Aghi M, Kramm C M, Chou T C, Breakefield X O, Chiocca E A
Molecular Neuro-Oncology Laboratories, Neurosurgical Service, Massachusetts General Hospital, Charlestown 02129, USA.
J Natl Cancer Inst. 1998 Mar 4;90(5):370-80. doi: 10.1093/jnci/90.5.370.
A bacterial enzyme, Escherichia coli cytosine deaminase, which converts the prodrug 5-fluorocytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects.
Rat 9L gliosarcoma cells were transfected with plasmids containing the E. coli cytosine deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells). The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured. The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored. The isobologram method of Loewe and the multiple drug-effect analysis method of Chou-Talalay were used to measure the interaction between the two prodrug-activating gene therapies. To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied.
The presence of transfected cytosine deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment.
一种细菌酶，即大肠杆菌胞嘧啶脱氨酶，可将前体药物5-氟胞嘧啶转化为有毒药物5-氟尿嘧啶；还有一种病毒酶，即单纯疱疹病毒胸苷激酶，可通过磷酸化作用将更昔洛韦从无活性的前体药物转化为细胞毒性剂，目前正积极研究将它们用于癌症的基因治疗。本研究的目的是确定联合使用这些前体药物激活基因疗法是否能增强抗癌效果。
用含有大肠杆菌胞嘧啶脱氨酶基因的质粒转染大鼠9L胶质肉瘤细胞（9L/CD细胞），用含有单纯疱疹病毒胸苷激酶基因的质粒转染（9L/TK细胞），或用两种表达质粒同时转染（9L/CD-TK细胞）。评估这些细胞系的药物敏感性；此外，还测量了按不同比例混合的9L和9L/CD-TK细胞的敏感性。监测前体药物治疗对裸鼠体内9L/CD-TK肿瘤生长（即大小和体积）的影响。采用Loewe的等效线图法和Chou-Talalay的多药效应分析法来测定两种前体药物激活基因疗法之间的相互作用。为阐明相互作用的机制，研究了不同前体药物处理后9L/CD-TK细胞中更昔洛韦的磷酸化情况。
9L胶质肉瘤细胞中存在转染后的胞嘧啶脱氨酶和胸苷激酶基因，在用相关前体药物处理后，无论在体外还是体内，细胞存活率均降低；这两种成分的作用似乎具有协同性，且在机制上与5-氟尿嘧啶处理后胸苷激酶增强更昔洛韦磷酸化有关。
