The role of a HSV thymidine kinase stimulating substance, scopadulciol, in improving the efficacy of cancer gene therapy

BACKGROUND: The most extensively investigated strategy of suicide gene therapy for treatment of cancer is the transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene followed by administration of antiviral prodrugs such as acyclovir (ACV) and ganciclovir (GCV). The choice of the agent that can stimulate HSV-TK enzymatic activity is one of the determinants of the usefulness of this strategy. Previously, we found that a diterpenoid, scopadulciol (SDC), produced a significant increase in the active metabolite of ACV. This suggests that SDC may play a role in the HSV-TK/prodrug administration system. METHODS: The anticancer effect of SDC was evaluated in HSV-TK-expressing (TK+) cancer cells and nude mice bearing TK+ tumors. In vitro and in vivo enzyme assays were performed using TK+ cells and tumors. The phosphorylation of ACV monophosphate (ACV-MP) was measured in TK- cell lysates. The pharmacokinetics of prodrugs was evaluated by calculating area-under-the-concentration-time-curve values. RESULTS: SDC stimulated HSV-TK activity in TK+ cells and tumors, and increased GCV-TP levels, while no effect of SDC was observed on the phosphorylation of ACV-MP to ACV-TP by cellular kinases. The SDC/prodrug combination altered the pharmacokinetics of the prodrugs. In accord with these findings, SDC enhanced significantly the cell-killing activity of prodrugs. The bystander effect was also significantly augmented by the combined treatment of ACV/GCV and SDC. CONCLUSIONS: SDC was shown to be effective in the HSV-TK/prodrug administration system and improved the efficiency of the bystander effect of ACV and GCV. The findings will be considerably valuable with respect to the use of GCV in lower doses and less toxic ACV. This novel strategy of drug combination could provide benefit to HSV-TK/prodrug gene therapy.     

Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene

The ability of herpes simplex virus type 1 thymidine kinase (HSV-tk)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-tk-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs via the transfer of phosphorylated GCV, the mechanism(s) of this bystander effect and the importance of gap junctions for the effect of prodrug/suicide gene therapy in primary human glioblastoma cells remains elusive. Surgical biopsies of malignant gliomas were used to establish explant primary cultures. Proliferating tumor cells were characterized immunohistochemically and found to express glial tumor markers including nestin, vimentin, glial fibrillary acidic protein (GFAP), S-100, and gap junction protein connexin 43 (Cx43). Western blot analysis revealed the presence of phosphorylated isoforms of Cx43 and Calcein/DiI fluorescent dye transfer showed evidence of efficient gap junction communication (GJC). In order to study the effect(s) of prodrug/suicide gene therapy in these cultures, human glioblastoma cell cultures were transfected with the HSVtk gene for transient or stable expression. Ganciclovir treatment of these cultures led to >90% of cells dead within 1 week. Eradication of cells could be inhibited by the addition of alpha-glycyrrhetinic acid (AGA), a GJC inhibitor. In parallel experiments, AGA decreased the immunodetection of phosphorylated Cx43 as analyzed by Western blot and inhibited fluorescent dye transfer. In conclusion, these observations are consistent with GJC as the mediator of the bystander effect in primary cultures of human glioblastoma cells by the transfer of phosphorylated GCV from HSVtk gene transfected cells to untransfected ones.     

Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene

The ability of herpes simplex virus type 1 thymidine kinase (HSV-tk)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-tk-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs via the transfer of phosphorylated GCV, the mechanism(s) of this bystander effect and the importance of gap junctions for the effect of prodrug/suicide gene therapy in primary human glioblastoma cells remains elusive. Surgical biopsies of malignant gliomas were used to establish explant primary cultures. Proliferating tumor cells were characterized immunohistochemically and found to express glial tumor markers including nestin, vimentin, glial fibrillary acidic protein (GFAP), S-100, and gap junction protein connexin 43 (Cx43). Western blot analysis revealed the presence of phosphorylated isoforms of Cx43 and Calcein/DiI fluorescent dye transfer showed evidence of efficient gap junction communication (GJC). In order to study the effect(s) of prodrug/suicide gene therapy in these cultures, human glioblastoma cell cultures were transfected with the HSVtk gene for transient or stable expression. Ganciclovir treatment of these cultures led to >90% of cells dead within 1 week. Eradication of cells could be inhibited by the addition of α-glycyrrhetinic acid (AGA), a GJC inhibitor. In parallel experiments, AGA decreased the immunodetection of phosphorylated Cx43 as analyzed by Western blot and inhibited fluorescent dye transfer. In conclusion, these observations are consistent with GJC as the mediator of the bystander effect in primary cultures of human glioblastoma cells by the transfer of phosphorylated GCV from HSVtk gene transfected cells to untransfected ones.     

HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells

Malignant glioma patients have a dismal prognosis with an urgent need of new treatment modalities. Previously developed gene therapies for brain tumors showed promising results in experimental animal models, but failed in clinical trials due to low transfection rates and insufficient expression of the transgene in tumor cells, as well as low bystander killing effects. We have previously shown that the histone deacetylase inhibitor 4-phenylbutyrate (4-PB) enhances gap junction communication between glioma cells in culture. In this study, we demonstrate an activation of recombinant HSV-tk gene expression, and a dramatic enhancement of gap junction-mediated bystander killing effect by administration of the HSV-tk prodrug ganciclovir together with 4-PB. These findings that 4-PB potentiates "suicide gene" expression as well as enhances gap junctional communication and bystander killing of tumor cells justify further testing of this paradigm as an adjunct to suicide gene therapy of malignant gliomas.     

Suicide gene therapy with Herpes simplex virus thymidine kinase and ganciclovir is enhanced with connexins to improve gap junctions and bystander effects

Connexins are proteins that form gap junctions between cells in various mammalian tissues. Because of their role in intercellular communication, connexins are important in the bystander cell death seen in Herpes simplex virus-thymidine kinase (HSV-TK) gene therapy for brain tumors. A selective review of connexin transduction/transfection studies with particular emphasis to central nervous system tumor cells is presented. In addition, specific references to studies with cell types that demonstrate low gap junction intercellular communication are presented. Data are included with the HT-29 colorectal tumor cell line to support the concept that enhancing gap junction protein expression in otherwise low gap junction communicating HT-29 cells increases bystander cell death and reduces tumor burden beyond what might be expected from HSV-TK and ganciclovir (GCV) treatment alone. Maximum in vitro bystander cell death was always produced when GCV treated co-cultures of TK-transduced and non-TK-transduced HT-29 cell lines were also transduced with connexin-43. When connexin was present in only one group of cells in the co-culture, there was more bystander cell death observed with connexin transduced into the non-TK-transduced cells, rather than the TK-transduced cells. The data presented reinforces conclusions made from earlier findings from cell line mixing experiments in which the non-TK-transduced cell population determined the level of bystander cell death (Burrows et al., 2002).     

A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma

BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease.     

Tanshinone IIA Increases the Bystander Effect of Herpes Simplex Virus Thymidine Kinase/Ganciclovir Gene Therapy via Enhanced Gap Junctional Intercellular Communication

The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy.     

Expression of MRP4 confers resistance to ganciclovir and compromises bystander cell killing

The multidrug resistance protein MRP4, a member of the ATP-binding cassette superfamily, confers resistance to purine-based antiretroviral agents. However, the antiviral agent ganciclovir (GCV) has not been shown to be a substrate of MRP4. GCV is important not only in antiviral therapy, but also in the selective killing of tumor cells modified to express herpes simplex virus thymidine kinase (HSV-TK). We therefore tested the effect of MRP4 on the cytotoxicity of GCV, on the ability of GCV to kill cells genetically modified to express HSV-TK, and on the bystander effect in which unmodified target cells are killed by GCV. Cells overexpressing MRP4 had markedly increased resistance to the cytotoxicity of GCV. Although, expression of recombinant HSV-TK increased the intracellular concentration of GCV nucleotide, cells were rescued by the cytoprotective effect of MRP4. In cells that overexpressed MRP4, intracellular accumulation of GCV metabolites was reduced, efflux of these metabolites was increased, and resistance to bystander killing was increased. Therefore, MRP4 can strongly reduce the susceptibility of HSV-TK-expressing cells to GCV, and its overexpression in adjacent cells protects them from bystander cell death. These findings indicate that a nucleotide transporter, such as MRP4, modulates the cellular response to GCV and thus may influence not only the efficacy of antiviral therapy, but also prodrug-based gene therapy, which is critically dependent upon bystander cell killing.     

