Cell density-dependent reduction of dihydroceramide desaturase activity in neuroblastoma cells

Abstract

This article is available online at http://www.jlr.orgIn the last two decades, sphingolipids and their precursors have been extensively studied for their cell signaling roles (recently reviewed in Ref. 1 ). Changes in sphingolipid levels are shown to play a role in regulating important cellular processes, such as cell proliferation, growth arrest, and cell death. Here we investigated the role of sphingolipids in cell-cycle arrest at high cell density, a phenomenon known as contact inhibition at saturation density ( 2 ). Density-dependent inhibition in cultured cells serves as a model to study the balance between cell proliferation and growth arrest in a multicellular organism. Cell-number control is important for maintenance of tissue homeostasis within multicellular organisms, and cell-cycle arrest is a critical aspect of that control mechanism ( 2, 3 ). Cell-cycle arrest at high saturation density is a complex process dependent on multiple factors, and earlier studies have shown that sphingolipids play a role in it. Gangliosides, GM 3 in particular, have been shown to promote growth inhibition in confl uent cells ( 2,4,5 ), and a recent study has revealed that abundance of another ganglioside, GM 1 , on the cellular membrane depends on the cell's population context ( 6 ). A more recent work showed that very long chain (VLC) ceramides (i.e., ceramides with fatty acid chain length C 22 -C 26 ) also play a role in cell-cycle arrest at contact inhibition in breast cancer cells ( 7 ).Changes in sphingolipids, resulting from various cellular insults or manipulation of the sphingolipid pathway, are often complex and include simultaneous changes of several sphingolipid species ( 1,8 ). Therefore, considering the interdependency of the sphingolipid pathway, we applied a metabolic approach to decipher the role of sphingolipids in density-dependent growth arrest in neuroblastoma cells. Neuroblastoma is the most common cancer in infants and understanding its biology is important for devising new therapeutic strategies.Abstract 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 signifi cantly 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 signifi cantly 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 confl uence, which was confi rmed by direct measurement of dihydroceramide desat...

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

“…Here, none of the modulating effects of new ASM isoforms are observed, as their mRNA expression remains nearly constant. These data stress the fact that sphingolipid metabolism is significantly influenced by cell confluence levels [25] . For sphingolipid research using cell culture models it is of highest importance to consider these influencing factors.…”

“…A further physiological cell model deals with the well-known phenomenon that sphingolipid metabolism significantly differs depending on cell confluence levels [25] . H4 cells were grown to varying levels of confluence and analysed for expression levels of each novel splice variant and their respective cellular ASM activity levels.…”

Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II

“…First, the SK1 specifi c inhibitor, PF543, did not affect HGC 27 cell proliferation at 8 and 24 h, although it avoided S1P production with no remarkable changes in other sphingolipid species. Second, the effect of dhCers at slowing or arresting the cell cycle with accumulation of cells in the G0/G1 phase is welldocumented ( 35,(83)(84)(85). Therefore, we suggest that the effect of SKI II on the cell cycle may be due to the accumulation of dhCer resulting from its Des1 inhibitory activity.…”

Inhibition of Sphingolipid Metabolism Enhances Resveratrol Chemotherapy in Human Gastric Cancer Cells

“…The DHCer desaturase enzymatic reaction was optimized, as previously described (Spassieva et al ., 2012). Briefly, the DHCer substrate was added to an Eppendorf tube and dried in a vacuum concentrator.…”