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Journal of Virology, April 2004, p. 4224-4233, Vol. 78, No. 8
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.8.4224-4233.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Bovine Herpesvirus Tegument Protein VP22 Enhances Thymidine Kinase/Ganciclovir Suicide Gene Therapy for Neuroblastomas Compared to Herpes Simplex Virus VP22

Zhaohua Qiu, Jerome S. Harms, Jun Zhu, and Gary A. Splitter*

Department of Animal Health and Biomedical Sciences, University of Wisconsin—Madison, Madison, Wisconsin 53706

Received 23 September 2003/ Accepted 12 December 2003


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ABSTRACT
 
Herpesvirus tegument protein VP22 can enhance the effect of therapeutic proteins in gene therapy, such as thymidine kinase (tk) and p53; however, the mechanism is unclear or controversial. In this study, mammalian expression vectors carrying bovine herpesvirus 1 (BHV-1) VP22 (BVP22) or herpes simplex virus type 1 (HSV-1) VP22 (HVP22) and equine herpesvirus type 4 (EHV-4) tk (Etk) were constructed in order to evaluate and compare the therapeutic potentials of BVP22 and HVP22 to enhance Etk/ganciclovir (Etk/GCV) suicide gene therapy for neuroblastomas by GCV cytotoxicity assays and noninvasive bioluminescent imaging in vitro and in vivo. BVP22 enhanced Etk/GCV cytotoxicity compared to that with HVP22 both in vitro and in vivo. However, assays utilizing a mixture of parental and stably transfected cells indicated that the enhancement was detected only in transfected cells. Thus, the therapeutic potential of BVP22 and HVP22 in Etk/GCV suicide gene therapy in this tumor system is not due to VP22 delivery of Etk into surrounding cells but rather is likely due to an enhanced intracellular effect.


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INTRODUCTION
 
Cancer is one of the most common worldwide diseases, with a high fatality rate. None of the conventional strategies (surgery, chemotherapy, and radiotherapy) can eliminate cancer cells completely. Furthermore, many serious side effects occur with therapies that interfere with the normal physiologic functions of the patient. Therefore, a need exists to develop more-promising approaches, one of which is thymidine kinase (tk)/ganciclovir (GCV) suicide gene therapy. Herpesvirus tk can effectively phosphorylate GCV, an antiviral nucleoside analog, to its monophosphate form, which is further phosphorylated by cellular kinases to its triphosphate form and incorporated into nascent DNA, causing chain termination and cell death (29). Concentrations that are lethal to cells expressing the herpesvirus tk gene but are nontoxic to normal mammalian cells can be achieved because GCV is a relatively poor substrate for mammalian tk. The effectiveness of the tk/GCV system for the treatment of cancer has been demonstrated in animal models of various types of cancer (12, 32). However, in human clinical trials, disease progression was seen in most patients upon long-term follow-up, although tumor regression occurred briefly after treatment (16). Therefore, a need exists to enhance tk/GCV suicide gene therapy for tumors.

Alphaherpesvirus virions are composed of three major structures: the capsid, tegument, and envelope (30). Located between the capsid and the envelope, the tegument is a highly stable macromolecular structure consisting of proteins critical to viral survival (4, 28). Not only are tegument proteins important viral structural proteins; they also play critical roles during infection. Some studies have shown that VP22, one of the tegument proteins, possesses novel trafficking ability: VP22 protein produced in one expressing cell traffics to the nuclei of neighboring nonexpressing cells (1, 3, 8, 9, 26). Further, VP22 chimeras can carry large effector proteins or nucleic acids while trafficking without altering the function of the attached proteins or nucleic acids (8, 17, 20, 21, 24, 33, 34, 35, 36, 37). The unique ability of VP22 and its fusion proteins to enter cells makes it a promising tool for gene delivery in gene therapy. The mechanism mediating the import of VP22 is unknown. However, the intercellular trafficking ability of VP22 has been controversial (10, 11). Some studies indicate that VP22 intercellular trafficking can be detected only in fixed cells, not in living cells (M. Lundberg and M. Johansson, Letter, Nat. Biotechnol. 19:713-714, 2001).

