HSV-TK和IL-12联合基因对宫颈癌的杀伤作用

目的观察单纯疱疹病毒胸苷激酶(HSV-TK)丙氧鸟苷(GCV)自杀基因系统联合IL-12基因对人宫颈癌hela细胞系体外杀伤作用。方法采用脂质体转染法将HSV-TK及IL-12联合基因转染hela细胞,并将其用于体外实验,观察GCV对转染联合基因的Hela的杀伤作用及旁观者效应等。结果加入GCV后体外培养联合基因转染的Hela细胞,24 h后细胞明显发生形态上改变,正常对照组细胞生长旺盛;随着GCV剂量的加大,转染联合基因的Hela细胞的存活率呈逐渐下降的趋势,GCV对转染细胞的杀伤作用较对正常细胞的杀伤作用明显增强。并观察到自杀基因系统具有明显的旁观者效应。结论脂质体可介导HSV-TK及IL-12联合基因转入人宫颈癌hela细胞并获得稳定表达,GCV对HSV-TK及IL-12联合基因转染的hela细胞具有明显的杀伤作用。     

Survivin启动子控制自杀基因HSV-TK真核表达载体对肝癌细胞杀伤活性的研究

目的研究自杀基因单纯疱疹病毒胸苷激酶(HSV-TK)对人肝癌细胞的特异性杀伤作用,推进该自杀基因治疗原发性肝癌的可行性,从而为肝癌靶向基因治疗奠定一定的基础。方法将pc DNA3.1-p Survivin-TK质粒转染人肝癌细胞Hep G2中,RT-PCR法检测自杀基因m RNA水平,Western blot法检测自杀基因蛋白量的表达;再次转染上述质粒到肝癌细胞Hep G2中,随着更昔洛韦(GCV)剂量的增加,来监测pc DNA3.1-p Survivin-TK重组载体对细胞的杀伤效果,CCK-8法检测GCV对肝癌细胞的毒性作用,流式细胞术检测细胞凋亡的情况。结果通过RT-PCR法、Western blot法表明了转染该重组质粒的Hep G2细胞可以成功表达TK基因;此外,在外源药物GCV作用下也实现了对肝癌细胞的特异杀伤,CCK-8法表明,随着药物浓度的增高质粒转染组细胞存活率逐渐下降;流式细胞术检测得知,细胞经GCV处理48 h后,转染pc DNA3.1-p Survivin-TK重组质粒的肝癌细胞组凋亡率约为(37.37±4.02)%,而未转染质粒组细胞凋亡率约为(1.00±0.62)%,两组间比较有统计学差异(P<0.01)。结论 HSV-TK/GCV自杀基因的毒性作用随着GCV浓度的增高而上升,并且HSV-TK/GCV系统对人肝癌细胞具有特异性靶向杀伤作用。     

放疗增强对转染pAdKDR／CD／TK自杀基因系统的SW480大肠癌细胞的杀伤作用

目的研究γ射线照射对含KDR启动子双自杀基因大肠癌细胞SW480的杀伤协同作用,和自杀基因及前药的放射增敏作用。方法重组腺病毒pAdKDR—TK、pAdKDR-CD和pAdKDR—TK／CD转染SW480细胞,观察7射线对SW480细胞基因转染率的增强作用和对前药GCV、5-FC、GCV＋5-FC杀伤SW480细胞的放射增敏效应,以及前药GCV、5-FC、GCV＋5-FC对γ射线杀伤SW480细胞的协同增效作用。结果转染双自杀基因的大肠癌细胞射线组比对照组转染率有明显升高（P〈O．01）,7射线对转染KDR—TK／CD基因的SW480细胞比未转染基因的对照组SW480细胞具有更强的杀伤作用（P〈0．001）。结论γ射线照射能明显提高双自杀基因的转染效率,强化了基因治疗的“旁观者效应”,自杀基因对γ射线的增敏作用。     

