Tet-On调节的自杀基因对人乳腺癌细胞的体外杀伤和旁观者效应研究

目的探讨Tet调控下的自杀基因HSVtk对人乳腺癌细胞的杀伤作用和旁观者效应。方法构建重组逆转录病毒载体pRevTRE／HSVtk，通过磷酸钙共沉淀法，分别将pRevTRE／HSVtk和pRevTet-On导入乳腺癌细胞株MCF-7。以RT—PCR法检测不同Dox浓度下转染乳腺癌细胞中HSVtk基因的表达，MTT法检测细胞生长抑制率，通过检测混合细胞生长克隆来评价Tet调控下的自杀基因的旁观者效应和对乳腺癌细胞的杀伤作用。结果建立了一株稳定的受多西环素（Doxcycline，Dox）诱导、表达HSVtk基因的乳腺癌细胞株MCF／TRE／tk／Tet—On。当Ganciclovir（GCV）及Dox浓度增加时，细胞存活率下降。MCF／TRE／tk／Tet—On在总细胞中的比例〉10％时，有明显的旁观者效应。结论在Tet—On调控下，导入乳腺癌细胞中的HSVtk基因产物能将药物前体GCV转变为毒性代谢物质，并诱发旁观者效应进一步杀灭乳腺癌细胞。     

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。     

HSVtk/GCV系统体外杀伤大肠癌LoVo细胞的实验研究

目的观察单纯疱疹病毒胸腺嘧啶核苷激酶/丙氧鸟苷(HSVtk+/GCV)系统对大肠癌LoVo细胞的体外杀伤作用,并研究其作用机理.方法用逆转录病毒载体介导HSVtk基因转染LoVo细胞获得阳性克隆HSVtk+/LoVo细胞,观察细胞的体外生长情况及GGV对HSVtk+/LoVo细胞的杀伤作用.经光镜、电镜观察细胞的死亡情况.结果 HSVtk+/LoVo细胞体外生长良好.GCV对HSVtk+/LoVo细胞随着浓度和作用时间的增加而增大,通过光镜、电镜观察到细胞的凋亡.结论 HSVtk+/GCV系统体外对大肠癌LoVo细胞有强大的杀伤作用,其作用机理与细胞调亡有关.     

经尿道灌注自杀基因治疗大鼠膀胱癌的实验研究

目的:探讨经尿道膀胱灌注脂质体和逆转录病毒载体介导的自杀基因(HSVtk),观察在羟基无环鸟苷(ganciclovir,GCV)作用下大鼠膀胱肿瘤的生长情况.方法:用N-甲基-亚硝基脲(MNU)膀胱灌注建立大鼠原位膀胱癌模型.经尿道膀胱灌注脂质体、逆转录病毒载体HSVtk基因,随后腹腔内注射GCV,连续6天.结果:经尿道膀胱灌注HSVtk基因后48h,RT-PCR检测出膀胱肿瘤中HSVtk基因表达.逆转录病毒组和脂质体组膀胱总重量与裸质粒组和对照组相比有显著差异,表明膀胱肿瘤的生长被抑制.TUNEL法原位检测肿瘤组织中凋亡细胞不仅出现在表层细胞,而且深层细胞大量凋亡,推测旁观者效应可能发挥了一定作用.结论:经尿道膀胱灌注脂质体DNA或重组逆转录病毒载体可以向肿瘤转导HSVtk基因.HSVtk/GCV系统能抑制大鼠原位膀胱肿瘤的生长,显示出抗肿瘤作用.     

