Induction of Neuronal Differentiation by p73 in a Neuroblastoma Cell Line

Abstract

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...

Regulation of telomerase activity by the p53 family member p73

“…In contrast to mice lacking p53, p73-negative mice exhibit severe developmental defects, including hydrocephalus, hippocampal dysgenesis, chronic infections and inflammation, and abnormalities in the pheromone sensory pathway . This phenotype indicates that p73 plays a fundamental role in various developmental processes and it has been reported that p73 expression is triggered along neuronal, myogenic and hematopoietic differentiation (De Laurenzi et al, 2000;Fontemaggi et al, 2001).…”

“…TAp73 expression is induced by cellular stress signals that partially overlap with activators of p53. In addition, the TAp73 promoter is activated by E2F1 and other proapoptotic genes such as adenoviral E1A and c-Myc, and during the course of cellular differentiation processes such as myogenic, hematopoietic and neuronal differentiation (De Laurenzi et al, 2000;Stiewe and Pu¨tzer, 2000;Fontemaggi et al, 2001;Zaika et al, 2001). Here, we have shown that induction of TAp73 expression by E2F1 triggers hTERT repression in H1299 cells.…”

The p53 family inhibitor ΔNp73 interferes with multiple developmental programs

“…22 Both induction of endogenous p73 by ATRA as well as ectopic expression of TAp73 isoforms have been shown to induce morphological and biochemical markers of neuronal differentiation in N1E-115 neuroblastoma cells. 18 Here we show, that DNp73a efficiently blocks ATRA-induced differentiation of SH-SY5Y neuroblastoma cells. Whereas ATRA-treatment induced extension of multiple neurites as a morphological marker and expression of neurofilament as a biochemical marker of neuronal differentiation in mock cells, this was significantly reduced in DNp73a transfectants (Figure 1g and h).…”

“…[16][17][18] Furthermore, exogenous expression of TAp73 is sufficient to induce morphological and biochemical markers of neuronal differentiation. 18 Whereas TAp73 enhances terminal differentiation of oligodendrocyte precursors, DNp73 inhibits this process.…”

“…[16][17][18] Furthermore, exogenous expression of TAp73 is sufficient to induce morphological and biochemical markers of neuronal differentiation. 18 Whereas TAp73 enhances terminal differentiation of oligodendrocyte precursors, DNp73 inhibits this process. 19 During nephrogenesis, DNp73 is expressed preferentially in proliferating nephron precursors, whereas TAp73 is predominantly expressed in the differentiation domain of the renal cortex.…”