Zhang R, Straus F H, DeGroot L J
Department of Medicine, The University of Chicago, Illinois 60637, USA.
Thyroid. 2001 Feb;11(2):115-23. doi: 10.1089/105072501300042749.
A replication defective adenovirus transducing thymidine kinase (TK) gene under the control of the rat thyroglobulin (rTg) promoter (AdrTgtk) was developed to evaluate its cell-specific killing activity in gene therapy. We also developed adenoviruses containing the TK gene driven by the cytomegalovirus (CMV) promoter (AdCMVtk), and luciferase (Luc) gene driven by the rTg or CMV promoter (AdrTgLuc or AdCMVLuc). Luc activity in FRTL-5, HepG2, COS1, rMTC, hMTC, Hela, GH3, T98G, and CA77 cells was measured after infection with AdrTgLuc or AdCMVLuc. FRTL-5 cells produce thyroglobulin (Tg), whereas all other cells are non-Tg-producing cell lines. Transduction by AdCMVLuc caused high Luc activity in all cell lines. However, infection with AdrTgLuc induced Luc activity only in FRTL-5 cells. AdCMVtk or AdrTgtk was used to transduce various cell lines to evaluate the different killing effect. After infection with AdCMVtk vector followed by ganciclovir (GCV) treatment, cell growth was strongly suppressed in all cell lines compared both to noninfected cells and to cells infected by AdCMVLuc in the presence of GCV. When FRTL-5 cells were infected with AdrTgtk followed by GCV treatment, more than 90% were killed, but only a minimal effect was observed in other cell lines, indicating that the Tg promoter transduced TK expression only in Tg-producing cells. When adenovirus is given intravenously, liver and spleen are the major organs infected. A high Luc activity was found in liver and spleen of AdCMVLuc treated animals. No Luc activity was found in liver and spleen of AdrTgLuc-treated animals, indicating that rTg does not transduce Luc expression in non-Tg-producing tissues in vivo. No significant changes of the serum transaminase levels and histologic abnormalities were found in animals treated with AdrTgtk/GCV compared with control animals. High levels of serum transaminases, lymphocyte infiltration, some Kupffer's cell prominence, and extensive single cell hypatocyte death were found in AdCMVtk/GCV-treated animals, indicating severe liver damage induced, as expected, by a noncell-specific promoter. These results indicate that transfer of TK gene driven by the rTg promoter has thyroid cell-specific killing ability in the presence of GCV, little in vivo toxicity, and should be useful in the future for treating thyroid Tg-producing cancers.
开发了一种复制缺陷型腺病毒，其在大鼠甲状腺球蛋白（rTg）启动子的控制下转导胸苷激酶（TK）基因（AdrTgtk），以评估其在基因治疗中的细胞特异性杀伤活性。我们还开发了含有由巨细胞病毒（CMV）启动子驱动的TK基因的腺病毒（AdCMVtk），以及由rTg或CMV启动子驱动的荧光素酶（Luc）基因的腺病毒（AdrTgLuc或AdCMVLuc）。用AdrTgLuc或AdCMVLuc感染后，测量FRTL-5、HepG2、COS1、rMTC、hMTC、Hela、GH3、T98G和CA77细胞中的Luc活性。FRTL-5细胞产生甲状腺球蛋白（Tg），而所有其他细胞都是不产生Tg的细胞系。AdCMVLuc转导在所有细胞系中均引起高Luc活性。然而，用AdrTgLuc感染仅在FRTL-5细胞中诱导Luc活性。使用AdCMVtk或AdrTgtk转导各种细胞系以评估不同的杀伤效果。用AdCMVtk载体感染后再用更昔洛韦（GCV）处理，与未感染细胞以及在GCV存在下用AdCMVLuc感染的细胞相比，所有细胞系中的细胞生长均受到强烈抑制。当FRTL-5细胞用AdrTgtk感染后再用GCV处理时，超过90%的细胞被杀死，但在其他细胞系中仅观察到最小的效果，这表明Tg启动子仅在产生Tg的细胞中转导TK表达。当静脉内给予腺病毒时，肝脏和脾脏是主要感染器官。在AdCMVLuc处理的动物的肝脏和脾脏中发现高Luc活性。在AdrTgLuc处理的动物的肝脏和脾脏中未发现Luc活性，这表明rTg在体内不产生Tg的组织中转导Luc表达。与对照动物相比，用AdrTgtk/GCV处理的动物的血清转氨酶水平和组织学异常没有显著变化。在AdCMVtk/GCV处理的动物中发现高水平的血清转氨酶、淋巴细胞浸润、一些枯否细胞突出以及广泛的单细胞肝细胞死亡，这表明如预期的那样，由非细胞特异性启动子诱导了严重的肝损伤。这些结果表明，由rTg启动子驱动的TK基因的转移在GCV存在下具有甲状腺细胞特异性杀伤能力，体内毒性小，并且将来应该可用于治疗产生甲状腺Tg的癌症。
