Quantitative analysis of p53-targeted gene expression and visualization of p53 transcriptional activity following intratumoral administration of adenoviral p53 in vivo.
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.
Ohtani
Shoichiro
S
Division of Surgical Oncology, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
Kagawa
Shunsuke
S
Tango
Yasuhisa
Y
Umeoka
Tatsuo
T
Tokunaga
Naoyuki
N
Tsunemitsu
Yousuke
Y
Roth
Jack A
JA
Taya
Yoichi
Y
Tanaka
Noriaki
N
Fujiwara
Toshiyoshi
T
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
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