PET imaging of endogenous p53 activation and validation of Cis-p53/TKGFP reporter system in cell cultures and sampled tumor tissue. (a) The p53-sensitive reporter vector contains an artificial p53-specific enhancer that activates expression of the TKeGFP reporter gene. (b–e) Transaxial PET images (GE Advance tomograph; General Electric Co., Milwaukee, Wisconsin, USA) through the shoulder (b and d) and pelvis (c and e) of two rats are shown (upper panel); the images are color-coded to the same radioactivity scale (percent dose/g). An untreated animal is shown on the left (b and c), and a BCNU-treated animal is shown on the right (d and e). Both animals have three subcutaneous tumor xenografts: U87 Cis-p53-TKGFP (test) in the right shoulder, U87 wild type (negative control) in the left shoulder, and RG2 TKGFP⁺ (positive control) in the left thigh. The untreated animal on the left shows localization of radioactivity only in the positive control tumor (RG2 TKGFP⁺, c); the test (U87 Cis-p53-TKGFP, b) and negative control (U87wt, b) tumors are at background levels. The BCNU-treated animal on the right shows significant radioactivity localization in the test tumor (right shoulder, d) and in the positive control (left thigh, e), but no radioactivity above background in the negative control (left shoulder, d). (f–i) Fluorescence microscopy and FACS analysis of a transduced U87 Cis-p53-TKGFP cell population in the noninduced (control) state (f and h), and 24 hours after a 2-hour treatment with 40 μg/ml BCNU (g and i). (j and k) Fluorescence microscopic images of U87 Cis-p53-TKGFP subcutaneous tumor samples obtained from untreated rats (j) and rats treated intraperitoneally with 40 mg/kg BCNU (k). The RT-PCR blots from in vitro (l) and in vivo (m) experiments show very low HSV1-tk expression in untreated U87 Cis-p53-TKGFP transduced cells and xenograft-bearing animals, respectively, and no HSV1-tk expression in wild-type U87 cells and tumor tissue, respectively. When U87 Cis-p53-TKGFP transduced cells and xenograft-bearing animals are treated with BCNU, there is a marked increase in HSV1-tk expression comparable to that in constitutively HSV1-tk–expressing RG2TK⁺ cells and xenografts. Adapted with permission from ref. 25.