We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21 WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21 WAF1 , even after removal of aphidicolin from the culture medium. The p21 WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21 WAF1 mRNA partially blocks expression of p21 WAF1 and promotes programmed cell death. These data indicate that p21 WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.We are using the SH-SY5Y neuroblastoma cell line as a model for neuronal terminal differentiation (33, 48). These cells express low levels of both the low-affinity nerve growth factor (NGF) receptor and the trkA NGF receptor (2, 48). SH-SY5Y cells treated with NGF and aphidicolin, a specific and reversible inhibitor of DNA polymerases ␣ and ␦, cease proliferation and extend long neurites (25, 33). The differentiated cells require NGF for survival and, in the presence of NGF, are stable for 4 to 6 weeks. These cells express neuronal markers and cytologically resemble sympathetic neurons. In contrast, NGF alone does not stop cell proliferation and induces only slight neurite extension (8, 55). Treatment with aphidicolin does not induce neurite extension, and the cells resume proliferation following removal of aphidicolin.Several mechanisms have been proposed for cessation of neuronal proliferation. One way by which growth arrest might occur is by down-regulating the expression of proteins required for progression of the cell cycle through the G 1 -S and G 2 -M transitions (24). cdc2 is the p34 cdc2 kinase, which in conjunction with cyclin B triggers mitosis (40). The kinases cdk2 and cdk4, in association with cyclins A, D1, D2, D3, and E, are required for the G 1 -S transition. Neuronal precursor cells, but not neurons in the adult brain, express cdc2, cdk2, and cyclins A and D2 (24,31,45,58,59). In contrast, cyclins D1 and E are expressed in both the developing and the adult brain (37, 58). Sympathetic neurons do not express cdc2, cdk2, or cyclin A but do express cdk4 and cyclins B, D1, D2, D3, and E (18). PC12 pheochromocytoma cells differentiated with NGF express decreased levels of cdc2 and cdk2 but continue to express cyclins A and E (6). Hence, although the details vary, down-regulation of cell cycle-associated proteins occurs in a variety of neuronal systems.A second potential mechanism for growth arrest involves induction of p21 WAF1, which binds to and inhibits cdc2, cdk2, and cdk4 (16,23,43). Transcription of p21 WAF1 is indu...

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