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Cooperative Effect of P21cip1/WAFА1 and 14-3-3R on Cell Cycle Arrest and Apoptosis Induction by P14arf

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Cooperative Effect of P21cip1/WAFА1 and 14-3-3R on Cell Cycle Arrest and Apoptosis Induction by P14arf Oncogene (2008) 27, 6707–6719 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Cooperative effect of p21Cip1/WAFÀ1 and 14-3-3r on cell cycle arrest and apoptosis induction byp14 ARF PG Hemmati1,2,3, G Normand1,3, B Gillissen1,2, J Wendt1,BDo¨ rken1,2 and PT Daniel1,2 1Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charite´, Campus Berlin-Buch, Germany and 2Clinical and Molecular Oncology, Max-Delbru¨ck-Center for Molecular Medicine, Berlin-Buch, Germany P14ARF (p19ARF in the mouse) plays a central role in the Introduction regulation of cellular proliferation. Although the capacity of p14ARF to induce a cell cycle arrest in G1 phase depends Genotoxic stress induced by chemodrugs or ionizing on a functional p53/p21-signaling axis, the G2 arrest irradiation triggers an arrest in the G2 phase of the cell triggered byp14 ARF is p53/p21-independent. Using iso- cycle to allow for the repair of damaged DNA. In most geneic HCT116 cells either wild-type or homozygously situations, this process depends on a functional p53/p21- deleted for p21, 14-3-3r or both, we further investigated signaling axis. In turn, cells deficient for p21 are the cooperative effect of p21 and 14-3-3r on cell cycle hampered in their ability to arrest in the G2 phase regulation and apoptosis induction byp14 ARF. In contrast following DNA damage and rapidly undergo mitotic to DNA damage, which induces mitotic catastrophe in catastrophe and subsequent cell death (Waldman et al., 14-3-3r-deficient cells, we show here that the expression 1996; Bunz et al., 1998; Bhonde et al., 2006b). In turn, of p14ARF triggers apoptotic cell death, as evidenced by forced expression of p21 strongly attenuates apoptosis nuclear DNA fragmentation and induction of pan-caspase sensitivities on DNA damage (Gorospe et al., 1996; activities, irrespective of the presence or absence of Martinez et al., 2002; Wendt et al., 2006). In addition to 14-3-3r. The activation of the intrinsic mitochondrial p21, 14-3-3s was shown to play a key role in regulating apoptosis pathwaybyp14 ARF was confirmed bycyto- G2/M checkpoint function (Hermeking et al., 1997). On chrome c release from mitochondria and induction of transcriptional upregulation by p53, 14-3-3s impairs caspase-9- (LEHDase) and caspase-3/7-like (DEVDase) mitotic entry by sequestering cyclin B1/cdc2 complexes activities. Moreover, 14-3-3r/p21 double-deficient cells (Hermeking et al., 1997; Chan et al., 1999; Hermeking, were exceedinglysensitive to apoptosis induction by 2003). While the expression of 14-3-3s blocks cells in the p14ARF as compared to wild-type cells or cells lacking G2 phase of the cell cycle, cells deficient for 14-3-3s are either gene alone. Notably, p14ARF-induced apoptosis was unable to maintain a stable G2 arrest, prematurely enter preceded byan arrest in the G2 phase of cell cycle,which mitosis and die by mitotic catastrophe (Chan et al., coincided with downregulation of cdc2 (cdk1) protein 1999). Notably, cells lacking both p21 and 14-3-3s are expression and lack of its nuclear localization. This highly sensitive to DNA damage-induced cell death, indicates that p14ARF impairs mitotic entrybytargeting indicating that p21 and 14-3-3s play complementary the distal DNA damage-signaling pathwayand induces roles and critically determine the response of the cells to apoptotic cell death, rather than mitotic catastrophe, out DNA damage (Chan et al., 2000; Hermeking, 2003). of a transient G2 arrest. Furthermore, our data delineate The p14ARF tumor suppressor (p19ARF in the mouse) is that the disruption of G2/M cell cycle checkpoint control encoded at the mammalian INK4a locus (Duro et al., criticallydetermines the sensitivityof the cell toward 1995; Mao et al., 1995; Quelle et al., 1995). Physiolo- p14ARF-induced mitochondrial apoptosis. gically, p14ARF (p19ARF) is rapidly upregulated on Oncogene (2008) 27, 6707–6719; doi:10.1038/onc.2008.193; oncogenic stress (de Stanchina et al., 1998; Radfar published online 22 September 2008 et al., 1998; Zindy et al., 1998). By stabilizing p53 through inhibition of hdm-2 (mdm-2), p14ARF triggers Keywords: p14ARF; 14-3-3s, p21; cell cycle; apoptosis; the expression of p53 target genes including p21 and mitotic catastrophe bax. Ectopic expression of p19ARF was shown to induce a p53-dependent arrest in the G1 and G2 phases of the cell cycle (Quelle et al., 1995). Similarly, the p19ARF- mediated induction of cycle arrest or apoptosis follow- Correspondence: Dr PT Daniel, Clinical and Molecular Oncology, ing oncogene expression depends on functional p53. Max-Delbru¨ ck-Center for Molecular Medicine, and University