Retroviral transfer of the beta-nerve growth factor gene into murine neuroectodermal tumor cells modulates cell proliferation rate, neurite formation, and NGF binding site expression

The response of wild-type and genetically engineered neuroectodermal tumor (NET) cells to exogenous and endogenously synthesized nerve growth factor ( NGF) was investigated. Differences in cell proliferation rate, neurite form ation, and expression of NGF binding sites were quantitatively determined. Ecotropic retroviral vectors were used to transfer the genes for beta-galac tosidase (beta-GAL) and NGF into wild-type C-1300 and Neuro-2A murine neuro blastoma (MNB) and rat pheochromocytoma (PC-12) cells. Conditioned media ob tained from NET cells infected with the NGF gene contained biologically act ive NGF, whereas media from beta-GAL infected cells did not. Infection with the NGF vector induced a short-term decrease in cell proliferation rate an d increased neurite formation in wild-type, substrate-adherent PC-12 and Ne uro-2A MNB cells (P > 0.05). Incubation of wild-type C-1300, Neuro-2A MNB, and PC-12 cells with NGF (0-200 ng/ml) for 5 days significantly reduced pro liferation rates in a concentration-dependent manner and increased neurite extrusion. All NGF-NET cells had a significantly diminished response to the antiproliferative action of exogenous NGF. Ligand binding assays with I-12 5-NGF demonstrated a marked reduction in the number of NGF binding sites on NGF-NET cells compared to wild type. The attenuated response of NGF-NET ce lls to exogenous NGF correlated positively with the down-regulation of NGF binding sites. In conclusion, beta-NGF gene transfer into wild-type NET cel ls induces the synthesis and secretion of NGF, temporarily decreases cell p roliferation rate, increases neurite extrusion, down-regulates NGF binding sites, and reduces NET cell responsiveness to NGF. A putative role for NGF may be the modulation of NET cell proliferation and differentiation. (C) 20 00 Wiley-Liss, Inc.