Characterization of nerve growth factor receptor in neural crest tumors using monoclonal antibodies.

Abstract

The nerve growth factor (NGF) receptor was characterized by using a new series of anti-receptor monoclonal antibodies (MAbs). These MAbs (i) showed significantly greater reactivity with a melanoma cell line expressing higher levels of NGF receptor, (ii) inhibited the binding of '251-labeled NGF to its receptor, and (iii) immunoprecipitated both metabolically labeled and '251-labeled NGF affinity-labeled receptor. These experiments defined the receptor as a 75-kDa cell-surface protein. The NGF receptor was visualized by immunoperoxidase staining in tissue sections of human nevi, melanomas, neurofibromas, a pheochromocytoma, and peripheral nerves. Uniform staining of the cytoplasm suggests that, in addition to cell-surface NGF receptors, there is a population of intracellular receptors.Nerve growth factor (NGF) has been the subject of extensive study because of its importance for regulation of development of sympathetic and sensory neurons and possibly other neural crest-derived cell types as well (1). Involvement of NGF in neural crest tumors such as melanoma has been suggested but never directly demonstrated. Efforts to identify oncogenes or oncogene-encoded products in malignant melanoma have been inconclusive (2), but the NGF receptor is a reasonable candidate particularly since, as we demonstrate in this study, it is expressed on melanoma cells in much greater quantities than on normal melanocytes. Progress in determining the relevance of this receptor to the transformed phenotype has been slow because the NGF receptor is not well characterized, nor are there good anti-receptor monoclonal antibodies (MAbs)

Nerve Growth Factor Stimulates Proliferation and Survival of Human Breast Cancer Cells through Two Distinct Signaling Pathways

“…K-252a (inhibitor of trk-tyrosine kinase activity) and PD98059 (inhibitor of MAPK cascade) were from Calbiochem (France). The mouse monoclonal anti-NGF receptor (p75 NTR ) antibody was from Euromedex (France) and was previously described for its ability to block the interaction between p75 NTR and NGF (20). The anti-lamin B (C-20), goat polyclonal IgG, and the polyclonal anti-p140 trkA (trk763) were from Santa Cruz Biotechnology.…”

Ruffling membrane, stress fiber, cell spreading and proliferation abnormalities in human Schwannoma cells

“…If permeablization was necessary, 0.2% Triton X-100 was added in the blocking buer (for anti-S100 and anti-paxillin). Primary antibodies were rabbit anti-S100b (1 : 6000, 1 h at room temperature; Dako), mouse antihuman p75 (conditioned medium, overnight at 48C; Ross et al, 1984) or anti-paxillin (1 : 1000 overnight at 48C; Transduction Labs). Cultures were then washed and incubated with a 1 : 200 dilution of TRITC-conjugated goat anti-rabbit IgG (Jackson Labs) (for anti-S100b) or TRITC-conjugated goat anti-mouse IgG (Jackson Labs) (for anti-p75 or anti-paxillin).…”

Efficient processing and expression of human nerve growth factor receptors in Xenopus laevis oocytes: effects on maturation.

“…We have used microinjection of receptor RNA in Xenopus laevis oocytes to obtain cell surface expression of the receptor. (12), and on some nonneuronal derivatives of the neural crest, such as melanoma cells (7) and proliferative melanocytes, PC12 cells, Schwann cells, and neurofibroma cells (9,17,24).We have previously reported gene transfer of genomic sequences encoding the human NGF receptor (5) and the isolation of NGF receptor cDNAs from a human melanoma library (13). The deduced amino acid sequence from a full-length cDNA indicated that the receptor is a transmembrane glycoprotein rich in cysteine residues and that it has a large number of negatively charged residues.…”

“…We have used microinjection of receptor RNA in Xenopus laevis oocytes to obtain cell surface expression of the receptor. (12), and on some nonneuronal derivatives of the neural crest, such as melanoma cells (7) and proliferative melanocytes, PC12 cells, Schwann cells, and neurofibroma cells (9,17,24).…”