ORIGINAL ARTICLE Reduction of apoptosis in Rb-deficient embryos via Abl knockout HL Borges 1 , IC Hunton and JYJ Wang Division of Hematology/Oncology, Department of Medicine, Moores Cancer Center, School of Medicine, University of California San Diego, La Jolla, CA, USA The retinoblastoma protein RB regulates cell prolifera- tion, differentiation and apoptosis. Homozygous knockout of Rb in mice causes embryonic lethality owing to placental defects that result in excessive apoptosis. RB bindstoanumberofcellularproteinsincludingthenuclear Ablproteinandinhibitsitstyrosinekinaseactivity. Ex vivo experiments have shown that genotoxic or inflammatory stress can activate Abl kinase to stimulate apoptosis. Employing the Rb-null embryos as an in vivo model of apoptosis, we have shown that the genetic ablation of Abl can reduce apoptosis in the developing central nervous system and the embryonic liver. These results are consistent with the inhibitory interaction between RB and Abl, and provide in vivo evidence for the proapoptotic function of Abl. Oncogene advance online publication, 18 December 2006; doi:10.1038/sj.onc.1210157 Keywords: central nervous system; fetal live; haploid insufficiency; knockout mice; tumor suppressor Introduction The human retinoblastoma susceptibility gene, Rb, encodes a nuclear protein that regulates cell cycle progression, terminal differentiation and programmed cell death (Chau and Wang, 2003; Liu et al., 2004; Nguyen and McCance, 2005). In mice, the constitutive knockout of Rb causes embryonic lethality resulting from defects in placental function (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992; de Bruin et al., 2003; Wu et al., 2003). The Rb-knockout embryos exhibit inappropriate cell cycle entry and massive apoptosis in the developing central nervous system (CNS), peripheral nervous system, lens, liver and muscles (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992). The excessive apoptosis, but not the ectopic S-phase, of Rb-null embryos is rescued when the embryos are developed on wild-type placentas (de Bruin et al., 2003; Wu et al., 2003). The mechanism by which RB inhibits cell prolifera- tion is well established. RB interacts with E2F to repress transcription of genes critical for cell cycle progression (Liu et al., 2004; Nguyen and McCance, 2005). The ectopic cell proliferation in Rb-null embryos is sup- pressed by the knockout of E2f1 or E2f3, providing in vivo evidence for the critical role of RB in the control of E2F activity (Tsai et al., 1998; Ziebold et al., 2001). The E2f-knockouts also reduced the apoptosis of Rb-null embryos, consistent with the proapoptotic functions of E2F (Tsai et al., 1998; Ziebold et al., 2001). Overproduction of E2F-1 in combination with the deprivation of survival signals can lead to activation of p53-dependent apoptosis through the intrinsic death pathway (Hickman et al., 2002; Sherr and McCormick, 2002). Consistently, the knockout of p53 or Apaf-1 also reduced apoptosis in the Rb-null embryos (Morgenbes- ser et al., 1994; Macleod et al., 1996; Guo et al., 2001). These results suggest the Rb-null placental defects can somehow activate the intrinsic apoptotic pathway in the Rb-null embryos. In cultured cells, E2F-1 upregulates the expression of p19Arf, which activates p53 through the sequestration of Mdm2 (Hickman etal., 2002; Sherr and McCormick, 2002). However, knockout of p19Arf did not rescue the apoptotic phenotype of Rb-null embryos (Tsai etal., 2002). This result suggests that p53 maybe activated by other pathways to induce apoptosis in the Rb-null embryos. The RB protein contains several distinct peptide- binding ‘pockets’ that mediate its interaction with a large number of viral and cellular proteins (Morris and Dyson, 2001; Chau and Wang, 2003). The interaction between RB and the C-terminal peptide of E2F-1, E2F-2 and E2F-3 has been elucidated by X-ray crystallography to involve the RB A/B domain (Lee et al., 2002; Xiao etal., 2003). The crystal structure of a shallow groove in the RB B-domain binding to the LxCxE-peptide found in viral oncoproteins such as E7 has also been solved (Lee et al., 1998; Liu et al., 2006). Recently, the three- dimensional structure of RB C-region in complex with an E2F/DP heterodimer has been reported (Rubin etal., 2005). The RB C-region also interacts with a Pro, Glu, Asn, Phe (‘PENF’) peptide motif found in several cellular proteins (Darnell et al., 2003), and a binding site for cyclin-dependent protein kinases (Adams et al., 1999). A majority of the RB-interacting proteins are Received 16 August 2006; revised 13 October 2006; accepted 17 October 2006 Correspondence: Dr JYJ Wang, Moores Cancer Center, Room 4328, School of Medicine, University of California, San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093-0820, USA. E-mail: jywang@ucsd.edu 1 Current address: Instituto de Cieˆncias Biome´dicas, Departamento de Anatomia, Universidade Federal do Rio de Janeiro, Centro de Cieˆncias da Sau´de, Rio de Janeiro, 21.941-590, Brazil. Oncogene (2006), 1–10 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc