In accordance with this function, Necdin overexpression shows growth inhibitory properties in NIH3T3 and SaOS cell lines. However, it is also expressed in myogenic precursors that have a high proliferating potential. Necdin is a p53 target gene and physically interacts with the p53 protein product suggesting a functional relationship. Furthermore, the expression of Necdin can protect cells from apoptosis in different models, including p53-induced apoptosis. Therefore we hypothesize that during carcinogenesis, and depending on the cellular context, Necdin possesses opposing functions and may act as a tumor suppressor based on its similarity with pRb proteins, or as an oncogene through its capacity to inhibit apoptosis and p53-dependent tumor suppressive cell fates. Results reported here support this dual functionality for Necdin. We show that despite the growth suppressive functions of Necdin, it was possible to derive growing cell populations expressing constitutively high levels of Necdin. These high levels of Necdin interfered with p53 activity and contributed to an ineffective growth arrest in response to stress. Overall, we provide evidence suggesting that upregulation of Necdin expression could provide advantages for p53 wild type cells during early carcinogenesis through its ability to decrease signaling from p53 pathways. Interestingly, we found higher Necdin expression to be associated with low malignancy potential ovarian tumors, where p53 mutations are rare, compared to high grade invasive ovarian cancers. Among all candidates identified, the gene encoding Necdin was selected for further study. Microarray analysis showed an upregulation of mRNA up to five-fold. In addition, a second probe set was associated with the Necdin gene and also revealed a 3.6-fold upregulation, although with a P value of 0.04. To further validate the microarray data, Necdin expression was analyzed on an extended set of six NIH3T3 sub-clones and nine independent PyLT-expressing NIH3T3 stable clones not included in our initial analysis. The higher expression levels of Nectin observed when PyLT is expressed, as determined by Northern blot analysis, correlated well with the data derived from microarray analyses. Moreover, a nonradioactive Dig-labeled probe gave only one specific band around the expected size of 1.6 kb, confirming the identity of the lower band in Figure 1 as Necdin. Some clones with variable levels of PyLT expression were also used to confirm that the variation measured at the RNA level was reproduced at the protein 
level for Necdin. Furthermore, when we derived a new heterogeneous Torin 1 population of NIH3T3 cells expressing PyLT, we again observed an upregulation of Necdin expression compared to a vectortransfected population control. Necdin variation could be seen as early as 72 hrs posttransfection of PyLT. These results show that elevated Necdin expression levels were a reproducible and MK-1775 955365-80-7 constant phenotype in PyLT-expressing NIH3T3 cells and not caused by a clonogenic effect, thus suggesting that Necdin may be involved in some of PyLT oncogenic functions. Necdin interacts with p53 and possibly modulates its activity, which raises the possibility that PyLT exerts its inhibitory effect on p53 through Necdin induction. Nutlin-3 is a small molecule antagonist of MDM2, which prevents the interaction between MDM2 and p53, thus promoting the accumulation of p53 in cells. It has been recently shown that nutlin-3 can efficiently induce cell cycle arrest or apoptosis in different cancer cell lines with functional p53. To assess the response induced in our model, the NIH3T3 cell line was treated with nutlin-3 and proliferation was followed by flow cytometry.