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

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-tk͞GFP (TKGFP) dual reporter gene was used to monitor transcriptional activation of p53-dependent genes. A retrovirus bearing the Cis-p53͞TKGFP 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-␤-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 molecularbiological 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.

Imaging in neurooncology

“…These experimental strategies have been reviewed in detail previously. 67 New developments aim toward 1) the detection of tumor cell migration in vivo, 160 2) the establishment of in vivo assays for direct imaging of tumor-specific signal transduction pathways (e.g., p53-, E2F-1 and HIF-1-␣ regulated pathways [161][162][163][164] ), 3) the design of labeled peptides binding specifically to the cell adhesion receptor integrin ␣(v)␤ 3 or other tumor-specific antigens and of labeled bone marrow-derived endothelial precursor cells to allow highly specific tumor visualization and the study of glioma angiogenesis and neovascularization, [165][166][167][168][169] 4) the generation and in vivo characterization of transgenic mice with gliomas induced by signaling through Ras and Akt pathways, 170 and 5) the construction of bifunctional imaging marker and therapeutic genes to allow direct assessment of therapeutic gene expression in culture and in vivo models by directly corresponding assays. 171,172 Especially the design of small tumor-specific antibody fragments is an attractive way for specific detection of Many of the current experimental protocols investigating new drug and treatment strategies for experimental gliomas include MRI, optical or PET imaging of either the distribution of therapeutic agents, [173][174][175] or therapyinduced tumor-changes, 171,176 -184 with the overall attempt of designing image-guided treatments.…”

The role of p53 in chemosensitivity and radiosensitivity

“…With the recent identification of tissue-specific p53 gene activation responses in vivo come a number of important insights into drug and radiation toxicity, as well as possible future efforts to further refine p53 blockade or altered specificity (Burns et al, 2001;El-Deiry, 2001;Fei et al, 2002). The emergence of imaging technologies for noninvasive detection of tumors, as well as the monitoring of molecular changes such as alterations in gene expression or protein-protein interaction, have already begun to impact on the role and activities of p53 in tumor therapy (Doubrovin et al, 2001;Massoud and Gambhir, 2003;. A number of open questions remain in the field, as does the promise of effective use of p53 in directing patient care either in terms of diagnosis or therapy.…”

Molecular imaging of gene therapy for cancer

“…A retroviral vector, Cis-p53/TK-GFP, was generated by placing the HSV-TK-GFP fusion gene 26 under control of an artificial cis-acting p53-specific enhancer element. 27 Following retroviral transduction of tumor cells in established oesteosarcoma xenografts, DNA damage induced upregulation of p53 transcriptional activity, which correlated with the expression of p53-dependent , indirect quantification of other biological properties of the hrR3 vector with the radiotracer assay used for imaging. Proliferating rat 9L gliosarcoma cells in culture were infected with hrR3 at a multiplicity of infection of 1.5 PFU/cell.…”