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

为了分析在体内表达p53肿瘤抑制因子（Ad-p53）的腺病毒载体的抗肿瘤作用机制，我们定量评估了p53靶向基因在经Ad-p53处理的裸鼠肿瘤中的表达，并可视化了p53的转录活性。裸鼠建立的人肺癌（H1299）异种移植物瘤内给药Ad-p53治疗。实时逆转录聚合酶链反应（RT-PCR）检测外源性p53及p53靶基因p21、MDM2、Noxa、p53AIP1的表达水平，并于治疗后第1、2、3、7、14天组织化学观察细胞凋亡诱导情况。除p53AIP1外，外源p53和p53靶基因mrna表达在Ad-p53治疗后1天达到最大值，随后迅速下降；治疗后2 ~ 3天细胞凋亡明显。我们开发了一种非侵入性和简单的方法来监测裸鼠体内Ad-p53的转录活性。我们建立了在p53-responsive p21 promoter（即p53R-GFP报告基因系统）控制下表达绿色荧光蛋白（GFP）报告基因的H1299细胞。将这些细胞移植到裸鼠体内，瘤内注射Ad-p53，通过3-CCD_camera实时观察外源p53的转录活性和瘤内GFP的表达。GFP在治疗后第3天表达量最大，在治疗后第7天显著降低。我们证明了Ad-p53治疗可以快速诱导p53靶向基因和肿瘤细胞凋亡。我们还成功地可视化了p53在体内的转录活性。利用实时定量RT-PCR技术定量分析p53靶向基因表达，利用p53R-GFP报告系统可视化新鲜异种移植物中p53转录活性，可能有助于评估Ad-pS3抗肿瘤作用的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