DN-p73 is activated after DNA damage in a p53-dependent manner to regulate p53-induced cell cycle arrest Stefania Vossio 1,4 , Emanuele Palescandolo 1,4 , Natalia Pediconi 1,4 , Francesca Moretti 1 , Clara Balsano 1,2 , Massimo Levrero* ,1,3 and Antonio Costanzo* ,1 1 Laboratory of Gene Expression, Fondazione Andrea Cesalpino, University of Rome ‘‘La Sapienza’’, Rome, Italy; 2 Dip. Medicina Interna, University of L’Aquila, L’Aquila, Italy; 3 Dip. Scienze Mediche Internistiche, University of Cagliari, Cagliari, Italy p53 and p73 genes are both activated in response to DNA damage to induce either cell cycle arrest or apoptosis, depending on the strength and the quality of the damaging stimulus. p53/p73 transcriptional activity must be tightly regulated to ensure that the appropriate biological response is achieved and to allow the cell to re- enter into the cell cycle after the damage has been repaired. In addition to multiple transcriptionally active (TA) isoforms, dominant negative (DN) variants, that lack the amino-terminal transactivation domain and function as trans-repressors of p53, p63 and p73, are expressed from a second internal promoter (P2-p73Pr). Here we show that, in response to a non apoptotic DNA damage induced by low doses of doxorubicin, p53 binds in vivo, as detected by a p53-specific chromatin immunoprecipitation assay, and activates the P2-p73 promoter. DN-p73a protein accumulates under the same conditions and exogenously expressed DN-p73a is able to counteract the p53-induced activation of the P2- p73Pr. These results suggest that DN-p73 may con- tribute to the autoregulatory loops responsible for the termination of p53/p73 responses in cells that do not undergo apoptosis. Accordingly, the activation of the P2- p73Pr is markedly enhanced in both p737/7 murine fibroblasts and in human cells in which p73 transcripts are selectively knocked-out by p73-specific small inter- fering RNAs. Oncogene (2002) 21, 3796 – 3803. DOI: 10.1038/sj/ onc/1205465 Keywords: p53; p73; oncogenes; tumor suppressor; apoptosis; cell cycle Damage to chromosomal DNA induces a complex cellular response designed to delay cell cycle progres- sion until the DNA is repaired (Zhou and Elledge, 2000). In the event of irreparable damage the cell undergoes apoptotic cell death in order to preserve genome integrity. Response to DNA damage is critical for cell viability. Unchecked DNA damage can lead to mutations, translocations and abnormal recombination events during S phase and also chromosome breakage and loss during mitosis. Failure to monitor or signal damaged DNA is a hallmark of cancer cells (Hartwell and Kastan, 1994). The choice between cell cycle arrest and apoptosis is influenced by both the quality and the strength of the DNA damage imposed to the cell. In general, cell cycle arrest is prevalent when cells are treated with low concentrations of DNA damaging agents and apoptosis is triggered in response to higher dosages. Several signal transduction pathways are activated and integrated within the cell to implement the appropriate decision (Rich et al., 2000). Many of these signals converge to regulate the activity of both the p53 tumor suppressor gene (Prives and Hall, 1999) and its family members p63 or p73 (Agami et al., 1999; Gong et al., 1999; Levrero et al., 1999, 2000; Yuan et al., 1999). Dierently from p53, p73 exists as a group of full length isoforms that arise from alternative splicings at the C-terminus (TA-p73 a-z) (De Laurenzi et al., 1998, 1999) and are all able to induce both cell cycle arrest and apoptosis when overexpressed. In addition, amino-terminally truncated isoforms (DN- p73s) that lack the transactivation domain and exert a dominant negative function towards p53, p73 and p63 activity have been described (Sayan et al., 2001). DN- p73 is expressed in vivo in the developing brain where it counteracts p53 dependent apoptosis (Pozniak et al., 2000). Similarly, truncated isoforms of the related protein p63 may act as dominant negative inhibitors of p53 and p73 dependent transcription (Yang et al., 1998) and protect keratinocytes from DNA damage induced apoptosis in vitro and in vivo (Liefer et al., 2000). Full-length TA-p73 and truncated DN-p73 mRNAs are transcribed from two dierent promoters named P1-p73Pr and P2-p73Pr, respectively. Whereas the regulation of the P1-p73 promoter has been characterized in several cellular systems and found to be activated in a E2F1-dependent manner (Irwin et al., 2000; Lissy et al., 2000; Pediconi et al., manuscript in preparation) very little is known about the transcrip- tional regulation of the DN-p73 proteins. To get insights into how the P2-p73Pr is regulated by Oncogene (2002) 21, 3796 – 3803 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc *Correspondence: A Costanzo and M Levrero, Laboratory of Gene Expression, Fondazione Andrea Cesalpino, University of Rome La Sapienza, Policlinico Umberto I, Viale del Policlinico 155, 00161 Rome, Italy; Email: levmax@tin.it 4 These authors contributed equally to the work Received 12 December 2001; revised 2 February 2002; accepted 12 March 2002