Polyamine biosynthesis inhibition enhances HSV-1 thymidine kinase/ganciclovir-mediated cytotoxicity in tumor cells

Cancer gene therapy with the aid of herpes simplex virus type 1 thymidine kinase gene (HSV-TK) and anti-herpes drug ganciclovir (GCV) has been widely used and its efficacy has been demonstrated in a variety of different malignant cells and animal tumor models. It is also commonly accepted, however, that this gene therapy regimen needs to be enhanced for a true clinical success. We studied whether polyamine biosynthesis inhibition by 2-difluoromethylornithine (DFMO), a clinically tested and well-tolerated chemotherapeutic drug, can increase the cytotoxicity of HSV-TK/GCV in 9L rat glioma cells. Our initial experiments showed that polyamine depletion actually protected the cells from cytotoxicity if GCV treatment was started too early after removal of DFMO. Analyses of cell growth, intracellular polyamine pools and cell cycle phase distribution suggested that later initiation of GCV treatment would be more beneficial due to increased proportion of cells in the middle of the cell cycle S phase. When the cells were exposed to GCV 3 or 4 days after removal of DFMO from growth medium, the cytotoxicity was increased up to 2.5-fold. We also verified whether cell cycle blockage per se could yield similar effect as DFMO. Our results from serum deprivation experiments showed that, despite of apparent cell growth and cell cycle phase distribution effects, serum starvation was weaker enhancer of HSV-TK/GCV cytotoxicity than DFMO. Finally, the general utility of HSV-TK/GCV + DFMO combination was tested in another tumor cell type, human prostate carcinoma cell line DU-145. DFMO sensitized these cells to HSV-TK/GCV cytotoxicity, but the effect was less prominent than in 9L cells. In conclusion, we have demonstrated that a correctly timed induction of DFMO-mediated polyamine biosynthesis inhibition can enhance the efficiency of HSV-TK/GCV gene therapy in vitro. The observed synergistic effect is potentially useful in clinical trials because, as opposed to use of other cell cycle-altering drugs, DFMO has already been tested in the treatment of human tumors and used as chemo preventive regimen with excellent tolerability.     

Polyamine depletion and cell cycle manipulation in combination with HSV thymidine kinase/ganciclovir cancer gene therapy

We have shown earlier that polyamine biosynthesis inhibition is accompanied by cell cycle alterations that can be utilized to enhance the efficacy of herpes simplex virus thymidine kinase - ganciclovir (HSV-TK/GCV) cancer gene therapy. In the present study, we asked 1) can the activated polyamine catabolism instead of biosynthesis inhibition be utilized to enhance the efficacy of HSV-TK/GCV gene therapy, and 2) can other known cell cycle inhibitors be used to make tumor cells more sensitive to this form of gene therapy? We show, using rat (9L) and human (U251-MG) glioma cell populations with 15% of HSV-TK-positive cells that DENSPM-induced activation of polyamine catabolism caused a profound polyamine deprivation in U251-MG cells, but there were no associated cell cycle effects in these cells. Consequently, we did not see any enhancement of the HSV-TK/GCV system. Aphidicolin, hydroxyurea, mimosine and resveratrol, but not lovastatin induced an apparent cell cycle arrest, followed by an intense but transient increase of the S phase cells after removal of the drug. This effect was shown to potentiate the HSV-TK/GCV cytotoxicity to some extent, especially in 9L cells and when the GCV treatment was started 0-24 h before the drug treatment. However, the enhancement was weaker than observed earlier with DFMO-induced cell cycle arrest and a considerable degree of the effect appeared to result from the growth-inhibitory actions of the drugs. In summary, we demonstrate that polyamine deprivation via DENSPM action is not associated with cell cycle effects and is not sufficient to cause enhancement of the HSV-TK/GCV system. Also, drugs with a rapid effect to the cell cycle are weak boosters of the HSVTK/GCV gene therapy, thus being less useful than DFMO for enhancement of this gene therapy form in animal studies and clinical trials.     

Recombinant interferon alpha2a synergistically enhances ganciclovir-mediated tumor cell killing in the herpes simplex virus thymidine kinase system.