慢病毒介导的突变型胸苷激酶对T淋巴细胞杀伤作用的实验研究

目的观察慢病毒介导的突变型单纯疱疹病毒胸苷激酶(HSV-sr39tk)及野生型HSV-tk对T细胞的体外杀伤作用,比较HSV-sr39tk/更昔洛韦(GCV)、HSV-sr39tk/阿昔洛韦(ACV)、HSV-tk/GCV、HSV-tk/ACV对T细胞存活率的影响。方法采用改良的磷酸钙沉淀法将包装质粒、包膜蛋白质粒和含目的基因的转移质粒共转染293T包装细胞,收集的病毒上清感染T细胞后,分别与不同浓度梯度的前体药物GCV和ACV作用4d后,MTT法测定细胞存活率。结果共转染293T细胞后获得较高滴度的慢病毒(2×106IU/ml)。前体药物GCV和ACV浓度在0～10.0μmol/L时转染HSV-sr39tk细胞(39tk+T细胞)存活率下降比较明显,GCV组细胞存活率由(96.04±3.23)%下降为(36.76±4.38)%,ACV组细胞存活率由(97.31±4.61)%下降为(43.75±8.99)%,而GCV和ACV浓度大于10.0μmol/L时,39tk+T细胞存活率下降趋势则减缓。统计显示39tk+T细胞对GCV、ACV均有敏感性(P值均<0.05);转染HSV-tk T细胞(tk+T细胞)对GCV有敏感性(P<0.05),而对ACV不具有敏感性(P>0.05);同一浓度时39tk+T细胞+GCV与tk+T细胞+GCV两组间T细胞存活率差异具有统计学意义(P<0.05)。结论慢病毒载体可高效、稳定地感染T细胞,同时不影响细胞的增殖,与野生型HSV-tk基因比较,表达突变型HSV-sr39tk的T细胞对GCV具有更高的敏感性,而且对ACV也具有敏感性。     

pAdKDR/CD/TK双自杀基因系统对肝癌细胞的杀伤作用的试验研究

目的探讨含KDR启动子的双自杀基因CD/TK对肝癌HepG2细胞的杀伤作用。方法观察不同前药对HepG2细胞、血管内皮细胞ECV304和LST174细胞的杀伤作用,评估KDR启动子的靶向杀伤作用。观察不同前药对转染不同自杀基因的HepG2细胞的杀伤作用,及旁观者效应。结果转染腺病毒的HepG2细胞及血管内皮ECV304细胞对前药具有较高的敏感性,而感染腺病毒的LST174细胞对前药不敏感(P0.05)。当转染细胞混合比例为50%时,应用前药后,转染CD/TK,CD,TK的HepG2细胞生存率分别为11.8±1.2%、41.3±3.7%、43.1±2.3%(P0.05)。结论含KDR启动子的双自杀基因CD/TK对肝癌HepG2细胞具有高度靶向杀伤作用;联合用药对转染双自杀基因的肝癌HepG2细胞具有协调增强杀伤作用。     

pAdKDR/CD/TK自杀基因系统对大肠癌SW480细胞体外杀伤作用的实验研究

目的研究腺病毒介导的KDR/CD/TK双自杀基因系统对大肠癌SW480细胞的杀伤作用。方法构建pAdKDR/CD/TK重组腺病毒,采用不同感染复数(MOI)的重组腺病毒感染大肠癌SW480细胞、ECV304细胞及LS174T细胞,再加入不同浓度前药(GCV、5-FC)培养后,以MTT法检测细胞生长抑制率,观察重组腺病毒联合GCV和5-FC对大肠癌SW480细胞、ECV304细胞和LS174T细胞的杀伤作用,了解前药GCV、5-FC、GCV/5-FC对3种细胞的杀伤作用及KDR启动子的靶向杀伤作用。结果 pAdKDR/CD/TK重组腺病毒对3种细胞具有相似的感染率,感染腺病毒的3种细胞中除LS174T细胞外,均检测到目的基因的表达。当转基因细胞的比率为50%时,SW480和ECV304细胞分别有(31.3±5.64)%、(33.5±5.4)%的存活,而LS174T细胞存活率仍为(98.1±0.95)%(P均<0.01)。单用GCV浓度为80μg/ml时,SW480细胞、ECV304细胞、LS174T细胞的生存率分别为(45.6±6.5)%、(45.9±5.4)%、(99.5±4.7)%,单用5-FC浓度为1000μg/ml时,三者生存率分别为(40.4±5.8)%、(45.3±4.7)%、(97.0±6.2)%,联合用药(80μg/ml+1000μg/ml)时,三者生存率分别为(20.5±0.46)%、(25.6±1.33)%、(97.3±3.96)%。联合用药分别与GCV及5-FC单独用药生存率比较,差异均有统计学意义(P均<0.05)。提示单独应用GCV及5-FC对SW480细胞、ECV304细胞有较强的杀伤作用,联合用药效果更佳。结论含KDR启动子的融合基因,具有选择性杀伤作用。前药GCV、5-FC对转染融合基因的大肠癌SW480细胞具有体外抑制作用,且有较强的旁观者效应。     