Although most VP22 and tk data have been obtained from studies with herpes simplex virus type 1 (HSV-1), bovine herpesvirus 1 (BHV-1) VP22 (BVP22) has a different phenotypic effect on cells, with a preponderance of nuclear localization compared to the localization of HSV-1 VP22 (HVP22) (14). Also, equine herpesvirus 4 (EHV-4) tk (Etk) reportedly has improved biotherapeutic potential compared to that of HSV-1 tk (Htk) (18). In the present study, we used GCV cytotoxicity assays and noninvasive bioluminescent imaging in vitro and in vivo to evaluate and compare the potentials of BVP22 and HVP22 to enhance Etk/GCV suicide gene therapy for neuroblastomas. We found that (i) Etk can increase the sensitivity of NXS2 neuroblastoma cells to GCV both in vitro and in vivo; (ii) in transiently transfected cells, both BVP22 and HVP22 can enhance the efficacy of Etk in vitro; (iii) in stably transfected cells, an in-frame BVP22 N-terminal fusion with Etk (Etk-BVP22) results in improved GCV activity in vitro compared to that with Etk only or with other fusions; (iv) both BVP22 and HVP22 can enhance Etk/GCV suicide gene therapy for neuroblastomas in vivo; (v) however, enhancement of the efficacy of Etk by BVP22 or HVP22 is not due to VP22 delivery of Etk into surrounding cells but likely is due to an enhanced intracellular effect.


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MATERIALS AND METHODS
 
Expression vectors and cells. NXS2 murine neuroblastoma cells (H-2a) (13), a gift from R. Reisfeld, were cultured with RPMI culture medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a CO2 incubator. Coding regions for BVP22, HVP22, Etk, Etk-BVP22 and Etk-HVP22 (in-frame VP22 N-terminal fusions), and BVP22-Etk and HVP22-Etk (in-frame VP22 C-terminal fusions) were cloned into the pIRESneo2 mammalian expression vector (Clontech, Palo Alto, Calif.) to construct recombinant plasmids (Fig. 1). These recombinant plasmids were transiently or stably transfected into NXS2 cells by using LipofectAMINE reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. Stably transfected cells were selected with the antibiotic G418 at 400 µg/ml (Qbiogene, Carlsbad, Calif.).



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  		FIG. 1. Schematic depiction of expression vectors. Coding regions for Etk, BVP22, HVP22, and their fusions were cloned into pIRESneo2 to construct recombinant plasmids pIRESneo2/BVP22, pIRESneo2/HVP22, pIRESneo2/Etk, pIRESneo2/Etk-BVP22, pIRESneo2/BVP22-Etk, pIRESneo2/Etk-HVP22, and pIRESneo2/HVP22-Etk. Expression of the inserts was driven by the CMV immediate-early enhancer/promoter.

NXS2 cell lines constitutively expressing luciferase were engineered by using the BD Retro-X system (BD Biosciences Clontech, San Diego, Calif.) to introduce the firefly luciferase gene. Two luciferase retrovectors were used: pLLRN and pLPCX/luc. The pLLRN retrovector was a control supplied by Clontech; it expresses luciferase by using the Rous sarcoma virus promoter and neomycin resistance. The pLPCX/luc retrovector was engineered as follows. The pGEM/luc plasmid (Promega, Madison, Wis.) was restriction enzyme digested and subcloned into the BamHI/XhoI sites of pENTR1A (Invitrogen). This construct was designated pENTR1A/luc. The BamHI/XhoI luciferase coding fragment of pENTR1A/luc was subsequently subcloned into the BglII/XhoI sites of pLPCX (Clontech). This construct was designated pLPCX/luc. The pLPCX/luc retrovector expresses luciferase by using the cytomegalovirus (CMV) immediate-early promoter and puromycin resistance. Retrovirus was produced according to the manufacturer's recommended protocol. NXS2 cell lines were subsequently transduced and selected by using either a G418 (400 µg/ml) or a puromycin (2.5 µg/ml) selection medium and were designated NXS2/LLRN and NXS2/luc. Cell lines were initially tested for luciferase expression by using a luciferase assay system (Promega) measuring cell lysate luciferase in a single-tube luminometer. Stable NXS2 cell lines expressing either luc/neo2, luc/BVP22, luc/HVP22, luc/Etk, luc/Etk-BVP22, or luc/Etk-HVP22 were constructed by transfecting NXS2/luc cells with the constructs shown in Fig. 1.

