[Cancer Biology & Therapy 2:2, 203-205, March/April 2003]; ©2003 Landes Bioscience
Commentary
Seeing is Believing Visualization of Transcriptional Activity of p53
*Correspondence to: Cancer Molecular Sciences, Pfizer Global Research and Development; Ann Arbor Laboratories; 2800 Plymouth Road; Ann Arbor, Michigan 48105 USA; Tel.: 734.622.1959; Fax: 734.622.5668; Email:
[email protected] Received 04/02/03; Accepted 04/02/03
KEY WORDS therapy,
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Bioluminescent Molecular Imaging of Endogenous and Exogenous p53-Mediated Transcription In Vitro and In Vivo Using an HCT116 Human Colon Carcinoma Xenograft Model
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Previously published online as a CB&T “Paper in Press” at: http://landesbioscience.com/journals/cbt/
Human p53 is a 393-amino acid nuclear protein that consists of three major domains: the N-terminal transactivation domain; a DNA binding domain in the center portion of the molecule; and a C-terminal oligomerization domain.1 Biochemically, p53 is a transcription factor that specifically binds to its consensus DNA binding sequence, 5´-PuPuPuC(A/T)(T/A)GPyPyPy N(0–13) PuPuPuC(A/T)(T/A)GPyPyPy-3’, located in the promoter or introns of the downstream target genes, and transactivates the expression of these genes.2 Biologically, p53 is a stress responsive protein and a tumor suppressor. Upon exposure to a variety of stimuli, including DNA damaging agents such as ionizing radiation, UV and anti-cancer drugs as well as hypoxia, oxidative stress, oncogene activation, cell adhesion, or rNTP depletion,3 p53 is activated through multiple mechanisms including phosphorylation and acetylation.4 Activated p53 acts as a guardian of the genome by inducing either growth arrest to allow the repair of cellular damage or apoptosis to eliminate irreparable cells.5,6 Major biological functions of p53 are mediated through transcriptional activation of its downstream target genes. For example, p53-induced growth arrest is achieved mainly by transactivation of Waf-1/p21 (for G1 arrest),7 of 14-3-3σ (for G2 arrest),8 and of PTGF-β.9 p53-induced apoptosis, on the other hand, is mediated by activation of genes involved in two major apoptotic pathways: the mitochondrial pathway including BAX, PIGs, NOXA, PUMA, p53AIP,10-15 and the death receptor pathway including KILLER/DR5, FAS/APO1, PIDD.16-18 Other p53-inducible genes that promote apoptosis include PERP,19 and APAF-1.20 p53 also activates DNA repair genes such as GADD45, p53R2, PCNA and DDB2/p4821-24 to regulate damage repair processes. In addition, p53 regulates angiogenesis and tumor metastasis by transcriptional activation or repression of a group of genes involved in angiogenesis and cell invasion/metastasis.5,25 Thus, dynamically monitoring p53 transcriptional activity would lead to a better understanding of biological processes regulated by p53. Two common approaches have been extensively used to monitor p53 transcriptional activity. The first is through the measurement of activity of CAT or luciferase reporter genes, driven either by a repetitive p53 consensus binding sequence or by a promoter fragment of known p53 target genes that contains such a binding site. The second is through the measurement of mRNA or protein levels of known p53 targets. All these approaches require cells or tissues being harvested at a given time point and a time-consuming process of sample preparation and measurement. Thus, they are quite impractical and inconvenient, particularly in in vivo animal studies and in the clinic. In this issue of Cancer Biology & Therapy, Wang and El-Deiry used a bioluminescent imaging technique26 to monitor the transcriptional activity of endogenous, as well as exogenous p53, in both in vitro and in vivo tumor models in a real-time, non-invasive, repetitive and quantitative manner.27 They engineered HCT116 colon carcinoma cells that harbor an endogenous p53 to stably express a PG13-luc, a well-characterized p53-responsive luciferase reporter.28 p53 activity was monitored by adding D-luciferin, the substrate of firefly luciferase, into the culture medium in an in vitro culture model or by tail-vein or intraperitoneal injection in an in vivo xenograft model, followed by bioluminescent imaging. Remarkably, although the p53 level in unstressed HCT116 cells is quite low, endogenous p53 activity can be readily detected as a bioluminescent signal in as little as a few thousand cultured cells or in 1 x 105 cells after subcutaneous injection into animals. This method, therefore, showed a much higher sensitivity in detecting p53 activity than the conventional approaches. Furthermore, the signal intensity, as reflected by p53 activity, was increased in a dosedependent manner up to 8-fold when exogenous p53 was expressed via adenoviral infection of a p53-expressing construct (Ad-p53) in cultured cells. Enhanced intensity was also observed in Ad-p53 infected cells after being inoculated into in vivo animals as well as in xenografted tumors developed from these cells, following a burst of Ad-p53 injection.27
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VISUALIZATION OF TRANSCRIPTIONAL ACTIVITY OF P53
Three imaging technologies are currently being used in biomedical research:29 1. Radionuclide techniques that uses single photon emission computed tomography (SPECT) and or positron emission tomography (PET); 2. Optical fluorescence imaging (OFI) that includes intravital microscopy of green fluorescent protein expression, bioluminescence, and near-infrared fluorescence; and 3. Magnetic resonance imaging (MRI).