缺氧对HRE-TK/GCV系统杀伤骨肉瘤细胞的增强作用

目的:探讨缺氧条件下,缺氧反应元件启动子-胸苷激酶/丙氧鸟苷(HRE-TK/GCV)系统对骨肉瘤细胞系MG63靶向性杀伤作用.方法:构建由HRE启动子驱动的含潮霉素磷酸转移酶-单纯疱疹病毒胸腺嘧啶激酶(Hygromycin phosphotransferase-thymidine kinase,HyTK)融合基因的真核表达质粒pBI-HRE-HyTK,并将其转染入骨肉瘤细胞系MG63.应用PCR和RT-PCR方法检测TK基因的整合及表达;用MTT法和混合共培养实验分别检测转基因细胞在缺氧或不缺氧状态下对GCV的敏感性以及HRE-TK/GCV系统的"旁观者效应";流式细胞仪检测细胞周期改变及电镜观察细胞超微结构变化探讨其作用机制.结果:成功构建了真核表达质粒pBI-HRE-HyTK,得到转基因细胞系MG63TK.检测到MG63TK细胞内TK基因的DNA和mRNA.MG63TK细胞在不缺氧状态下对GCV不敏感,当GCV浓度达50μg/ml时,仅30%左右细胞被杀死;而缺氧状态下,GCV浓度1μg/ml时,50%左右细胞被杀死,GCV浓度50μg/ml时,90%以上细胞被杀死.混合共培养实验中,缺氧状态下,HRE-TK/GCV系统旁观者效应明显增强,MG63细胞存活率显著低于不缺氧状态下.缺氧和GCV共同作用使转基因细胞DNA合成抑制,细胞周期阻滞于G0G1期,细胞发生凋亡和坏死.结论:缺氧可以增强HRE-TK/GCV系统对体外培养骨肉瘤细胞系选择性的杀伤作用和旁观者效应,该系统为骨肉瘤靶向性基因治疗提供了新的有效途径.     

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。     

细胞之间介质传导的pCEAcd-tk/前药体系的旁观者效应

目的 :探讨融合自杀基因 pCEAcd tk/前药体系的旁观者效应的机理。 方法 :质粒 pCEAcd tk提取后 ,脂质体介导的方法转染到SPCA 1细胞中 ,与未转染的SPCA 1细胞混合 (2∶8)后接种到 96孔培养板中 (每孔 3× 10 3 ) ,加前体药物 5 氟胞嘧啶 [5 Fluorocytesine ,5 FC(5 μmol/L) ]和 [丙氧鸟苷 (Ganciclovir ,GCV(10 μmol/L) ],培养 5天 ,MTT法测定细胞存活率而观察旁观者效应。利用细胞种植密度的稀密和取转导了 pCEAcd tk融合基因的SPCA 1细胞 (处理细胞 )加前体药物的培养液和细胞裂解液对未转染细胞SPCA 1的毒性探讨旁观者效应的传递方式。结果 :融合基因pCEAcd tk/前药体系的旁观者效应在细胞密度较低时更明显 ,处理细胞的细胞培养液具有细胞毒性。结论 :融合自杀基因 pCEAcd tk/前药体系的旁观者效应的传递方式之一是通过细胞之间的某些介质传递。     

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。     

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.     

VP22的细胞间传递作用对HSV-tk/GCV的肿瘤细胞杀伤促进效应

目的：探讨VP22的细胞间传递作用对HSV－tk/GCV杀伤肿瘤的促进作用。方法：将VP22与报告基因－绿色荧光蛋白基因（GFP）或前药酶基因－单纯疱疹病毒胸苷激酶基因（HSV－tk）构建在同一载体上,进行DNA序列分析鉴定;将此融合工体转染293T或COS7细胞,免疫荧光分析确定转染情况和细胞传递作用;在前药GCV不同浓度、转染与未转染细胞比例不同时,MTT法检测细胞增殖情况;利用转染细胞增养液上清培养未转染细胞,确定旁观者效应是否由培养液转移。结果：PCR及序列分析显示融合基因插入载体正确;对293T或COS7细胞转染率可达25％－305,且证明融合蛋白已被表达并在细胞传递;HSV－tk与VP22融合后在前药GCV浓度低至0．1μg/ml时就可显示出细胞杀伤效应增强,并随着表达VP22－HSV－tk细胞比例的增加,细胞杀伤作用增强。结论：VP22介导的自杀基因产物的细胞间传递作用可解决自自杀基因的低效细胞毒作用,明显增强细胞杀伤效应。     