Northern blot analysis. The expression of Etk, BVP22, and HVP22 in transiently transfected or stably transfected cells was assayed by Northern blot analysis. RNA was purified by using RNeasy (Qiagen, Valencia, Calif.), and preparation of the gel and sample RNA, electrophoresis, and transfer of RNA to the membrane were performed by using NorthernMax (Ambion, Austin, Tex.). Probe generation, hybridization, stringency washes, and substrate development were carried out by using North2South direct horseradish peroxidase labeling and detection (Pierce, Rockford, Ill.).

GCV cytotoxicity assay in transiently transfected cells. NXS2 cells were plated at a density of 1,000 per well in flat-bottom, tissue culture-treated 96-well plates. One day later, cells were transiently transfected with the constructs shown in Fig. 1. One day posttransfection, the GCV cytotoxicity assay was performed. Briefly, cells were treated with GCV (InvivoGen, San Diego, Calif.) at a concentration of 0, 0.01, 0.1, 1, 10, or 100 µg/ml in a final volume of 100 µl of RPMI with 10% FBS for 3 days. The medium was changed with addition of fresh GCV, and the cells were incubated for another 3 days. The surviving cells were detected by a CellTiter 96 AQueous assay (Promega). All data points were measured at least in triplicate in three separate experiments. Percent survival was calculated as (optical density at 490 nm [OD490] of test wells - OD490 of empty wells)/(OD490 of untreated wells - OD490 of empty wells) x 100.

GCV cytotoxicity assay in stably transfected cells. NXS2 cells were stably transfected with the constructs shown in Fig. 1. Cells were plated at a density of 1,000 per well in flat-bottom, tissue culture-treated 96-well plates. One day later, the GCV cytotoxicity assay was performed as described above.

GCV cytotoxicity assay for total cells in a mixture of stably transfected and parental cells. NXS2 parental cells (at 0, 20, 40, 60, 80, 90, or 100%) were mixed with cells stably transfected with neo2, BVP22, HVP22, Etk, Etk-HVP22, HVP22-Etk, Etk-BVP22, or BVP22-Etk and were then plated at a density of 1,000 total cells per well in flat-bottom, tissue culture-treated 96-well plates. One day later, the GCV cytotoxicity assay was performed as described above.

GCV cytotoxicity assay for parental cells in a mixture of stably transfected and parental cells. NXS2/LLRN cells (at 0, 20, 40, 60, 80, 90, or 100%) were mixed with cells stably transfected with neo2, BVP22, HVP22, Etk, Etk-HVP22, HVP22-Etk, Etk-BVP22, or BVP22-Etk and were then plated at a density of 1,000 total cells per well in black flat-bottom, tissue culture-treated 96-well plates. The cells were treated with GCV twice as described above. Bright-Glo luciferase assay reagent (100 µl; Promega) was added to wells 5 to 10 min before bioluminescent imaging using a cryogenically cooled IVIS system (Xenogen Corp., Alameda, Calif.). The signal intensity was quantified as the sum of all photon counts detected within each well. All data points were measured at least in triplicate in three separate experiments. The percentage of surviving parental cells was calculated as (photon counts for test wells)/(photon counts for untreated wells) x 100.

GCV cytotoxicity assay for stably transfected cells in mice with neuroblastoma cells. Thirty A/J mice (H-2a; weight, 20 ± 1 g) were randomly divided into six groups (five mice per group) and injected intradermally on the lower back with 2 x 106 NXS2 cells stably transfected with luc/neo2, luc/BVP22, luc/HVP22, luc/Etk, luc/Etk-BVP22, or luc/Etk-HVP22. Starting at 10 days after implantation, mice were treated intraperitoneally (i.p.) with GCV (50 mg/kg of body weight) once a day for 14 consecutive days. Tumors were evaluated by bioluminescent images acquired 10, 17, and 24 days after tumor implantation. Briefly, beetle luciferin (Promega) was dissolved to 30 mg/ml in phosphate-buffered saline. Mice were anesthetized and subsequently injected i.p. with beetle luciferin at 150 µg/g of body weight. Images were acquired by the IVIS system 10 to 20 min after luciferin administration. The signal intensity was quantified as the sum of all photon counts detected within the region of interest by using Living Image (version 2.20) software. Mice were killed when tumors were >15% of body weight.

