title: Cell density-dependent reduction of dihydroceramide desaturase activity in neuroblastoma cells.
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 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.
authors.affiliation: Department of Medicine, Medical University of South Carolina, Charleston, SC 29425.
authors.forename: Stefka D
authors.initials: SD
authors.lastname: Spassieva
authors.affiliation: Department of Pediatrics Division of Hematology/Oncology, Medical University of South Carolina, Charleston, SC 29425; and.
authors.forename: Mehrdad
authors.initials: M
authors.lastname: Rahmaniyan
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425; and.
authors.forename: Jacek
authors.initials: J
authors.lastname: Bielawski
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425; and.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Clarke
authors.affiliation: Department of Pediatrics Division of Hematology/Oncology, Medical University of South Carolina, Charleston, SC 29425; and.
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Kraveka
authors.affiliation: Department of Medicine, Medical University of South Carolina, Charleston, SC 29425; Division of General Internal Medicine, Ralph H. Johnson Veterans Affairs Hospital, Charleston, SC 29401. Electronic address: obeidl@musc.edu.
authors.forename: Lina M
authors.initials: LM
authors.lastname: Obeid
mesh_terms: D002452:Cell Count; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D006801:Humans; D009447:Neuroblastoma / Q000473:pathology*; D010088:Oxidoreductases / Q000378:metabolism*; D019163:Reducing Agents / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000494:pharmacology
chemical_list: D002518:Ceramides; D003470:Culture Media; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; C109343:dihydroceramide; D010088:Oxidoreductases; C108138:dihydroceramide desaturase