title: Quantitative analysis of p53-targeted gene expression and visualization of p53 transcriptional activity following intratumoral administration of adenoviral p53 in vivo.
abstract: 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.
authors.affiliation: Division of Surgical Oncology, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
authors.forename: Shoichiro
authors.initials: S
authors.lastname: Ohtani
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Kagawa
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Tango
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Umeoka
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Tokunaga
authors.forename: Yousuke
authors.initials: Y
authors.lastname: Tsunemitsu
authors.forename: Jack A
authors.initials: JA
authors.lastname: Roth
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Taya
authors.forename: Noriaki
authors.initials: N
authors.lastname: Tanaka
authors.forename: Toshiyoshi
authors.initials: T
authors.lastname: Fujiwara
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D045744:Cell Line, Tumor; D002999:Clone Cells; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000235:genetics; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012694:Serine / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; C498702:PMAIP1 protein, human; C408009:Pmaip1 protein, mouse; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins; D012694:Serine; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2