Migratory neural stem cells for improved thymidine kinase-based gene therapy of malignant gliomas

Gene therapy of glioma based on viral delivery of herpes simplex virus type I thymidine kinase (HSV-TK) has failed in the clinic because of low transduction efficacy. To circumvent this problem, this study evaluated highly migratory HSV-TK-transduced neural stem cells (NSC) for their ability to kill untransduced glioma cells by a gap junction-mediated bystander effect. The admixture of HSV-TK-transduced NSC to U87MG and LN-18 human malignant glioma cell lines at ratios of 1:10 or 1:1 eliminated more than 50% or 90% of glioma cells in the presence of ganciclovir (25 microM). Glioma cell cytotoxicity required cell-cell contact. Similarly, tumor cell cytotoxicity was observed in two of three primary glioblastoma cell cultures, and the presence of this bystander effect correlated with the expression of connexin 43 in the untransduced glioma target cells. In conclusion, we delineate a role for migratory HSV-transfected NSC to eliminate glioma cells purely by means of the bystander effect.     

重组含HSV—TK基因腺病毒的构建及对肿瘤细胞的杀伤作用

High-titer replication-defective recombinant adenovirus expressing HSV-TK gene was constructed. Firstly, shuttle plasmid pAdCMVTK containing HSV-TK gene and CMV promoter was constructed and then recombined with right arm of adenovirus DNA. Secondly, the positive plaques containing recombinant adenovirus were identified and selected out by PCR and Southern blotting after infection into human embryo kidney 293 cells. The titer of recombinant adenovirus AdCMVTK was determined by plaque forming assay and it was as high as 10(12) pfu/ml. Tumor cells were infected with AdCMVTK and then treated with GCV. Cytotoxic effects were assayed with MTT method. HeLa, A549 and LoVo cells infected with AdCMVTK (M. O. I. = 100) became sensitive to the prodrug GCV, with IC50 less than 4 mumol/L. Significant bystander effect was observed. Results here show that the AdCMVTK/GCV system might be potential in the gene therapy for cancer.     

Selective gene therapy for human lung adenocarcinoma by tumor-specific expression of herpes simplex virus thymidine kinase gene

According to the fact that CEA gene expressed only in lung adenocarcinoma but not in normal lung cells, a retroviral expression vector (pCEATK) of the herpes simplex virus thymidine kinase (HSV-TK) gene regulated by CEA promoter was constructed and introduced into CEA-producing human lung adenocarcinoma cells GL and non-CEA-producing HeLa cells. The expression of pCEATK and Ganciclovir (GCV) sensitivity of the transfected cells were tested in vitro and in vivo . pCEATK expressed only in CEA-producing GL cells but not in non-CEA-producing HeLa cells. The sensitivity to GCV of pCEATK-transfected GL was 992 times higher compared with that of the parental cell line and there was obvious "bystander effect" in vitro. HeLa cells transfected wtih pCEATK were still resistant to GCV. Injection of GCV resulted in significant regression of pCEATK-transfected GL tumors in nude mice. In addition, all mice with any fraction of GL cells expressing HSV-TK exhibited a significant reduction in tumor growth, including mice     

The anti-tumor effect of human monocyte-derived dendritic cells loaded with HSV-TK/GCV induced dying cells

