This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ohtani, S. Articles by Fujiwara, T. Search for Related Content PubMed PubMed Citation Articles by Ohtani, S. Articles by Fujiwara, T.

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.