Loss of retinoblastoma (Rb) tumor suppressor function leads to deregulated cell proliferation and contributes to the development of most human cancers. In response to Rb inactivation, the p53 tumor suppressor is activated and this usually results in the elimination of the cell by apoptosis. This """"""""guardian of Rb"""""""" function for p53 plays a critical role in suppressing tumorigenesis. Experimental evidence demonstrates that the signaling pathway from Rb inactivation to p53 activation requires the E2F1 transcription factor. Deregulation of E2F1, by overexpression or Rb inactivation, results in p53 accumulation and the induction of apoptosis. It has been widely speculated that the alternative reading frame (ARF) tumor suppressor, an inhibitor of mdm2, mediates the activation of p53 by E2F1. This suggestion is based on the findings that E2F1 transcriptionally activates the ARF gene promoter and that other oncogenes, such as Myc, require ARF to induce p53-dependent apoptosis. In preliminary data we demonstrate that ARF is in fact dispensable for E2F1-induced apoptosis. Instead, the induction of p53-dependent apoptosis by E2F1 is correlated with the caffeine-sensitive phosphorylation of p53. Moreover, the ataxia-telangiectasia mutated (ATM) kinase, which has been implicated in activating p53 in response to DNA damage, is found to be required for E2F1-induced p53 phosphorylation and apoptosis. In contrast, the ability of E2F 1 to stimulate the expression of target genes and to promote S phase entry is unaffected by the absence of ATM. The product of the Nijmegen breakage syndrome gene, NBS1, is also shown to be required for E2F1 to induce the phosphorylation of p53 and apoptosis. NBS1 is part of the Mre11/Rad50 DNA repair complex and has recently been shown to directly bind E2F1. These findings expand the known functions for ATM and NBS1 and significantly alter the current model for how cell cycle deregulation activates p53. Our hypothesis is that deregulated E2F1 activity stimulates ATM in an NBS1-dependent manner to activate p53 and perhaps other checkpoint response factors. This E2F1-ATM pathway may respond to both cell cycle deregulation and DNA damage to induce apoptosis and suppress tumorigenesis. A major goal of these studies will be to molecularly define the roles of ATM and NBS1 in the signaling pathway between E2F1 and p53. A role for p73 and c-Abl in E2F1-induced, ATM-dependent apoptosis will also be explored. Finally, the functional relationship between E2F1, ATM and NBS1 in modulating tumor development will be examined using murine models.
