Serine palmitoyltransferase (SPT) is the rate-limiting enzyme in de novo ceramide generation and is composed of two subunits, LCBI and LCBII. RNA for LCBI and LCBII is highly expressed in 17 of 17 human neuroblastoma cell lines by quantitative TaqMan RT-PCR. To study the role of LCBI and LCBII in the de novo ceramide increase induced by the synthetic retinoid N- (4-hydroxyphenyl) retinamide (4-HPR), we designed small interfering RNA (SiRNA) constructs for LCBI and LCBII. Using Lipofectamine 2000, we transfected SiRNA LCBI and SiRNA LCBII into the CHLA-90 neuroblastoma cell line. CHLA-90 transfected with SiRNA LCBI or LCBII for 48 hrs were treated with 4-HPR (10 μM ) for 6 hrs and de novo (14C palmitic acid label) ceramide levels were analyzed by thin layer chromotagraphy (TLC). RT- PCR showed that LCBI gene and LCBII mRNA expression were down-regulated > 70% by their respective SiRNA, but not by the other SiRNA. This was confirmed at the protein level for LCBI. However, de novo ceramide production was only decreased (∼10-fold compared to the vehicle control) by LCBII, and not LCBI, SiRNA. Suppression of de novo ceramide production by 4-HPR (∼7 fold) was still observed in LCBII SiRNA-transfected CHLA-90 for 16 hrs. We also used Gene Pulser Xcell Electroporation System to transfect SiRNA LCBII into the SK-N-RA neuroblastoma cell line and decreased LCBII mRNA ( > 80%) and decreased 4-HPR induction of de novo ceramide synthesis by > 65%. Here we provide the first evidence from human neuroblasoma cell lines that de novo ceramide increase induced by 4-HPR was mainly regulated by LCBII subunit of SPT. These data indicate that SiRNA for LCBII, but not LCBI can be used to block 4-HPR-stimulated de novo ceramide synthesis in human neuroblastoma cells.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]