Med- Nonetheless, data from several in vitro models suggest ical Center Charite´ , Campus Berlin-Buch, Lindenberger Weg 80, 13125 that p14ARF (p19ARF) regulates cell cycle arrest and Berlin-Buch, Germany. apoptosis in a p53-independent manner as well (Eymin E-mail: [email protected] 3These authors contributed equally to this work. et al., 2001, 2003; Martelli et al., 2001; Hemmati et al., ARF Received 24 September 2007; revised 5 December 2007; accepted 11 2002; Normand et al., 2005). Furthermore, p14 was April 2008; published online 22 September 2008 shown to trigger the DNA damage-signaling pathway 14-3-3r and p21 in p14ARF-induced cell cycle arrest and apoptosis PG Hemmati et al 6708 through ATR/Chk-1 or ATM in a p53-independent of p21 protein in the 14-3-3s KO cells were not induced manner (Li et al., 2004; Rocha et al., 2005; Rocha and further on p14ARF expression. As expected, 14-3-3s Perkins, 2005; Eymin et al., 2006a, b), indicating that levels were induced on infection with Ad-p14ARF in the p14ARF may control cellular proliferation in a way much WT cells. In contrast, p21 KO cells exhibited high basal more complex than initially thought. levels of 14-3-3s protein, which were not further Although the G1 cell cycle arrest induced by p14ARF inducible by p14ARF. This constitutively high expression depends on a functional hdm-2/p53/p21-signaling axis, of 14-3-3s in the p21 KO cells may result from the expression of p14ARF in p53- and/or p21-deficient cells increased p53, which was reported to be hyperfunctional triggers an arrest in the G2 phase through downregulat- (Blagosklonny et al., 2002). Taken together, these results ing cdc2 (cdk1) expression and functional activity (Eymin indicate that the loss of 14-3-3s does not affect the et al., 2003, 2006a; Hemmati et al., 2005; Normand et al., ability of p14ARF to induce a G2/M cell cycle arrest in 2005). This is in line with the finding that p14ARF triggers p53 WT cells. the DNA damage-signaling pathway in a p53/p21- As compared to WT, p21 KO or 14-3-3s KO cells, independent manner (Rocha et al., 2003, 2005; Rocha there was no decrease in the relative number of cells in and Perkins, 2005; Eymin et al., 2006b). As 14-3-3s is the S phase of the cell cycle on expression of p14ARF in involved in the sequestration of cyclin B1/cdc2 com- the DKO cells (Figure 1). This suggests that these cells plexes, the loss of 14-3-3s, either alone or in combination are impaired in the S phase control and still proliferate with loss of p21, may sensitize for p14ARF-induced following p14ARF expression. To confirm this hypothesis, cell death by deregulating G2/M checkpoint function HCT116 cells were infected with Ad-p14ARF (25 MOI) (Hermeking et al., 1997; Graves et al., 2001; Hermeking, for 48 h, pulse-labeled with 5-bromo-2-deoxyuridine 2003). We therefore investigated whether the absence of (BrdU) for 30 min and analysed by flow cytometry 14-3-3s, alone or in conjunction with loss of p21, alters (Figure 2a). As shown in Figure 2b, quantification of the cell cycle behavior and apoptosis sensitivities on BrdU-positive cells as percent of cells in S phase p14ARF expression. revealed that mock-treated or control vector-infected cells deficient for either p21, 14-3-3s or both displayed an overall higher degree of BrdU incorporation as compared to the WT cells. In particular, cells lacking Results p21, that is p21 KO and p21/14-3-3s DKO cells, exhibited the highest rates of BrdU incorporation. Induction of G2/M arrest by p14ARF is independent of p21 Although DKO cells were impaired to undergo growth and 14-3-3s arrest on p14ARF expression, a substantial decrease in the HCT116 cells, either wild-type (WT) or homozygously relative number of BrdU-positive cells was observed in deleted for either 14-3-3s (14-3-3s KO), p21 (p21 KO) the WT, p21 KO or 14-3-3s KO cells on p14ARF or both p21 and 14-3-3s (double knockout (DKO)), expression. This suggests that the combined loss of both were infected with an adenoviral vector expressing p21 and 14-3-3s impairs the S phase control following p14ARF (Ad-p14ARF) at 25 multiplicity of infection expression of p14ARF. (MOI) in parallel with mock-treated (control) or control Expression of p14ARF induced the accumulation of vector-infected cells (Ad-lacZ; 25 MOI) and assayed for cells in the G2/M phase irrespective of the presence or cell cycle distribution after 48 h (Figures 1a and b). absence of 14-3-3s and/or p21, as evidenced by an Although WT cells arrested in the G1 phase of the cell increase in the number of cells with a 4N DNA content. cycle showed a corresponding decrease in the relative To differentiate between cells arrested in the G2 phase number of cells in the S phase, p21 KO, 14-3-3s KO or versus M phase, we employed the bivariate analysis of DKO cells accumulated in the G2/M phase on expres- DNA content and expression of mitotic protein mono- sion of p14ARF.
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