细菌内同源重组法制备CD腺病毒及其体外杀瘤作用的初步研究

目的观察应用重组IFN-α2a能否增强单纯疱疹病毒胸腺嘧啶核苷激酶基因（HSV-tk）/丙氧鸟苷（GCV）系统对卵巢癌细胞系SKOV3的杀伤效应。方法利用逆转录病毒载体将潮霉素磷酸转移酶和HSV-tk的融合基因（hytk）导入SKOV3细胞,测定GCV对SKOV3/hytk细胞的杀伤作用和旁观者效应。观察IFN-α2a与HSV-tk/GCV协同作用对旁观者效应的影响,并且在联合应用GCV和IFN-α2a后,进行细胞周期分析。结果GCV对SKOV3/hytk细胞有明显的杀伤作用,并且可以同时杀伤周围未转基因的细胞,联合应用IFN-α2a与HSV-tk/GCV可以明显增强旁观者效应（P<0.05）,使处于S期的SKOV3/hytk细胞较对照组和使用单药组明显增多（P<0.01）。结论IFN-α2a与HSV-tk/GCV联合应用可以产生协同作用,增强HSV-tk/GCV系统在体外对卵巢癌细胞系SKOV3的杀伤效应,具有良好的应用前景。     

转染连接蛋白基因对人脑胶质瘤细胞间隙连接通讯及其生长变化的研究

目的 研究连接蛋白（Cx)基因对人脑胶质瘤细胞的细胞间隙连接通讯（GJIC）及其增殖的抑制作用,探索以Cx43基因治疗胶质瘤的可行性。方法 将含Cx43cDNA的质粒,以脂质体介导转染Cx43表达缺失的TJ905人胶质母细胞瘤细胞,通过Northern印染杂交、原位杂交及免疫组化染色检测Cx43mRNA及蛋白表达,划痕标记荧光染料示踪技术（SLDT）检测GJIC,MTT法测定细胞增殖率,核仁组成区嗜银蛋白（AgNOR)染色检测细胞增殖活性,TUNEL法检测细胞凋亡。结果 转染后TJ905细胞有不同程度的Cx43mRNA和蛋白表达及GJIC恢复。Cx43表达水平高的克隆细胞增殖明显下降,细胞凋亡并未增加。结论 Cx43基因及GJIC在恶性胶质瘤的发生发展过程中起重要作用,可能成为恶性胶质瘤基因治疗的优选靶的之一。     

Cx43基因转染胶质瘤细胞抑制细胞增殖与下调若干重要生长因子

目的：研究连接蛋白43(Cx43)基因对胶质瘤细胞增殖的抑制作用及其可能的机制。方法：以脂质体介导，将Cx43cDNA转染于内源性Cx43表达缺失的鼠C6胶质瘤细胞和人TJ905胶质母细胞瘤细胞。采用MTT法及AgNORs(核仁组成区嗜银蛋白)平均数检测细胞增殖、。TUNEL法检测细胞凋亡、划痕标记染料示踪技术检测细胞间隙连接通讯(GJIC)。应用Western蛋白印迹及免疫组化法检测CX43cDNA转染细胞前后若干与胶质瘤发生密切相关的重要生长因子及受体表达，其中包括IGF1、bFGF、PDGF、IGFBP3、EGFR。结果：胶质瘤细胞转染Cx43cDNA后细胞增殖被抑制，细胞凋亡并未增加；GJIC明显恢复。BFGF、PDGF，IGF1、IGFBP3表达显著下调，但EGFR表达无变化。结论：Cx43基因对胶质瘤细胞增殖的抑制作用与GJIC恢复以及bFGF，PDGF，IGF1，IGFBP3表达下调相关，CX43可望成为胶质瘤基因治疗的靶基因。     

腺病毒介导的肿瘤血管靶向的体外自杀基因治疗研究

本工作在血管内皮细胞中分别建立了HSV-tk/GCV和EC-cd/5-FC自杀基因系统,用于抑制实体肿瘤中血管生成的研究.构建含自杀基因的重组腺病毒AdCMVtk和AdCMVcd,滴度均1×10~(12)pfu/ml.病毒感染小鼠血管内皮细胞lGll(MOI=100),并鉴定自杀基因的表达,测定原药对它们的杀伤.生长抑制试验表明,GCV对lGll/Ad-tk细胞杀伤的IG_(50)为0.2μmol/ L,5-FC对lGll/Ad-cd细胞杀伤的IC_(50)为20μmol/L.电镜下显示被原药杀伤的血管内皮细胞有明显的细胞凋亡现象.同时混合细胞实验证明两系统均存在明显的旁杀伤效应.从而在体外水平证明了HSV-tk/GCV和EC-cd/5-FC自杀基因系统杀伤增生血管内皮细胞的有效性.     

