Cooperative effect of p21Cip1/WAF−1 and 14-3-3σ on cell cycle arrest and apoptosis induction by p14ARF

P14ARF (p19ARF in the mouse) plays a central role in the regulation of cellular proliferation. Although the capacity of p14ARF to induce a cell cycle arrest in G1 phase depends on a functional p53/p21-signaling axis, the G2 arrest triggered by p14ARF is p53/p21-independent. Using isogeneic HCT116 cells either wild-type or homozygously deleted for p21, 14-3-3σ or both, we further investigated the cooperative effect of p21 and 14-3-3σ on cell cycle regulation and apoptosis induction by p14ARF. In contrast to DNA damage, which induces mitotic catastrophe in 14-3-3σ-deficient cells, we show here that the expression of p14ARF triggers apoptotic cell death, as evidenced by nuclear DNA fragmentation and induction of pan-caspase activities, irrespective of the presence or absence of 14-3-3σ. The activation of the intrinsic mitochondrial apoptosis pathway by p14ARF was confirmed by cytochrome c release from mitochondria and induction of caspase-9- (LEHDase) and caspase-3/7-like (DEVDase) activities. Moreover, 14-3-3σ/p21 double-deficient cells were exceedingly sensitive to apoptosis induction by p14ARF as compared to wild-type cells or cells lacking either gene alone. Notably, p14ARF-induced apoptosis was preceded by an arrest in the G2 phase of cell cycle, which coincided with downregulation of cdc2 (cdk1) protein expression and lack of its nuclear localization. This indicates that p14ARF impairs mitotic entry by targeting the distal DNA damage-signaling pathway and induces apoptotic cell death, rather than mitotic catastrophe, out of a transient G2 arrest. Furthermore, our data delineate that the disruption of G2/M cell cycle checkpoint control critically determines the sensitivity of the cell toward p14ARF-induced mitochondrial apoptosis.