2002 Fiscal Year Final Research Report Summary
Molecular Analysis of Resistance to p53 Gene Therapy in Human Lung Cancer
| Project/Area Number |
13671390
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Thoracic surgery
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| Research Institution | OKAYAMA UNIVERSITY |
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Principal Investigator |
FUJIWARA Toshiyoshi Okayama University Hospital, Center for Gene and Cell therapy, Associate Professor, 医学部・歯学部附属病院, 助教授 (00304303)
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| Project Period (FY) |
2001 – 2002
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| Keywords | p53 / Gene Therapy / Lung cancer / Adenovirus vector / Resistance to Therapy |
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| Research 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-transcriptase polymerase chain reaction (RT-PCR) and induction of apoptosis was observed histochemically on days 1, 2, 3, 7 and 14 after treatment. Expression of mRNAs of exogenous p53 and p53-targeted genes (except p53AIP1) was at its maximum 1 day after Ad-p53 treatment, 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 intratu
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moral 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 promotes (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 it decreased remarkably by 7 days after treatment. We demonstrated that Ad-p53 treatment rapidly induced p53-targeted genes and apoptosis in tumors. We also succeeded in visualizing p53 transcriptional activity in vivo. 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-pS3 and novel therapeutic approaches. Less
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[Publications] Ohtani, S., Kagawa, S., Tnago, Y., Umeoka, T., Tokunaga, N., Tsunemitsu, Y., Roth, J.A., Taya, Y.S., Tanaka, N., Fujiwara, T.: "Quantitative analysis of p53-targeted gene expression and visualization of p53 transcriptional activity following intratumoral administration of adenoviral p53 in vivo."Cancer Ther.. 3. 93-100 (2004)
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