HSV-TK/GCV系统在体外对胃癌MNK-45细胞株的增殖抑制作用研究

目的　探讨单纯疱疹病毒胸苷激酶基因/丙氧鸟苷（HSV-TK/GCV）自杀基因系统对胃癌MNK-45细胞增殖、侵袭和迁移的抑制效应。方法　分别采用MTS试验、CCK8试验、Transwell试验、Western blot试验和RT-PCR方法检测HSV-TK/GCV系统对胃癌MNK-45细胞在24，48，72和96h的增殖、侵袭和迁移的改变；对MNK-45细胞凋亡相关蛋白mRNA水平的变化。结果　HSV-TK/GCV系统能够有效抑制胃癌MNK-45细胞的增殖效应，并能够减弱胃癌MNK-45细胞的侵袭和迁移能力，诱导MNK-45细胞凋亡相关蛋白Caspase3、Bax蛋白水平和mRNA的表达增加，抑制Bcl-2蛋白表达水平的降低，促进凋亡的发生。结论　HSV-TK/GCV系统能够有效抑制胃癌MNK-45细胞增殖、侵袭和迁移，诱导MNK-45细胞凋亡的发生。     

CDglyTK自杀基因系统治疗小鼠慢性粒细胞白血病皮下移植瘤的研究

为了探讨逆转录病毒介导的CDglyTK自杀基因系统时K562细胞的体内外杀伤作用，将逆转录病毒介导的CDglyTK自杀基因转染入K562细胞，体外实验用MTT法观察5-氟胞嘧彰丙氧鸟苷（5-fluorocytosine／ganciclovir，5-FC／GCV）时K562／CDglyTK细胞的生长抑制率。体内实验时将K562／CDglyTK细胞和K562细胞接种于裸鼠皮下，使用GCV和5-FC后，观察裸鼠肿瘤体积的变化及裸鼠的生存率。体外实验表明，GCV联合5-FC时K562／CDglyTK细胞具有明显的杀伤作用；体内实验结果显示，皮下注射K562细胞和K562／CDglyTK细胞后小鼠成瘤率无明显区别；使用5-FC／GCV可明显抑制裸鼠体内的肿瘤形成；经5-FC／GCV治疗后K562／CDglyTK组的肿瘤体积较对照组明显缩小，裸鼠生存率也较时照组明显提高。结论：双自杀基因在体内外对K562细胞均有杀伤作用。     

超声微泡靶向破坏介导HSV-TK/GCV抑制BALB/c-nu小鼠卵巢癌的实验研究

目的 探讨超声微泡靶向破坏介导下HSV-TK基因联合更昔洛韦对裸鼠卵巢癌的杀伤效应.方法 挑选成功种植卵巢癌细胞且状态良好的雌性BALB/c-nu小鼠40只,随机分为4组:A组,HSV-TK+超声微泡(MBs)+超声辐射(US)组;B组,HSV-TK+超声辐射(US)组;C组,HSV-TK组;D组,磷酸盐缓冲液(PBS)组.分别利用Western-Blot和real time RT-PCR检测各组瘤体组织内TK蛋白和mRNA的表达,TUNEL法检测不同组的肿瘤细胞凋亡,并比较各组之间的肿瘤生长状况及裸鼠生存时间.结果 A组TK蛋白、mRNA表达及肿瘤细胞凋亡率高于其他各组(P＜0.05),平均肿瘤体积A组显著低于其他各组(P＜0.05),但生存时间上A组同其他各组之间差异并无统计学意义.结论 超声微泡靶向破坏介导HSV-TK联合更昔洛韦可促进裸鼠卵巢癌细胞的凋亡,并抑制肿瘤生长速度,但短期观察并不能延长裸鼠生存时间.     