The herpes simplex virus thymidine kinase (HSV-TK) gene is being developed in the treatment of many different types of tumors. The HSV-TK gene sensitizes tumor cells to the antiviral drug ganciclovir (GCV) and mediates the bystander effect in which unmodified tumor cells are killed as well. Although this approach has shown a significant antitumor effect, the need to potentiate this therapy exists. The results of this study indicate that recombinant interferon alpha2a (1FNalpha2a) acts synergistically with GCV to kill HSV-TK-expressing PA1 human ovarian tumor cells. Furthermore, it enhances the bystander killing of nearby unmodified tumor cells that do not express the HSV-TK gene. Previous studies have suggested that in vitro and in vivo bystander effects may be mediated by different mechanisms. However, IFNalpha2a enhanced bystander killing in both systems, with the survival of mice bearing preexisting tumors being significantly prolonged when they were treated with IFNalpha2a and HSV-TK/GCV compared with either treatment alone. Mechanism studies have shown that treatment with IFNalpha2a and GCV caused an increase in cells in S phase 24 hours after therapy in the HSV-TK-expressing cells, but the mechanism of action of IFNalpha2a does not seem to be related to an increase in DNA damage, because GCV incorporation was not increased after treatment with IFNalpha2a. These findings suggest that IFNalpha2a may be a useful adjunctive therapy for the HSV-TK/GCV system.     

Sequential Histopathological Changes in vivo after Suicide Gene Therapy of Gastric Cancer Induced by N-Methyl-N'-nitro-N-nitrosoguanidine in Rats

Gastrointestinal cancer is the most important clinical target of gene therapy. Suicide gene therapy, such as with the herpes simplex virus type 1 thymidine kinase ( HSV-TK ) gene, has been shown to exert antitumor efficacy in various cancer models in vitro. We previously reported in situ gene transfer and gene therapy for gastric cancer induced by N -ethyl-. N '-nitro-. N -nitrosoguanidine (ENNG) in dogs. Here, we describe the sequential histopathological changes after suicide gene therapy of N -methyl-. N '-nitro-. N nitrosoguanidine (MNNG)-induced gastric cancer in rats. Gastric tumors were induced by MNNG in 38/73 (52%) of Wistar strain rats. The suicide gene therapy group (14 rats) was subjected to in situ gene transfer with a recombinant adenovirus vector carrying the HSV-TK gene driven by CAG promoter (Ad.CAGHSV-TK) in gastric tumor, followed by the antiviral drug ganciclovir (GCV). To observe the histopathological changes at various tunes after HSV-TK/GCV gene therapy, groups of animals were sacrificed at 3, 8, and 30 days after gene transfer. Apoptosis in the gastric tumors was detected by the TUNEL method to assess the efficacy of HSV-TK/GCV gene therapy, and it was marked in the 8- and 30-day treatment groups compared to the sham operation controls ( P <0.001). Various histopathological changes, degeneration of cancer tissue and fibrosis after necrosis and apoptosis were significantly greater in the 30-day treatment group. The HSV-TK gene was detectable in peripheral blood by PCR until 30 days after gene transfer. These results may be useful in devising a method of suicide gene therapy for humans.     

Drug resistance to ganciclovir observed in suicide gene therapy is due to the loss of integrated herpes simplex virus-thymidine kinase gene

Human pancreatic cancer cells (AsPC-1) expressing the herpes simplex virus-thymidine kinase (HSV-TK) gene were inoculated into nude mice and ganciclovir (GCV) was administrated for the treatment. At the initial course of treatment we observed a notable reduction of tumor masses. However, therapeutic effect of GCV to the recurrent tumors decreased significantly. This reduced sensitivity of the AsPC-1/HSV-TK cells to GCV was also confirmed by an in vitro test. Examination of HSV-TK gene in the drug resistant cells showed a loss of the integrated gene. These data indicate that repeated GCV administration results in the loss of integrated HSV-TK gene and confers GCV resistance.     

Effects of retroviral-mediated herpes simplex virus thymidine kinase gene transfer to murine neuroblastoma cell lines in vitro and in vivo

Selective introduction of genes conferring chemosensitivity on proliferating tumor cells can be used to treat cancer. We investigated the efficacy of retrovirus-mediated gene transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene to murine neuroblastoma cell lines (neuro-2a) in vitro and in vivo. Retrovirus-mediated HSV-TK gene transfer to the neuro-2a cells resulted in sensitivity to ganciclovir (GCV) in vitro. In A/J mice, tumors produced from HSV-TK transduced neuro-2a cells regressed after GCV treatment. Intratumoral injection of recombinant retrovirus expressing HSV-TK gene also inhibited growth of the tumor established in A/J mice. These results demonstrate that HSV-TK gene therapy might be a feasible approach for inhibiting the growth of neuroblastoma.     

