Activating the p53 DNA damage response

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The mechanism by which p53 activation occurs in response to DNA damage has remained unresolved. The potential role of DNA-PK and an unidentified factor(s) ("Factor X") in signaling DNA damage and activating p53 has been suggested (Woo et al., 1998). Here data are obtained in vitro indicating that "factor X" is the checkpoint kinase 2 (Chk2) protein. Experimental evidence points to a pathway in which DNA-PK and Chk2 act synergistically and sequentially to activate p53 sequence specific DNA binding in vitro, and specifically play a role in the activation of a latent population of p53. In response to genotoxic stress, mammalian cells can activate cell cycle checkpoint pathways to arrest the cell for repair of DNA or induce apoptosis to eliminate damaged cells. Based on the in vitro data obtained, the ability of DNA-PK and Chk2 in mediating these p53 responses was investigated. It has been suggested that Chk2 functions downstream of ATM in G 1 cell cycle arrest. Using Chk2 -/- mouse embryonic fibroblasts (MEFs), it has been observed that unlike ATM-/- or p53 -/- MEFs, these cells behave like normal MEFs in manifesting p21 induction and G 1 arrest upon exposure to ionizing radiation. Therefore Chk2 is not involved in p53 mediated G 1 arrest. The role of Chk2 and DNA-PK were then examined in the p53 dependent apoptotic response using adenovirus ElA expressing MEFs. It is shown that Chk2 -/- and DNA-PK-/-, like p53 -/­cells, did not undergo DNA damage induced apoptosis while ATM -/- cells behaved like normal cells in their ability to invoke an apoptotic response. The role of the serine-15 residue on p53 mediated apoptosis was investigated using p53 S 15A knock in MEFs expressing E 1 A and these cells exhibited an attenuated apoptotic response indicating the contribution of this residue in mediating this response. Finally, the ability of these cells to undergo apoptosis in the absence of protein synthesis suggests that pre-existing, "latent", p53 is sufficient for executing this response. Taken together, these studies indicate that Chk2 and DNA-PK are involved in the activation of latent p53, independently of ATM, and specifically in triggering DNA damage-induced apoptosis.
Bibliography: p. 135-167
Some pages are in colour.
Jack, M. T. (2004). Activating the p53 DNA damage response (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/24522