双自杀基因慢病毒转移载体系统的构建及其对K562细胞的杀伤作用

为了探讨双自杀基因慢病毒转移载体系统对K562细胞的杀伤作用,采用分子生物学方法构建了含大肠杆菌胞嘧啶脱氨酶(E.coli CD)和单纯疱疹病毒胸苷激酶(HSV-TK)的双自杀基因慢病毒转移载体,将慢病毒基因转移系统中的3种成分质粒(目的基因转移载体、包装结构及包膜结构质粒)用脂质体分为2组(实验组、对照组)共转染入病毒包装细胞293T,在荧光显微镜下观察转染效果,在透射电镜下观察上清中的病毒颗粒.大量收集病毒上清,浓缩后感染K562细胞,荧光显微镜下观察感染效果,RT-PCR鉴定目的基因在K562细胞中的整合转录.给予前体药物5-氟胞嘧啶(5-FC)和(或)无环鸟苷(GCV)后,用MTT法测定体外杀伤效应,扫描电镜下观察使用前体药物后K562细胞的变化.结果表明成功构建了双自杀基因慢病毒转移载体,脂质体法可有效将上述慢病毒表达质粒转入293T细胞.荧光显微镜下观察到对照组大量绿色荧光蛋白(green fluorescenceprotein,GFP)表达.透射电镜下观察到大量浓缩后病毒颗粒.荧光显微镜下观察到此基因转移载体系统对293T细胞及K562细胞的感染率均很高,单独使用GCV或5-FC对转染自杀基因的K562细胞的生长抑制率(growth inhibition ration,GIR)分别为48.73%、50.16%,与未转染K562细胞比较明显升高,有显著性差异(P＜0.01),联合使用5-FC和GCV时K562细胞的GIR为87.69%,比单独使用5-FC或GCV时明显升高,有显著性差异(P＜0.01).结论双自杀基因慢病毒基因转移载体系统可将双自杀基因高效转移至K562细胞,是一种有效的基因转移载体系统.     

Adeno-Associated Viral-Mediated Gene Transfer to Hepatoma: Thymidine Kinase/Interleukin 2 Is More Effective in Tumor Killing in Non-Ganciclovir (GCV)-Treated Than in GCV-Treated Animals

Interleukin 2 (IL-2) enhancement of herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV)-induced tumor killing was studied by cloning the human interleukin 2 gene into an HSV-TK-bearing adeno-associated viral (AAV) vector (TK/IL-2). The mouse hepatocellular carcinoma cell line Hepa 1-6 was used as a model in this study. We found that TK/IL-2-transduced Hepa 1-6 cells were more susceptible to ganciclovir treatment than tumor cells transduced with only TK in both nude mice and immunocompetent C57L/J mice. TK/IL-2-transduced tumors also showed shrinkage without GCV treatment. The tumor-killing effect of AAV-mediated TK/IL-2 gene transfer was further studied by inoculating animals with TK/IL-2- or TK-transduced tumor cells mixed with unmodified cells with or without GCV treatment. Although tumor growth in each group was inhibited, the best result was obtained from the TK/IL-2-transduced group without GCV treatment. In this group, 10% of the transduced tumor cells could eradicate the whole tumor in 50% of the animals tested as well as provide long-term protection against tumor cell rechallenge. When this group was treated with GCV, the antitumor effect of TK/IL-2 was reduced. We attribute this to the early ablation of transgene-bearing tumor cells by GCV treatment, which thus reduces the duration of IL-2 expression. We conclude that (i) TK/IL-2 plus GCV treatment generates a stronger tumor-killing effect than HSV-TK plus GCV and (ii) tumor killing of TK/IL-2 is more effective in non-GCV-treated animals than in GCV-treated animals.     

Enhanced ganciclovir killing and bystander effect of human tumor cells transduced with a retroviral vector carrying a herpes simplex virus thymidine kinase gene mutant

Gene transfer of the herpes simplex virus thymidine kinase (TK) gene associated with ganciclovir (GCV) treatment can lead to death of TK-expressing cells, and of neighboring TK- cells because of the bystander effect. Thus, a small proportion of TK+ cells in a tumor can lead to its complete regression after GCV treatment. However, a lack of efficacy of gene transfer into tumors associated with low GCV sensitivity and poor bystander effect of human cancer cells currently limit the clinical use of this suicide gene therapy approach. To increase the potency of suicide gene therapy, we have tested the GCV sensitivity and the bystander effect of TK mutants that have been previously described. After retroviral transduction of the TK mutants into human tumor cells of various origins, we have found a strong killing effect of GCV with cells expressing the mutants TK30 or TKF161C. The GCV sensitivity of several human tumor cell types expressing TK30 was 9- to 500-fold higher than cells containing wild-type TK. Furthermore, TK30-expressing cells were able to kill bystander cells much more efficiently than TK-expressing cells. Thus, TK30 mutant is a promising candidate for suicide gene therapy clinical trials.     