Inhibition of glioma cells in vitro and in vivo using a recombinant adenoviral vector containing an astrocyte-specific promoter

Gene therapy using the herpes simplex virus thymidine kinase (HSV-TK) gene in combination with the drug ganciclovir (GCV) is a promising approach for the treatment of cancer-inducing gliomas, a tumor with a poor prognosis. In an attempt to limit the toxic effects on normal tissues, we constructed a recombinant adenoviral vector, Adgfa2TK, in which the HSV-TK gene is driven by the promoter for the gene encoding glial fibrillary acidic protein, an intermediate filament protein expressed primarily in astrocytes. Infection by Adgfa2TK of a glial cell line (C6) and a non-glial cell line (MDA-MB-231) revealed markedly increased expression of HSV-TK in glial cells as determined by Western blot. In comparison, high HSV-TK protein levels were produced in both cell lines after infection with a control virus, AdCMVTK, in which the constitutive cytomegalovirus viral promoter was used to direct HSV-TK expression. Infection of two glial cell lines (C6, U251) and two non-glial cell lines (HepG2, MDA-MB-231) with Adgfa2TK followed by GCV treatment revealed high toxicity in glial cell lines (50% growth inhibitory concentration: <2 microg/mL of GCV) with little or no toxicity (50% growth inhibitory concentration: >75 microg/mL) in the non-glial cell lines. In vivo, injection of Adgfa2TK into C6 tumors grown in nude mice followed by intraperitoneal GCV treatment significantly repressed tumor growth compared with the controls. Adgfa2TK may be useful for directing expression of the HSV-TK gene to gliomas.     

Characterization of HIV-1 TAT peptide as an enhancer of HSV-TK/GCV cancer gene therapy

Cancer suicide gene therapy based on herpes simplex virus type I thymidine kinase (HSV-TK) and ganciclovir (GCV) suffers from the lack of efficacy in clinical use, which is mostly due to low gene-transfer efficiency and absence of bystander effect in tumors. We have previously demonstrated the enhancement of GCV cytotoxicity by fusing the HSV-TK with the cell penetrating peptide from HIV-1 transactivator protein transduction domain (TAT PTD). Despite the earlier promising results, we found that the triple fusion protein HIV-1 transactivator protein transduction domain-thymidine kinase suicide gene-green fluorescent protein marker gene (TAT-TK-GFP) increased GCV cytotoxicity only in 3/12 of different human tumor cell lines. Extended GCV exposure enhanced the cytotoxic effect of HSV-TK/GCV gene therapy, but the difference between TK-GFP and TAT-TK-GFP was not statistically significant. The modest improvement on cell killing mediated by TAT PTD in Chinese hamster ovary cells appeared to be associated with cell-surface heparan sulfate proteoglycan (HSPG) composition. However, TAT-mediated increased cell death did not correlate with the density of cell-surface HSPG expression in different tumor cell lines. In conclusion, although some degree of enhancement by TAT was shown in certain tumor cells in vitro, it is unlikely that TAT peptide linked to a suicide protein could be a useful booster of in vivo gene therapy trials.     