丹参酮Ⅱ A增强HSV-tk/GCV旁观者效应及其与Cx43 mRNA表达的关系

目的　研究丹参酮ⅡA(TanshinoneⅡA ,Tan)对HSV tk/GCV旁观者效应的增强作用及其与间隙连接蛋白Cx4 3转录表达的关系。方法　应用polybrene转染、荧光定量RT PCR等技术 ,观察Tan对宫颈癌细胞ME180 (ME)、ME/TK转化细胞旁观者效应及诱导Cx4 3mRNA表达的作用。结果　在HSV tk/GCV系统中 ,Tan显著地提高了ME/TK细胞对GCV的敏感性。在 2 μg/mlGCV作用下 ,以1.3× 10 9mol/LTan与不加Tan的作用相比较 ,在ME与ME/TK不同比例混合细胞的各组中 ,细胞的存活率明显降低 ,差异均有显著性 (P <0 .0 5 )。并观察到Tan对GCV旁观者效应的增强作用 ,在一定范围内为 1.3× 10 -8mol/L和 1.3× 10 -9mol/L。RT PCR结果表明 ,经 1.3× 10 -8mol/L和 1.3× 10 -9mol/LTan处理的ME细胞 ,其Cx4 3mRNA的相对拷贝数比值增高约 8.83倍及 8.4 7倍。结论　在宫颈癌ME180细胞中 ,Tan在 1.3× 10 -8mol/L和 1.3× 10 -9mol/L范围内具有明显增强HSV tk/GCV旁观者效应的作用。Tan在转录水平诱导Cx4 3mRNA表达上调 ,与旁观者效应的增强作用密切相关。     

HSV-tk/GCV基因治疗在卵巢癌中的研究进展

卵巢癌自杀基因治疗多采用HSV-tk/GCV系统.该系统的旁观者效应可在一定程度上弥补基因转染率低的缺陷,通过缝隙连接胞间通信(GJIC)增强剂可提高该系统的旁观者效应,通过选择构建高效能载体系统、应用靶向性调控机制或与其他方法联合治疗可增强疗效.该系统治疗卵巢癌具有广阔的发展前景.     

HSV-tk／GCV基因治疗在卵巢癌中的研究进展

卵巢癌自杀基因治疗多采用HSV-tk／GCV系统。该系统的旁观者效应可在一定程度上弥补基因转染率低的缺陷，通过缝隙连接胞间通信(GJIC)增强剂可提高该系统的旁观者效应，通过选择构建高效能载体系统、应用靶向性调控机制或与其他方法联合治疗可增强疗效。该系统治疗卵巢癌具有广阔的发展前景。     

全反式维甲酸联合TK基因治疗对骨肉瘤旁观者效应的研究

目的观察全反式维甲酸（ATRA）联合脂质体介导的自杀基因／丙氧鸟苷（TK／GCV）系统对人骨肉瘤U-2细胞旁观者效应的影响。方法重组质粒plRES—EGFP—TK培养和转染人骨肉瘤U-2细胞,荧光倒置显微镜和流式细胞仪观察转染后的细胞生长状态,MTT法检测plRES—EGFP．TK／GCV系统对人骨肉瘤U-2细胞的杀伤力。ELISA检测细胞培养液中连接蛋白（CX）43的表达情况。结果pl—RES—EGFP—TK／GCV系统对人骨肉瘤U-2细胞旁观者效应在TK^＋：TK-细胞比例为5：5时明显出现（P〈0．05）,而联合ATRA后,在TK^＋：TK-细胞比例为3：7时明显出现（P〈0．05）,且在细胞生存率均在50％时,TK^＋细胞比例下降20％。ATRA（10μmol／L）处理人骨肉瘤U-2细胞24h、48h、72h后,CX43蛋白的浓度分别为（0．559±0．018）ng／mL、（0．654±0．023）ng／mL、（0．826．-t-O．065）ng／mL,均P〈0．01。结论全反式维甲酸可能通过增强CX43的表达,提高plRES—EGFP—TK／GCV系统对人骨肉瘤U-2细胞的杀伤效应。     