Synergy between the herpes simplex virus tk/ganciclovir prodrug suicide system and the topoisomerase I inhibitor topotecan

An established principle of antineoplastic chemotherapy is that multidrug regimens are generally superior to single-agent therapy. This prompted us to elucidate whether the topoisomerase inhibitor topotecan (TPT) could enhance the efficacy of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system for the treatment of cancer. We assessed the interaction between these two treatments in murine MC38 and human HT-29 colon carcinoma cell lines that were genetically modified to constitutively express HSV-tk, sensitizing them to GCV. Synergistic cell killing was observed in a clonogenic assay over most of the cytotoxic dose range by the median-effect principle of Chou and Talalay (Adv. Enzyme Regul. 1984; 22:27-55). Subcutaneous tumor models, using the same cell lines in C57BL/6 and athymic nude mice, respectively, demonstrated that the combination of GCV and TPT resulted in statistically significant enhanced survival relative to single-agent treatment. In addition, nude mice bearing HT-29 tumor xenografts were treated with an Ad5 E1b Mr 55,000 attenuated replication-competent adenovirus expressing HSV-tk (Ad.TK(RC)) either alone or in combination with GCV and/or TPT. These experiments demonstrated that Ad.TK(RC) followed by GCV and TPT was more efficacious than any other treatment tested. Our results suggest that for antineoplastic therapy, molecular chemotherapy based on the HSV-tk/GCV system combined with traditional chemotherapy is a logical and practical future direction to pursue. Suicide gene therapy is the approach whereby genetically altering a cell makes it susceptible to an otherwise relatively nontoxic prodrug. By this approach it is possible to achieve relatively high concentrations of the toxic metabolites in the transduced cells while maintaining low systemic levels of the active drug. The most often used metabolic suicide gene transfer system is the HSV-tk/GCV paradigm, which is currently being used in cancer therapy or as a safety modality. The low response rate observed in the early clinical HSV-tk cancer trials may be due to failure in achieving adequate transduction efficiency and/or prodrug concentration within the tumor. The combination of such suicide gene prodrug systems with adjunctive drugs resulting in synergistic cytotoxicity might improve the clinical utility of this approach.     

Contribution of a combination of ponicidin and acyclovir/ganciclovir to the antitumor efficacy of the herpes simplex virus thymidine kinase gene therapy system

We have previously reported that ponicidin (PND), isolated from Rabdosia ternifolia, potentiates the cell-killing activity of antiherpes prodrugs acyclovir (ACV) and ganciclovir (GCV) in human cancer cells expressing herpes simplex virus thymidine kinase (HSV-TK). To extend these in vitro results to in vivo situations, HSV-TK-expressing HeLa cells were injected into nude mice. The in vivo growth of TK(+) HeLa cells was significantly inhibited by coadministration of PND and ACV, or of PND and GCV, compared with single use of ACV or GCV in spite of lower doses of 1 or 0.25 mg/mouse, respectively. These results indicate that there is a good correlation between this in vivo efficacy and previously reported in vitro efficacy. Because of the insufficiency of incorporation of genes into tumors, bystander cell killing has attracted special interest. In the present study, we determined the ability of PND to potentiate the bystander effects of ACV and GCV in both in vitro and in vivo systems. In vitro combined use of PND with ACV or GCV rendered tumor cells more sensitive to the prodrugs, demonstrating a 1.8- to 97-fold or 2.8- to 26-fold reduction in IC(50) compared with ACV or GCV only, respectively, in 1 to 20% of HSV-TK(+) cells. In the in vivo experiments using nude mice injected with 3 or 10% HSV-TK(+) cells, tumor volume was lower in mice treated with a combination of PND and ACV/GCV than in those treated with ACV or GCV only. No toxicity of PND was seen in mice even at a dose 10-fold higher than that used in the in vivo experiments. These novel strategies could provide benefit to ablative cancer gene therapy by making it feasible to use toxic GCV at lower doses and relatively nontoxic ACV.     