缺氧/辐射双敏感性启动子调控HSV-TK表达对肺癌移植瘤的作用

背景与目的放射-基因治疗是近年来国际上肿瘤治疗的新策略,由于实体瘤常处于缺氧状态而对放射敏感性低,放射-基因治疗尚未达理想疗效。本研究拟构建缺氧/辐射双敏感性启动子,增强缺氧条件下放射诱导的HSV-TK表达水平,提高肺癌放射-基因治疗效果。方法利用基因重组构建HRE-Egr启动子及其调控的HSV-TK表达载体;脂质体介导重组质粒转染肺癌A549细胞,分为对照组、放射组(6Gy)、缺氧组(1%氧浓度)和放射合并缺氧组,Northernblot法检测转染细胞中HSV-TK表达,四甲基偶氮唑蓝(MTT)法检测放射、缺氧和GCV处理后的细胞存活率。建立BALB/c裸鼠肺癌移植瘤模型,检测放射合并质粒转染后移植瘤体积变化并计算抑瘤率。结果对照组细胞仅可检测到HyTK的低水平表达(21U),放射组和缺氧组HyTK基因表达水平均显著升高(分别为227U和94U),放射合并缺氧组(769U)显著高于放射组。在缺氧条件下,放射合并GCV处理后细胞存活率为(7.2±1.8)%,显著低于常氧组的(32.7±4.6)%。放射联合GCV可明显抑制HRE-Egr启动子转染肺癌移植瘤,抑瘤率达91.2%。结论HRE-Egr启动子具有辐射/缺氧双重敏感性,并使放射后的HSV-TK表达水平在缺氧下得到显著增强。放射联合HRE-Egr启动子可显著抑制肺癌移植瘤的生长。     

Exogenous wt-p53 enhances the antitumor effect of HSV-TK/GCV on C6 glioma cells

Objective To study on the antitumor effect of combining wt-p53 gene with suicide gene therapy (HSV-tk+GCV) for malignant gliomas. Methods AdCMV-p53 was transfected into C6 glioma cells at MOI of (Multiplicity of infection) 0(G100), 10(TPG1), 100(TPG2), then AdCMV-tk was transducted to C6 glioma cells of G100, TPG1 and TPG2, respectively, at MOI of 100. The C6 glioma cells tranfected with both AdCMV-p53 and AdCMV-tk were exposed to various concentration of GCV. The cell survival rate was measured by MTT assay in vitro. Rat glioma model was established by injecting 5 × 10⁵ C6 glioma cells into right caudate nucleus of SD rats. AdCMV-p53 and AdCMV-tk were injected into glioma on day 5 and 6, respectively. On day 7, ganciclovir (GCV) was administrated intraperitoneally at 15 mg/kg/day for 14 days. The survival time of all rats was observed. The growth of intracerebral tumors was monitored dynamically by enhanced MRI. Cell apoptosis was evaluated by TUNEL method. Expression of HSV-tk gene was identified by in situ hybridization and expression of exogenous p53 gene was detected with Western blotting. Results In vitro, wt-p53 significantly enhanced antitumor effect of HSV-tk/GCV. The concentration of GCV for ID50 of TPG2 cells (0.001 μg/ml GCV) was 10 times lower than that for the cells of tk-GCV group (MOI = 100), while the concentration of GCV for ID100 of TPG2 (0.01 μg/ml GCV) and TPG1(0.1 μg/ml GCV) was 100 and 10 times lower than that for the cells of tk-GCV group (MOI = 100), respectively. Apoptosis of C6 glioma cells also could be induced by transfection with wt-p53 gene slightly. For in vivo study, the survival time of tumor-bearing rats treated with HSV-TK/GCV or wt-p53 combined with HSV-TK/GCV was significantly prolonged and the intracerebral tumors were regressed and disappeared earlier in the combined gene therapy group than those in the HSV-TK/GCV therapy group as shown in enhanced MRI. However, only half dose of GCV for the rats treated with both wt-p53 and HSV-TK/GCV was needed to obtain the same efficacy as those rats treated with HSV-TK/GCV alone. These results indicate that the transfection of wt-p53 potentiates the effect of HSV-TK/GCV therapy. Conclusions The combination of HSV-tk/GCV system with wt-p53 gene transduction is optimal for clinical therapeutic trials of suicide gene therapy for malignant gliomas.     

HSVTK/GCV自杀基因系统治疗乳腺癌的研究

 目的　探讨HSV TK/GCV自杀基因系统对小鼠乳腺癌细胞系MA782 / 5S 810 2体外及体内杀伤作用及其产生的旁观者效应。方法　采用脂质体转染法将GINaTK载体转入包装细胞PA317。取病毒上清液感染小鼠乳腺癌细胞MA782 / 5S 810 2 ,得到带有HSV TK基因的MA782 / 5S 810 2 /TK细胞 ,并将其分别用于体外和体内实验。结果　载体HSV TK导入了PA317细胞。体外实验结果显示 ,当MA782 / 5S 810 2 /TK细胞数占混合细胞 10 %时 ,低浓度 (10 μg/ml)的GCV就可将 5 0 %左右的肿瘤细胞杀死。体内实验结果显示GCV可明显抑制MA782 / 5S 810 2 /TK细胞在BALB/C小鼠体内的肿瘤形成。实验组肿瘤组织与对照组相比存在明显的病理学改变。结论　逆转录病毒可介导HSV TK基因转入小鼠乳腺癌细胞MA782 / 5S 810 2并获稳定表达 ,HSV TK/GCV自杀基因系统在体内外对乳腺癌细胞均有杀伤作用 ,且存在明显的旁观者效应。     

