of the mechanisms used by FusOn-H2 to induce apoptosis in bystander tumor cells. Whatever the explanation, the combination of virus-induced apoptosis and the accompanying bystander effect would be expected to enhance the antitumor activity of FusOn-H2 in vivo. Indeed, direct comparison of FusOn-H2 with Baco-1, an oncolytic HSV-1-derived virus that depends on its replicative potency to destroy tumor cells, showed a clear advantage for FusOn-H2 in terms of tumor growth inhibition (Figure 5).
It may also be important that FusOn-H2 induced the apoptotic death of tumor cells much more rapidly than Baco-1 could kill the same type of cells by direct replication-dependent oncolysis. Specifically, a complete CPE was apparent within 24 h in cells infected with FusOn-H2, while an obvious CPE was lacking in Baco-1-infected cells even at 48 h post infection, despite heavy virus infection as indicated by GFP expression. The more rapid induction of tumor cell death by FusOn-H2 compared with Baco-1 may confer a therapeutic advantage because of the host's innate immunity, which responds immediately after the initiation of virus infection and might well clear slowly acting oncolytic viruses such as Baco-1 before they are able to exert their full cytopathic effect against tumor cells.
It has been reported that an HSV-1-based oncolytic virus, NV1066, which harbors a deletion in one copy of the duplicate γ34.5 genes, not only induces apoptosis in a human gastric cancer cell line, but also produces a bystander effect of apoptotic cell death.26 However, in our experiments, Baco-1, a similarly constructed HSV-1-based oncolytic virus, did not induce apoptosis in the four tumor cell lines that were tested. The discrepancy probably stems from the difference in tumor cells used in these two studies. Indeed, we have found that FusOn-H2 does not induce apoptosis in some human tumor cells, such as A549 lung carcinoma and U20S osteosarcoma cells (data not shown). The underlying mechanism for this sensitivity is unclear but may be linked to the mutational status of p53. That is, tumor cells with wild-type p53 may be relatively insensitive to the induction of apoptosis by FusOn-H2 or any oncolytic virus for that matter. Experiments to explore this lead are under way.
We and others have recently demonstrated that the incorporation of cell membrane fusion activity into an oncolytic virus can significantly increase the antitumor activity of that virus.27, 28, 29 FusOn-H2 is one of the viruses reported to have a fusogenic phenotype in a variety of tumor cells.12, 17 These findings, in the context of the current study, suggest that the antitumor activity of FusOn-H2 derives from the combined effect of three distinct killing mechanisms: direct cytolysis owing to virus replication, syncytia formation and apoptosis. The relative contribution of each mechanism appears to depend on the tumor cell context. For example, in tumor cells with p53 gene mutation, FusOn-H2-induced apoptosis is likely to be dominate, while in those with wild-type p53, the contribution of this mechanism would likely be negligible or, at best, minor compared with the contributions of direct cytolysis and syncytia formation. An oncolytic agent with multiple killing mechanisms has definite advantages over the one that depends on a single mechanism to kill tumor cells. Most obvious is the ability to forestall or prevent the emergence of treatment resistance, the most challenging problem in clinical cancer management. The intrinsic instability of the cancer cell genome means that resistance to specific anticancer agents can be readily generated by random mutations that develop as the cancer cells undergo dysregulated division. Although resistant tumor cells may be present before therapy, they most often arise during treatment, resulting in disease progression despite continued administration of anticancer agents. One such example can be found in the response of chronic myeloid leukemia (CML) to Gleevec, a small molecule inhibitor of the BCR-ABL kinase.30 Although treatment of early stage CML with Gleevec has been quite successful, later stages of the disease (blast crisis) resist such therapy due to the emergence of drug-resistant cells.31 Thus, combined therapy with several agents that target different cellular pathways or a single therapeutic agent with multiple antitumor mechanisms is needed to prevent this type of drug resistance. We suggest that the multiple killing mechanisms of FusOn-H2 qualify this mutant HSV-2 as a potentially valuable anticancer agent. For example, it might be applied early in treatment to eradicate or reduce the size of inoperable solid tumors, with only a minimal risk of generating resistant tumor cells.
Materials and methods
Cell lines and viruses
African green monkey kidney (Vero) cells, SW403 and SW480 human colon cancer cell lines and the A549 line of human lung carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD, USA). EC9706, a human esophageal cancer cell line,32 was provided by Dr Mingrong Wang (Chinese Academy of Medical Sciences). SKOV3 cells, a human ovarian cancer cell line, were provided by Dr Robert Bast (M.D. Anderson Cancer Center). U20S cells, a human osteosarcoma line, were provided by Dr Lawrence Donehower. All of the cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS).
