Further analysis by immunohistochemistry demonstrated elevated expression of pChk2, γH2AX (S139), phosphorylated p53 (on serine 15 and 20) in β2SP+/− mice at different timepoints in comparison to wildtype mice (Fig. 4A,B, Supporting Table 2). Moreover, peak phosphorylated p53 expression correlated with elevated p21 expression, suggesting activation of the DNA damage response and downstream target gene regulation by p53, leading to growth arrest and delayed liver regeneration. Given the elevated levels of p53 in β2SP+/− mouse livers following PHx, we further investigated whether

down-regulation of p53 influences cell cycle progression of β2SP+/− MEFs and primary hepatocytes Nutlin-3 cost in which β2SP levels were reduced by shRNA-mediated Angiogenesis inhibitor knockdown. Under serum-starved

conditions, β2SP−/− mutant (MT) MEFs showed a marked reduction of the G0/G1 population in comparison to wildtype control MEFs (Fig. 5), consistent with in vivo data obtained from β2SP+/− PHx mouse livers. There was no significant difference between wildtype MEFs transfected with p53 shRNA and untransfected wildtype MEFs. These results suggest that, whereas p53 induction is associated with reduced β2SP levels in vivo following PHx, p53 is not the exclusive mediator of aberrant cell cycle in β2SP+/− hepatocytes. It is likely that factors other than p53 also contribute to the defects in cell cycle progression observed in regenerating β2SP+/− hepatocytes. β2SP has been previously demonstrated to be a key adaptor protein in TGF-β signaling and a key tumor-suppressor protein in foregut cancers: nearly 40% of β2SP+/− mice develop hepatocellular carcinoma by 15 months of age and nearly 90% of β2SP+/−/Smad4+/− double heterozygote mutants develop gastric tumors.13, 20 Moreover, FACS analysis of β2SP−/− MEFs following serum starvation and release demonstrated reduction of the G1 population and faster entry into S phase in mutant MEFs compared with wildtype.16 These observations suggest that β2SP plays a key role in

cell cycle progression and tumorigenesis. To further investigate this role in vivo, we examined liver regeneration Loperamide following PHx. Interestingly, our results demonstrated that diminished expression of β2SP is associated with increased DNA damage, activation of the DNA damage response proteins p53 and p21, and G2/M-phase arrest following PHx. TGF-β, which is capable of inducing an antiproliferative gene response at any point in the cell cycle, is most effective at inhibiting cell cycle progression during G1 by way of inhibition of cyclins D, E, and A or by way of Cdk4 synthesis.21-24 Disruption of TGF-β signaling in liver regeneration, by way of conditional knockout of the TGF-β type II receptor (TBRII), has similarly demonstrated a nearly 2-fold increase and acceleration of S-phase entry following hepatectomy.