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¹ Division of Surgical Oncology, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan; ² Center for Gene and Cell Therapy, Okayama University Hospital, Okayama, Japan; ³ First Department of Surgery, Shiga University of Medical Science, Shiga, Japan; ⁴ Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, University of Texas MD Anderson Cancer Center, Houston, TX; and ⁵ Radiobiology Division, National Cancer Center Research Institute, Tokyo, Japan

Requests for Reprints: Toshiyoshi Fujiwara, Division of Surgical Oncology, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Phone: 81-86-235-7257; Fax: 81-86-221-8775. E-mail: toshi_f{at}md.okayama-u.ac.jp

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 Ser⁴⁶ in p53-sensitive H1299 cells in vitro but not in p53-resistant H226Br cells, suggesting that phosphorylation of Ser⁴⁶ 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.

Grant support: Supported in part by Ministry of Education, Science, and Culture and Ministry of Health and Welfare of Japan [Health Sciences Research Grants (Research on Human Genome and Gene Therapy)], National Cancer Institute and NIH PO1 CA78778 (J. A. Roth) SPORE 2P50 CA70907 (J. A. Roth), and W. M. Keck Foundation (J. A. Roth).

Received 2/18/03; revised 9/13/03; accepted 9/30/03.

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