OBJECTIVETo assess the effects of both TrkA and p75(NTR) on nerve growth factor (NGF)-induced differentiation of neuroblastoma cells.

METHODSRetroviral vectors were constructed to express the high affinity NGF receptor (TrkA) and low affinity NGF receptor (p75(NTR)). Neuroblastoma cell line IMR-32 was transfected by the vectors expressing either TrkA or p75(NTR) or both by using lipofectmine trade mark reagent separately or cotransfected at the same time. Southern blot, Northern blot, RT-PCR and flow cytometry were used to determine the success of the transfection. MTT technique was to monitor the cell proliferation. Colony formation in soft agar and tumor forming assay in nude mice were used to test the biological characteristics of the tumor cells. Terminal-deoxynucleotidytransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used to test the apoptosis of the tumor cells.

RESULTSStable transformant cell lines expressing TrkA, p75(NTR) or both genes were established. Studies on these transformant cell lines have shown different NGF responses. The p75(NTR) transfection only resulted in the mild differentiation response, and transfection of TrkA gene caused remarkable neurite extension, up-regulation of neurofilament and decreased expression of N-myc oncogene after NGF treatment. The cotransfection of the two genes into this cell line resulted in the more rapid and more apparent morphological changes than single TrkA transfected cells after NGF treatment. The cotransfected cells underwent apoptosis after withdrawal of NGF.

CONCLUSIONSThe results indicate that coexpression of both low- and high-affinity NGF receptors are not only more efficient in restoration of NGF-induced differentiation pathway, but also be able to activate the pro-apoptotic activity of low-affinity NGF receptor and make the tumor cells become NGF-dependent and irreversibly differentiated.