Abe A, Takeo T, Emi N, Tanimoto M, Ueda R, Yee J K, Friedmann T, Saito H
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
Proc Soc Exp Biol Med. 1993 Jul;203(3):354-9. doi: 10.3181/00379727-203-43611.
To investigate the possibility of killing tumor cells by the expression of an exogenously introduced toxic gene, we have constructed a novel retroviral vector (LTRNL) which has the polyA signal deleted herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene. The vector becomes toxic by treating cells expressing HSV1-tk with the antiherpetic drugs acyclovir or ganciclovir (GCV). Cells of the human leukemia lines (K562, MEG-01) were infected with this vector and two transduced cell lines (K562/LTRNL, MEG-01/LTRNL) were established. Southern blot analysis confirmed the integration of the HSV1-tk transgene in these cells and Northern blot analysis exhibited the expression of 4.8-kb viral mRNA containing the HSV1-tk gene. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the in vitro cytotoxic effects of GCV to these cells demonstrated that concentrations of about 2.5 microM for K562/LTRNL and 1.25 microM for MEG-01/LTRNL cells resulted in 50% inhibition of cell growth after 72 hr. Subcutaneous tumors of MEG-01/LTRNL in KSN nude mice, but not those of uninfected MEG-01 cells, showed durable regressions after exposure of the mice to 40 mg/kg of GCV given subcutaneously once a day for 15 days. This study indicates that the LTRNL-infected human leukemia cells exhibit inducible susceptibility to GCV.
为了研究通过表达外源性导入的毒性基因来杀伤肿瘤细胞的可能性，我们构建了一种新型逆转录病毒载体（LTRNL），该载体缺失了多聚腺苷酸信号的单纯疱疹病毒1型胸苷激酶（HSV1-tk）基因。用抗疱疹药物阿昔洛韦或更昔洛韦（GCV）处理表达HSV1-tk的细胞后，该载体变得具有毒性。用人白血病细胞系（K562、MEG-01）的细胞感染此载体，建立了两个转导细胞系（K562/LTRNL、MEG-01/LTRNL）。Southern印迹分析证实了HSV1-tk转基因在这些细胞中的整合，Northern印迹分析显示含有HSV1-tk基因的4.8 kb病毒mRNA的表达。对GCV对这些细胞的体外细胞毒性作用进行的MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐）测定表明，对于K562/LTRNL细胞，约2.5 microM的浓度以及对于MEG-01/LTRNL细胞，1.25 microM的浓度在72小时后导致50%的细胞生长抑制。将KSN裸鼠皮下接种MEG-01/LTRNL细胞，但未接种未感染的MEG-01细胞，在小鼠每天皮下注射40 mg/kg GCV，共15天后，皮下肿瘤出现持久消退。本研究表明，LTRNL感染的人白血病细胞对GCV表现出诱导敏感性。
