Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands.
Virol J. 2011 Apr 11;8:162. doi: 10.1186/1743-422X-8-162.
The survival of glioma patients with the current treatments is poor. Early clinical trails with replicating adenoviruses demonstrated the feasibility and safety of the use of adenoviruses as oncolytic agents. Antitumor efficacy has been moderate due to inefficient virus replication and spread. Previous studies have shown that truncation of the adenovirus i-leader open reading frame enhanced cytopathic activity of HAdV-5 in several tumor cell lines. Here we report the effect of an i-leader mutation on the cytopathic activity in glioma cell lines and in primary high-grade glioma cell cultures.
A mutation truncating the i-leader open reading frame was created in a molecular clone of replication-competent wild-type HAdV-5 by site-directed mutagenesis. We analyzed the cytopathic activity of this RL-07 mutant virus. A cell-viability assay showed increased cytopathic activity of the RL-07 mutant virus on U251 and SNB19 glioma cell lines. The plaque sizes of RL-07 on U251 monolayers were seven times larger than those of isogenic control viruses. Similarly, the cytopathic activity of the RL-07 viruses was strongly increased in six primary high-grade glioma cell cultures. In glioma cell lines the RL-07 virus was found to be released earlier into the culture medium. This was not due to enhanced viral protein synthesis, as was evident from equivalent E1A, Fiber and Adenovirus Death Protein amounts, nor to higher virus yields.
The cytopathic activity of replicating adenovirus in glioblastoma cells is increased by truncating the i-leader open reading frame. Such mutations may help enhancing the antitumor cytopathic efficacy of oncolytic adenoviruses in the treatment of glioblastoma.
目前治疗胶质瘤患者的生存率较差。复制型腺病毒的早期临床试验证明了使用腺病毒作为溶瘤剂的可行性和安全性。由于病毒复制和传播效率低下，抗肿瘤疗效一直较为温和。先前的研究表明，截断腺病毒 i-启动子开放阅读框可增强 HAdV-5 在几种肿瘤细胞系中的细胞病变活性。在这里，我们报告了 i-启动子突变对胶质瘤细胞系和原发性高级别胶质瘤细胞培养物中细胞病变活性的影响。
通过定点诱变在复制型野生型 HAdV-5 的分子克隆中创建了截断 i-启动子开放阅读框的突变。我们分析了这种 RL-07 突变病毒的细胞病变活性。细胞活力测定显示 RL-07 突变病毒在 U251 和 SNB19 神经胶质瘤细胞系中的细胞病变活性增加。RL-07 在 U251 单层上的斑块大小是同源对照病毒的七倍。同样，RL-07 病毒在六种原发性高级别神经胶质瘤细胞培养物中的细胞病变活性也大大增强。在神经胶质瘤细胞系中，RL-07 病毒较早释放到培养基中。这不是由于病毒蛋白合成增强所致，从等量的 E1A、纤维蛋白和腺病毒死亡蛋白的量可以明显看出，也不是由于病毒产量增加所致。
截断 i-启动子开放阅读框可增强复制型腺病毒在神经胶质瘤细胞中的细胞病变活性。这种突变可能有助于提高溶瘤腺病毒在治疗神经胶质瘤中的抗肿瘤细胞病变疗效。
