Abstract 5261: Transfection of nerve growth factor cDNA into SK-N-BE(2)C neuroblastoma modulates TrkA and p75 receptor expression and activation

Abstract

Abstract The diverse biological role of nerve growth factor (NGF) as a necessary modulator of neuroectodermal cell survival, proliferation and differentiation has been previously well described in both normal and neoplastic cell lines. However, recent studies demonstrate that neuroectodermal tumors, such as neuroblastoma, contain genetic alterations that result in limited secretion of active NGF, as well as expression abnormalities of its receptors TrkA and p75. To better define the differential effects of exogenous and endogenously synthesized NGF, the in vitro response of wild-type and genetically engineered neuroblastoma cells was investigated. Replication-defective NGF-expressing retroviral vectors were prepared using the calcium phosphate co-precipitation method and used to transfect SK-N-BE(2)C cells. Presence of the NGF cDNA was confirmed by RT-PCR, and secretion of NGF into the conditioned media by western immunoblotting. NGF transfection resulted in decreased cell proliferation as determined by nonradioactive cell proliferation assay. Wild type and NGF transfected (BE(2)C-NGF) cells were then incubated with exogenous NGF at 200ng/mL for varying time periods up to 24 hours. The expression of TrkA and p75, as well as proteins of their associated survival and apoptosis cascades were determined by Western immunoblotting of whole cell lysates. p75 expression in the NGF transfected cell line was equivalent to the parental wild type cell, but TrkA expression was significantly lower in BE(2)C-NGF. At baseline, BE(2)C-NGF cells demonstrate significant down-regulation of phospho-cRAF and its downstream effector phospho-p42/44 in comparison to the wild type cell line. Additionally, NGF stimulation further down-regulated phospho-c-RAF expression in the NGF transfected cell line, with maximal down-regulation occurring at 24 hours. BE(2)C-NGF cells also demonstrate significant baseline activation of phospho-p38 in comparison to the wild type cell line, with expression further up-regulated with NGF stimulation at 5 minutes and 1 hour. In conclusion, nerve growth factor cDNA gene transfer into wild type SK-N-BE(2)C neuroblastoma results in decreased activation of the p-42/44 survival pathway and concomitant up-regulation of the p-38 apoptotic pathway, leading to a overall decreased rate of proliferation. This effect is likely the result of a decreased TrkA to p75 receptor ratio, and further understanding of these pathways may facilitate the discovery of novel therapeutic targets for neuroblastoma treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5261.