GCV cytotoxicity assay for parental cells in mice with neuroblastoma cells. Fifteen A/J mice were randomly divided into three groups (five mice per group). NXS2 cells stably transfected with Etk, Etk-BVP22, or Etk-HVP22 were mixed with NXS2/LLRN at a ratio of 1:1 and intradermally injected into the mice at 2 x 106 cells per mouse. Ten days after implantation, mice were treated with GCV, and then tumors were evaluated by bioluminescent imaging as described above.

Statistics. The two-tailed t test was used for statistical analysis of GCV cytotoxicity. Differences were considered significant at a P value of <0.05.


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RESULTS
 
Construction of mammalian expression vectors for tk, BVP22, HVP22, and their fusion genes. To investigate the abilities of BVP22 and HVP22 to enhance Etk/GCV suicide gene therapy for neuroblastoma in vitro and in vivo, coding regions for Etk-BVP22 and Etk-HVP22 (N-terminal fusions) and for BVP22-Etk and HVP22-Etk (C-terminal fusions) were cloned to construct recombinant mammalian expression plasmids. Plasmids were also constructed for Etk, BVP22, and HVP22 (Fig. 1). After transient or stable transfection of NXS2 cells, mRNAs for Etk, BVP22, and HVP22 (approximately 2.7 to 2.8 kb), as well as their fusion genes (approximately 3.7 to 3.8 kb), were detected by Northern blot analysis using Etk, BVP22, or HVP22 cDNA probes generated by PCR with plasmid templates. Results of Northern blot analysis of stably transfected cells are shown in Fig. 2.



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  		FIG. 2. Northern blot analysis for the transcription of Etk, BVP22, HVP22, and their fusion genes in stably transfected NXS2 cells. RNA was purified from NXS2 cells stably transfected with neo2, Etk, BVP22, Etk-BVP22, BVP22-Etk, HVP22, Etk-HVP22, or HVP22-Etk, separated by electrophoresis, blotted, and probed with Etk, BVP22, and HVP22 cDNAs. Short arrows indicate mRNAs for single genes (Etk, BVP22, and HVP22). Long arrows indicate mRNAs for fusion genes (Etk-BVP22, BVP22-Etk, Etk-HVP22, and HVP22-Etk).

GCV induced cytotoxicity in transiently and stably transfected NXS2 cells. NXS2 cells formed normal monolayers before and after transfection. The GCV dosage was evaluated for induction of cytotoxicity in NXS2 cells. In both transiently (Fig. 3A) and stably (Fig. 4A) transfected cells, GCV at 10 µg/ml resulted in fewer surviving cells after transfection with tk or fusions than after transfection with BVP22, HVP22, or neo2 only (P < 0.05). Therefore, GCV at 10 µg/ml was used in subsequent GCV cytotoxicity assays in vitro. In transiently transfected cells, both BVP22 and HVP22 enhanced the efficacy of Etk (Fig. 3B). In stably transfected cells, an in-frame BVP22 N-terminal fusion with Etk (Etk-BVP22) resulted in improved GCV activity compared to that with Etk only or with other fusions (P < 0.01) (Fig. 4B).



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  		FIG. 3. GCV cytotoxicity assay in transiently transfected cells. NXS2 cells were planted in 96-well plates at 1,000 cells/well and were transiently transfected with either pIRESneo2, pIRESneo2/BVP22, pIRESneo2/HVP22, pIRESneo2/Etk, pIRESneo2/Etk-HVP22, pIRESneo2/HVP22-Etk, pIRESneo2/Etk-BVP22, or pIRESneo2/BVP22-Etk. Starting at 1 day following transfection, cells were exposed to GCV at 0, 0.01, 0.1, 1, 10, or 100 µg/ml for 6 days. Cell survival was measured by a CellTiter 96 AQueous assay. All data points are averages ± standard deviations from three separate experiments. (A) Survival of cells treated with increasing GCV concentrations. (B) Survival of cells treated with GCV at 10 µg/ml.



