Molecular Analysis of Resistance to p53 Gene Therapy in Human Lung Cancer

为了分析表达p53肿瘤抑制因子的腺病毒载体（Ad-p53）在体内的抗肿瘤作用的机制，我们定量评估了p53靶向基因的表达和可视化的p53在用Ad-p53治疗的裸鼠肿瘤中的转录活性。通过瘤内施用Ad-p53来治疗在裸鼠中建立的人肺癌（H1299）异种移植物。在治疗后第1、2、3、7和14天，通过实时逆转录聚合酶链反应（RT-PCR）定量外源性p53和p53靶基因p21、MDM 2、Noxa和p53 AIP 1的表达水平，并通过组织化学观察细胞凋亡的诱导。外源性p53和p53靶基因（p53 AIP 1除外）的mRNA表达在Ad-p53处理后1天达到最大值，然后迅速下降;处理后2-3天原位细胞凋亡明显。我们开发了一种非侵入性和简单的方法来监测外源性p53的转录活性， 关于我们 Ad-p53在裸鼠中的道德施用。我们建立了H1299细胞，其在p53应答性p21启动子的控制下表达绿色荧光蛋白（GFP）报告基因（即，p53 R-GFP报告系统）。肿瘤内注射Ad-p53后，用3-CCD_camera真实的检测肿瘤内GFP的表达，并观察外源性p53的转录活性。GFP在处理后3d表达最高，处理后7 d表达明显下降。我们证明，Ad-p53治疗迅速诱导肿瘤中的p53靶向基因和凋亡。我们还成功地在体内可视化p53转录活性。通过实时定量RT-PCR定量分析p53靶基因的表达，并通过使用p53 R-GFP报告系统在新鲜异种移植物中可视化p53转录活性，可能有助于评估Ad-pS3的抗肿瘤作用机制和新的治疗方法。少

To analyze the mechanism of the antitumor effect of an adenoviral vector expressing the p53 tumor suppressor (Ad-p53) in vivo, we quantitatively assessed p53-targeted gene expression and visualized transcriptional activity of p53 in tumors in nude mice treated with Ad-p53. Human lung cancer (H1299) xenografts established in nude mice were treated by intratumoral administration of Ad-p53. The levels of expression of exogenous p53 and p53-targeted genes p21, MDM2, Noxa, and p53AIP1 were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and induction of apoptosis was observed histochemically on days 1, 2, 3, 7 and 14 after treatment. Expression of mRNAs of exogenous p53 and p53-targeted genes (except p53AIP1) was at its maximum 1 day after Ad-p53 treatment, then decreased rapidly ; apoptosis was evident in situ 2-3 days after treatment. We developed a noninvasive and simple method for monitoring the transcriptional activity of exogenous p53 following intratu … More moral administration of Ad-p53 in nude mice. We established H1299 cells that express the green fluorescent protein (GFP) reporter gene under the control of p53-responsive p21 promotes (i.e., the p53R-GFP reporter system). Xenografts of these cells in nude mice were treated by intratumoral administration of Ad-p53, and the transcriptional activity of exogenous p53 could be visualized as intratumoral GFP expression in real time by 3-CCD_camera. Expression of GFP was maximal 3 days after treatment, and it decreased remarkably by 7 days after treatment. We demonstrated that Ad-p53 treatment rapidly induced p53-targeted genes and apoptosis in tumors. We also succeeded in visualizing p53 transcriptional activity in vivo. Quantitative analysis of p53-targeted gene expression by real-time quantitative RT-PCR and visualization of p53 transcriptional activity in fresh xenografts by using the p53R-GFP reporter system may be useful in assessing the mechanisms of the antitumor effects of Ad-pS3 and novel therapeutic approaches. Less