Efficacy of the bystander effect in the herpes simplex virus thymidine kinase-mediated gene therapy is influenced by the expression of connexin43 in the target cells

Tumoricidal "bystander effect" observed in the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy was studied between different rat glioma cell lines (9L and C6 cells) under both in vitro and in vivo conditions. For that purpose, mixed populations of wild-type cells (9Lwt and C6wt) and respective HSVtk gene-transduced cells (9Ltk and C6tk) were examined for their sensitivity to GCV. A potent in vitro bystander effect was observed in 9Lwt/9Ltk and 9Lwt/C6tk combinations but not in C6wt/9Ltk and C6wt/C6tk combinations. In vivo bystander effect studied in a subcutaneous tumor model in athymic nude mice was also potent in 9Lwt/9Ltk and 9Lwt/C6tk combinations. Because the expression of connexin43, a major protein in the connexin family gene products, in 9L cells is much higher than that in C6 cells, the results suggest that the amount of connexin in target (wild-type) cells but not in effector (HSVtk gene-bearing) cells is important for the generation of the bystander effect. This hypothesis was further confirmed by the observation that in vitro bystander effect in C6wt/C6tk combination was potentiated by transduction of the connexin43 gene to the target cells.     

可调控自杀基因的SCID小鼠乳腺癌基因治疗的研究

目的：探讨经强力霉素（Dox)诱导后，丙氧鸟苷（GCV）对SCID小鼠乳腺癌的调控性治疗作用。方法：重组逆转录病毒载体pRevTRE/HSVtk与pRevTet-On共转染乳腺癌细胞MCF－7，接种SCID小鼠成瘤后，腹腔内分别注射生理盐水（NS），GCV及Dox GCV治疗15d。观测肿瘤体积和组织病理改变及用RT－PCR分析肿瘤组织有无HSVtk的表达。结果：乳腺癌SCID小鼠Dox GCV治疗15d后，肿瘤体积明显减小，生长受抑，组间比较有显著性差异（P＜0．05）；HE染色发现治疗组肿瘤有局部坏死，炎性细胞浸润。RT－PCR结果显示Dox诱导后肿瘤组织HSVtk表达较明显。结论：在Dox的诱导作用下，GCV对可调控性自杀基因乳腺癌SCID小鼠有显著治疗作用。     

Cytotoxicity of HSVtk and hrTNF-alpha fusion genes with IRES in treatment of gastric cancer

The efficacy of the suicide gene therapy by using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system for the treatment of cancer is limited because of the insufficient gene transfer and the low killing activity. To enhance the antitumor activity, we probed into whether recombinant ritroviral expression vector PLXSN expressing both HSVtk and TNF-alpha genes could potentiate the destruction of SGC7901. The PL(tk-TNF-alpha)SN harboring HSVtk and TNF-alpha genes in sequence was constructed with a bicistronic unit including the internal ribosomal entry site, the recombinant retroviruses were transferred into SGC7901 cells by lipofectamine, and pEGFP and Western blot analysis were used to detect the expression of fusion genes in transfected SGC7901 cells, and then apoptosis of the transfected cells were detected by using the TdT-mediated dUTP nick end labeling, flow cytometric analysis and transmission electron microscopy. In vitro study, the transfected gastric cancer cells were maintained in the GCV-contained medium, to assay the cell killing effect and bystander effect. In vivo experiments, retroviral serum plasmids were transfected into tumor-bearing nude mice, to observe the changes of tumor volumes and survival of the mice. In vitro there was no significant difference of cell survival rate between the three groups. However, in vivo results showed that tk/GCV, tk-TNF-alpha/GCV and TNF-alpha could inhibit the tumor growth, and the obvious anti-tumor effect was shown in tk-TNF-alpha/GCV group, and TNF-alpha obviously enhanced the anti-tumor effect in vivo. The pathologic examination showed necrosis of the cancer in the treated groups.     

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.     

