The p53-related p73 and p63 genes encode proteins that share considerable structural and functional homology with p53. Despite similarities, their deletion in mice has different outcomes, implying that the three genes may play distinct roles in vivo. Here we show that endogenous p73 levels increase in neuroblastoma cells induced to differentiate by retinoic acid and that exogenously expressed p73, but not p53, is sufficient to induce both morphological (neurite outgrowth) and biochemical (expression of neurofilaments and neural cell adhesion molecule (N-CAM); down-regulation of N-MYC and up-regulation of pRB) markers of neuronal differentiation. This activity is shared, to different extents, by all p73 isoforms, whereas the transcriptionally inactive mutants of p73 isoforms are ineffective. Conversely, blockage of endogenous p73 isoforms with a dominant negative p73 results in the abrogation of retinoid-induced N-CAM promoter-driven transcription. Our results indicate that the p73 isoforms activate a pathway that is not shared by p53 and that is required for neuroblastoma cell differentiation in vitro.The p53 homologue p73 (1) is expressed in human cells as several alternatively spliced forms that show different abilities to homo/heterodimerize with each other and with p53 (1-3). Few studies have directly investigated p73 functions, and its assumed activities are largely based on analogy with its p53 homologue. Indeed, p73 isoforms share with p53 the ability to transactivate the p21 promoter, thus enforcing cell cycle arrest at the G 1 /S transition (1, 2, 4), and to induce apoptosis when overexpressed (4). p73 apoptotic functions are also recruited in response to DNA damage, through a p53-independent pathway that requires the activation of the nuclear tyrosine kinase c-Abl by either ATM or a proficient MLH-1 mismatch repair gene (5-7). Unlike p53, mutant p73 genes have rarely been found in human tumors (8 -10). There is evidence that p73 may play a role in differentiation. The carboxyl terminus of both p63 and p73 contains a sterile ␣ motif found in a variety of signaling proteins that is known to play a role in development and differentiation (11). The p73 gene has been mapped to a region (1p36.33) that is frequently deleted in neuroblastomas (undifferentiated neuronal tumors (12, 13)), suggesting that loss or altered expression of p73 in vivo may be a determinant of the undifferentiated phenotype of neuroblastoma. Moreover, neurons from p53 null mice and neurons from wild type mice cultured with p53 antisense oligonucleotides in vitro show accelerated spontaneous differentiation (14). This suggests the hypothesis that loss of the p53 heterodimeric partner of p73 may liberate more uncomplexed or homodimerized p73 and allow expression of its differentiation-inducing function.Accordingly, we have investigated the role of p73 in the differentiation of neuroblastoma cell lines, a well studied model of neuronal differentiation and death (15-17). MATERIALS AND METHODSCell Culture-N1E-115 cells were grown in Dulbecco...

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Purpose: We assessed the relevance of Slug (SNAI2) for apoptosis resistance and invasion potential of neuroblastoma cells in vitro and in vivo. Experimental Design: We evaluated the effect of imatinib mesylate on invasion and analyzed the genes modulated by imatinib mesylate treatment in neuroblastoma cells. Slug expression, inhibited by imatinib mesylate treatment, was knocked down in neuroblastoma cells by RNA interference, and the effects on invasion and apoptosis were evaluated in vitro. A pseudometastatic model of neuroblastoma in severe combined immunodeficient mice was used to assess the effects of Slug silencing alone or in combination with imatinib mesylate treatment on metastasis development. Results: Microarray analysis revealed that several genes, including Slug, were down-regulated by imatinib mesylate. Slug expression was detectable in 8 of 10 human neuroblastoma cell lines. Two Slug-expressing cell lines were infected with a vector encoding a microRNA to Slug mRNA. Infected cells with reduced levels of Slug were tested for the expression of apoptosis-related genes (p53, Bax, and Bcl-2) identified previously as Slug targets. Bcl-2 was down-regulated in Slug-interfered cells. Slug down-regulation increased sensitivity to apoptosis induced by imatinib mesylate, etoposide, or doxorubicin. Invasion of Slug-silenced cells was reduced in vitro. Animals injected with Slug-silenced cells had fewer tumors than controls and the inhibition of tumor growth was even higher in animals treated with imatinib mesylate. Conclusions: Slug down-regulation facilitates apoptosis induced by proapoptotic drugs in neuroblastoma cells and decreases their invasion capability in vitro and in vivo. Slug inhibition, possibly combined with imatinib mesylate, may represent a novel strategy for treatment of metastatic neuroblastoma.

In metastatic cancer cells, the process of invasion is regulated by several transcription factors that induce changes required for migration and resistance to apoptosis. Slug (SNAI2, Snail2) is involved in epithelial mesenchymal transition in physiological and in pathological contexts. We show here that in embryonic kidney, colon carcinoma, chronic myeloid leukemia-blast crisis,