Sequential histopathological changes in vivo after suicide gene therapy of gastric cancer induced by N-methyl-N'-nitro-N-nitrosoguanidine in rats.

Gastrointestinal cancer is the most important clinical target of gene therapy. Suicide gene therapy, such as with the herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, has been shown to exert antitumor efficacy in various cancer models in vitro. We previously reported in situ gene transfer and gene therapy for gastric cancer induced by N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) in dogs. Here, we describe the sequential histopathological changes after suicide gene therapy of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric cancer in rats. Gastric tumors were induced by MNNG in 38 / 73 (52%) of Wistar strain rats. The suicide gene therapy group (14 rats) was subjected to in situ gene transfer with a recombinant adenovirus vector carrying the HSV-TK gene driven by CAG promoter (Ad.CAGHSV-TK) in gastric tumor, followed by the antiviral drug ganciclovir (GCV). To observe the histopathological changes at various times after HSV-TK / GCV gene therapy, groups of animals were sacrificed at 3, 8, and 30 days after gene transfer. Apoptosis in the gastric tumors was detected by the TUNEL method to assess the efficacy of HSV-TK / GCV gene therapy, and it was marked in the 8- and 30-day treatment groups compared to the sham operation controls (P < 0.001). Various histopathological changes, degeneration of cancer tissue and fibrosis after necrosis and apoptosis were significantly greater in the 30-day treatment group. The HSV-TK gene was detectable in peripheral blood by PCR until 30 days after gene transfer. These results may be useful in devising a method of suicide gene therapy for humans.     

Synergistic effect of resveratrol and HSV-TK/GCV therapy on murine hepatoma cells

Despite its low transfer efficiency, suicide gene therapy with HSV-TK is known for its bystander killing effect. The connexin-based gap junction is believed to mediate the bystander effect. Recently, we found that resveratrol, a polyphenol compound, increased the expression of Cx26 and Cx43, which are connexins and important constituents of gap junctions, in murine hepatoma cells. Hypothetically, the resveratrol-induced upregulation of gap junctions may improve the bystander effect that HSV-TK/GCV has on hepatoma cells. Our present investigation revealed that resveratrol could enhance intercellular communication at the gap junctions in CBRH7919 hepatoma cells and thereby enhance the bystander killing effect of GCV on CBRH7919^TK cells. However, inhibition of gap junction using its long-term inhibitor alpha-glycyrrhetinic acid had a negative influence on the bystander effect of gene therapy with HSV-TK/GCV. In addition, combined resveratrol and GCV treatment in tumor-bearing mice with CBRH7919^TK and CBRH7919^WT cells at a ratio of 2:3 resulted in a significant decrease in the volume and weight of the tumor in comparison to GCV or only resveratrol. The present results demonstrate that resveratrol can enhance the bystander effect exerted by the HSV-TK/GCV system by enhancing connexin-mediated gap junctional communication.     

Multimodal cancer treatment mediated by a replicating oncolytic virus that delivers the oxazaphosphorine/rat cytochrome P450 2B1 and ganciclovir/herpes simplex virus thymidine kinase gene therapies.

Multimodal therapy is generally more effective than single-agent treatment for cancer. rRp450 is an engineered herpes simplex viral mutant that replicates in and kills tumor cells in a relatively selective fashion. It also expresses, in infected cells, the cyclophosphamide (CPA)-sensitive rat cytochrome P450 2B1 (CYP2B1) and the ganciclovir (GCV)-sensitive herpes simplex virus thymidine kinase (HSV-TK) transgenes. We show that cultured rat 9L and human U87deltaEGFR glioma cells, infected and lysed by rRp450, also exhibit supra-additive sensitivity to both CPA and GCV, as determined by Chou-Talalay synergy analysis. DNA cross-linking, assayed by ethidium bromide fluorescence, was significantly inhibited in the presence of GCV, suggesting that interactions between the CPA/CYP2B1 and GCV/HSV-TK gene therapies occurred at the level of DNA repair. In vivo, regression of 9L s.c. tumor volumes in athymic mice was achieved only by the multimodal treatment allowed by rRp450 viral oncolysis combined with CPA/CYP2B1 and GCV/HSV-TK gene therapies, whereas all other treatment combinations produced only tumor growth retardation.     

