1. ¹Department of Cancer Biology, Lerner Research Institute, Cleveland, OH, USA
2. ²Department of Environmental Health Sciences, Case Western Reserve University, Cleveland, OH, USA
3. ³Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA
4. ⁴Department of Radiation Oncology, Cleveland Clinic, Cleveland, OH, USA

Correspondence: Dr A Almasan, Departments of Cancer Biology and Radiation Oncology, Cleveland Clinic, NB40, 9500 Euclid Avenue, Cleveland, OH 44195, USA. E-mail: almasaa@ccf.org

Received 3 April 2006; Revised 3 July 2006; Accepted 18 July 2006; Published online 9 October 2006.

Abstract

The retinoblastoma (pRB) family proteins regulate the E2F transcription factors; their complexes regulate critical transitions through the cell cycle. The function of these pRB family/E2F complexes, which includes p130/E2F4, in response to genotoxic agents, is not well understood. We investigated the role of E2F4 in the genotoxic stress response. Following radiation treatment, E2F4 colocalized with p130 in the nucleus during a radiation-induced stable G₂-phase arrest. Arrested cells had significantly decreased expression of Cyclins A2 and B1 and decreased phosphorylation of mitotic protein monoclonal-2 (MPM-2) mitotic proteins. Small interference RNA (siRNA)-mediated knockdown of E2F4 sensitized cells to subsequent irradiation, resulting in enhanced cellular DNA damage and cell death, as determined by caspase activation and decreased clonogenic cell survival. Downstream E2F4 targets potentially involved in the progression from G₂ into M phase were identified by oligonucleotide microarray expression profiling. Chromatin immunoprecipitation localized E2F4 at promoter regions of the Bub3 and Pttg1 mitotic genes following irradiation, which were among the downregulated genes identified by the microarray. These data suggest that in response to radiation, E2F4 becomes active in the nucleus, enforces a stable G₂ arrest by target gene repression, and thus provides increased cell survival ability by minimizing propagation of cells that have irreparable DNA damage.