Diverse responses to retinoid in morphological differentiation, tumorigenesis and n‐myc expression in human neuroblastoma sublines

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Abstract
We established the subline RT‐BMV‐C6 from the parent human neuroblastoma cell line RT‐BM by a process that required repeated subculture of cells, which were prone to disaggregation. RT‐BMV‐C6 and the parent cloned line, RT‐BM‐I, had an identical marker chromosome, confirming that both lines were derived from a common progenitor. In the analysis of surface antigen expression, RT‐BMV‐C6 did not react with UJ‐127‐11, Leu7 or KP‐NAC2 MAbs to which RT‐BM‐I showed positive binding. The levels of both N‐myc amplification and expression in RT‐BMV‐C6 were twice as high as the level obtained in RT‐BM‐I. Colony‐forming efficiency in soft agar was 2.0 ± 0.8% for RT‐BMV‐C6 and 3 times greater than that for RT‐BM‐I (0.6 ± 0.1%). When 100 × 106 cells of RT‐BM‐I and RT‐BMV‐C6 were inoculated into nude mice, tumor incidence was significantly higher for RT‐BMV‐C6 (6/6; 100%) than for RT‐BM‐I (0/6; 0%). Our data show that N‐myc is closely related to tumorigenicity in NB. When RT‐BM‐I and RT‐BMV‐C6 were co‐cultured with a new synthetic retinoid, polyprenoic acid (E5166), and dibutyryi cyclic AMP, RT‐BM‐I was induced to neuronal differentiation, defined by the formation of neuronal processes and expression of neurofilaments, whereas RT‐BMV‐C6 was not. However, when exposed to E5166, N‐myc expression of RT‐BMV‐C6 was more strongly reduced than that of RT‐BM‐I, and colony formation of RT‐BMV‐C6 was significantly inhibited as compared to RT‐BM‐I. These findings suggest that the reduction of N‐myc expression might closely correlate with growth inhibition accompanying neuronal differentiation of neuroblastoma cells.