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  		FIG. 4. GCV cytotoxicity assay in stably transfected cells. NXS2 cells stably transfected with either neo2, BVP22, HVP22, Etk, Etk-HVP22, HVP22-Etk, Etk-BVP22, or BVP22-Etk were plated at a density of 1,000 cells per well in 96-well plates. Starting 1 day later, cells were exposed to GCV at 0, 0.01, 0.1, 1, 10, or 100 µg/ml for 6 days. Cell survival was measured by a CellTiter 96 AQueous assay. All data points are averages ± standard deviations from three separate experiments. (A) Survival of cells treated with increasing GCV concentrations. (B) Survival of cells treated with GCV at 10 µg/ml.

GCV induced cytotoxicity in a mixture of parental and stably transfected cells but not in parental cells alone. After parental cells were mixed with stably transfected cells, a GCV cytotoxicity assay of total cells (Fig. 5) indicated that Etk improved the sensitivity of the total cells to GCV and that VP22 enhanced the efficacy of Etk. BVP22 resulted in more cell killing than HVP22, and the in-frame VP22 N-terminal fusions with Etk (Etk-BVP22 and Etk-HVP22) resulted in enhanced cell killing compared to that with the C-terminal fusions (BVP22-Etk and HVP22-Etk). However, luciferase-transfected parental cells (NXS2/LLRN) showed similar survival rates in all the groups (Fig. 6). The similar survival of all cell groups suggests that the enhancement of Etk efficacy by VP22 observed in the experiments described above occurred only in the transfected cells, not in the parental cells.



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  		FIG. 5. GCV cytotoxicity assay for total cells in a mixture of stably transfected and parental cells. NXS2 parental cells (at 0, 20, 40, 60, 80, 90, or 100%) were mixed with cells stably transfected with either neo2, BVP22, HVP22, Etk, Etk-HVP22, HVP22-Etk, Etk-BVP22, or BVP22-Etk and were then plated at a density of 1,000 total cells per well in 96-well plates. Starting 1 day later, cells were exposed to GCV at 10 µg/ml for 6 days. Cell survival was measured by a CellTiter 96 AQueous assay. All data points are averages ± standard deviations from three separate experiments.



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  		FIG. 6. GCV cytotoxicity assay for parental cells in a mixture of stably transfected and parental cells. NXS2/LLRN cells (at 0, 20, 40, 60, 80, 90, or 100%) were mixed with cells stably transfected with either neo2, BVP22, HVP22, Etk, Etk-HVP22, HVP22-Etk, Etk-BVP22, or BVP22-Etk and were then plated at a density of 1,000 total cells per well in black 96-well plates. Starting 1 day later, cells were exposed to GCV at 10 µg/ml in 100 µl of medium per well for 6 days. Bright-Glo luciferase assay reagent was added to each well 5 to 10 min before bioluminescent imaging using the IVIS system. The signal intensity was quantified as the sum of all photon counts detected within each well. All data points are averages ± standard deviations from three separate experiments.

Etk-VP22 fusions promote GCV-induced tumor regression in vivo more efficiently than Etk alone. To study whether the results we obtained in vitro could also be observed in vivo, NXS2/luc cells were stably transfected with either neo2, BVP22, HVP22, Etk, Etk-BVP22, or Etk-HVP22. Luciferase expression was detected in NXS2/luc cells before and after stable transfection with Etk, VP22, or fusion genes by a Bright-Glo luciferase assay (Promega) using the IVIS system. The introduction of Etk, VP22, or fusion genes did not significantly affect luciferase expression in cells. The stably transfected cells were implanted into A/J mice. Bioluminescent images acquired before initiation of GCV treatment (10 days after tumor implantation) showed that most mice had small palpable tumors of similar sizes. After GCV treatment, bioluminescent images were acquired once a week to evaluate tumor growth. NXS2 tumors containing Etk-BVP22 or Etk-HVP22 were significantly smaller than those containing Etk only, which, in turn, were significantly smaller than those containing only BVP22, HVP22, or neo2 (Fig. 7). These data indicated that, in agreement with the results obtained in vitro, both BVP22 and HVP22 could promote Etk/GCV suicide gene therapy in vivo. No tumor metastases to different body organs were observed for the duration of the experiment.




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  		FIG. 7. GCV cytotoxicity assay for transfected NXS2 cells in mice with neuroblastomas. NXS2 cells transfected with either luc/neo2, luc/BVP22, luc/HVP22, luc/Etk, luc/Etk-BVP22, or luc/Etk-HVP22 were implanted into mice at 2 x 106 cells per mouse. Starting at day 10 after implantation, mice were treated with GCV (50 mg/kg of body weight) i.p. once a day for 14 consecutive days. Tumors were evaluated by bioluminescent images acquired 10, 17, and 24 days post-tumor implantation. (A) Bioluminescent imaging of luciferase activity in mice into which tumors had been implanted. (B) Luciferase activity determined from mice as photons in total region of interest (ROI) in each group.