Death receptor-independent cytochrome c release and caspase activation mediate thymidine kinase plus ganciclovir-mediated cytotoxicity in LN-18 and LN-229 human malignant glioma cells

Suicide gene therapy using viral transfer of herpes simplex virus type I (HSV-1) thymidine kinase (TK) and subsequent ganciclovir (GCV) chemotherapy was the first approach used in clinical trials of somatic gene therapy for glioblastoma. The molecular pathways mediating TK/GCV-induced cell death remain to be elucidated. Here, we report that adenoviral (Ad)-TK/GCV-induced death is p53-independent and does not involve altered CD95 or CD95L expression. Ectopic expression of the preferential caspase 8 inhibitor, crm-A, inhibits Ad-CD95L-induced cell death but has no effect on TK/GCV cytotoxicity. LN-18 glioma cells selected for resistance to death receptor-mediated cell death do not acquire cross-resistance to TK/GCV. TK/GCV triggers mitochondrial cytochrome c release and activation of caspases 3, 7, 8 and 9 in a death receptor-independent manner. These events are associated with the loss of BCL-X(L). Forced expression of a BCL-X(L) transgene, or co-exposure to a pseudosubstrate caspase inhibitor, zVAD-fmk, inhibit TK/GCV cytotoxicity. Double-transfected cell lines expressing crm-A and enhanced green fluorescent protein (eGFP) show that the bystander effect in vitro is also death receptor- and caspase 8-independent. TK/GCV therapy does not kill glioma cells in synergy with cancer chemotherapy drugs, including lomustine, temozolomide and topotecan. In contrast, there is strong synergy of TK/GCV and CD95L. Thus, TK/GCV-induced cell death involves a mitochondria-dependent loop of caspase acvtivation that can be synergistically enhanced by death receptor agonists such as CD95L. TK/GCV-mediated sensitization of glioma cells to CD95L expressed on immune effector cells or parenchymal brain cells might account for the immune system's and bystander effects of TK/GCV therapy observed in rodent glioma models in vivo.     

自杀基因抑制K562细胞成瘤的动物实验研究

目的探讨自杀基因胞嘧啶脱氨酶（CD）和胸苷激酶（TK）在小鼠体内对K562细胞的抑制作用。方法将在慢病毒介导下已转染CDglyTK的K562细胞种植于裸鼠皮下,观察其成瘤情况和对前体药物[环氧鸟苷（GCV）、5-氟胞嘧啶（5-FC）3的敏感性。结果种植K562细胞和K562／CDglyTK细胞的裸鼠分别在（7．0土1．2）和（7．1土0．9）d后长出肉眼可见的瘤块。两组裸鼠生存期分别是（52．2土5．3）和（54．2±3．7）d,差异无统计学意义（P〉O．05）;接种K562／CDglyTK细胞的小鼠经GCV和5-FC处理后,其裸鼠分别在（26．9土I．7）、（25．7土I．9）d后肉眼可见肿瘤生长,对照组接种K562细胞后（6．9±I．7）d生长出肉眼可见肿瘤,两者差异有统计学意义（P〈0．01）。结论自杀基因在体内对K562细胞有明显的抑制作用,能增强前体药物GCV和5-FC对肿瘤细胞的药物作用。     

In vitro and in vivo enhancement of ganciclovir-mediated bystander cytotoxicity with gemcitabine.

To improve bystander cell killing with HSV-TK/GCV, we have utilized dFdCyd to reduce endogenous dGTP, which competes with GCVTP for incorporation into DNA. In this study we demonstrate the ability of dFdCyd to enhance GCV-mediated bystander cytotoxicity in cultured SW620 human colon carcinoma cells as well as in a murine xenograft model. In vitro, dFdCyd reduced cellular dGTP levels and produced a fourfold increase in the GCVTP:dGTP ratio. This elevated GCVTP:dGTP ratio resulted in a twofold increase in GCVMP incorporation into DNA in bystander cells cocultured with HSV-TK-expressing cells. The combination of GCV and dFdCyd was determined to be synergistic by isobologram analysis of bystander cytotoxicity. Tumors in mice treated with GCV and dFdCyd exhibited a significant growth delay requiring 40 days to obtain approximately 10 times their initial size compared to tumors in PBS- or single-drug-treated animals, which grew rapidly, increasing to a similar size in just 19 to 24 days. In addition, complete tumor regression was observed only in animals treated with both drugs. Furthermore, dFdCyd alone or in combination with GCV produced no evidence of toxicity or significant weight loss. These data suggest that dFdCyd may improve the clinical efficacy of HSV-TK/GCV gene therapies.