Abstract 2252: Gadd45b deficiency impairs G2/M cell cycle progression leading to premature senescence

Abstract

Abstract Gadd45a, Gadd45b, and Gadd45g encode for small (18 kD), evolutionarily conserved proteins that play a pivotal role in diverse cellular functions such as cell cycle arrest, DNA repair, and are regulated by the nature of the stress stimulus encountered, its magnitude, and the cell type. Here, we show that Gadd45b negatively regulates stress-induced senescence. By using the 3T3 growth protocol to study tissue culture-induced senescence; we observed that Gadd45b KO MEFs display reduced growth rates at early passages (by passage 5) compared to wild type (WT) MEFs. Notably, this is in contrast with Gadd45a KO MEFs that show enhanced growth rate and escape senescence. Furthermore, senescence associated (SA) βgal staining showed increased blue stained cells at earlier passages in Gadd45b KO MEFs compared to WT MEFs. When MEFs were cultured at 3% oxygen, Gadd45b KO MEFs at 3% oxygen showed increased growth rate compared to MEFs at 21% oxygen, but growth was still retarded compared to WT MEFs at 21%, indicating that Gadd45b KO MEFs have increased sensitivity to oxidative stress leading to increased senescence. Additionally, we also observed that Gadd45b levels in WT MEFs increased with increasing passage number. Propidium Iodide staining and FACS analysis showed that Gadd45b KO MEFs arrest at the G2M phase of cell cycle. At passage 7, the majority (75%) of Gadd45b KO MEFs were observed to arrest at the G2M phase, whereas only 15% of WT MEFs arrested at G2M. This finding is in striking contrast to other senescent MEFs, which normally arrest at G1. Furthermore, FACS analysis of phospho-histone H3 (ser10) stained cells (mitotic) showed that, Gadd45b KO MEFs have less phospho-histone H3-positive cells than WT MEFs indicating that Gadd45b KO MEFs are arrested in G2 phase rather than M phase. Recent data also indicate that Gadd45b KO MEFs show increased phospho-histone H2Ax and phospho-p53 staining compared to WT MEFs, suggesting that loss of Gadd45b leads to increased DNA damage signaling. Interestingly, other stressors such as sub-lethal H2O2 as well as UV irradiation, known to increase oxidative stress, were observed to trigger increased premature senescence in Gadd45b KO MEFs compared to WT MEFs. Notably, staining embryos for SA βgal, we show that embryos from Gadd45b KO mice exhibit increased SA βgal staining compared to WT embryos, thus providing in vivo evidence for increased senescence in Gadd45b KO mice. Taken together, these data provide the impetus to further elucidate the role of Gadd45b in G2 arrest and premature senescence. Note: This abstract was not presented at the meeting. Citation Format: Andrew Magimaidas, Barbara Hoffman, Dan Liebermann. Gadd45b deficiency impairs G2/M cell cycle progression leading to premature senescence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2252. doi:10.1158/1538-7445.AM2014-2252