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Loss of P21 Disrupts P14arf-Induced G1 Cell Cycle Arrest but Augments P14arf-Induced Apoptosis in Human Carcinoma Cells

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Loss of P21 Disrupts P14arf-Induced G1 Cell Cycle Arrest but Augments P14arf-Induced Apoptosis in Human Carcinoma Cells Oncogene (2005) 24, 4114–4128 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc Loss of p21 disrupts p14ARF-induced G1 cell cycle arrest but augments p14ARF-induced apoptosis in human carcinoma cells Philipp G Hemmati1,3, Guillaume Normand1,3, Berlinda Verdoodt1, Clarissa von Haefen1, Anne Hasenja¨ ger1, DilekGu¨ ner1, Jana Wendt1, Bernd Do¨ rken1,2 and Peter T Daniel*,1,2 1Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charite´, Campus Berlin-Buch, Berlin-Buch, Germany; 2Max-Delbru¨ck-Center for Molecular Medicine, Berlin-Buch, Germany The human INK4a locus encodes two structurally p16INK4a and p14ARF (termed p19ARF in the mouse), latter unrelated tumor suppressor proteins, p16INK4a and p14ARF of which is transcribed in an Alternative Reading Frame (p19ARF in the mouse), which are frequently inactivated in from a separate exon 1b (Duro et al., 1995; Mao et al., human cancer. Both the proapoptotic and cell cycle- 1995; Quelle et al., 1995; Stone et al., 1995). P14ARF is regulatory functions of p14ARF were initially proposed to usually expressed at low levels, but rapid upregulation be strictly dependent on a functional p53/mdm-2 tumor of p14ARF is triggered by various stimuli, that is, suppressor pathway. However, a number of recent reports the expression of cellular or viral oncogenes including have implicated p53-independent mechanisms in the E2F-1, E1A, c-myc, ras, and v-abl (de Stanchina et al., regulation of cell cycle arrest and apoptosis induction by 1998; Palmero et al., 1998; Radfar et al., 1998; Zindy p14ARF. Here, we show that the G1 cell cycle arrest et al., 1998). In turn, induction of p14ARF mediates the induced by p14ARF entirely depends on both p53 and p21 in accumulation of p53 via sequestration and subsequent human HCT116 and DU145 carcinoma cells. In con- degradation of its natural antagonist mdm-2 through trast, neither loss of p53 nor p21 impaired apoptosis the ubiquitin/proteasome pathway (Pomerantz et al., induction by p14ARF as evidenced by nuclear DNA frag- 1998; Stott et al., 1998). This prolongation of p53 half- mentation, phosphatidyl serine exposure, and caspase acti- life leads to the activation of its downstream target genes vation, which included caspase-3/7- and caspase-9-like such as p21 and Bax (Sherr, 2001). Thus, activation of activities. However, lack of functional p21 resulted in the the p14ARF/p53 signaling cascade is viewed as an accumulation of cells in G2/M phase of the cell cycle and important fail-safe mechanism that protects cells from markedly enhanced p14ARF-induced apoptosis that was, excessive and uncontrolled growth triggered through nevertheless, efficiently inhibited by the cell permeable hyperproliferative stimuli (Voorhoeve and Agami, 2003; broad-spectrum caspase inhibitor zVAD-fmk (valyl-alanyl- Zindy et al., 2003). Consequently, p14ARF was recog- aspartyl-(O)-methyl)-fluoromethylketone). Thus, loss of nized as potent tumor suppressor as functional inactiva- cell cycle restriction point control in the absence of p21 tion of p14ARF accelerates tumorigenesis and promotes may interfere with p14ARF-induced apoptosis. Finally, these chemoresistance by disabling p53 (Eischen et al., 1999; data indicate that the signaling events required for G1 cell Schmitt et al., 1999). cycle arrest and apoptosis induction by p14ARF dissociate In contrast to the initial notion that most if not all upstream of p53. biological activity of p14ARF depends on a functional Oncogene (2005) 24, 4114–4128. doi:10.1038/sj.onc.1208579 p53/mdm-2 signaling axis, a number of recent reports Published online 7 March 2005 indicate that p14ARF as well as its murine homologue p19ARF and p53 act in overlapping pathways rather than Keywords: p14ARF; p53; p21; cell cycle; apoptosis in a strictly sequential manner (Carnero et al., 2000). Consequently, murine p19ARF was shown to cause a G1 cell cycle arrest independently from p53, mdm-2, and Rb, (Weber et al., 2000) as well as p21 (Modestou et al., 2001) and p27 (Groth et al., 2000). In the same vein, we Introduction demonstrated that apoptosis induction by p14ARF is independent from p53 and Bax (Hemmati et al., 2002). The mammalian INK4a locus encodes two structurally Furthermore, a genome-wide screen identified a number unrelated proteins, the cyclin-dependent kinase inhibitor of genes putatively involved in the p53-independent regulation of cellular proliferation by p19ARF (Kuo et al., *Correspondence: P Daniel, Clinical and Molecular Oncology, 2003). Most intriguingly, it was reported recently that University Medical Center Charite´ , Campus Berlin-Buch, Linden- c-Myc, itself a potent trigger of p14ARF (p19ARF) berger Weg 80, Berlin-Buch 13125, Germany; expression, is part of a p53-independent inhibitory E-mail: [email protected] 3These authors contributed equally to this work feedbackloop (Qi et al., 