Different mechanisms for inhibition of cell proliferation via cell cycle proteins in PC12 cells by nerve growth factor and staurosporine

Abstract

PC12 cells have previously been shown to cease cell division during nerve growth-factor (NGF)-induced differentiation by affecting specific cell cycle proteins. Staurosporine, a protein kinase inhibitor, also causes PC12 cell differentiation, independently of neurotrophins or plasma membrane receptors. We have investigated the relationship of the tumor suppressor protein, p53, and other cell cycle proteins to the antiproliferative effects of NGF and staurosporine in PC12 cells. NGF treatment of PC12 cells stimulated an increase of p53 protein in the nucleus and, more slowly, an increase in total cellular p53 protein. Levels of the cyclin-kinase inhibitor p21/WAF1, cyclin D1, and cyclin G, all downstream transcriptional targets of p53, increased after short times of NGF treatment. Cessation of replication and differentiation occurred more rapidly in defined medium (2 days) than in serum medium (6 days), in correspondence with the more rapid changes in both p53 and p21/WAF1 levels in defined medium (1 hour) than in serum (1 day). Levels of p34cdc2 and p33cdk2 kinase dropped after 6 to 10 days treatment with NGF in serum, close to the time of terminal differentiation. Staurosporine, on the other hand, inhibited DNA replication of PC12 cells in a time- and dose-dependent fashion by affecting cyclin-dependent kinases. Staurosporine had no effect on the protein levels of p53, p21/WAF1, or cyclin G. The kinase activity of both p34cdc2 and p33cdk2 were inhibited in vitro with IC50 values of 20 nM and 75 nM, respectively. In vivo p34cdc2 kinase activity was inhibited within 1 day, before the decrease in the levels of p34cdc2 protein at days 2 to 3. In contrast, in vivo p33cdk2 kinase activity only decreased in concert with protein levels. Although both NGF and staurosporine inhibit DNA replication concomitant with induction of differentiation by affecting the activity of p34cdc2 and p33cdk2, the mechanism of the two agents is quite different. NGF achieves inhibition of activity of these cyclin-dependent kinases by signalling through the TrkA receptor to the tumor suppressor protein p53 and then to p21/WAF1. In contrast, staurosporine directly inhibits the activity of p34cdc2 and p33cdk2 by binding to them and also indirectly by alteration of their phosphorylation through other regulatory kinases.

Nerve Growth Factor Potentiates p53 DNA Binding but Inhibits Nitric Oxide-Induced Apoptosis in Neuronal PC12 Cells

“…As shown here, NGF-activated p53 binds to the p21 Waf1/Cip1 promoter and activates its transcription. Furthermore, p21 Waf1/Cip1 protein levels were highly increased with NGF alone as previously described [9,57]. NGF may therefore prime PC12 cells for survival by increasing levels of the antiapoptotic factors p21 Waf1/Cip1 and Bcl-2 during differentiation.…”

ΔNp73 Modulates Nerve Growth Factor-Mediated Neuronal Differentiation through Repression of TrkA

“…To confirm this, the induction of growth-associated protein 43 (GAP-43) and p21 was examined by Western blot analysis. GAP-43 is a neuronal differentiation marker whose expression directly correlates with the extent of differentiation, while p21 is required for the cell cycle arrest mediated by NGF during differentiation (14,41). We showed that GAP-43 was induced in an NGF-dependent manner ( Fig.…”

A Novel, Nerve Growth Factor-activated Pathway Involving Nitric Oxide, p53, and p21WAF1 Regulates Neuronal Differentiation of PC12 Cells

“…These contradictory findings may be related to differ- ences in culture conditions which affect both the extent of differentiation and the expression of cell cycle-associated proteins. For measurements of p21 WAF1 promoter activity, we treated the PC12 cells for 3 days in defined medium, which considerably speeds both NGF-induced differentiation (27) and induction of p21 WAF1 (62). The other group treated their cells for only 2 days in serum-containing medium.…”