VP22 does not enhance Etk/GCV cytotoxicity in parental cells in vivo. To investigate whether the enhancement of Etk/GCV suicide gene therapy by VP22 in vivo, observed above, is due to VP22 intercellular trafficking, NXS2 cells stably transfected with Etk, Etk-BVP22, or Etk-HVP22 were mixed with parental NXS2 cells expressing luciferase (NXS2/LLRN) at a ratio of 1:1 and then injected into mice. NXS2/LLRN cell growth was evaluated based on bioluminescent images acquired before and after GCV treatment. As with the results in vitro, tumors in all three groups grew at similar rates (Fig. 8), suggesting that VP22 did not carry Etk molecules from transfected cells to adjacent parental cells in vivo.




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  		FIG. 8. GCV cytotoxicity assay for parental cells in mice with neuroblastomas. NXS2 cells transfected with either Etk, Etk-BVP22, or Etk-HVP22 were mixed with NXS2/LLRN cells at a ratio of 1:1 and intradermally injected into mice at 2 x 106 cells per mouse. Starting at day 10 after implantation, mice were treated with GCV, and then tumors were evaluated by bioluminescent images acquired 10, 17, and 24 days after tumor implantation. (A) Bioluminescent imaging of luciferase activity in mice into which tumors had been implanted. (B) Luciferase activity determined from mice as photons in total region of interest (ROI) in each group.


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DISCUSSION
 
Recent studies have demonstrated that tk/GCV suicide gene therapy can provide effective treatment for several kinds of tumors with high cell-cell gap junction communications through which a bystander effect can be achieved (2, 5, 15). However, many tumor types, such as neuroblastoma and medulloblastoma, have poor cell-cell gap junction communications, a property that has greatly limited the effect of tk/GCV suicide gene therapy (23, 25, 27, 31). Therefore, a need exists to enhance the tk/GCV effect regardless of any gap junction-dependent bystander effect. HVP22 was reported to enhance tk/GCV suicide gene therapy both in vitro and in vivo by delivering functional tk protein from an expressing cell into many neighboring cells regardless of the gap junction (6). However, other studies reported that VP22 intercellular trafficking could be detected only in fixed cells, not in living cells (Lundberg and Johansson, letter). To better understand the utility of VP22 in gene therapy, we performed assays using VP22 and tk constructs to evaluate GCV-induced cytotoxicity in NXS2 cells both in vitro and in vivo. Mammalian expression vectors containing VP22 fused with Etk, which is 12-fold more sensitive to GCV than Htk (18), were used to evaluate the biotherapeutic potential of VP22 in tk/GCV suicide gene therapy. Enhancement of the efficacy of Etk by VP22 could be seen in both transiently and stably transfected NXS2 cells in vitro. After stable transfection, all the cells should express the transfected genes, suggesting that the enhancement of Etk efficacy by VP22 is not due to VP22 delivery of Etk into surrounding cells. To detect the intercellular trafficking ability of VP22, we also performed GCV cytotoxicity assays in a mixture of stably transfected and parental cells in vitro. In the assay for total cells (Fig. 5), as in the assays with transiently (Fig. 3) and stably (Fig. 4) transfected cells, Etk could improve the sensitivity of NXS2 cells to GCV, and VP22 could enhance the efficacy of Etk. However, neither Etk nor its fusions with VP22 had any effect on the parental cells (Fig. 6). Studies have shown that neuroblastoma cells have poor cell-cell gap junction communications, which are considered to be important in mediating the bystander effect in tk/GCV suicide gene therapy (31). Our work suggests that Etk/GCV treatment has no bystander effect on NXS2 cells and that VP22 is unable to deliver Etk from expressing cells to the surrounding nonexpressing cells (Fig. 6). Thus, the present results indicate that the enhancement of Etk/GCV suicide gene therapy by VP22 is not due to VP22 carrying functional Etk protein to nontransfected cells, but rather is likely an enhanced intracellular effect.

