We applied a metabolic approach to investigate the role of
sphingolipids in cell density-induced growth arrest in
neuroblastoma cells. Our data revealed that
sphingolipid metabolism in
neuroblastoma cells significantly differs depending on the cells' population context. At high cell density, cells exhibited G0/G1 cell-cycle arrest and reduced
ceramide,
monohexosylceramide, and
sphingomyelin, whereas
dihydroceramide was significantly increased. In addition, our metabolic-labeling experiments showed that
neuroblastoma cells at high cell density preferentially synthesized very long chain (VLC)
sphingolipids and dramatically decreased synthesis of
sphingosine-1-phosphate (S1P). Moreover, densely populated
neuroblastoma cells showed increased message levels of both anabolic and catabolic
enzymes of the
sphingolipid pathway. Notably, our metabolic-labeling experiments indicated reduced
dihydroceramide desaturase activity at confluence, which was confirmed by direct measurement of
dihydroceramide desaturase activity in situ and in vitro. Importantly, we could reduce
dihydroceramide desaturase activity in low-density cells by applying conditional media from high-density cells, as well as by adding
reducing agents, such as DTT and
L-cysteine to the media. In conclusion, our data suggest a role of the
sphingolipid pathway, dihydroceramides desaturase in particular, in confluence-induced growth arrest in
neuroblastoma cells.