Non-small cell lung cancer as a target disease for herpes simplex type 1 thymidine kinase-ganciclovir gene therapy

Lung cancer is a group of diseases that are difficult to cure and new treatment modalities, like gene therapy are actively tested to find alternatives for currently used strategies. Herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) method is one of the most frequently utilized forms of gene therapy and it has been tested on lung cancer, but no systematic study with comparison of different lung cancer types has been published. In this study, we examined in vitro and in vivo how good targets non-small cell lung cancer (NSCLC) cell lines representing adenocarcinoma, squamous cell lung cancer and large cell lung cancer are for adenovirus-mediated HSV-TK/GCV gene therapy. By using an adenovirus vector carrying a fusion gene of HSV-TK and green fluorescent protein (GFP), we found that: a) adenoviruses were efficient gene transfer vehicles for all types of NSCLCs; b) all adenocarcinoma and large cell lung cancer cells were good targets for HSV-TK/GCV therapy, whereas one of the squamous cell carcinoma cell lines was not responsive to the treatment; c) bystander effect played a major role in the success of this gene therapy form; d) subcutaneous tumors representing all three NSCLC types were efficiently treated with adenovirus-mediated HSV-TK/GCV gene therapy. In summary, this form of gene therapy appeared to be efficient treatment for human NSCLC and these results warrant further studies with primary lung cancer cells and orthotopic lung tumor models.     

Unrepairable DNA double-strand breaks initiate cytotoxicity with HSV-TK/ganciclovir

The herpes simplex virus thymidine kinase (HSV-TK) is the most widely used suicide gene in cancer gene therapy due to its superior anticancer activity with ganciclovir (GCV) compared with other HSV-TK substrates, such as 1-β-D-arabinofuranosyl thymine (araT). We have evaluated the role of DNA damage as a mechanism for the superiority of GCV. Using γ-H2AX foci as an indicator of DNA damage, GCV induced ≥ sevenfold more foci than araT at similar cytotoxic concentrations. The number of foci decreased after removal of either drug, followed by an increase in Rad51 foci indicating that homologous recombination repair (HRR) was used to repair this damage. Notably, only GCV produced a late and persistent increase in γ-H2AX foci demonstrating the induction of unrepairable DNA damage. Both drugs induced the ATR damage response pathway, as evidenced by Chk1 activation. However, GCV resulted in greater activation of ATM, which coincided with the late induction of γ-H2AX foci, demonstrating the presence of DNA double-strand breaks (DSBs). The increase in DSBs after Rad51 induction suggested that they occurred as a result of a failed attempt at HRR. These data demonstrate that the late and unrepairable DSBs observed uniquely with GCV account for its superior cytotoxicity and further suggest that inhibition of HRR will enhance cytotoxicity with HSV-TK/GCV.     

Gene therapy utilizing the Cre/loxP system selectively suppresses tumor growth of disseminated carcinoembryonic antigen-producing cancer cells.

