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

我们评价了P53基因导入和热疗对恶性胶质瘤细胞的联合作用。所用细胞系为U251-MG、U373-MG(突变型P53)和D54、U87-MG(野生型P53)。1)首先确定了Ad/P53载体的最佳摩尔浓度。2)采用液体孵箱，在43℃条件下进行2小时的热休克处理。3)在体外实验中，两种处理在所有细胞系中都表现出相加效应。4)在细胞周期分析中，单纯热休克对U373-MG和D54细胞的分布没有影响。然而，单独导入P53后，亚G1期和G1期细胞数均增加。联合治疗的效果与单独导入P53的效果相似。5)DNA片段化分析显示，单独导入P53可诱导细胞凋亡，但不能单独诱导热休克诱导细胞凋亡。联合处理诱导的细胞凋亡率较高，可能是由热休克引起的。无论肿瘤细胞的P53状态如何，热休克都能观察到这种增强效应。6)U373-MG和U251-MG细胞的凋亡诱导依赖于caspase，而D54细胞不依赖于caspase。7)p21蛋白只有在P53基因导入后才有高表达。8)作为体内实验，建立了U373-MG裸鼠皮下移植瘤模型。9)对以下4组进行治疗。A)对照载体(CTR)和37℃热休克(HS)，b)CTR和43℃HS，c)p53载体(P53V)和37℃HS，d)p53V和43℃HS。结果表明，与对照组(A)相比，(B)、(C)、(D)组对肿瘤的生长无明显抑制作用。为了达到更有效的热疗效果，今后的实验应考虑A)重复热休克，B)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，D)使用具有较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.