Trial of novel thermo-gene therapy for malignant brain tumors-Aiming for enhancement of hyperthermia using p53 introduction-

我们评估了p53导入和热疗对恶性胶质瘤细胞的联合作用。使用的细胞系是U251-MG、U373-MG（突变型p53）和D54、U87-MG（野生型p53）。1）首先，确定Ad/p53载体的最佳MOl。2)使用液体培养箱在43℃下施加热休克2 h。3）在体外实验中，两种处理对所用细胞系均表现出相加效应。4）细胞周期分析中，单独热休克对U373-MG和D54细胞的分布无明显影响。然而，单独引入p53，亚G1和G1均增加。联合治疗的效果与单独导入p53的效果相似。5）在DNA片段化实验中，单独导入p53可诱导细胞凋亡，而单独热休克则不能诱导细胞凋亡。联合处理可诱导较高比例的细胞凋亡，这可能是热休克所致。无论肿瘤细胞的p53状态如何，都观察到热休克的这种增强作用。6)U373-MG和U251-MG细胞的凋亡诱导依赖于caspase，而D54细胞的凋亡诱导不依赖于caspase。7）p53基因导入后p21蛋白表达增加。8）作为体内实验，我们建立了U373-MG裸鼠皮下移植瘤模型。9)治疗分为以下四组。A）对照载体（CTR）和37℃热休克（HS），B）CTR和43℃ HS，c）p53载体（p53 V）和37℃ HS，d）p53 V和43℃ HS。结果表明，与对照组（a）相比，（B）、（c）、（d）组对肿瘤生长无明显抑制作用，为进一步提高热疗效果，应考虑采用热休克重复和针电极加热。关于基因转移，还应考虑C）四环素控制下的基因表达，以及D）使用具有更高转移效率的脂质体。

We evaluated combination effect of p53 introduction and hyperthermia on malignant glioma cells. Cell lines used are U251-MG, U373-MG (mutant p53), and D54, U87-MG (wild-type p53). 1)First, optimal MOl of Ad/p53 vector was determined. 2)Heat shock was applied at 43℃ for 2h using liquid incubator. 3)In in vitro experiment, additive effect of both treatments was observed in all the cell lines used. 4)In cell cycle analysis, no change of distribution was observed by heat shock alone in either U373-MG or D54 cells. However, there was an increase in both sub-G1 and G1 by p53 introduction alone. The effect of combination treatment was similar to that of p53 introduction alone. 5)In DNA fragmentation assay, apoptosis was induced by p53 introduction alone but not by heat shock alone. Higher percentage of apoptosis was induced by the combination treatment, which was likely to be caused by heat shock. This enhancing effect by heat shock was observed regardless of p53 status of tumor cells. 6)Apoptosis induction was caspase-dependent in U373-MG and U251-MG cells, but was caspase-independent in D54 cells. 7)p21 protein was over-expressed only after p53 introduction. 8)As in vivo experiment, we established U373-MG subcutaneous tumor model using nude mice. 9)Treatments were given to the following four groups. A)control-vector(CTR) and 37℃ heat shock(HS), b)CTR and 43℃ HS, c)p53-vector(p53V) and 37℃ HS, d)p53V and 43℃ HS. Results showed that no significant inhibitory effect on tumor growth was observed in groups (b),(c),and (d)compared with control-group (a).For future experiments, to achieve more efficient hyperthermia, A)repeating of heat shock, and B)heating with needle electrode, should be considered. Regarding gene transfer, C)gene expression under the control of tetracycline, and D) use of liposome with higher transfer efficiency, should be also be considered.