Nerve growth factor (NGF)-mediated protection of neural crest cells from antimitotic agent-induced apoptosis: the role of the low-affinity NGF receptor

Abstract

Prevention by nerve growth factor (NGF) of apoptotic death in neural cells has been variously ascribed to binding of NGF to its low-affinity (p75) or high-affinity (trkA) receptor or to a cooperative interaction between the two. In a series of studies using, in turn, neuroblastoma cell lines that express only p75, mutant NGF species that bind selectively to either p75 or trkA, and a polyclonal antibody that binds to the NGF-binding domain of p75, we demonstrate that NGF binding to p75 is both necessary and sufficient for the abrogation of apoptosis in neuroblastoma cells treated with antimitotic agents.

Fig. 1.

Effects of NGF (42 nm) on antimitotic agent-induced human neuroblastoma cell death. A, SH-SY5Y cells were pretreated with NGF for 24 hr, then exposed for 1 hr to 3.2 nm NCS in the presence of NGF.B, NGP cells were pretreated similarly with NGF, then exposed for 1 hr to 2.4 nm NCS in the presence of NGF. In both cases, NGF was maintained in the culture medium for the duration of the experiment. The relationship between cell adherence and apoptosis and the methods for determining adherent cell counts have been described in detail previously (Falcione et al., 1993; Hartsell et al., 1995). In this and the following figures, error bars represent the SEM of three determinations within a single representative experiment and, where not apparent, are smaller than the size of the symbol. All experiments were performed twice, and each repetition gave comparable results to those shown. □, NCS alone; ♦, NCS + NGF; ○, NGF alone; ▵, control.

Fig. 2.

Effects of p75- and trkA-selective mutant NGF species (4.2 nm) on antimitotic agent-induced SH-SY5Y cell death. Mutant NGF species produced by site-directed mutagenesis were obtained from John W. Winslow (Genentech) and have been fully characterized as described previously (Shih et al., 1994). Mutant 6 (A) is p75-selective; mutants 21 (B) and 24 (C) are trkA-selective. In all cases, the duration of NCS (1.6 nm) treatment was 1 hr, and mutant NGF exposure began 24 hr before NCS treatment and continued throughout the duration of the experiment. □, NCS alone; •, NCS + mutant NGF; ⋄, control; ▵, mutant NGF alone.

Fig. 3.

Effects of antibody 9651 (1:1000) on the protective effects of NGF (42 nm) in antimitotic agent-treated SH-SY5Y cells. Antibody 9651 was obtained from Moses V. Chao (Cornell University Medical College). This antibody specifically blocks NGF binding to the extracellular domain of p75 (Huber and Chao, 1995). Cells were incubated with antibody for 1 hr before the addition of NGF, and both antibody and NGF were maintained in the medium throughout the duration of the experiment. NCS (1.6 nm) treatment was 1 hr in duration, beginning 24 hr after the addition of NGF to the medium. □, Control; ⋄, NGF alone; ⊕, NGF + antibody 9651; ▴, NCS alone; ⊞, NGF + NCS; •, NGF + antibody 9651 + NCS; ○, antibody 9651 alone; ⋄+, antibody 9651 + NCS.