Until recently, the ability of ARF (human p14(ARF), murine p19(ARF)) tumour-suppressor protein, encoded by the INK4A/ARF locus, to inhibit cell growth in response to various stimuli was related to its ability to stabilize p53 through the so-called ARF/MDM2/p53 pathway. However, recent data have demonstrated that ARF is not implicated in this unique p53-dependent pathway. By use of transient and stable expression, we show here that human p14(ARF) inhibits the growth of human tumoral cells lacking functional p53 by inducing a transient G(2) arrest and subsequently apoptosis. This p14(ARF)-induced G(2) arrest was correlated with inhibition of CDC2 activity, inactivation of CDC25C phosphatase and induction of the CDK inhibitor p21(WAFI). Apoptosis was demonstrated using Hoechst 33352 staining, proteolytic activation of caspase-3 and PARP cleavage. Similar results were obtained in experiments with cells synchronized by hydroxyurea block. Importantly, we were able to reproduce these effects 'in vivo' by showing that p14(ARF) inhibits the growth of p53 nullizygous human tumours in nude mice and induces the regression of p53 -/- established tumours. In these experiments, tumoral regression was associated with inhibition of cell proliferation as well as induction of apoptosis confirming the data obtained in cell lines.

p14ARF is a tumor suppressor that controls a well-described p53/Mdm2-dependent checkpoint in response to oncogenic signals. Here, new insights into the tumor-suppressive function of p14 ARF are provided. We previously showed that p14 ARF can induce a p53-independent G 2 cell cycle arrest. In this study, we demonstrate that the activation of ATM/ATR/CHK signaling pathways contributes to this G 2 checkpoint and highlight the interrelated roles of p14 ARF and the Tip60 protein in the initiation of this DNA damage-signaling cascade. We show that Tip60 is a new direct p14 ARF binding partner and that its expression is upregulated and required for ATM/CHK2 activation in response to p14 ARF . Strikingly, both p14 ARF and Tip60 products accumulate following a cell treatment with alkylating agents and are absolutely required for ATM/CHK2 activation in this setting. Moreover, and consistent with p14 ARF being a determinant of CHK2 phosphorylation in lung carcinogenesis, a strong correlation between p14 ARF and phospho-CHK2 (Thr68) protein expression is observed in human lung tumors (P < 0.00006). Overall, these data point to a novel regulatory pathway that mediates the p53-independent negative-cell-growth control of p14 ARF . Inactivation of this pathway is likely to contribute to lung carcinogenesis.