Frank Susanne, Steffens Sabine, Fischer Ute, Tlolko Aurelia, Rainov Nikolai G, Kramm Christof M
Department of Pediatric Hematology and Oncology, University Children's Hospital, Heinrich Heine University, Duesseldorf, Germany.
Cancer Gene Ther. 2002 Feb;9(2):178-88. doi: 10.1038/sj.cgt.7700422.
The time course of cytotoxicity induction and the bystander effect of the rabbit cytochrome P450 4B1 (cyp4B1)/4-ipomeanol (4-IM) or 2-aminoanthracene (2-AA) pharmacogene therapy systems were investigated and compared with the herpes simplex virus type 1 thymidine kinase/ganciclovir (HSV-tk/GCV) system. Experiments were performed in rat 9L gliosarcoma cells stably expressing cyp4B1 (9L-4B1), HSV-tk (9L-tk), or their egfp (enhanced green fluorescent protein) fusion genes. Cyp4B1-mediated activation of 2-AA showed a high cell killing efficiency within only 48 hours with an onset after already 15 minutes of prodrug exposure. Residual 9L-4B1 cells were mostly damaged sublethally upon 2-AA treatment showing an S phase arrest by cell cycle analysis. 4-IM treatment of 9L-4B1 cells generated an overall weaker cell killing, especially after prodrug exposure times of less than 48 hours. Residual cells surviving 4-IM treatment showed a G2/M arrest and restarted proliferation after prodrug treatment was stopped. HSV-tk/GCV pharmacogene therapy resulted in a slower cytotoxicity induction than cyp4B1/2-AA treatment with a significantly lower cell killing efficiency after 24 and 48 hours. HSV-tk/GCV-mediated cytotoxicity was widely similar to the cytotoxicity induced by cyp4B1/4-IM with the exception of a continuous 48-hour prodrug exposure where 4-IM treatment showed a significantly higher cell killing rate. Cells surviving HSV-tk/GCV suicide gene therapy were not viable and showed an S-phase arrest. Whereas HSV-tk/GCV induced a strong bystander effect, only moderate bystander cell death depending on cell-to-cell contact was demonstrated in 9L/9L-4B1 cocultures upon 2-AA treatment and was even absent with 4-IM, thereby contrasting with earlier reports. The absence of a strong bystander effect may limit, on one hand, the overall utility of the cyp4B1 systems for cancer gene therapy. On the other hand, the weak bystander effect together with the fast induction of cytotoxicity may provide marked advantages for the use of the cyp4B1 systems as biosafety enhancers for gene marking or replacement studies and donor lymphocyte infusions after allogeneic bone marrow transplantation.
研究了兔细胞色素P450 4B1（cyp4B1）/4-异亚丙基丙酮（4-IM）或2-氨基蒽（2-AA）药物基因治疗系统诱导细胞毒性的时间进程和旁观者效应，并与单纯疱疹病毒1型胸苷激酶/更昔洛韦（HSV-tk/GCV）系统进行了比较。实验在稳定表达cyp4B1（9L-4B1）、HSV-tk（9L-tk）或其增强绿色荧光蛋白（egfp）融合基因的大鼠9L胶质肉瘤细胞中进行。Cyp4B1介导的2-AA激活在仅48小时内就显示出高细胞杀伤效率，在前药暴露15分钟后就开始起效。2-AA处理后，残留的9L-4B1细胞大多受到亚致死性损伤，细胞周期分析显示其处于S期停滞。9L-4B1细胞经4-IM处理后产生的总体细胞杀伤作用较弱，尤其是在前药暴露时间少于48小时的情况下。经4-IM处理后存活的残留细胞显示出G2/M期停滞，在前药处理停止后重新开始增殖。HSV-tk/GCV药物基因治疗导致细胞毒性诱导比cyp4B1/2-AA处理慢，在24小时和48小时后细胞杀伤效率显著较低。HSV-tk/GCV介导的细胞毒性与cyp4B1/4-IM诱导的细胞毒性广泛相似，不同之处在于连续48小时的前药暴露，此时4-IM处理显示出显著更高的细胞杀伤率。经HSV-tk/GCV自杀基因治疗后存活的细胞无法存活，并显示出S期停滞。虽然HSV-tk/GCV诱导了强烈的旁观者效应，但在9L/9L-4B1共培养物中，2-AA处理后仅表现出中度的依赖细胞间接触的旁观者细胞死亡，而4-IM处理时甚至没有旁观者效应，这与早期报道形成对比。旁观者效应不强烈一方面可能会限制cyp4B1系统在癌症基因治疗中的整体效用。另一方面，微弱的旁观者效应与快速诱导的细胞毒性可能为将cyp4B1系统用作基因标记或替代研究以及异基因骨髓移植后供体淋巴细胞输注的生物安全性增强剂提供显著优势。
