Increased Necdin could paradoxically promote growth or survival. A possible role for Necdin in DNA damage response was suggested by the upregulation of Necdin following different genotoxic stresses. By using nutlin-3, we showed that p53 activation clearly induced Necdin in a dose dependent manner, supporting a previous report that identified Necdin as a p53 target gene. Moreover, we show that modulation of the Necdin level affects p53-dependent growth arrest. Indeed, we demonstrate that an increase in Necdin expression results in a delayed cell cycle arrest while inversely targeting Necdin by shRNA accelerates this arrest. The interaction of Necdin with p53 suggests that this delay in growth arrest is probably associated with a direct inhibitory effect of Necdin over p53. We noted that Necdin affected p21 induction following p53 activation in our model supporting previous results. Therefore, interference with p53 transcriptional Sorafenib moa activity may represent the mechanism underlying the cell cycle arrest variations caused by Necdin. However, 
we believe that other mechanisms may be involved since p21 mediated-arrest mostly relies on functional Rb and in PyLT-expressing cells, the Rb proteins are kept inactive by their interaction with PyLT. As p53 induction upon genotoxic stress is associated with multiple additional signaling events, we directly addressed p53 stimulation by exposure to nutlin-3. This specific stimulation results in a functional induction of p53, although the posttranslational SCH727965 CDK inhibitor phosphorylation of p53 observed with genotoxic stress are absent or barely detectable with nutlin-3. This suggests that phosphorylation may not be critical for interaction of Necdin with p53 and that Necdin does not interfere with the phosphorylation status to modulate p53 activity. In addition to phosphorylation, other modifications contribute to p53 activity, including acetylation, which is increased upon nutlin-3 stimulation. The deacetylase Sirt1 is a negative regulator of p53 activation and Necdin interactions with this protein potentiate its activity upon genotoxic stress. However, we did not address the status of these post-translationals modifications in our model. Additionally, it is important to note that p53 responses can differ with particular drugs depending on the dose employed, the duration of the treatment, and the metabolic state of the cell. Others mechanisms can explain Necdin inhibitory effects over p53. Necdin binds the N-terminal transactivation domain of p53. Some proteins share this binding site, among them SOCS1, which contribute to p53 activation. It is possible that Necdin competes with activating proteins such as SOCS1 for p53 binding, leading to a decrease in p53 response. Others examples exist where the response to p53 activation varies according to the presence or absence of specific cellular partners. The capacity of p53 to translocate to the mitochondria where it plays a transcription-independent function in apoptosis is now well documented. Tid1 is a p53-interacting protein that helps this localization from the nucleus to mitochondria. Both cytoplasmic and nuclear cellular partners have been revealed for Necdin and expression of these partners has been shown to cause Necdin relocalisation in the cell. Perhaps interference with p53 activation may arise from the ability of Necdin to relocate p53 in other cellular compartment. All these mechanisms are consistent with the notion that Necdin can inhibit p53 function and require further investigation. Combining our data on p53 inhibition by Necdin with the knowledge that Necdin is a direct p53 response gene suggests that Necdin is part of a negative feedback loop controlling p53 activity.