CD-TK和HSV-TK两种自杀基因体系对前列腺癌细胞的杀伤作用

背景与目的：自杀基因治疗是一种有前景的基因治疗方法,本实验通过观察单纯疱疹病毒胸苷激酶(herpes simplex virus-thymidine kinase,HSV—TK)和CD-TK两种自杀基因体系对前列腺癌细胞PC-3m的杀伤效果,探讨该基因治疗在前列腺癌治疗中的应用价值。方法：应用逆转录病毒载体将HSV—TK基因和CD—TK融合基因分别转染：PC-3m细胞,经RT—PCR鉴定后,MTT法检测丙氧鸟苷(ganciclovir,GCV)、5-氟胞嘧啶(5-flucytosine,5-FC)前体药物单一或联合应用对转染后：PC-3m细胞的杀伤作用,以未转染的PC-3m细胞作对照。结果：GCV对转染后PC-3m细胞有较强的细胞毒作用,半数抑制浓度为8．34μg／ml,但旁观者效应不明显;5-Fc则有显著旁观者效应,当混育细胞中转染细胞比例是5％时即可出现;联合用药具有协同作用,两药相互作用指数小于1．0。结论：CD—TK自杀基因体系对前列腺癌细胞具有显著杀伤作用,明显优于单用HSV—TK基因体系,深入研究意义较大。     

人FAS启动子驱动的HSV-TK重组腺病毒靶向杀伤SKBR3乳腺癌细胞的研究

目的:构建脂肪酸合成酶(FAS)启动子驱动下的单纯疱疹病毒胸苷激酶(HSV-TK)重组腺病毒,研究其对乳腺癌细胞SKBR3的靶向杀伤作用。方法:以AdEasyTM腺病毒系统为载体,构建FAS启动子驱动下的HSV-TK重组腺病毒载体Ad-FAS-TK,将线性化的Ad-FAS-TK在AD-293细胞中包装,经过大量扩增和纯化,得到重组腺病毒。MTT法检测重组腺病毒Ad-FAS-TK与前体药物更昔洛韦(GCV)对SKBR3细胞的靶向杀伤作用。TUNEL法检测细胞凋亡。结果:成功构建FAS启动子驱动下的HSV-TK重组腺病毒载体Ad-FAS-TK,经包装、扩增和纯化得到约1010pfu/ml的重组腺病毒。MTT法和TUNEL检测结果显示,FAS启动子驱动下的HSV-TK重组腺病毒与GCV能够诱导SKBR3细胞凋亡,产生靶向细胞毒作用。结论:FAS启动子驱动下的HSV-TK重组腺病毒联合GCV对SKBR3细胞具有靶向杀伤作用,FAS启动子可以作为肿瘤靶向基因治疗的工具。     

Potentiation of ganciclovir toxicity in the herpes simplex virus thymidine kinase/ganciclovir administration system by ponicidin

The herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) administration system is commonly used in gene therapy trials. We have evaluated the effect of ponicidin, a diterpenoid isolated from a plant, Rabdosia ternifolia, on the cell-killing activity of the anti-herpes drugs acyclovir (ACV) and GCV. Ponicidin preferentially activated HSV-1-specific TK but not cellular kinases. In HSV-infected cells, ponicidin significantly accumulated the phosphorylated metabolites of GCV and suppressed the extracellular release of GCV. These data suggested that the cytotoxicities of ACV and GCV in HSV-TK-expressing cells might be potentiated by ponicidin. After transfected with the HSV-1 TK gene, COS-1 and several human cancer cells became highly sensitive to the cytotoxic properties of the nucleoside analogs. When ponicidin at the concentration without antiviral activities (0.2 microg/mL) was combined with ACV or GCV, the cytotoxic levels in HSV-TK-expressing cells were enhanced by 3- to 87-fold and 5- to 52-fold, respectively, compared with the nucleoside alone. When the stability of the bioactivity of ponicidin in the blood of mice was evaluated, the substance showed relatively long-lasting effects on the potentiation of the anti-herpetic and cytotoxic activities of GCV after intravenous administration. These data suggest that the combined use of ponicidin with GCV will be effective for cancer gene therapy, because high cytotoxicity in viral TK-expressing cells should yield more rapid and enhanced tumor elimination.     

人MUC4启动子驱动的HSV-TK重组腺病毒靶向杀伤SGC-7901胃癌细胞的实验研究

目的:构建人粘蛋白( MUC4)启动子驱动下的单纯疱疹胸苷激酶基因(HSV-TK)重组腺病毒,研究其对SGC-7901胃癌细胞的靶向杀伤作用.方法:克隆MUC4启动子区625 bp活性序列,构建重组荧光素酶报告基因载体pGL3-MUC4,检测其在SGC-7901胃癌细胞及NIH3T3成纤维细胞中的转录活性.以AdEasyTM腺病毒系统为载体,构建MUC4启动子驱动下的HSV-TK重组腺病毒rAdeno-MUC4-TK,感染SGC-7901及NIH3T3,经更昔洛韦(GCV)处理,MTT法检测细胞活力,TUNEL法检测细胞凋亡.结果:成功扩增出大小为625 bp的MUC4启动子序列.pGL3-MUC4在SGC-7901细胞中具有强转录活性,转录活性高于强启动子SV40 6.6倍,而NIH3T3成纤维细胞系中几乎无转录活性.构建重组腺病毒rAdeno-MUC4-TK,MTT法和TUNEL检测结果显示,其与GCV联合能够诱导SGC-7901细胞凋亡,产生靶向细胞毒作用.结论:人MUC4启动子驱动下的HSV-TK重组腺病毒联合GCV对SGC-7901胃癌细胞具有靶向杀伤作用,MUC4启动子可以作为胃癌靶向基因治疗的工具.     