These technologies have been used to monitor expression of various genes,30-37 enzymatic activities or proteolytic processing,38,39 proteinprotein interactions,40-41 virus infections,42 cancer treatment responses,43 and apoptosis.44 The in vivo transcriptional regulation of p53-dependent genes was recently reported using GFP fluorescent reporter and PET techniques.36 Although PET offers some technical advantages over bioluminescence, such as relatively higher sensitivity, deeper tissue penetration, and full quantitative capability,29 it has an inherently lower spatial resolution, and involves the use of radioactive materials and relative complicated probe labeling methodologies. In addition, compared to GFP fluorescence, the luciferase bioluminescence used by Wang and El-Diery offers some advantages, such as inherently low background and real-time activity measurements due to a quicker turnover of luciferase enzyme in the presence of substrate.26 What are the potential applications of this p53 imaging technique? It appears that the failures in current cancer therapy are mainly attributable to the development of resistance to apoptosis in cancer cells. Mutant p53-bearing cancer cells, which account for 50% of human cancers,1 are in general more resistant to apoptosis induced by anti-cancer therapies than those containing wt p53.45,46 Thus, rational cancer therapy calls for agents that use p53 to kill cancer cells.47 One approach to achieve this is through reactivating p53 in cancer cells with mutant p53. To this end, two small molecular weight compounds, CP-31398 and PRIMA have been identified and characterized. These compounds are reported to change p53 conformation from mutant to wild type, stabilize p53 protein, and restore p53 tumor suppressor function in cancer cells harboring a mutant p53.48-50 On the other hand, p53 activation, followed by induction of apoptosis in normal cells appears to be responsible for many side effects of cancer chemotherapies. A small molecular weight compound, PFTα, was identified and reported to inhibit p53 activity, and to protect mice from the lethal side effects of anticancer treatments.51 The in vitro and in vivo bioluminescent imaging models developed by Wang and El-Deiry, or similar models to be established in p53 mutant cells, can be used to evaluate the efficacy of these p53 regulatory compounds by monitoring induction or inhibition of p53 activity in a real-time and non-invasive way. The in vivo model can be particularly be useful in monitoring whether drugs are active and if so, their active half-life in tumors. The data can then be used to design dose regimen to achieve a maximal efficacy with a minimal toxicity. On the drug discovery side, the in vitro model can be used for cell-based, high-throughput screening for compounds that either inhibit p53 or activate p53. Active compounds can be further evaluated in in vivo models as described. Furthermore, these models can be used to evaluate whether a particular anti-cancer agent acts via activation of p53, followed by induction of apoptosis, in order to monitor the cancer therapeutic response and to assess treatment efficacy. Thus, noninvasive bioluminescent imaging to monitor real-time in vivo activity of p53 provides an opportunity for obtaining more specific information about physiological, as well as pathological, processes affected by p53. 204
It should also significantly reduce the cost of p53-based anticancer drug discovery by allowing the use of fewer animals in testing, facilitating design of optimal dosing regimens and helping scientists to make early decisions on advancing or terminating a compound based upon target modulation. References 1. Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 1994; 54:4855-78. 2. el Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B. Definition of a consensus binding site for p53. Nat Genet 1992; 1:45-9. 3. Giaccia AJ, Kastan MB. The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev 1998; 12:2973-83. 4. Ko LJ, Prives C. p53: puzzle and paradigm. Genes Dev 1996; 10:1054-72. 5. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000; 408:307-10. 6. Stewart ZA, Pietenpol JA. p53 Signaling and cell cycle checkpoints. 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