Cytotoxicity of HSVtk and hrTNF-alpha fusion genes with IRES in treatment of gastric cancer

The efficacy of the suicide gene therapy by using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system for the treatment of cancer is limited because of the insufficient gene transfer and the low killing activity. To enhance the anti-tumor activity, we probed into whether recombinant retroviral expression vector PLXSN expressing both HSVtk and TNF-alpha genes could potentiate the destruction of SGC7901. The pL(tk-TNF-alpha)SN harboring HSVtk and TNF-alpha genes in sequence was constructed with a bicistronic unit including the internal ribosomal entry site, the recombinant retroviruses were transferred into SGC7901 cells by lipofectamine, and pEGFP and Western blot analysis were used to detect the expression of fusion genes in transfected SGC7901 cells, and then apoptosis of the transfected cells were detected by using the TdT-mediated dUTP nick end labeling, flow cytometric analysis and transmission electron microscopy. In vitro study, the transfected gastric cancer cells were maintained in the GCV-contained medium, to assay the cell killing effect and bystander effect. In vivo experiments, retroviral serum plasmids were transfected into tumor-bearing nude mice, to observe the changes of tumor volumes and survival of the mice. In vitro there was no significant difference of cell survival rate between the three groups. However, in vivo results showed that tk/GCV, tk-TNF-alpha/GCV and TNF-alpha could inhibit the tumor growth, and the obvious anti-tumor effect was shown in tk-TNF-alpha/GCV group, and TNF-alpha obviously enhanced the anti-tumor effect in vivo. The pathologic examination showed necrosis of the cancer in the treated groups.     

Treatment of rat experimental brain tumors by herpes simplex virus thymidine kinase gene-transduced allogeneic tumor cells and ganciclovir

Transfer of the herpes simplex virus thymidine kinase (HSVtk) gene, followed by administration of ganciclovir (GCV), generates the "bystander effect," in which HSVtk-negative wild-type cells are killed by GCV, as are HSVtk-expressing cells. Our previous study demonstrated that intracranial 9L gliomas could be efficiently treated due to this bystander effect by injecting the 9L glioma cells transduced with the HSVtk gene in the vicinity of the preimplanted wild-type 9L glioma and then administering GCV. For a possible clinical application of the bystander effect-mediated cell killing, we tested HSVtkgene-transduced allogeneic C6 glioma cells (C6tk) instead of syngeneic 9L glioma cells transduced with the HSVtk gene. Fisher rats were implanted intracranially with wild-type 9L glioma cells, subsequently injected with C6tk cells at the same brain coordinate, and thereafter treated with GCV or saline. When the rats were treated with GCV, a significant retardation of tumor growth was observed by serial magnetic resonance imaging, although this growth retardation was less prominent than that observed with 9L glioma cells transduced with the HSVtk gene; consequently, survival was prolonged (P < .01). Tumors that received C6tk cells contained almost no HSVtk-positive cells after treatment with GCV. Rejection of allogeneic tumor cells, although possibly incomplete in the brain, can also contribute to the safety of this therapeutic strategy.     

The role of cellular- and prodrug-associated factors in the bystander effect induced by the Varicella zoster and Herpes simplex viral thymidine kinases in suicide gene therapy

