Tumor promoter receptors regulating neurite formation in cultured human neuroblastoma cells.

The mouse skin tumor promoter phorbol-12,13-dibutyrate (PDBU) reversibly enhanced neurite outgrowth in SH-SY5Y human neuroblastoma cells, whether serum was present or absent. The half-maximum response in serum occurred at 10 nM. The binding of [20-3H]phorbol-12,13-dibutyrate [( 3H]PDBU) was studied. Five mouse skin tumor promoters, which included teleocidin, mezerein, and three structural congeners of phorbol, enhanced neurite outgrowth and inhibited binding of [3H]PDBU, but two nonpromoting phorbol congeners did neither. Saccharin and cyclamate, promoters in rat bladder, did not inhibit [3H]PDBU binding. Binding of 10 nM [3H]PDBU at 37 degrees was maximal within 5 min and stable for at least 5 hr. Down modulation of binding was not detected. Following binding at 37 degrees, the dissociation rate in excess PDBU was biphasic, whether measured at 37 degrees or at 4 degrees. The Scatchard curve was also consistent with two types of sites, about 2.5 X 10(5) sites/cell with Kd = 8.5 nM and 1.2 X 10(6) sites with Kd = 125 nM. Negative cooperativity was not observed. In short-term assays, nerve growth factor (NGF) did not alter [3H]PDBU binding, and phorbol ester promoters did not alter 125I-NGF binding. Furthermore, [3H]PDBU binding was unaltered following growth of cells for 1 week in PDBU or NGF, conditions under which neurite outgrowth was continuously enhanced, and other phenotypic expressions of differentiation are known to be increased. Specific [3H]PDBU binding sites were present in five neuroblastoma cell lines, two of which are responsive and three unresponsive by neurite outgrowth to promoters and NGF, suggesting the possibility of a common lesion in distal steps in the unresponsive lines.