Analysis of Molecular Mechanism of ARF-dependent apoptotic cell death

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The tumor suppressor p19ARF, p14ARF in humans, is encoded by the Ink4a/ARF locus (Quelle, D. E.et al., (1995) Cell 83, 993-100) and mutated, deleted, or silenced in many forms of cancer. pl9ARF induces growth arrest by antagonizing the activity of the p53-negative regulator, Mdm2, thereby permitting a p53 transcriptional response (Kamijo, T. et al., (1997) Cell 91, 649-659; Kamijo T. et al., (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8292-829'). Although ARF activation and p53 stabilization alone are not sufficient to induce apoptosis in all cell types, upregulation ofp53 in this way strongly sensitizes cells to die in response to genotoxic stresses or additional apoptotic signals induced by oncogenes themselves. In this project, we first analyzed the mechanism of ARF-dependent apoptosis and demonstrated that ARF induces mitochondria-dependent apoptosis in p53 wild-type, ARF/p16-null cells. We also found that ARF evokes cytocbrome c release from mitochondria, decreases mitochondria mem … More brane potential and activates pro-caspase-9 to induce apoptosis. Our findings suggest that this apoptotic cellular modulation is brought about by up-regulation of the pro-apoptotic Bc1-2 family proteins Bax and Bim, and down-regulation of anti apopt3tic Bcl-2, in mitochondrial fractions. Additionally, ARF seems to down-tegulate Bc1-2 in a p53-dependent manner, while up-regulating Bax/Bim via a p53-independent pathway (Nakazawa Y, Kamuo, Koike K, Noda T. J Biol Chem. 278(30) : 27888-95. 2003)A recent study showed that triple knockout (TKO) mice lacking ARF, p53, and MDM2 develop multiple and more aggressive tumors per animal than mice lacking either gene alone (Weber, J. D et al., (1999) Nat. Cell Biol. 1, 20-26), indicating that ARF may suppress tumor progression by a p53/MDM2-independent manner. Moreover, previous reports presented that murine and human ARFs interact with MDM2 via NH2-terminal domains, but the functional domain of ARF and the molecular mechanism of p53/MDM2-independent tumor suppression has thus far not been identified. Taken together these findings, we next addressed the molecular mechanism of ARF-induced apoptotic cell death in a p53-independent manner using a p53/Rb-inactivated cell line, and, surprisingly, found that the COOH-terminal of ARF has the ability to induce p53-independent cell death. This is the first report that describes the apoptotic cell death-function of the p19ARF COOH-terminal. Furthermore, we address the responsible caspase cascade for p19ARF COOH-terminal induced apoptosis and relationship between the activated caspases and MAPKs controlling p19ARF COOH-terminal-induced apoptosis. For further investigation of ARF-induced cell death, we made p53/Rb-inactivated cells by introducing SV4OT into ARF-null MEFs. Re-introduction of ARF into the p53/Rb-inactivated cells up-regulated mitochondria-dependent apoptotic cell death accompanied cytochrome c release from mitochondria and activation of the intrinsic pathway caspases, i.e. caspase-9,-3 and-7. Interestingly, ARF bound SV4OT in the ARF-induced cells and re-activation of p53 was seemed to be mediated by the SV4OT inactivation by ARF, resulting in the mitochondrial dysfunction (Nakazawa Y and Kamijo T, submitted). Less