Steady-state polypeptide modulations associated with nerve growth factor (NGF)-induced terminal differentiation and NGF deprivation-induced apoptosis in human neuroblastoma cells.

L M Jensen; Y Zhang; E M Shooter

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PubMed · 1527053

Steady-state polypeptide modulations associated with nerve growth factor (NGF)-induced terminal differentiation and NGF deprivation-induced apoptosis in human neuroblastoma cells.

L M Jensen·Y Zhang·E M Shooter

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Abstract

Human neuroblastoma SH-SY5Y cells differentiate terminally in culture upon exposure to nerve growth factor (NGF) for 4-5 weeks. The neuronal phenotypic properties acquired in response to prolonged NGF treatment include morphological differentiation, cessation of mitotic activity, neuronal marker expression, increased membrane electrical potentials, and a survival dependence upon NGF for trophic support (Jensen, L.M. (1987) Dev. Biol. 120, 56-64). Thus, differentiated cultures survive indefinitely in the continued presence of NGF, however, withdrawal of NGF from differentiated cultures effects the loss of cellular viability within 3-6 days. Here, we show that death of differentiated SH-SY5Y cells caused by NGF deprivation is characteristic of apoptosis. To compare the differentiation promoting and the neurotrophic properties of NGF, whole SH-SY5Y cell extracts were analyzed by two-dimensional polyacrylamide gel electrophoresis using isoelectric focusing and nonequilibrium pH gradient electrophoresis gels in the first dimension. Steady-state levels of polypeptides extracted from whole-cell lysates of naive (untreated) cells, terminally differentiated cells, and NGF-deprived differentiated cells were compared. Over 1,000 spots from each were analyzed using computer-aided spot matching and densitometry. We noted 25 polypeptides that decreased during differentiation, including 15 that decreased by a factor of 10 or more. The levels of five polypeptides were induced from very low or undetectable levels in naive cells. Withdrawal of NGF from terminally differentiated cells produced alterations in steady-state protein patterns substantially distinct from those occurring during differentiation. While levels of most proteins do not appear affected early after NGF withdrawal, others rapidly return to levels comparable with those of the naive state and some changes occurring with differentiation are enhanced further upon NGF withdrawal. Three polypeptides were regulated uniquely by NGF withdrawal, including two that were induced, on average, 20- and 28-fold and another that was depressed more than 7-fold after NGF deprivation, before cell death. These data indicate that NGF elicits both constitutive and nonconstitutive changes in gene expression and suggest that the differentiation promoting and the neurotrophic properties of NGF correlate with the regulation of different gene products.

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Cell Death Cell Differentiation Cell Nucleus DNA Damage Electrophoresis, Gel, Two-Dimensional Humans In Vitro Techniques Nerve Growth Factors Nerve Tissue Proteins Neurons Tumor Cells, Cultured Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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L M Jensen, Y Zhang, E M Shooter. 1992-09-25. Steady-state polypeptide modulations associated with nerve growth factor (NGF)-induced terminal differentiation and NGF deprivation-induced apoptosis in human neuroblastoma cells.. https://pubmed.ncbi.nlm.nih.gov/1527053/

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