尼莫地平增强HyTK/ACV自杀基因对脑胶质瘤细胞的杀伤作用

目的 :探讨尼莫地平增强阿昔洛韦 (ACV)介导的转HyTK基因治疗诱导脑胶质瘤细胞的死亡方式及旁观者效应机制。方法 :构建含HyTK基因的逆转录病毒载体LNSHyTK ,并转染人脑胶质瘤BT32 5和SCW细胞。单用ACV和联合尼莫地平应用体外试验观察对BT32 5 tk和SCW tk的杀伤效果。同时应用转基因前和转基因后瘤细胞共培养观察其旁观者效应。结果 :低浓度尼莫地平联合ACV作用下转tk基因细胞的凋亡率明显高于单独使用ACV ,并明显提高旁观者效应。结论 :尼莫地平能增强HyTK ACV自杀基因对脑胶质瘤细胞的杀伤作用 ,为钙拮抗剂应用于转tk基因治疗提供了实验依据。     

尼莫地平增强HyTK／ACV自杀基因对脑胶质瘤细胞的杀伤作用

目的：探讨尼莫地平增强阿昔洛韦(ACV)介导的转HyTK基因治疗诱导脑胶质瘤细胞的死亡方式及旁观者效应机制。方法：构建含HyTK基因的逆转录病毒载体LNSHyTK，并转染人脑胶质瘤BT325和SCW细胞。单用ACV和联合尼莫地平应用体外试验观察对BT325／tk和SCW／tk的杀伤效果。同时应用转基因前和转基因后瘤细胞共培养观察其旁观者效应。结果：低浓度尼莫地平联合ACV作用下转tk基因细胞的凋亡率明显高于单独使用ACV，并明显提高旁观者效应。结论：尼莫地平能增强HyTK／ACV自杀基因对脑胶质瘤细胞的杀伤作用，为钙拮抗剂应用于转tk基因治疗提供了实验依据。     

Retrovirus-mediated transfer of the herpes simplex virus thymidine kinase and connexin26 genes in pancreatic cells results in variable efficiency on the bystander killing: implications for gene therapy.

Currently, there is no effective treatment for pancreatic cancer and prodrug-activating gene therapy with the herpes simplex virus thymidine kinase gene (HSV-tk) in combination with ganciclovir (GCV) has been suggested as a candidate approach against this disease. In the present study, we have evaluated the efficacy of the HSV-tk/GCV treatment in a panel of pancreatic tumor cells (NP-9, NP-18, NP-31) and the potentiation of the cytotoxic effect in combination with the overexpression of the connexin 26 gene (Cx26). Pancreatic cells transduced with a retrovirus containing the HSV-tk gene showed different sensitivities to GCV that seemed to be independent of HSV-tk expression levels. The extent of the bystander effect also varied among the pancreatic tumor cells and correlated with the level of gap junction intercellular communication (GJIC). Transduction of the pancreatic tumor cells with a retrovirus carrying the connexin 26 gene resulted in high levels of connexin 26 expression and in an increase in the GJIC that correlated to an extent in the bystander effect in both NP-9Cx26 and NP-18Cx26 cells. Neither an increment in GJIC nor an increase in the bystander killing was detected in NP-31Cx26. The bystander effect in NP-18 Cx26 cells was also prevented by the long term inhibitor of GJIC, 18-alpha-glycyrrhetinic acid (AGA). Together, these results demonstrate that pancreatic tumor cells are highly different as regards the susceptibility to HSV-tk/GCV treatment. Moreover, they indicate that overexpression of the Cx26 gene does not always correspond to an increase in GJIC although they clearly suggest the role of GJIC in mediating the bystander effect.     

Stimulation of intercellular communication of poor-communicating cells by gap-junction-competent cells enhances the HSV-TK/GCV bystander effect in vitro

We have previously shown that gap-junctional intercellular communication (GJIC) appears to play a role in the bystander effect that is observed in anticancer suicide gene therapy mediated by herpes simplex virus (HSV) thymidine kinase (tk) and ganciclovir (GCV). We now report that when connexin-expressing (Cx+) cells are present within a noncommunicating population of cells (Cx-), there is GJIC between the Cx+ and Cx- cells and that due to this stimulation of GJIC, the bystander effect also occurs when the 2 cell types are mixed. We transfected HeLa cells, which do not express any detectable level of connexin, with Cx43. The Cx+ and Cx- HeLa cells were further transfected with the tk gene, giving 4 phenotypes: Cx+tk-, Cx+tk+, Cx-tk+ and Cx-tk-. We observed GJIC between Cx+ and Cx- cells, but not between Cx- and Cx- cells, regardless of the tk genotype. Similarly, we observed the HSV-tk/GCV bystander effect in Cx+tk-/Cx-tk+ and Cx+tk+/Cx-tk- cocultures. The extent of the bystander effect in cocultures of Cx+tk- and Cx-tk+ cells was stronger than in cocultures of Cx+tk+ and Cx-tk- cells when each mixture had the same ratio of Cx+ and tk+ cells. These results suggest that Cx-expressing HeLa cells stimulate GJIC capacity between them and non-Cx-expressing HeLa cells, which mediates the bystander effect in mixtures of Cx+ cells and Cx- cells in vitro. Thus, Cx expression even in only a limited fraction of tumor cells may enhance the efficacy of the HSV-tk/GCV strategy by inducing a bystander effect.     