Transduction of human pancreatic tumor cells with vesicular stomatitis virus G-pseudotyped retroviral vectors containing a herpes simplex virus thymidine kinase mutant gene enhances bystander effects and sensitivity to ganciclovir

We examined the suitability of Moloney murine leukemia virus (MLV) 4070A-, cat endogenous virus (CEV) RD114-, or vesicular stomatitis virus G (VSV-G)-pseudotyped retroviruses containing the humanized enhanced green fluorescent protein (hEGFP) or one of two herpes simplex virus thymidine kinase (HSV-TK) genes to transduce and provide gene expression in human pancreatic tumor cells. Fluorescence-activated cell sorter analysis demonstrated that VSV-G-pseudotyped hEGFP vector infected a greater percentage of cells and generated more robust gene expression than MLV 4070A- or CEV RD114-pseudotyped vectors. Dot blot and Southern blot analysis of genomic DNA revealed up to 10-fold more gene copies in G418-selected VSV-G hEGFP vector-transduced cells compared with genomic DNA from cells transduced with MLV 4070A or CEV RD114 pseudotypes. Cells transduced with VSV-G pseudotypes of HSV-TK(WT) or the HSV-TK30 vectors were 5- to 10-fold more sensitive to ganciclovir (GCV) than other pseudotype-transduced cells. A 40- to 61-fold difference in sensitivity to GCV was observed between cells transduced with VSV-G HSV-TK30 vector and cells transduced with MLV 4070A HSV-TK(WT) vector in vitro. A 13-fold reduction in tumor volume was observed in severe combined immunodeficient mice inoculated with PancTuITK30 cells compared with mice inoculated with PancTuITK(WT) cells during GCV treatment. We conclude that the choice of glycoprotein envelope and the potency of a particular suicide gene were therapeutically additive and increased the number of HSV-TK-positive cells and sensitivity toward GCV in human pancreatic tumors cells for prodrug gene therapy.     

脂质体/DNA复合物介导HSV-TK基因在膀胱癌细胞的表达和生物活性

目的：探讨脂质体／HSV－TK重组DNA对膀胱癌细胞的转导和细胞内的活性表达。方法：用脂质体／DNA复合物交HSV－TK基因逆转录病毒重组子（pLXSN-TK)导入膀胱癌细胞株BIU87。携带HSV－TK基因的BIU87细胞（BIU87－TK）由G418筛选获得。经过Southern杂交和细胞原位杂交检测后,观测ACV对BIU87－TK细胞存活的影响。结果：Southern印迹杂交证实HSV－TK基因存在于BIU87－TK细胞染色体中。细胞原位杂交检测HSV－TKmRNA的表达。外源DNA的导入以及HSV－TK和Neo 基因表达不会改变细胞生长特性。然而,在ACV作用下,转导HSV－TK基因的膀胱癌细胞的存活率受到影响,ACV可显著抑制癌细胞生长。结论：脂质体／TK重组DNA复合物可用于膀膛癌细胞的转基因治疗。     

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.     

人MUC4启动子驱动HSV-TK腺病毒靶向杀伤胃癌细胞的研究

目的：构建人MUC4启动子驱动下的HSV-TK重组腺病毒,研究其对胃癌细胞的靶向杀伤作用。方法：免疫荧光法检测MUC4在胃癌细胞系中的表达。克隆MUC4启动子区625bp活性序列,利用重组荧光素酶检测系统检测其在SGC-7901胃腺癌细胞及NUGC4胃印戒细胞癌细胞中的转录活性。以AdEasyTM腺病毒系统为载体,构建MUC4启动子驱动下的HSV-TK重组腺病毒,与前体药物GCV联合,检测其对上述两种胃癌细胞的细胞毒作用。结果：MUC4蛋白在两种胃癌细胞的胞浆和胞膜均有表达,而在成纤维细胞中无表达。克隆的MUC4启动子片段在SGC-7901和NUGC4细胞中具有强转录活性,转录活性高于强启动子SV40,而NIH3T3成纤维细胞系中几乎无转录活性。MUC4启动子驱动下的HSV-TK重组腺病毒与GCV联合能够诱导SGC-7901和NUGC4胃癌细胞凋亡,产生特异性靶向细胞毒作用。结论：人MUC4启动子驱动下的HSV-TK重组腺病毒联合GCV对SGC-7901和NUGC4胃癌细胞具有靶向杀伤作用,MUC4启动子可以作为胃癌靶向基因治疗的工具。     

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.     

