Mediation of Nerve Growth Factor-driven Cell Cycle Arrest in PC12 Cells by p53

Abstract

Upon stimulation with nerve growth factor (NGF), PC12 cells extend neurites and cease to proliferate by influencing cell cycle proteins. Previous studies have shown that neuritogenesis and a block at the G 1 /S checkpoint correlate with the nuclear translocation of and an increase in the p53 tumor suppressor protein. This study was designed to determine if p53 plays a direct role in mediating NGF-driven G 1 arrest. A retroviral vector that overexpresses a temperature-sensitive p53 mutant protein (p53ts) was used to extinguish the function of endogenous p53 in PC12 cells in a dominant-negative manner at the nonpermissive temperature. NGF treatment led to transactivation of a p53 response element in a luciferase reporter construct in PC12 cells, whereas this response to NGF was absent in PC12(p53ts) cells at the nonpermissive temperature. With p53 functionally inactivated, NGF failed to activate growth arrest, as measured by bromodeoxyuridine incorporation, and also failed to induce p21/WAF1 expression, as measured by Western blotting. Since neurite outgrowth proceeded unharmed, 50% of the cells simultaneously demonstrated neurite morphology and were in S phase. Both PC12 cells expressing SV40 T antigen and PC12 cells treated with p53 antisense oligonucleotides continued through the cell cycle, confirming the dependence of the NGF growth arrest signal on a p53 pathway. Activation of Ras in a dexamethasone-inducible PC12 cell line (GSRas1) also caused p53 nuclear translocation and growth arrest. Therefore, wild-type p53 is indispensable in mediating the NGF antiproliferative signal through the Ras/MAPK pathway that regulates the cell cycle of PC12 cells. NGF,1 a neurotrophic polypeptide, belongs to a closely related family of neurotrophins composed of brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5. These paracrine hormones activate the development, maintenance, and regeneration of neurons in the nervous system (1). NGF signals the development of sympathetic, sensory, and a population of central nervous system neurons through its high affinity receptor, TrkA.The induction of neuronal differentiation invokes two interrelated cellular processes: progression through the stages of neurite outgrowth and cell cycle arrest (2). The rat pheochromocytoma cell line PC12, derived from a transplantable chromaffin tumor, provides a model system for the NGF-mediated conversion to a neuronal phenotype (3). PC12 cells contain both the tyrosine kinase (TrkA) and low affinity (p75 NTR ) NGF receptors (4, 5). Differentiation requires the TrkA receptor and proceeds through the Ras/MAPK pathway (6, 7). NGF decreases the growth rate of PC12 cells (8) and, in the short term, causes synchronized PC12 cells to accumulate in the G 1 phase of the cell cycle with a decrease in DNA synthesis (9). Continued exposure to NGF arrests the population in G 1 with an increased number in the G 2 /M phase also (10). Long-term treatment of PC12 cells with NGF promotes terminal differentiation, in which the PC12 cells resemble symp...

S100B Increases Proliferation in PC12 Neuronal Cells and Reduces Their Responsiveness to Nerve Growth Factor via Akt Activation

“…2, B-D, and 3, A and B), was accompanied by augmented levels of p21 WAF1 , similar to exposure of S100B Ϫ PC12 cells to a factor (i.e. NGF) known to cause PC12 cell proliferation arrest via up-regulation of p21 WAF1 (22)(23)(24) (also see Fig. 2, B and D).…”

“…The possibility that this might be the case is also demonstrated by the finding that a 4 -6-day treatment with NGF, which is known to cause a rapid and transient activation of p53 and produce PC12 cell proliferation arrest and differentiation (22)(23)(24) (also see Figs. 2, 3, D and F, and 5E), determined p53 nuclear translocation in ϳ30% of S100B ϩ PC12 cells, whereas no such event occurred in S100B Ϫ cells (Fig.…”

“…However, NGF up-regulated p21 WAF1 expression (Fig. 4A), likely via p53 activation (22)(23)(24) (also see Fig. 7B).…”

“…Thus, PC12 cells that had experienced S100B expression and accumulation could not respond fully to the differentiating effect of NGF until S100B was present in relatively high amounts. S100B Increases p21 WAF1 Levels, Extent of Rb Phosphorylation, and Levels of p21 WAF1 -Cyclin D1 Complex-NGF was shown to promote PC12 cell proliferation arrest and, in part, differentiation via stimulation of p53 transcriptional activity and up-regulation of the p53 downstream effector protein p21 WAF1 , an inhibitor of cyclin A-and E-dependent cdks (22)(23)(24). After 6 days of culture, NGF up-regulated p21 WAF1 expression in S100B Ϫ PC12 cells as expected (Fig.…”

Epigenetic setting and reprogramming for neural cell fate determination and differentiation

“…It is possible that p53 and CBP/p300 function as scaffolds for epigenetic settings. Studies of neuronal precursor-like PC12 and neuroblastoma cells have shown that p53 gene expression is induced and required during neuronal differentiation and maturation [83]. p53 was also reported to bind to the nerve rstb.royalsocietypublishing.org Phil.…”