Quantitative analysis of p53-targeted gene expression and visualization of p53 transcriptional activity following intratumoral administration of adenoviral p53 in vivo

• Published in: Molecular cancer therapeutics Vol. 3; no. 1; pp. 93 - 100
• Main Authors: Ohtani, Shoichiro, Kagawa, Shunsuke, Tango, Yasuhisa, Umeoka, Tatsuo, Tokunaga, Naoyuki, Tsunemitsu, Yousuke, Roth, Jack A, Taya, Yoichi, Tanaka, Noriaki, Fujiwara, Toshiyoshi
• Format: Journal Article
• Language: English
• Published: United States American Association for Cancer Research 01.01.2004
• Subjects: Adenoviridae - genetics; Animals; Apoptosis; Cell Line, Tumor; Clone Cells; Cyclin-Dependent Kinase Inhibitor p21; Cyclins - genetics; Female; Flow Cytometry - methods; Gene Expression; Genetic Therapy - methods; Genetic Vectors - administration & dosage; Genetic Vectors - genetics; Green Fluorescent Proteins; Humans; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Fluorescence; Nuclear Proteins - genetics; Phosphorylation; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins c-bcl-2 - genetics; Proto-Oncogene Proteins c-mdm2; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction - methods; Serine - metabolism; Transcription, Genetic - genetics; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - metabolism; Xenograft Model Antitumor Assays
• ISSN: 1535-7163; 1538-8514
• DOI: 10.1158/1535-7163.93.3.1

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 apoptosis was observed histochemically on days 1–3, 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 46 in p53 -sensitive H1299 cells in vitro but not in p53 -resistant H226Br cells, suggesting that phosphorylation of Ser 46 is involved in p53 -dependent 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 system may be useful in assessing the mechanisms of the antitumor effects of Ad- p53 and novel therapeutic approaches.