To investigate the factors influencing the bystander effect--a key element in the efficacy of suicide gene therapy against cancer--we compared the effect triggered by four extremely efficient gene/prodrug combinations, i.e., VZVtk/BVDU, the thymidine kinase of Varicella zoster virus associated with (E)-5-(2-bromovinyl)-2'-deoxyuridine; VZVtk/BVaraU, the same enzyme associated with (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil; HSVtk/BVDU, the association of the Herpes simplex virus thymidine kinase with BVDU; and the classical HSVtk/GCV (ganciclovir) paradigm. The cells used, the human MDA-MB-435 breast cancer, and the rat 9L glioblastoma lines were equally sensitive in vitro to these four associations. In both cell types, the combinations involving pyrimidine analogues (BVDU, BVaraU) displayed a smaller bystander killing than the combination involving the purine analogue (GCV). In addition, the bystander effect induced by all the tk/prodrug systems was reduced in MDA-MB-435 cells in comparison to 9L cells; albeit, the viral kinases were produced at a higher level in the breast cancer cells. All systems induced apoptotic death in the two cell types, but the MDA-MB-435 cells, deprived of connexin 43, were noncommunicating in striking contrast with the 9L cells. That functional gap junctions have to be increased in order to improve the breast cancer cell response to suicide gene therapy was demonstrated by transducing the Cx43 gene: this modification enhanced the bystander effect associated in vitro with GCV treatment and, by itself, decreased the tumorigenicity of the untreated cells. However, the noncommunicating MDA-MB-435 cells triggered a significant bystander effect both in vitro and in vivo with the HSVtk/GCV system, showing that communication through gap junctions is not the only mechanism involved.     

Comparative in vitro and in vivo cytotoxic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and its arabinosyl derivative, (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), against tumor cells expressing either the Varicella zoster or the Herpes simplex virus thymidine kinase

The inhibitory effects of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and its arabinosyl derivative (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU) on the growth of both MDA-MB-435 human breast carcinoma and 9L rat gliosarcoma cells expressing the thymidine kinase (tk)-encoding gene of the Varicella zoster virus (VZV) or the Herpes simplex virus (HSV) were evaluated. In vitro, BVDU and BVaraU effectively killed both cell types expressing VZVtk, with 50% inhibitory concentration values ranging from 0.06 to 0.4 microM, whereas ganciclovir (GCV) lacked activity. On HSVtk+ cells, BVDU had high cytotoxic activity, with 50% inhibitory concentration values that were similar to those of GCV, whereas BVaraU was inactive. In vivo, BVDU applied intraperitoneally caused a 50% tumor growth inhibition in nude mice inoculated subcutaneously with VZVtk+ as well as HSVtk+ mammary tumor cells. In mice and at variance with the in vitro results, BVaraU had very little activity against the VZVtk+ mammary cells; GCV had the highest activity on the HSVtk+ cells, resulting in a 50% eradication of the tumors. With the 9L rat gliosarcoma model, the VZVtk/BVDU system completely failed to inhibit the development of VZVtk+ glioma tumors induced subcutaneously in syngeneic rats, although BVDU had a similar 45-minute half-life in both rats and mice. Factors other than degradation of the prodrug and related to the mode of action of these analogs are possibly involved in the observed discrepancies between the in vitro and in vivo results.     

Bystander-mediated regression of osteosarcoma via retroviral transfer of the herpes simplex virus thymidine kinase and human interleukin-2 genes

Current treatment of osteosarcoma produces disappointing outcomes, and innovative therapies must be investigated. We have used retroviral vectors to transfer the herpes simplex virus thymidine kinase (HSVtk) and interleukin-2 genes to human osteosarcoma cells. Each gene was stably transduced and expressed; the HSVtk gene effectively conferred ganciclovir (GCV) susceptibility to transduced cells. A strong bystander effect was observed in vitro, whereby nontransduced tumor cells in proximity to transduced cells acquired susceptibility to GCV killing. Human osteosarcoma cells were used to develop a series of experiments in athymic nude mice to treat experimental osteosarcoma. Subcutaneously implanted mixtures of tumor cells and HSVtk vector producer cells developed into tumors that completely regressed upon administration of GCV. Subcutaneously implanted mixtures of transduced and wild-type cells showed a potent bystander effect upon administration of GCV, with complete tumor ablation when as little as 10% of the cells were HSVtk+. A significant (P < .05) antitumoral response was seen against primary tumors composed of unmodified cells when a secondary tumor of transduced cells was implanted at a distance of 1 cm, suggesting a diffusible bystander factor. The presence of interleukin-2-transduced cells improved the efficacy of treatment. A significant (P < .03) antitumoral response was seen in the treatment of established osteosarcomas by the injection of HSVtk vector producer cells.