FusOn-H2 was derived from the wild-type HSV-2 strain 186 (wt186). Details of its construction have recently been published.12 Baco-1 is an HSV-1-based oncolytic virus. It possesses deletions in both copies of the γ34.5 gene, and the sequence of an artificial bacterial chromosome and the GFP gene are inserted into the UL46/4 locus.33, 34 Viral stocks were prepared by infecting Vero cells with 0.01 plaque-forming units (PFU) per cell. Viruses were harvested 2 days later and purified as described.34 The purified viruses were titrated, divided into aliquots and stored at −80°C until use.
Hoechst dye staining of infected cells and quantitation of chromatin condensation
Cells seeded into 24-well plates were infected on the next day with FusOn-H2, wt186 or Baco-1 at 10 PFU/cell or were mock infected. Twenty-four hours after infection, the cells were stained with Hoechst dye 33358 (Sigma-Aldrich, St Louis, MO, USA) at a final concentration of 1 μg/ml for 30 min at 37°C and then examined under a fluorescent microscope.
DNA laddering assay
Cells were seeded into six-well plates at 50% density and infected 1 day later with viruses at 10 PFU/cell. Twenty-four hours after virus infection, the cells were harvested and their DNA was extracted with DNAzol reagent (Invitrogen, Carlsbad, CA, USA). The extracted DNA was treated with RNase (100 μg/ml) before phenol:chloroform extraction and ethanol precipitation. The DNA was then loaded onto 1% agarose gels for electrophoresis and visualization under UV light after staining with ethidium bromide.
Correlation of EGFP expression with chromatin condensation
Cells seeded in 12-well plates were infected the next day with FusOn-H2 at 1 PFU/cell. Hoechst dye staining for chromatin condensation was performed exactly as described above. The overlay of micrographs from the same field with different fluorescent lights was facilitated with Spot Image Software (Diagnostic Instrument Inc., Sterling Heights, MI, USA). The GFP-positive and GFP-negative apoptotic cells were separately counted in the same fields (∼100 apoptotic cells were counted per field). A total of three fields were included in calculations to verify the bystander apoptotic effect of FusOn-H2 infection.
The TUNEL assay for apoptosis was performed on tumor tissue with the in situ cell death detection kit manufactured by Roche Applied Science (Penzberg, Germany), according to the manufacturer's instructions. Briefly, paraffin-embedded tumor sections were dewaxed and then rehydrated by a standard procedure. Then, the slides were incubated with freshly prepared permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) on ice for 8 min. After washing with PBS, the slides were covered with TUNEL reaction mixture and incubated at 37°C for 60 min in a humidified atmosphere in the dark. DNA fragmentation was detected with a fluorescent microscope.
Female Hsd athymic (nu/nu) mice (obtained from Harlan, Indianapolis, Indiana) were kept under specific pathogen-free conditions and used in experiments when they attained the age of 5–6 weeks. EC9706 cells were harvested from subconfluent cultures by a brief exposure to 0.25% trypsin and 0.05% EDTA. After trypsinization was stopped with medium containing 10% FBS, the cells were washed once in serum-free medium and resuspended in PBS. On day 0, 5 × 106 EC9706 cells were inoculated into the right flank of nude mice. Two weeks after tumor cell implantation, when the tumors reached approximately 5 mm in diameter, mice received a single intratumor injection of 3 × 106 PFU of FusOn-H2 or Baco-1 in a volume of 100 μl, or the same volume of PBS. The tumors were measured weekly and their volumes were determined by the formula tumor volume (mm3)=(length (mm)) × (width (mm))2 × 0.52. For the TUNEL assay, mice were euthanized by CO2 exposure 3 days after receiving intratumor injection of 1 × 107 PFU of FusOn-H2 or Baco-1 viruses. Tumor tissues were explanted and sectioned for TUNEL staining as described above.
Quantitative data are reported as means±standard deviations. Statistical analyses were performed by Student's t-test or one-way ANOVA. P-values less than 0.05 were considered significant.
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We thank Dr Mingrong Wang (Chinese Academy of Medical Sciences) for the gift of EC9706 cells and Dr Jonathan Prigge for critical reading of the manuscript. This project was supported in part by a grant from Department of Defense Ovarian Cancer Research Program (DAMD17-03-1-0434).
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