Bystander effect in suicide gene therapy is directly proportional to the degree of gap junctional intercellular communication in esophageal cancer

Gap junctional intercellular communication (GJIC) has been shown to be involved in the bystander effect through herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) gene therapy. In this study, we examined the expression of connexins, the components of gap junction, and the degree of GJIC in esophageal cancer cell lines and compared the bystander effect in cells with different capacities of GJIC. We found loss in connexin 26 expression and reduced connexin 43 in esophageal cancer. GJIC capacity varied among cell lines and was dependent on the connexin 43 expression in the cell-cell contact areas. In mixing assay, the extent of the bystander effect was tightly correlated with the degree of GJIC capacity. The effects of retinoic acid and cAMP on the bystander effect were also investigated. Treatment with retinoic acid, but not with cAMP, was associated with augmented bystander killing by increase in GJIC in some esophageal cancer cell lines. Our results indicated that the degree of GJIC was predictive to identify a tumor as suitable for gene therapy with the HSV-tk/GCV system. Also GJIC chemically-enhanced with retinoic acid might be useful to improve response in suicide gene therapy.     

HSV—tk／GCV系统对神经胶质瘤的自杀基因治疗研究

目的在ｉｎｖｉｔｒｏ和ｉｎｖｉｖｏ水平建立致死神经胶质瘤的ＨＳＶ－ｔｋ／ＧＣＶ自杀基因系统,并验证该系统的有效性。方法用携带单纯疱疹病毒胸苷激酶（ＨＳＶ－ｔｋ）基因的重组逆转录病毒,感染大鼠神经胶质瘤细胞Ｃ６,筛选稳定表达ｔｋ的克隆细胞Ｃ６／ｔｋ;经生长抑制试验,比较Ｃ６／ｔｋ细胞对三种核苷类似物ＧＣＶ、ＢＶｄＵ和ＡＣＶ的敏感性;Ｃ６／ｔｋ细胞在裸鼠皮下成瘤,用ＧＣＶ进行治疗并观察疗效。结果ｔｋ基因整合入Ｃ６细胞并在Ｃ６／ｔｋ细胞中稳定表达;生长抑制试验表明,ＧＣＶ是最为有效的原药,Ｃ６／ｔｋ细胞对ＧＣＶ高度敏感,ＩＣ５０＜０．２μｍｏｌ／Ｌ,而野生型Ｃ６细胞和转染空载病毒载体的Ｃ６／０细胞对ＧＣＶ的ＩＣ５０≥１００μｍｏｌ／Ｌ,相差５００多倍。裸鼠ｉｎｖｉｖｏ实验得到相应结果,治疗组肿瘤受到明显抑制和杀伤。结论在ｉｎｖｉｔｒｏ和ｉｎｖｉｖｏ水平,表达ｔｋ基因的肿瘤细胞均被ＧＣＶ有效杀伤,这表明ＨＳＶ－ｔｋ／ＧＣＶ自杀基因系统有可能成为基因治疗脑瘤的有效方法。     

缝隙连接通讯抑制剂对Cx43cDNA转染后化疗旁观者效应的影响

背景与目的:有研究通过转染Cx43cDNA观察到C6细胞化疗过程中存在旁观者效应,并推测缝隙连接细胞间通讯可能为化疗旁观者效应机制之一,本研究旨在探讨脑胶质瘤化疗旁观者效应与缝隙连接细胞间通讯功能的关系。方法:(1)质粒扩增,提取纯化及酶切鉴定:氯化钙法将质粒转入JM109菌株扩增,提取纯化后酶切鉴定;(2)基因转染:LipofectAMINE2000介导C6细胞质粒DNA转染,G418筛选,半定量RT-PCR及划痕荷载染料传输实验(SLDT)鉴定。(3)将转染后细胞分为实验组和对照组:将VM-26处理细胞与VM-26未处理细胞按不同比例在无VM-26环境混合培养,培养液中加入缝隙连接细胞间通讯功能抑制剂18α-次甘草酸(AGA)为实验组,不加AGA为对照组,以MTT法及流式细胞仪技术观察AGA对Cx43cDNA转染后化疗旁观者效应的影响。结果:(1)获得转染了相应质粒的细胞株C6-Cx43及C6-Non;(2)C6-Cx43细胞Cx43mRNA表达显著增加,染料传输能力较C6-Non明显增强;(3)C6胶质细胞缝隙连接细胞间通讯功能低下;(4)当转染Cx43cDNA,明显上调C6细胞缝隙连接细胞间通讯功能后...     

