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

为了分析表达P53抑癌基因的腺病毒载体(Ad-P53)体内抗肿瘤作用的机制，我们定量检测了P53靶向基因的表达和P53在裸鼠体内的可视转录活性。建立人肺癌(H1299)裸鼠移植瘤模型，瘤内注射Ad-P53。分别于治疗后第1、2、3、7、14天用实时定量逆转录聚合酶链式反应(RT-PCR)检测外源性P53及P53靶基因p21、MDM2、Noxa和p53AIP1的表达水平，并观察其对细胞凋亡的诱导作用。除p53AIP1外，外源性P53和P53靶向基因的mRNAs在Ad-P53处理后1d达到高峰，然后迅速下降，在处理后2~3d可见明显的细胞凋亡。我们开发了一种非侵入性且简单的方法来监测…后外源性P53的转录活性。裸鼠体内对Ad-P53更道德的管理。我们建立了在p53-响应性p21启动子(即p53R-GFP报告系统)控制下表达绿色荧光蛋白(GFP)报告基因的H1299细胞。瘤内注射Ad-P53后，3-CCDCamera可实时显示瘤内GFP的转录活性。GFP在治疗后3d达高峰，治疗后7d明显下降。我们证明了Ad-P53治疗能迅速诱导肿瘤中的P53靶向基因和细胞凋亡。我们还成功地在体内可视化了P53的转录活性。实时定量RT-PCR定量分析P53靶向基因的表达，p53R-GFP报告系统显示新鲜移植瘤中P53的转录活性，可能有助于评估Ad-PS3的抗肿瘤作用机制和新的治疗方法。较少

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