Mutation Research 494 (2001) 73–85 Identification of genotoxic stress in human cells by fluorescent monitoring of p53 expression Ariel Quiñones ∗ , Nikolai G. Rainov Department of Neurosurgery, Martin-Luther-University, Magdeburger-Strasse 16, D-06112 Halle, Germany Received 16 January 2001; received in revised form 5 April 2001; accepted 12 April 2001 Abstract The tumor suppressor protein p53 is induced upon DNA damage essentially by post-translational regulatory mechanisms, which lead to a substantial increase of p53 levels. To exploit this essential property of p53, we developed a novel reporter system for monitoring accumulation and subcellular translocation of p53 protein, which is able to function as a simple test for detecting mutagenic and genotoxic stress in human cells. For this purpose, we constructed a plasmid with a specific translational TP53::EGFP gene fusion and selected stable transfected clones in the human cell line HEK293, in which p53 is functionally stabilized due to the expression of the transgenic adenoviral E1A oncoproteins. HEK293-TP53::EGFP clones may be used as a living cell system for monitoring not only of the induction of p53 protein in the cell, but also of its subcellular localization. Using this human reporter cell system, we examined levels of p53 by fluorescence microscopy and by FACS analysis following treatment with several classes of genotoxic and carcinogenic compounds. All tested DNA damaging agents caused a significant increase of intracellular p53-EGFP levels in a concentration-dependent manner. On the other hand, non-genotoxic carcinogens and stress conditions that cannot damage DNA were not able to induce p53-EGFP accumulation. The induction effect caused by genotoxic stress was found to be dependent on the endogenous p53 status, because it was not observed in p53-deficient cell lines. This corroborates the notion that p53 may be used as an universal sensor for genotoxic stress and demonstrates the usefulness of HEK293-p53-EGFP cells as a reporter system for identification of mutagens and genotoxic carcinogens in human cells by means of visualizing and monitoring intracellular p53 levels and localization. © 2001 Elsevier Science B.V. All rights reserved. Keywords: p53; DNA-damage; Genotoxic stress; Mutagens; Carcinogens; EGFP 1. Introduction The tumor suppressor gene TP53 encodes a tetrameric nuclear phosphoprotein, p53, which plays a central role in the control of the cell cycle, in the reg- ulation of apoptosis and in maintaining the genomic integrity of the cell (for reviews see: [1–6]). The ∗ Corresponding author. Tel.: +49-345-5574050; fax: +49-345-557-1412. E-mail address: ariel.quinones@medizin.uni-halle.de (A. Quiñones). human p53 protein consists of 393 amino acids (aa) and has been functionally divided into four principal domains: the N-terminal transactivation domain (aa 1–43) [7], the central DNA-binding core domain (aa 102–292) [8], which also exhibits 3 ′ –5 ′ exonuclease activity [9], the tetramerization domain (aa 325–355) [10,11] and the C-terminal regulatory domain that exhibits sequence-unspecific DNA- and RNA-binding properties (aa 363–393) [11]. Furthermore, a princi- pal and two secondary nuclear localization signals have been found flanking the tetramerization do- main, respectively (NLS, aa 316–324, 369–375, and 1383-5718/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII:S1383-5718(01)00179-6