title: Bioluminescence detection of cells having stabilized p53 in response to a genotoxic event.
abstract: Inactivation of p53 is one of the most frequent molecular events in neoplastic transformation. Approximately 60% of all human tumors have mutations in both p53 alleles. Wild-type p53 activity is regulated in large part by the proteosome-dependent degradation of p53, resulting in a short p53 half-life in unstressed and untransformed cells. Activation of p53 by a variety of stimuli, including DNA damage induced by genotoxic drugs or radiation, is accomplished by stabilization of wild-type p53. The stabilized and active p53 can result in either cell-cycle arrest or apoptosis. Surprisingly, the majority of tumor-associated, inactivating p53 mutations also result in p53 accumulation. Thus, constitutive elevation of p53 levels in cells is a reliable measure of p53 inactivation, whereas transiently increased p53 levels reflect a recent genotoxic stress. In order to facilitate noninvasive imaging of p53 accumulation, we here describe the construction of a p53-luciferase fusion protein. Induction of DNA damage in cells expressing the fusion protein resulted in a time-dependent accumulation of the fusion that was noninvasively detected using bioluminescence imaging and validated by Western blot analysis. The p53-Luc protein retains p53 function because its expression in HCT116 cells lacking functional p53 resulted in activation of p21 expression as well as induction of apoptosis in response to a DNA damaging event. Employed in a transgenic animal model, the proposed p53-reporter fusion protein will be useful for studying p53 activation in response to exposure to DNA-damaging carcinogenic agents. It could also be used to study p53 stabilization as a result of inactivating p53 mutations. Such studies will further our understanding of p53's role as the "guardian of the genome" and its function in tumorigenesis.
authors.affiliation: Department of Radiation Oncology, University of Michigan School of Medicine, Ann Arbor 48109, USA. alnawaz@umich.edu
authors.forename: Alnawaz
authors.initials: A
authors.lastname: Rehemtulla
authors.forename: Neelam
authors.initials: N
authors.lastname: Taneja
authors.forename: Brian D
authors.initials: BD
authors.lastname: Ross
mesh_terms: D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004249:DNA Damage; D005260:Female; D005786:Gene Expression Regulation / Q000528:radiation effects; D017930:Genes, Reporter; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D008163:Luminescent Measurements; D009154:Mutation; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D051736:Proto-Oncogene Proteins c-mdm2; D011839:Radiation, Ionizing; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects
chemical_list: C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D008156:Luciferases; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2