The INK4a-ARF locus encodes two distinct tumor suppressors, p16INK4a and p19 ARF . Whereas p16 INK4arestrains cell growth through preventing phosphorylation of the retinoblastoma protein, p19 ARF acts by attenuating Mdm2-mediated degradation of p53, thereby stabilizing p53. Recent data indicate that Mdm2 shuttles between the nucleus and the cytoplasm and that nucleo-cytoplasmic shuttling of Mdm2 is essential for Mdm2's ability to promote p53 degradation. Therefore, Mdm2 must export p53 from the nucleus to the cytoplasm where it targets p53 for degradation. We show here that coexpression of p19 ARF blocks the nucleo-cytoplasmic shuttling of Mdm2. Moreover, subnuclear localization of Mdm2 changes from the nucleoplasm to the nucleolus in a shuttling time-dependent manner, whereas p19 ARF is exclusively located in the nucleolus. In heterokaryons containing Mdm2 and p19 ARF , the longer the Mdm2 shuttling is allowed, the more Mdm2 protein colocalizes with p19 ARF in the nucleolus, implying that Mdm2 moves from the nucleoplasm to the nucleolus and then associates with p19 ARF there. Furthermore, whether or not Mdm2 colocalizes with p19 ARF in the nucleolus, p19 ARF prevents Mdm2 shuttling. This observation suggests that Mdm2 might be exported through the nucleolus and p19 ARF could inhibit the nuclear export of Mdm2 by tethering Mdm2 in the nucleolus. Taken together, p19 ARF could stabilize p53 by inhibiting the nuclear export of Mdm2.

The inhibition of KSP causes mitotic arrest by activating the spindle assembly checkpoint. While transient inhibition of KSP leads to reversible mitotic arrest, prolonged exposure to a KSP inhibitor induces apoptosis. Induction of apoptosis by the KSP inhibitor couples with mitotic slippage. Slippage-refractory cells show resistance to KSP inhibitor-mediated lethality, whereas promotion of slippage after mitotic arrest enhances apoptosis. However, attenuation of the spindle checkpoint confers resistance to KSP inhibitor-induced apoptosis. Furthermore, sustained KSP inhibition activates the proapoptotic protein, Bax, and both activation of the spindle checkpoint and subsequent mitotic slippage are required for Bax activation. These studies indicate that in response to KSP inhibition, activation of the spindle checkpoint followed by mitotic slippage initiates apoptosis by activating Bax.

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The Hdm2 oncoprotein inhibits p53 functions by two means: (i) it blocks p53's transactivation activity and (ii) it targets p53 for degradation in a proteasomedependent manner. Recent data indicate that Hdm2 shuttles between the nucleus and the cytoplasm and that the regulation of p53 levels by Hdm2 requires its nuclear export activity. Two different models are consistent with these observations. In the first, Hdm2 binds to p53 in the nucleus and shuttles p53 from the nucleus to the cytoplasm, and then it targets p53 to the cytoplasmic proteasome. Alternatively, Hdm2 and p53 could be exported separately from the nucleus and then associate in the cytoplasm, where Hdm2 promotes the degradation of p53. To distinguish between these two models, several Hdm2 mutants were employed. Hdm2NLS lacks the ability to enter the nucleus, whereas Hdm2NES is deficient in nuclear export. Hdm2NLS, Hdm2NES, or the combination of both mutants were unable to promote p53 degradation in the cotransfected 2KO cells (which were null for both the p53 and mdm2 genes), although wild-type Hdm2 efficiently reduced p53 levels under the same conditions. This observation is not a result of the differences in expression levels or stability between Hdm2 and these mutants. Moreover, coexpression of these mutants had no effect on wild-type Hdm-2-induced p53 destabilization. Thus, Hdm2 must shuttle p53 from the nucleus to the cytoplasm to target it for degradation in the cytoplasm.

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