Li J H, Lax S A, Kim J, Klamut H, Liu F F
Department of Research, Princess Margaret Hospital/Ontario Cancer Institute, University of Toronto, Canada.
Int J Radiat Oncol Biol Phys. 1999 Feb 1;43(3):607-16. doi: 10.1016/s0360-3016(98)00432-5.
Nasopharyngeal carcinoma (NPC) is a malignant disease of the head/neck region, with a 5-year survival level of approximately 65%. To explore gene therapy as a novel approach which might improve outcome, we have shown previously that introduction of human recombinant wild-type p53 mediated by the adenoviral vector (Ad5CMV-p53) was cytotoxic in two human nasopharyngeal carcinoma (NPC) cell lines (CNE-1 and CNE-2Z). The current work was designed to determine whether this strategy, combined with ionizing radiation (XRT), was more effective than either treatment alone.
CNE-1, CNE-2Z, and a normal human nasopharyngeal fibroblast strain, KS1, were infected with 2- and 6-plaque-forming units (pfu)/cell of Ad5CMV-p53, respectively. These doses were isoeffective for beta-galactosidase activity in the CNE-1 and CNE-2Z cells. XRT was administered 24 h post-infection, and Western blot analyses were conducted for p53, p21WAF1/CIP1, bax, and bcl-2 2 days after XRT. Cell survival was assessed using a clonogenic assay. Presence of DNA ladders reflecting apoptosis was detected using DNA agarose gel electrophoresis, and cell cycle was analyzed using flow cytometry.
The combination of Ad5CMV-p53 plus XRT (2, 4, and 6 Gy) resulted in an approximately 1-log greater level of cytotoxicity compared to that observed with XRT alone for both NPC cell lines. The two modalities appear to be interacting in a synergistic manner in cancer cells, but not in KS1 fibroblasts. XRT alone stimulated minimal p53 expression in control cells; Ad5CMV-p53 alone induced significant recombinant p53 expression, which was not further enhanced by the addition of XRT. Similar observations were made for p21WAF1/CIP1 expression. No changes were observed for bax or bcl-2 expression with any of these treatments. Apoptosis was induced following 4 Gy of XRT alone, but was observed after only 2 Gy when combined with Ad5CMV-p53. Cell cycle analysis indicated that Ad5CMV-p53 infection did not perturb the cell cycle beyond that observed with XRT alone.
p53 gene therapy and XRT appears to interact in a synergistic manner; underscoring the significant potential of this novel strategy in the treatment of NPC.
鼻咽癌（NPC）是头颈部的一种恶性疾病，5年生存率约为65%。为了探索基因治疗作为一种可能改善治疗结果的新方法，我们之前已经表明，由腺病毒载体（Ad5CMV-p53）介导的人重组野生型p53的导入对两个人鼻咽癌（NPC）细胞系（CNE-1和CNE-2Z）具有细胞毒性。当前的研究旨在确定这种策略与电离辐射（XRT）联合使用是否比单独使用任何一种治疗方法更有效。
分别用2个和6个空斑形成单位（pfu）/细胞的Ad5CMV-p53感染CNE-1、CNE-2Z和一种正常人鼻咽成纤维细胞系KS1。这些剂量对CNE-1和CNE-2Z细胞中的β-半乳糖苷酶活性具有等效效果。在感染后24小时给予XRT，并在XRT后2天对p53、p21WAF1/CIP1、bax和bcl-2进行蛋白质印迹分析。使用克隆形成试验评估细胞存活率。使用DNA琼脂糖凝胶电泳检测反映凋亡的DNA梯带的存在，并使用流式细胞术分析细胞周期。
与单独使用XRT相比，Ad5CMV-p53与XRT（2、4和6 Gy）联合使用对两种NPC细胞系均导致细胞毒性水平提高约1个对数。这两种方式在癌细胞中似乎以协同方式相互作用，但在KS1成纤维细胞中并非如此。单独使用XRT刺激对照细胞中p53的表达极少；单独使用Ad5CMV-p53诱导显著的重组p53表达，添加XRT后并未进一步增强。对p21WAF1/CIP1表达也有类似的观察结果。这些处理中的任何一种对bax或bcl-2表达均未观察到变化。单独使用4 Gy的XRT后诱导凋亡，但与Ad5CMV-p53联合使用时，仅2 Gy后就观察到凋亡。细胞周期分析表明，Ad5CMV-p53感染对细胞周期的干扰不超过单独使用XRT时观察到的情况。
p53基因治疗和XRT似乎以协同方式相互作用；突出了这种新策略在鼻咽癌治疗中的巨大潜力。