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.     

人MUC4启动子驱动的HSV—TK重组腺病毒靶向杀伤SGC-7901胃癌细胞的实验研究

目的：构建人粘蛋白（MUC4）启动子驱动下的单纯疱疹胸苷激酶基因（HSV-TK）重组腺病毒,研究其对SGC一7901胃癌细胞的靶向杀伤作用。方法：克隆MUC4启动子区625bp活性序列,构建重组荧光素酶报告基因载体pGL3MUC4,检测其在sGD790l胃癌细胞及NIH3T3成纤维细胞中的转录活性。以AdEasy。”腺病毒系统为载体,构建Muc4启动子驱动下的HsV—TK重组腺病毒rAdeno—MUC4一TK,感染SGC7901及NIH3T3,经更昔洛韦（GCV）处理,MTT法检测细胞活力,TUNEL法检测细胞凋亡。结果：成功扩增出大小为625bp的MUC4启动子序列。pGL3一MUC4在SGC一790l细胞中具有强转录活性,转录活性高于强启动子SV406．6倍,而NIH3T3成纤维细胞系中几乎无转录活性。构建重组腺病毒rAdeno—MUC4一TK,MTT法和TUNEI。检测结果显示,其与GCV联合能够诱导SGC-7901细胞凋亡,产生靶向细胞毒作用。结论：人MUC4启动子驱动下的HSV—TK重组腺病毒联合GCV对SGC一7901胃癌细胞具有靶向杀伤作用,MUC4启动子可以作为胃癌靶向基因治疗的工具。     

自杀基因系统HSV-TK/GCV联合声动力疗法对人肺癌细胞NCI-446的体外杀伤实验

目的 探讨自杀基因系统HSV-TK/GCV联合声动力疗法对人小细胞肺癌NCI 446细胞的体外杀伤作用.方法 利用荧光和RT PCR检测方法,筛选出含HSV-TK基因的阳性克隆株NCI-446/TK.用MTT法检测不同浓度的GCV对NCI-446/TK细胞的杀伤效应,以及不同浓度混合NCI-446和NCI-446/TK细胞后的旁观者效应.同时,将HSV-TK/GCV自杀基因系统与声动力疗法进行联合,通过MTT法测定细胞存活率,并以流式细胞术检测细胞周期及凋亡率.结果 当GCV浓度为0.2 mg/L时,筛选出的阳性克隆株NCI-446/TK细胞的生长受到明显的抑制(P＜0.01),并且随着GCV浓度的递增,抑制效应越明显.当NCI-446/TK细胞占总细胞的10％时,有46％的混合培养细胞生长受到抑制(P＜0.01),其抑制效应随NCI-446/TK细胞所占比例的增加而增加.将HSV-TK/GCV自杀基因系统与声动力疗法进行联合后,其细胞存活率明显低于单一治疗组(P＜0.01),并且G0/G1期细胞所占比例及凋亡率均高于单一治疗组.结论 HSV-TK/GCV自杀基因系统的杀伤效应随着GCV浓度的递增而增强,并且有明显的旁观者效应.与声动力疗法联合后,其作用效果优于单一治疗组.     

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.     

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.     

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.     

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。     

Application of the Cre recombinase/loxP system further enhances antitumor effects in cell type-specific gene therapy against carcinoembryonic antigen-producing cancer.

A considerable number of studies of cancer have shown that the cell type-specific promoter is an effective tool for selective expression of foreign genes in tumor cells. However, few reports have demonstrated significant in vivo antitumor effects using this strategy thus far, possibly because the low activity of such a promoter results in insufficient expression of genes in cancer cells as well as in insignificant antitumor effects, even when the cells are infected by highly efficient gene transfer methods. To overcome this problem, we used the Cre/loxP system for the cell type-specific gene therapy against carcinoembryonic antigen (CEA)-producing cancer. We constructed a pair of recombinant Ads. One expresses the Cre recombinase (Cre) gene under the control of the CEA promoter (Ad.CEA-Cre). The other contains the herpes simplex virus thymidine kinase (HSV-TK) gene separated from the strong CAG promoter by insertion of the neomycin resistance (neo) gene (Ad.lox-TK). The HSV-TK gene of the latter Ad is designed to be activated through excisional deletion of the neo gene by Cre enzyme released from the former one only when CEA-producing cells are infected simultaneously with these Ads. Coinfection by these Ads rendered a human CEA-producing cancer cell line 8.4-fold more sensitive to ganciclovir (GCV) compared with infection by Ad.CEA-TK alone, the HSV-TK gene of which is directly regulated by the CEA promoter. On the other hand, coinfection with these Ads did not significantly change the GCV sensitivity of non-CEA-producing cells. Intratumoral injection of Ad.CEA-Cre combined with Ad.lox-TK followed by GCV treatment almost completely eradicated CEA-producing tumors established in the subcutis of athymic mice, whereas intratumoral injection of Ad.CEA-TK with GCV administration at most retarded the growth of inoculated tumors. These results suggest distinct advantages of the Cre/loxP system applied in the conventional cell type-specific gene therapy against cancer.     

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.