Imaging transcriptional regulation of p53-dependent genes with positron emission tomography in vivo

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Abstract
A noninvasive method for molecular imaging of the activity of different signal transduction pathways and the expression of different genes in vivo would be of considerable value. It would aid in understanding the role specific genes and signal transduction pathways have in various diseases, and could elucidate temporal dynamics and regulation at different stages of disease and during various therapeutic interventions. We developed and assessed a method for monitoring the transcriptional activation of endogenous genes by positron-emission tomography (PET) imaging. The HSV1-tk/GFP (TKGFP) dual reporter gene was used to monitor transcriptional activation of p53-dependent genes. A retrovirus bearing the Cis-p53/TKGFP reporter system was constructed in which the TKGFP reporter gene was placed under control of an artificial cis-acting p53-specific enhancer. U87 glioma and SaOS-2 osteosarcoma cells were transduced with this retrovirus and used to establish xenografts in rats. We demonstrated that DNA damage-induced up-regulation of p53 transcriptional activity correlated with the expression of p53-dependent downstream genes, such as p21, in U87 (wild-type p53), but not in SaOS-2 osteosarcoma (p53 -/-) cells. We showed that PET, with [(124)I]FIAU (2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-[(124)I]iodouracil) and the Cis-p53TKGFP reporter system, is sufficiently sensitive to image the transcriptional regulation of genes in the p53 signal transduction pathway. These imaging results were confirmed by independent measurements of p53 activity and the expression levels of downstream genes (e.g., p21) by using conventional molecular-biological assays. PET imaging of p53 transcriptional activity in tumor xenografts by using the Cis-p53TKGFP reporter system may be useful in assessing novel therapeutic approaches.