Nerve growth factor (NGF) is a neurotrophic factor known to affect the survival and differentiation of neuroblasts and some neuroblastoma cell lines. Treatment of human neuroblastoma cell line, SHSY5Y, with NGF induces reversible neuronal differentiation, but does not slow the growth of these cells. Recently, we found that terminal differentiation of SHSY5Y cells can be induced by treatment with NGF plus a pulse of the DNA polymerase inhibitor, aphidicolin. The resulting cells display long neurites, do not divide and are quite stable in the presence of NGF, but die if NGF is removed from the medium. Aphidicolin without NGF reversibly stops growth, but does not induce neuronal differentiation. The goal of this proposal is to understand the connection between differentiation and cell proliferation in the context of signal transduction by NGF. Our first Specific Aim is to determine which effects of aphidicolin are critical for terminal differentiation. We will determine for how long cells must be exposed to NGF and aphidicolin to induce commitment to differentiation and whether drugs, other than aphidicolin, which block the cell cycle at G1/S or at M phase also enhance neuronal differentiation. Our second Specific Aim is to determine whether aphidicolin enhances NGF signal transduction and whether NGF modulates the activities of cell cycle-dependent kinases. Our third Specific Aim is to determine which of the cell cycle-associated proteins is downregulated or modified by phosphorylation and is responsible for the cessation of cell proliferation by terminally differentiated SHSY5Y cells. Our fourth Specific Aim is to clone and characterize master regulatory genes which regulate neuronal differentiation. We will use PCR with primers based on the most conserved regions of the basic helix-loop- helix and POU-domain gene families. One partial bHLH cDNA already has been cloned. Also, we will screen a cDNA library for mRNA's preferentially expressed in the differentiated cells using probes enriched by subtractive hybridization. Our fifth Specific Aim is to prepare recombinant cell cultures in which the gene products identified in Specific Aims 3 and 4 are upregulated or downregulated. Thereby, we will determine the roles that these gene products play in regulating differentiation and cell proliferation.
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