Recent advances in biotechnology have enabled in vivo imaging of luciferase reporter proteins in living mice by use of a cooled charge-coupled device (CCD) camera. Because bioluminescent imaging has minimal background activity, this technology is very sensitive for detecting light emitted from luciferases. Importantly, bioluminescent imaging measures only live cells, because luciferase requires O2 and ATP to catalyze light from its substrate, luciferin. This feature renders bioluminescent imaging a particularly attractive method for measuring tumor cell growth as opposed to other techniques that measure total tumor volume, including necrotic areas. We introduced this novel technology into our studies on the biotherapeutic potential of VP22 in Etk/GCV suicide gene therapy in vivo. Mice were implanted with NXS2 cells transduced with Etk or its fusions as well as luciferase and were then treated with GCV. The tumors were measured as bioluminescent images, which reflected the number of living cells. Etk-VP22 fusions promoted GCV-induced tumor regression more efficiently than Etk alone in vivo. However, when NXS2 parental cells expressing luciferase (NXS2/LLRN) were mixed with cells transfected with Etk or its fusions, they showed similar growth rates in mice regardless of the genes introduced in transfected cells. This suggests that VP22 could not carry Etk molecules from transfected cells to adjacent parental cells in vivo, a finding similar to our in vitro findings.

The potential of VP22 for gene therapy has been supported by a number of studies; however, the mechanism of action is not clear. Others have reported increased cell death of nontransfected cells cocultured with cells expressing VP22 fused with p53 or tk (6, 21); however, additional studies have been unable to confirm that VP22 can deliver functional therapeutic proteins from transfected to nontransfected cells (10). For example, no cytotoxic effect was observed in cells incubated with a fusion protein of VP22 and the diphtheria toxin A fragment (dtA), an extremely potent inhibitor of protein synthesis, indicating that transport of dtA into cells by VP22 was inefficient (10). Similarly, in the present study, VP22 enhanced Etk/GCV suicide gene therapy for neuroblastomas both in vitro and in vivo; however, no delivery of Etk into nontransfected cells by VP22 was detected. Thus, our findings suggest that VP22 promotes suicide gene therapy of transfected cells but may not traffic to nontransfected cells under the conditions studied. Although VP22 offers a potential benefit in gene therapy, a great need exists to understand how this benefit is conferred. In our present study, no cell-to-cell trafficking was detected; therefore, the benefit of VP22 appears to result from a direct effect on the transfected cells.

Although VP22 intercellular trafficking is a point of controversy, VP22 nuclear localization has been confirmed by many labs, including ours (7, 14, 22). VP22 may enhance the tk/GCV system by increasing the transport of tk protein molecules into nuclei, where incorporation of phosphorylated GCV into nascent DNA occurs, causing chain termination and cell death. In this study, BVP22 was found to possess greater potential to enhance Etk/GCV suicide gene therapy than HVP22 both in vitro and in vivo. BVP22 and HVP22 possess only 28.7% amino acid homology, with numerous motif differences. Further, BVP22 localizes predominately to the nucleus during BHV-1 infection, while HVP22 localizes primarily to the cytoplasm early during viral infection and translocates to the nucleus during cell division (7, 14, 19). The greater potential of BVP22 to enhance tk/GCV suicide gene therapy may result from its transporting the tk protein into nuclei more effectively and efficiently than HVP22.

In other studies on the interaction between BVP22 and host cells in our lab, we found that BVP22 altered the transcription of certain cellular genes, including Bax and Bcl-2 (unpublished data). BVP22 may make NXS2 cells more sensitive to Etk/GCV cytotoxicity by regulating specific cellular gene transcription. Further studies on the mechanism by which VP22 enhances tk/GCV suicide gene therapy for neuroblastomas are needed in order to better understand the biotherapeutic potential of VP22 in tk/GCV suicide gene therapy.


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ACKNOWLEDGMENTS
 
This work was supported by NIH grant R01GM/AI 60986.

We thank Angiela Mathison for helpful discussions regarding the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: AHABS, 1656 Linden Dr., Madison, WI 53706. Phone: (608) 262-0359. Fax: (608) 262-7420. E-mail: Splitter{at}svm.vetmed.wisc.edu. Back


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Journal of Virology, April 2004, p. 4224-4233, Vol. 78, No. 8
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.8.4224-4233.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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