Qi V, Weinrib L, Ma N, Li J H, Klamut H, Liu F F
Department of Research, Princess Margaret Hospital/Ontario Cancer Institute, University Health Network, Toronto, Canada.
Int J Hyperthermia. 2001 Jan-Feb;17(1):38-47. doi: 10.1080/02656730150201589.
It has previously been demonstrated that Ad5CMV-p53 gene transfer, either used alone or delivered concomitantly with ionizing radiation, resulted in cytotoxicity mediated by apoptosis in nasopharyngeal carcinoma (NPC) cell lines. In this study, a novel approach was evaluated of combining Ad5CMV-p53 gene therapy with hyperthermia (HT), in the CNE-1 NPC cell line, which harbours a mutation in codon 249 of the p53 gene.
CNE-1 cells were infected using either Ad5CMV-p53 or Ad5CMV-B-gal, followed, 24 h later, by HT (43 degrees C x 0-2 h). Protein was extracted for Western blot analysis, and apoptosis was evaluated using acridine-orange ethidium bromide staining, followed immediately by fluorescent microscopy examination for the proportion of cells displaying morphologic features of apoptosis.
Ad5CMV-p53 gene therapy combined with HT resulted in a dose-dependent cytotoxicity with less than 1% clonogenic survival when 10 pfu/cell of Ad5CMV-p53 was combined with 2 h heating at 43 degrees C. Western blotting demonstrated that treatment with Ad5CMV-p53 resulted in the rapid expression of p53, which was minimally affected by HT. The inducible form of hsp70 was maximally expressed at 48 h post-HT, with minimal effect when cells were additionally treated with Ad5CMV-p53. Clonogenic cytotoxicity was associated with the development of apoptosis, with up to 70% of CNE-1 cells displaying morphologic features of apoptosis after the combination treatments.
Based on the shapes of the clonogenic survival curves, Ad5CMV-p53 gene therapy and HT appear to interact in an additive manner, suggesting the therapeutic potential of this combined treatment approach for patients with NPC.
先前已经证明，单独使用Ad5CMV-p53基因转移或与电离辐射同时进行，可导致鼻咽癌（NPC）细胞系中由凋亡介导的细胞毒性。在本研究中，评估了一种将Ad5CMV-p53基因治疗与热疗（HT）相结合的新方法，该方法应用于携带p53基因第249密码子突变的CNE-1 NPC细胞系。
用Ad5CMV-p53或Ad5CMV-β-半乳糖苷感染CNE-1细胞，24小时后进行热疗（43℃×0 - 2小时）。提取蛋白质进行蛋白质印迹分析，并使用吖啶橙-溴化乙锭染色评估细胞凋亡，随后立即通过荧光显微镜检查显示凋亡形态特征的细胞比例。
当每细胞10个空斑形成单位（pfu）的Ad5CMV-p53与43℃加热2小时相结合时，Ad5CMV-p53基因治疗与热疗相结合导致剂量依赖性细胞毒性，克隆形成存活率低于1%。蛋白质印迹显示，Ad5CMV-p53处理导致p53快速表达，热疗对其影响最小。热休克蛋白70（hsp70）的诱导型在热疗后48小时最大程度表达，当细胞额外用Ad5CMV-p53处理时影响最小。克隆形成细胞毒性与凋亡的发生相关，联合治疗后高达70%的CNE-1细胞显示凋亡形态特征。
根据克隆形成存活曲线的形状，Ad5CMV-p53基因治疗和热疗似乎以相加方式相互作用，表明这种联合治疗方法对NPC患者具有治疗潜力。
