Quantitative analysis of p 53-targeted gene expression and visualization of p 53 transcriptional activity following intratumoral administration of adenoviral p 53 in vivo
Quantitative analysis of p 53-targeted gene expression and visualization of p 53 transcriptional activity following intratumoral administration of adenoviral p 53 in vivo
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腺病毒 p 53 体内瘤内给药后 p 53 靶向基因表达的定量分析和 p 53 转录活性的可视化
DOI：
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
T. Fujiwara
中科院分区：
文献类型：
--
作者：
S. Ohtani;S. Kagawa;Yasuhisa Tango;T. Umeoka;Naoyuki Tokunaga;Y. Tsunemitsu;J. Roth;Y. Taya;N. Tanaka;T. Fujiwara
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。用实时定量逆转录-聚合酶链式反应(RT-PCR)检测外源性P53和P53靶基因p21、MDM2、Noxa和p53AIP1的表达水平，并在治疗后1-3、7、14天用组织化学方法观察细胞凋亡的诱导情况。外源性P53和P53靶向基因(p53AIP1除外)的mRNA在Ad-P53处理后1d达到高峰，然后迅速下降，在处理后2~3d在原位可见明显的细胞凋亡。我们建立了一种非侵入性、简便的方法来监测裸鼠瘤内注射Ad-P53后外源性P53的转录活性。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。用瘤内注射Ad-P53治疗裸鼠移植瘤，用3-CCD摄像机实时观察外源性P53的转录活性。GFP的表达在治疗后3d最高，治疗后7d明显下降。我们证明了Ad-P53治疗能快速诱导肿瘤中的P53靶向基因和细胞凋亡，并成功地在体内显示了P53的转录活性。我们还发现，Ad-P53感染在体外诱导了对P53敏感的H1299细胞中P53的Ser的磷酸化，但在对P53耐药的H2 26BR细胞中却没有，提示Ser的磷酸化参与了P53依赖的细胞凋亡。我们的数据表明，实时定量RT-PCR定量分析p53靶向基因的表达，并利用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-P53的抗肿瘤作用机制和新的治疗方法。摩尔癌症治疗。2004；3(1)：93-100]
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 transcription-PCR (RT-PCR) and induction of apoptosiswas observed histochemically on days1–3, 7, and 14 after treatment. Expression of mRNA of exogenous p53 and p53-targeted genes (except p53AIP1) was at its maximum 1 day after Ad-p53 treatment and 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 intratumoral 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 promoter (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 decreased remarkably by 7 days after treatment. We demonstrated that Ad-p53 treatment rapidly induced p53-targeted genes and apoptosis in tumors and succeeded in visualizing p53 transcriptional activity in vivo. We also found that Ad-p53 infection induced phosphorylation of p53 at Ser in p53-sensitive H1299 cells in vitro but not in p53-resistant H226Br cells, suggesting that phosphorylation of Ser is involved in p53dependent apoptosis. Our data indicate that 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 systemmaybe useful in assessing themechanisms of the antitumor effects of Ad-p53 and novel therapeutic approaches. [Mol Cancer Ther. 2004;3(1):93–100]
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影响因子：
11.2
作者：
T. Fujiwara;E. A. Grimm;T. Mukhopadhyay;Wei-wei Zhang;Laurie B. Owen-Schaub;J. Roth
通讯作者：
T. Fujiwara;E. A. Grimm;T. Mukhopadhyay;Wei-wei Zhang;Laurie B. Owen-Schaub;J. Roth
DOI：
10.1093/jnci/86.19.1458
发表时间：
1994-10-05
期刊：
JOURNAL OF THE NATIONAL CANCER INSTITUTE
影响因子：
--
作者：
FUJIWARA, T;CAI, DW;ROTH, JA
通讯作者：
ROTH, JA
影响因子：
10.5
作者：
Timothy A. Chan;Paul M. Hwang;H. Hermeking;K. Kinzler;B. Vogelstein
通讯作者：
Timothy A. Chan;Paul M. Hwang;H. Hermeking;K. Kinzler;B. Vogelstein
DOI：
10.1073/pnas.96.25.14517
发表时间：
1999-12-07
影响因子：
11.1
作者：
Yu, J;Zhang, L;Vogelstein, B
通讯作者：
Vogelstein, B
影响因子：
56.9
作者：
HOLLSTEIN, M;SIDRANSKY, D;HARRIS, CC
通讯作者：
HARRIS, CC