Recent clinical trials of cancer gene therapy have shown encouraging results for controlling localized tumors. However, to control metastatic or disseminated tumor cells, further modification of vectors is required to enhance specificity and infectivity against targets. We investigated whether utilization of the Cre recombinase(Cre)/loxP system contributes to enhanced antitumor effects together with minimal adverse reactions in specific gene therapy against disseminated carcinoembryonic antigen (CEA)-producing cancer cells in the peritoneal cavity of mice. CEA-producing cancer would be a good therapeutic target because it is found in lung, stomach and colon sites, which account for most cancers. We constructed a pair of recombinant adenoviral vectors (Ads), one of which expresses the Cre gene under the control of the CEA promoter (Ad.CEA-Cre); the other expresses the herpes simplex virus thymidine kinase (HSV-TK) gene (Ad.lox-TK), or the beta-galactosidase gene (beta-gal) by Cre (Ad.lox-beta-gal). Intraperitoneal coinjection of Ad.CEA-Cre and Ad.lox-beta-gal into mice with peritonitis carcinomatosa by CEA-producing tumor cells showed selective expression of the beta-gal gene in tumor foci. Coinfection of Ad.CEA-Cre and Ad.lox-TK followed by ganciclovir (GCV) administration significantly suppressed the total tumor weight in the peritoneal cavity of the mice to 13% of that of the untreated mice and 22% of that of the mice treated with Ad.CEA-TK/GCV, an Ad that expressed the HSV-TK gene driven by the CEA promoter alone. Moreover, treatment with Ad.CEA-Cre and Ad.lox-TK/GCV completely suppressed tumors in 4 of 10 (40%) mice without significant weight loss, although 2 of 10 mice treated with Ad.CAG-TK/GCV, an adenovirus vector that strongly but nonspecifically expressed the TK gene, died due to severe side effects including diarrhea, weight loss and liver dysfunction. These findings suggest that cell type-specific gene therapy using the Cre/loxP system is effective against disseminated cancer cells without significant side effects.     

Purified herpes simplex thymidine kinase retroviral particles. II. Influence of clinical parameters and bystander killing mechanisms

High-titer, purified herpes simplex virus thymidine kinase (HSV-TK) retroviral particles, followed with intraperitoneal ganciclovir (GCV) were tested in Fischer rats bearing 1-week established 9L gliosarcomas. 9L cells were infused intracranially through a cannula on day 0, given intracranial infusions of HSV-TK retroviral particles on days 7-12, and given 5 or 10 daily doses of intraperitoneal GCV starting at day 14. Tumor volumetric studies performed on rat brains at day 26 after tumor infusion revealed significant differences in experimental groups receiving HSV-TK retroviral particles plus 10-day GCV or the 100% transduced 9L-TK group receiving GCV versus control groups treated with either buffer, HSV-TK vector, or either 5- or 10-day regimens of GCV alone. The duration of GCV administration and the volume of tumor burden influenced efficacy. Adjuvant dexamethasone did not significantly affect efficacy. Experiments in which 9L rechallenge of animals treated with 9L-TK/GCV or 9L tumors treated with HSV-TK vector/GCV indicated that a host immune response was involved in rejecting the rechallenge tumor. Outcome was dependent upon the site and size of the rechallenge inoculum. In vitro, bystander effects were significant but were especially marked when cell-to-cell contact was maintained. Cumulatively, the data indicate that both the bystander effect and the host immune response are responsible for the efficacy observed in the suicide gene therapy paradigm using purified HSV-TK retroviral particles and GCV to treat smaller tumor burden in rats with 9L gliosarcoma.     

Herpes simplex virus thymidine kinase and granulocyte macrophage colony-stimulating factor combination gene therapy in a murine CT26 cell colon cancer model

We evaluated the antitumor effects of combination gene therapy on CT26 mouse colon cancer cells, using the genes for herpes simplex virus thymidine kinase gene HSV-TK combined with granulocyte macrophage colony-stimulating factor (GM-CSF) compared with HSV-TK alone. Cells, unmodified or retrovirally transduced with HSV-TK or GM-CSF, were inoculated subcutaneously into syngeneic BALB/c mice in various combinations. HSV-TK and GM-CSF were also delivered using different routes (in separate cells vs doubly transfected single cells). Both HSV-TK (with i.p. ganciclovir - GCV - treatment) and GM-CSF genes had independent antitumor effects, and given together they caused significant reduction in tumor volumes compared with the HSV-TK gene alone (P < 0.001). Following GCV treatment, however, the treated/control ratios for tumor volumes were not different between tumors containing either HSV-TK alone or both genes (0.27 vs 0.25, respectively). Thus, the presence of GM-CSF did not increase the bystander effect of HSV-TK. Tumors receiving genes transferred in separate cells tended to be more consistently suppressed after GCV treatment than when both genes were transferred in the same cells, although this was not statistically significant. Thus, combination GM-CSF and HSV-TK gene therapy produced greater therapeutic efficacy than HSV-TK alone, but the bystander effect was not enhanced by GM-CSF.