2004). Received 14 June 2004; revised 26 January 2005; accepted 28 January Therefore, we aimed at further investigating and 2005; published online 7 March 2005 dissecting the signaling pathways involved in cell cycle p21 in p14ARF-induced cell cycle arrest and apoptosis PG Hemmati et al 4115 arrest and apoptosis induction by p14ARF. To this end, Whereas an arrest in G1 phase of the cell cycle upon we employed the colorectal cancer cell line HCT116 and expression of p14ARF entirely depends on the presence of its isogeneic sublines homozygously deleted for either p53 (and p21, see below), recent studies indicate that p53 p53 (HCT116-p53À/À) or p21 (HCT116-p21À/À), the is dispensable for the induction of p14ARF-triggered two key regulators of cell cycle arrest and cell death. apoptotic cell death (Hemmati et al., 2002; Eymin et al., We show here that the arrest in G1 phase of the cell cycle 2003). To demonstrate that apoptosis induction by upon expression of p14ARF strictly depends on the p14ARF is indeed independent from p53, apoptotic DNA presence of both p53 and p21. In contrast, p14ARF- fragmentation (Figure 2) and activation of caspases mediated apoptosis is not impaired upon deletion of (Figure 3) were studied in p53-proficient versus p53- either p53 or p21, involves the activation of caspases deficient HCT116 cells. To this end, HCT116-WT and including caspase-3/7- and caspase-9-like activities, and HCT116-p53À/À cells were transduced with Ad-p14ARF in is subject to inhibition by zVAD-fmk(valyl-alanyl- parallel with mock-treated (control) or control vector- aspartyl-(O)-methyl)-fluoromethylketone). Similarly, p53- infected (Ad-lacZ) cells and subjected to flow-cytometric mutated, p21-deficient DU145 prostate cancer cells were analysis of DNA fragmentation 72 h after infection. As not impaired in their ability to undergo apoptotic cell shown in Figure 2a, expression of p14ARF induced death upon p14ARF expression. Notably, loss of p21 apoptotic DNA fragmentation, that is, cells displaying resulted in the accumulation of cells in G2/M phase of the a sub-G1 DNA content, in both p53-proficient and cell cycle and markedly enhanced p14ARF-induced apop- p53-deficient HCT116-WT cells to an identical extent. tosis. This delineates a novel aspect of p14ARF-induced This effect increased in a dose-dependent manner cell death, which may be substantially augmented in the irrespective of the presence or absence of p53 absence of proper G1 restriction point control by loss (Figure 2b). Similarly, there was no difference in the of p21. induction of pancaspase activities by p14ARF in both HCT116 sublines (Figure 3a and b), indicating that p14ARF-induced apoptosis is independent from p53. To corroborate that apoptosis induction by p14ARF is p53 independent, DU145 prostate cancer cells, which Results are p53 mutated and p21 deficient, were infected with Ad-p14ARF and subjected to flow-cytometric detection of Apoptosis induction by p14ARF is not impaired by loss of p53 and/or p21 apoptotic DNA fragmentation (Figure 4a and b). In analogy to HCT116 cells, we observed a dose-dependent In contrast to the initial notion that most if not all increase in the number of apoptotic cells (Figure p14ARF activity strictly depends on a functional p53/ 4a), which was initiated at 48 h and reached substantial mdm-2 signaling axis, a number of recent reports levels at 72–96 h after transduction with Ad-p14ARF established that p14ARF is capable of mediating p53- (Figure 4b). In parallel, a dose-dependent increase of independent effects as well. To investigate this discre- pancaspase activities was detected in DU145 cells 72 h pancy and to further dissect the p14ARF signaling after infection (Figure 4c). As depicted in Figure 4d, cascade, we employed a previously constructed and caspase activation occurred between 48 and 72 h after functionally characterized adenoviral vector system transduction with Ad-p14ARF and further increased after (Ad-p14ARF) for the transient expression of p14ARF in a 96 h. This underscores the notion that p14ARF-induced number of genetically well-defined cell lines. These apoptosis is independent from p53. included HCT116 colorectal cancer cells lacking either p53 or p21 and DU145 prostate cancer cells carrying Loss of p21 sensitizes to apoptosis induction by p14ARF distinct defects in p53 and p21. To this end, HCT116 parental cells wild type for both Data obtained in p53-deficient DU145 and HCT116 p53 and p21 (HCT116-WT) and the isogeneic sublines cells indicated that p14ARF induces apoptosis irrespective homozygously deleted for either p53 (HCT116-p53À/À) of the presence or absence of functional p53. Further- or p21 (HCT116-p21À/À) were transduced with Ad- more, lackof p21 induction in either cell line suggests p14ARF and assayed for transgene expression and that p14ARF-triggered apoptosis is p21 independent. To activation of the p53 pathway (Figure 1). Expression further address this issue, we investigated p21-deficient of p14ARF was detectable by immunofluorescence in all HCT116 cell in parallel with p21-proficient HCT116- HCT116 sublines 24 h after infection with 25 MOI of WT cells.
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