Herpes simplex virus thymidine kinase (HSV-TK) gene and dendritic cells (DC) have been used as the pioneering in cancer therapy. HSV-TK gene can induce apoptosis and necrosis in tumor cells in the presence of the non-toxic prodrug ganciclovir (GCV). We investigated the anti-tumor effect of DC vaccination by introducing dying cells from HSV-TK gene treatment as an adjuvant. HepG₂-TK cell line was established by transfecting human hepatoma cell line HepG₂ (HLA-A₂ positive) with HSV-TK gene. Dying tumor cells were generated by culturing HepG₂-TK cells with GCV. After engulfed dying cells efficiently, immature DCs (imDC) derived from human monocytes were fully matured and elicited marked proliferation and cytotoxicity against HLA matched HepG₂ cells in autologous peripheral blood mononuclear cells (PBMC). It also implied that HepG₂ specific CTLs played an important role in the cytotoxicity which was primarily depended on Th1 responses. Given the feasibility of inducing dying cells by HSV-TK/GCV in vivo, our results suggest an effective method in clinical human hepatocellular carcinoma (HCC) treatment by an in vitro model of applying HSV-TK gene modified human tumor cells integrated with DC vaccination.     

Connexin Over-Expression Differentially Suppresses Glioma Growth and Contributes to the Bystander Effect Following HSV-Thymidine Kinase Gene Therapy

Neoplastic transformation is frequently associated with a loss of gap junctional intercellular communication and reduced expression of connexins. The introduction of connexin genes into tumor cells reverses the proliferative characteristics of such cells. However, there is very little comparative information on the effects of different connexins on cancer cell growth. We hypothesized that Cx26, Cx32, or Cx43 would display differential growth suppression of C6 glioma cells and uniquely modulate the bystander effect following transduction of C6 cells with HSVtk followed by suicide gene therapy. The bystander phenomenon is the death of a greater number of tumor cells than are expressing the HSVtk gene, presumably due to the passage of toxic molecules through gap junction channels. To test this hypothesis, we used retroviral vectors to infect C6 glioma cells producing connexin-expressing and HSVtk-expressing cell lines. All three connexin-expressing cell lines grew significantly slower than GFP-infected or native C6 cells. Cx32 and Cx26 were significantly more effective at mediating the bystander effect in cocultures of C6-connexin cells with C6-HSVtk cells. These studies indicate that connexins have unique properties that contribute to their tumor suppressive function.     

Connexin over-expression differentially suppresses glioma growth and contributes to the bystander effect following HSV-thymidine kinase gene therapy

Neoplastic transformation is frequently associated with a loss of gap junctional intercellular communication and reduced expression of connexins. The introduction of connexin genes into tumor cells reverses the proliferative characteristics of such cells. However, there is very little comparative information on the effects of different connexins on cancer cell growth. We hypothesized that Cx26, Cx32, or Cx43 would display differential growth suppression of C6 glioma cells and uniquely modulate the bystander effect following transduction of C6 cells with HSVtk followed by suicide gene therapy. The bystander phenomenon is the death of a greater number of tumor cells than are expressing the HSVtk gene, presumably due to the passage of toxic molecules through gap junction channels. To test this hypothesis, we used retroviral vectors to infect C6 glioma cells producing connexin-expressing and HSVtk-expressing cell lines. All three connexin-expressing cell lines grew significantly slower than GFP-infected or native C6 cells. Cx32 and Cx26 were significantly more effective at mediating the bystander effect in cocultures of C6-connexin cells with C6-HSVtk cells. These studies indicate that connexins have unique properties that contribute to their tumor suppressive function.     

Tentative novel mechanism of the bystander effect in glioma gene therapy with HSV-TK/GCV system.

Although many works support gap junctional intercellular communication (GJIC) having a close relation to bystander cell killing in herpes simplex virus thymidine kinase (HSV-TK) gene and ganciclovir (GCV) treatment, our previous work suggested that other factors involved in bystander effect besides GJIC exist. To confirm our primary work, we evaluated the mode of the bystander cell (C6) co-cultured with TK-positive cells (TF10.2) in our designed "insert plates" in which two cell lines could be separated but share the same medium. Another method that we used was adding the supernatant from the medium of GCV-treated TF10.2 cells to the wild type C6. Growth inhibition of the bystander cells was observed despite the absence of GJIC. In addition, apoptotic cell death of TK+ cells and bystander cells was obvious. These studies suggested that other pathways besides cell-cell contacts may play a role in bystander cell killing; the factors released from TK-positive cells could induce apoptosis of bystander cells.     

