Hamstra D A, Rice D J, Fahmy S, Ross B D, Rehemtulla A
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor 48105-0582, USA.
Hum Gene Ther. 1999 Aug 10;10(12):1993-2003. doi: 10.1089/10430349950017356.
The use of cytosine deaminase (CD) in conjunction with 5-fluorocytosine (5-FC) has been studied for cancer gene therapy as a means of achieving tumor-specific generation of the toxic metabolite 5-fluorouracil (5-FU). Since 5-FC is frequently used as an antifungal agent, and because it has little or no efficacy as an antibacterial agent, we hypothesized that yeast CD (YCD) might be more efficient at utilizing 5-FC as a substrate and hence be a better choice for a CD/5-FC gene therapy strategy than the typically utilized bacterial CD (BCD). To that end Saccharomyces cerevisiae CD was cloned from yeast genomic DNA and expressed in vitro. Functional analysis of BCD and YCD expressed in COS-1 cells indicated that BCD and YCD both utilized cytosine with equal efficacy; however, 5-FC was an extremely poor substrate for BCD, with an apparent catalytic efficiency 280-fold lower than that observed for YCD. Retroviral infection of tumor cell lines in vitro indicated that the IC50 of 5-FC was 30-fold lower in YCD-infected cultures as compared with cultures infected with BCD retrovirus. In addition, when SCCVII murine squamous cell carcinoma cells were infected in vitro at low rates of infection (< or =10%) there was no significant cytotoxicity toward BCD-expressing cells while there was potent cytotoxicity to both YCD-expressing cells and "bystander cells" even at this low level of expression. Finally, stable BCD- or YCD-expressing SCCVII clones were developed and used in an orthotopic immune-competent model of head and neck cancer. Subsequent treatment with 5-FC followed by monitoring of tumor growth by noninvasive magnetic resonance imaging (MRI) and survival of animals indicated a growth delay during the course of 5-FC treatment for BCD-expressing tumors, which quickly regrew at the end of treatment. In contrast, YCD-expressing tumors exhibited not only a growth delay, which was of longer duration, but also in some cases frank tumor regression and complete cures occurred.
胞嘧啶脱氨酶（CD）与5-氟胞嘧啶（5-FC）联合用于癌症基因治疗，作为实现毒性代谢物5-氟尿嘧啶（5-FU）肿瘤特异性生成的一种手段，已得到研究。由于5-FC常被用作抗真菌剂，且作为抗菌剂几乎没有疗效，我们推测酵母CD（YCD）可能更有效地将5-FC用作底物，因此对于CD/5-FC基因治疗策略而言，它比通常使用的细菌CD（BCD）是更好的选择。为此，从酵母基因组DNA中克隆了酿酒酵母CD并在体外表达。对在COS-1细胞中表达的BCD和YCD进行功能分析表明，BCD和YCD利用胞嘧啶的效率相同；然而，5-FC是BCD的极差底物，其表观催化效率比YCD低280倍。体外对肿瘤细胞系进行逆转录病毒感染表明，与感染BCD逆转录病毒的培养物相比，5-FC在YCD感染的培养物中的半数抑制浓度（IC50）低30倍。此外，当SCCVII小鼠鳞状细胞癌细胞在体外以低感染率（≤10%）感染时，对表达BCD的细胞没有明显的细胞毒性，而即使在这种低表达水平下，对表达YCD的细胞和“旁观者细胞”都有强大的细胞毒性。最后，构建了稳定表达BCD或YCD的SCCVII克隆，并将其用于头颈癌原位免疫活性模型。随后用5-FC进行治疗，然后通过无创磁共振成像（MRI）监测肿瘤生长和动物存活情况，结果表明，表达BCD的肿瘤在5-FC治疗过程中出现生长延迟，但在治疗结束后迅速复发。相比之下，表达YCD的肿瘤不仅出现了持续时间更长的生长延迟，而且在某些情况下还出现了明显的肿瘤消退并实现了完全治愈。
