br Introduction Embryonic stem cells

Introduction Embryonic stem cells (ESCs) are self-renewing pluripotent cells, derived from the inner cell mass of Go 6983 (Smith, 2001). The transcriptional control of pluripotency is well characterized. Master transcription factors, such as Pou5f1 (Oct4), Sox2, and Nanog, maintain pluripotency and suppress lineage-affiliated transcripts. Another distinct feature of ESCs is their fast proliferation rate with a much truncated G1 phase. Such characteristics impose a burden for ESCs to maintain genome stability. ESCs are hyper-sensitive to DNA damage, resulting in high levels of apoptosis (Hong and Stambrook, 2004; Qin et al., 2007). The molecular underpinnings are not well understood. The molecular mechanisms that control ESC cell-cycle progression and genome stability may differ from those in somatic cells. For example, p53 is a well-known tumor suppressor and frequently mutated in cancer (Freed-Pastor and Prives, 2012; Isobe et al., 1986; Kruse and Gu, 2009). The major function of p53 is to maintain genome stability. In response to cellular stresses, p53 is stabilized and activated, resulting in cell-cycle arrest, apoptosis, or senescence by directly trans-activating downstream target genes such as p21, Mdm2, Noxa, and Puma (Vousden and Prives, 2009). However, the role of p53 in ESCs upon DNA damage is controversial, with some studies showing p53-dependent apoptosis while others show opposite results (Aladjem et al., 1998; de Vries et al., 2002). In addition, the role of the p53 protein family member, p73, is not clear in ESCs. The Trp73 gene encodes two major isoforms, TAp73 andΔNp73, transcribed from alternative promoters (Sayan et al., 2010). TAp73 can induce cell death via trans-activation of target genes (Irwin et al., 2000), whereas the amino-terminal truncated ΔNp73 has an anti-apoptotic function (Nakagawa et al., 2002). Retinoblastoma protein (RB) is a well-characterized tumor suppressor that negatively regulates G1/S transition in somatic cells. RB is highly expressed in ESCs but does not seem to be functional in affecting cell-cycle progression, presumably due to its hyper-phosphorylation. However, ESCs require the RB protein family to initiate the differentiation program (Conklin et al., 2012).
Results
Discussion In this study, we investigated the role of p53, which plays a key role in guarding genome stability, in ESCs. Consistent with a previous report that p53 is important for Adriamycin (a trade name of doxorubicin)-induced apoptosis in mouse ESCs (Li et al., 2015), we found that doxorubicin or CDDP robustly activates p53/p73/RB, and cells were arrested at G2/M followed by massive apoptosis in mouse ESCs. Notably, chemical inhibition of CDK1 in ESCs can trigger DNA damage, leading to G2/M arrest and subsequent apoptosis (Huskey et al., 2015). This observation is consistent with our results that G2/M arrest precedes apoptosis. We demonstrate that both p53 and p73 play a pivotal role in promoting apoptosis induced either by DNA damage or by differentiation, whereas they are dispensable in cell-cycle G2M arrest in mouse ESCs in response to DNA damage. ESCs can differentiate into a variety of somatic cells in vitro, providing an excellent system to dissect molecular mechanisms under various cellular contexts. We show that both TAp73 and ΔNp73, two isoforms transcribed from the Trp73 gene using alternative transcription start sites, are upregulated in a p53-dependent manner upon doxorubicin treatment in ESCs, in keeping with a report that p53 can bind the promoter of ΔNp73 (Murray-Zmijewski et al., 2006). Previous studies have shown that TAp73 promotes apoptosis, whereas ΔNp73 functions as a dominant-negative inhibitor of p53 and inhibits TAp73 transcriptional activity to attenuate apoptosis. In this study, we found that p53-induced TAp73 is largely responsible for doxorubicin-induced apoptosis in mouse ESCs, while ectopic expression of ΔNp73 suppresses this process. Strikingly, we found that induction of p73 upon ESC differentiation is independent of p53. Thus, the molecular pathway in the regulation of p73 is different upon DNA damage or upon differentiation in mouse ESCs. Consistently, knockdown of p53 alone (and thus reduction of p73 expression) is sufficient to block doxorubicin-induced apoptosis, while simultaneous knockdown of both p53 and p73 completely abolishes differentiation-induced apoptosis. It is plausible that regulation of p73 during differentiation using a p53-independent pathway may be because p73 functions in developmental processes such as neurogenesis. It will be of interest to investigate how p73 is regulated during differentiation, and how this molecular pathway overrides the regulation by p53.