Study of the Mechanism of Bystander Effect of KDR-CDglyTK System Mediated by Adenovirus for the Treatment of Gastric Cancer

We evaluated the relationship between cellular gap junction and bystander effect in gastric cancer SCG7901 cell killing by adenovirus-mediated KDR-CDglyTK system. SCG7901 and HeLa cells were transfected (MOI-100) with recombinant adenovirus carrying fusion gene. Transfection efficiency/fusion gene mRNA expression were determined; intercellular communications in the presence of apigenin were measured by fluorescence recovery after photobleaching (FRAP). Transfected/non-transfected cells were mixed at 5:95/10:90 % ratios, respectively, incubated for 24 h with/without apigenin, and for 72 h with GCV and 5-FU. Cell survival was determined by MTT method. The transfected SCG7901/HeLa cells expressed GFP. Fluorescence intensity in SCG7901 cells decreased after photobleaching but recovered over time. Fluorescence intensity in HeLa cells also decreased after photobleaching with no recovery. Following apigenin treatment, fluorescence intensity differed between SCG7901 and HeLa cells. Survival rates of SCG7901 cells differed among prodrug, apigenin, and prodrug + apigenin groups compared with controls, but no significant difference was observed for HeLa cells. We concluded that the intercellular communication in SCG7901 cells related with the gap junctions, while there was no such communication in HeLa cells. The bystander killing effect related with the intercellular gap junctions and was increased by apigenin with no such effect seen in HeLa cells.     

PBI-SUR-TK载体靶向介导HSV-TK自杀基因诱导肝癌细胞凋亡

目的:构建Survivin启动子调控的表达载体,并检测在启动子调控下HSV-TK自杀基因对肝癌细胞HepG₂和正常肝细胞HL-7702凋亡的影响。方法:合成含TK基因的质粒PBI-SUR-TK,利用脂质体Lipofectamine 2000将其导入肝癌细胞和肝细胞。然后分别运用RT-PCR和Western blot特异性检测基因和蛋白的表达情况;利用CCK8方法检测细胞增殖情况,流式细胞仪上机检测细胞凋亡情况。结果:肝癌细胞转染组有更多的TK基因表达产物,增殖情况减弱,凋亡情况明显。结论:Survivin启动子驱动的HSV-TK/GCV自杀基因系统对肝癌可能有一定的治疗作用。     

Replication-competent, oncolytic herpes simplex virus type 1 mutants induce a bystander effect following ganciclovir treatment

Cells expressing herpes simplex virus (HSV) thymidine kinase (tk) are killed by ganciclovir (GCV). Adjacent cells without HSV-tk also die, a phenomenon known as the 'bystander effect'. However, there is no evidence that replication-competent HSV induces a bystander effect in the presence of GCV. Therefore, we investigated the bystander effect in HEp-2 cells infected with replication-competent, oncolytic HSV-1 mutants, hrR3 and HF10. In cells infected at a multiplicity of infection (MOI) of 3, GCV did not induce apoptosis. At low MOIs of 0.3 and 0.03, however, a number of adjacent, uninfected cells apoptosed following GCV treatment. Irrespective of GCV treatment, HEp-2 cells expressed minimal levels of connexin 43 (Cx43). However, Cx43 expression was enhanced by GCV in response to infection with HF10 at an MOI of 0.3, but not at an MOI of 3. Expression of other proteins involved in gap junctions, including Cx26 and Cx40, was not augmented under these conditions. The PKA and PI3K signal transduction pathways are likely involved in enhanced Cx43 expression as inhibitors of these pathways prevented Cx43 upregulation. These results suggest that infection with replication-competent HSV-1 induces the bystander effect in cells treated with GCV because of efficient intercellular transport of active GCV through abundant gap junctions.