Hsu H, Rainov N G, Quinones A, Eling D J, Sakamoto K M, Spear M A
Gene Therapy Program, Translation Cancer Research Program, UCSD Cancer Center, Univ. of California at San Diego, San Diego, CA, USA.
Anticancer Res. 2003 May-Jun;23(3B):2723-8.
Cytochrome p450 isozyme CYP4B1 converts the inert prodrug 4-ipomeanol (4-IM) into toxic alkylating metabolites. Induction of cytotoxicity by 4-IM combined with ionizing radiation (IR) in cells transfected with a fusion protein of rabbit cytochrome CYP4B1 under control of the radiation inducible EGR1 promoter was investigated. The capability of activated 4-IM to sensitize cells to IR was also assessed.
Survival fractions of cells, determined by MTT assays, stably transfected with EGR1-CYP4B1 were compared with that of cells transfected with a control plasmid after IR followed by 4-IM. Radiosensitization was tested by comparing clonogenic survival curves of cells transfected with the CYP4B1 cassette under a CMV promoter instead of EGR-1, irradiated with or without 4-IM.
MTT assays for cytotoxicity indicated a decrease in relative survival fractions (survival with 4-IM/survival without 4-IM) of the EGR1-CYP4B1 transfected cells with increasing radiation dosage, but not of control cells. Clonogenic assays revealed decreased survival fractions with increasing radiation doses (CYP4B1 transfected and control cells) and 4-IM concentrations (CYP4B1 transfected cells), but showed no significant differences in slope of survival curves with 4-IM.
The results indicate IR potentiates the cytotoxic activity of the EGR1-CYP4B1/4-IM transgene system, but activated 4-IM does not sensitize cells to IR. Thus, the EGR1-CYP4B1/4-IM system is a viable radiation-gene therapy system that may allow for improved spatial and temporal control of cytotoxicity by therapeutic radiation fields.
细胞色素P450同工酶CYP4B1可将惰性前药4-异亚丙基丙酮（4-IM）转化为有毒的烷基化代谢产物。研究了在辐射诱导型EGR1启动子控制下，用兔细胞色素CYP4B1融合蛋白转染的细胞中，4-IM与电离辐射（IR）联合诱导的细胞毒性。还评估了活化的4-IM使细胞对IR敏感的能力。
通过MTT法测定，将稳定转染EGR1-CYP4B1的细胞在IR后再给予4-IM后的存活分数，与转染对照质粒的细胞的存活分数进行比较。通过比较在巨细胞病毒（CMV）启动子而非EGR-1控制下转染CYP4B1盒的细胞在有或无4-IM照射下的克隆存活曲线，来测试放射增敏作用。
细胞毒性的MTT分析表明，随着辐射剂量增加，转染EGR1-CYP4B1的细胞的相对存活分数（有4-IM时的存活/无4-IM时的存活）降低，但对照细胞没有。克隆分析显示，随着辐射剂量（转染CYP4B1的细胞和对照细胞）和4-IM浓度（转染CYP4B1的细胞）增加，存活分数降低，但在有4-IM时存活曲线的斜率没有显著差异。
结果表明IR增强了EGR1-CYP4B1/4-IM转基因系统的细胞毒性活性，但活化的4-IM不会使细胞对IR敏感。因此，EGR1-CYP4B1/4-IM系统是一种可行的放射基因治疗系统，可能允许通过治疗性辐射场改善细胞毒性的空间和时间控制。