A cell type-specific and gap junction-independent mechanism for the herpes simplex virus-1 thymidine kinase gene/ganciclovir-mediated bystander effect.

Tumor cells expressing the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene are killed by nucleoside analogues such as ganciclovir (GCV). GCV affects not only the cells expressing HSV-tk but also neighboring cells that do not express the gene; this phenomenon commonly is called "bystander effect." GCV metabolites transfer via gap junctional intercellular communication (GJIC) accounts for the bystander effect in different cell lines, but other mechanisms have also been described. In this study, we analyzed the mechanisms of the bystander effect in two cell lines exhibiting different capacities of communication (DHD/K12 and 9L). The 9L cells exhibited a very good bystander effect, which was completely blocked by a long-term inhibitor of GJIC, 18 alpha-glycyrrhetinic acid. DHD/K12 cells exhibited a moderate bystander effect that was not abolished by 18 alpha-glycyrrhetinic acid or 1-octanol, another strong inhibitor of GJIC. Interestingly, we also observed a bystander effect in cultures where HSV-tk-expressing DHD/K12 cells were physically separated from their untransfected counterparts but grown in the same medium. Moreover, the transfer of filtered conditioned medium from GCV-treated HSV-tk-expressing DHD/K12 cells to DHD/K12 parental cells induced a decrease of survival in a concentration-dependent manner, suggesting that the bystander effect in this cell line was mediated by a soluble factor.     

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.     

缝隙连接蛋白43对双自杀基因系统旁观者效应的影响

Objective： To observe the influence of connexin 43 （Cx43） on the bystander effect induced by cytosine deaminase （CD） and herpes simplex virus thymidine kinase （HSV-tk） coexpression suicide genes system in human cholangiocarcinoma QBC939 cells and transplantation tumors in nude mice. Methods： In vitro, the CD＋tk＋ and CD＋tk＋Cx＋ cells were respectively treated with 5-fluorocytosine （5-Fc） and Ganciclovir （GCV）. The cytotoxic effect was evaluated by MTT method. In order to investigate the influence of Cx43 on the bystander effect, the size of transplantation tumors of the CD＋tk＋ and CD＋tk＋Cx＋ cells was measured before and after application of 5-Fc and GCV. Results： CD and tk genes were stably expressed in transfected QBC939 cells. The increased expression of Cx43 was determined by testing for the presence of Cx43 mRNA by RT-PCR and the presence of Cx43 protein by Western Blot in CD＋tk＋Cx＋ cells. The killing effect of 5-Fc and GCV on CD＋tk＋Cx＋ cells was more effective than that on CD＋tk＋ cells both in vitro and in vivo. Conclusion： Double suicide genes system CD/5-Fc＋tk/GCV could induce remarkable killing effect on cholangiocarcinoma cells in vitro and transplantation tumors in vivo. The cotransfection of Cx43 gene could enhance the bystander effect and hence the inhibition of carcinoma cells.     

Bystander effect in glioblastoma cells with a predominant cytoplasmic localization of connexin43

Herpes simplex virus thymidine kinase (TK) gene transfer followed by ganciclovir (GCV) administration is an approach investigated for glioblastoma treatment. The bystander effect (BE) enhances the cytotoxic effect of this strategy by allowing the diffusion of phosphorylated GCV from TK-expressing cells toward neighboring TK negative cells. This transfer of toxic metabolites is mainly mediated via gap junctions that are composed of connexins. Downregulation and/or cytoplasmic localization of connexins are common in tumors, and should be detrimental to the success of the TK/GCV strategy. In this study, we investigated the level of expression, the localization and the functionality of connexin43 (Cx43) in three glioblastoma cell lines. We showed that Cx43 was predominantly located in lysosomes and late endosomes, with only few gap junctions present at the cell surface. Surprisingly, the gap-junctional intercellular communication (GJIC) and the BE capacity were preserved, and in two of the cell lines analyzed, it was at least twice as high as compared to a control HeLa transfectant that expresses high levels of Cx43 at the cell membrane. Experiments performed in the presence of alpha-glycyrrhetinic acid or small interfering RNA confirmed that Cx43 was responsible for the GJIC and the BE. Our results indicate for the first time that the very limited numbers of gap junctions present in glioblastoma cells are highly functional. We thus conclude that the TK/GCV strategy is still a valuable therapeutic option to be developed for the treatment of glioblastoma patients.