GLIAL-CELL LINE-DERIVED NEUROTROPHIC FACTOR NEURTURIN-INDUCED DIFFERENTIATION AND ITS ENHANCEMENT BY RETINOIC ACID IN PRIMARY HUMAN NEUROBLASTOMAS EXPRESSING C-RET, GFR-ALPHA-1, AND GFR-ALPHA-2

Neuroblastomas often undergo spontaneous differentiation and/or regres sion in vivo, which is at least partly regulated by the signals throug h neurotrophins and their receptors, Recently, glial cell line-derived neurotrophic factor (GDNF) and a second family member, neurturin (NTN ), have been found to mediate their signals by binding to a heterotetr americ complex of c-Ret tyrosine kinase receptors and glycosylphosphat idylinusitol-linked proteins, GFR alpha-1 (GDNFR-alpha) or GFR alpha-2 (TrnR2/GDNFR-beta/ NTNR-alpha/RETL2). Here, we studied the effect of GDNF and NTN on human neuroblastomas in the short-term primary culture system, as well as the expression of c-Ret, GFR alpha-1, GFR alpha-2, GDNF, and NTN. GDNF (1-100 ng/ml) induced morphological differentiati on in 34 of 38 primary neuroblastomas and an accompanying increase in c-Fos induction. These effects were markedly enhanced by treatment wit h 5 mu M all-bans-retinoic acid. Although GDNF alone induced a rather weak differentiation independent of the disease stages, the enhancemen t of neurite outgrowth induced by treatment with both GDNF and all-tra ns-retinoic acid was significantly correlated with younger age (less t han 1 year; P = 0.0039), non-stage 4 diseases (P = 0.0023), a single c opy of N-myc (P = 0.027), and high levels of TRK-A expression (P = 0.0 062). To examine the expression levels of GFR alpha-1, we cloned a sho rt form of the human GFR alpha-1 gene with a 15-bp deletion by screeni ng a human adult substantia nigra cDNA library. Many primary neuroblas tomas expressed c-Ret, GFR alpha-1, and GFR alpha-2 as well as their l igands, GDNF and NTN, suggesting the presence of a paracrine or autocr ine signaling system within the tumor tissue. The effect of NTN on pri mary culture cells of neuroblastoma was similar to that of GDNF, These imply that the GDNF(NTN)/c-Ret/GFR alpha-1(GFR alpha-2) signaling may have an important role in regulating the growth, differentiation, and cell death of neuroblastomas.