In this study, endogenous long chain ceramides were measured in 32 human head and neck squamous cell carcinoma (HNSCC) and 10 nonsquamous head and neck carcinoma tumor tissues, as compared with adjacent noncancerous tissues, by liquid chromatography/ mass spectroscopy. Interestingly, only one specific ceramide, C 18:0 -ceramide, was selectively down-regulated in the majority of HNSCC tumor tissues. On the other hand, in nonsquamous tumor tissues, this selectivity for C 18 -ceramide was not detected. These data suggested the hypotheses that decreased levels of C 18 -ceramide might impart a growth advantage to HNSCC cells and that increased generation of C 18 -ceramide may be involved in the inhibition of growth. These roles were examined by reconstitution of C 18 -ceramide at physiologically relevant concentrations in UM-SCC-22A cells (squamous cell carcinoma of hypopharynx) via overexpression of mammalian upstream regulator of growth and differentiation factor 1 (mUOG1), a mouse homologue of longevity assurance gene 1 (mLAG1), which has been shown to specifically induce the generation of C 18 -ceramide. Liquid chromatography/mass spectroscopy analysis showed that overexpression of the mLAG1/ mUOG1 resulted in increased levels of only C 18:0 -ceramide by ϳ2-fold, i.e. concentrations similar to those of normal head and neck tissues. Importantly, increased generation of C 18 -ceramide by mLAG1/mUOG1 inhibited cell growth (ϳ70 -80%), which mechanistically involved the modulation of telomerase activity and induction of apoptotic cell death by mitochondrial dysfunction. In conclusion, this study demonstrates, for the first time, a biological role for LAG1 and C 18 -ceramide in the regulation of growth of HNSCC.

These authors contributed equally to this work. SummaryThe nectrotrophic fungus Alternaria alternata f.sp. lycopersici infects tomato plants of the genotype asc/asc by utilizing a host-selective toxin, AAL-toxin, that kills the host cells by inducing programmed cell death. Asc-1 is homologous to genes found in most eukaryotes from yeast to humans, suggesting a conserved function. A yeast strain with deletions in the homologous genes LAG1 and LAC1 was functionally complemented by Asc-1, indicating that Asc-1 functions in an analogous manner to the yeast homologues. Examination of the yeast sphingolipids, which are almost absent in the lag1Dlac1D mutant, showed that Asc-1 was able to restore the synthesis of sphingolipids. We therefore examined the biosynthesis of sphingolipids in tomato by labeling leaf discs with L-[3-3 H]serine. In the absence of AAL-toxin, there was no detectable difference in sphingolipid labeling between leaf discs from Asc/Asc or asc/asc leaves. In the presence of pathologically significant concentrations of AAL-toxin however, asc/asc leaf discs showed severely reduced labeling of sphingolipids and increased label in dihydrosphingosine (DHS) and 3-ketodihydrosphingosine (3-KDHS). Leaf discs from Asc/Asc leaves responded to AAL-toxin treatment by incorporating label into different sphingolipid species. The effects of AAL-toxin on asc/asc leaflets could be partially blocked by the simultaneous application of AAL-toxin and myriocin. Leaf discs simultaneously treated with AAL-toxin and myriocin showed no incorporation of label into sphingolipids or long-chain bases as expected. These results indicate that the presence of Asc-1 is able to relieve an AAL-toxin-induced block on sphingolipid synthesis that would otherwise lead to programmed cell death.

Sphingolipids, lipids with a common sphingoid base (also termed long chain base) backbone, play essential cellular structural and signaling functions. Alterations of sphingolipid levels have been implicated in many diseases, including neurodegenerative disorders. However, it remains largely unclear whether sphingolipid changes in these diseases are pathological events or homeostatic responses. Furthermore, how changes in sphingolipid homeostasis shape the progression of aging and neurodegeneration remains to be clarified. We identified two mouse strains, flincher (fln) and toppler (to), with spontaneous recessive mutations that cause cerebellar ataxia and Purkinje cell degeneration. Positional cloning demonstrated that these mutations reside in the Lass1 gene. Lass1 encodes (dihydro)ceramide synthase 1 (CerS1), which is highly expressed in neurons. Both fln and to mutations caused complete loss of CerS1 catalytic activity, which resulted in a reduction in sphingolipid biosynthesis in the brain and dramatic changes in steady-state levels of sphingolipids and sphingoid bases. In addition to Purkinje cell death, deficiency of CerS1 function also induced accumulation of lipofuscin with ubiquitylated proteins in many brain regions. Our results demonstrate clearly that ceramide biosynthesis deficiency can cause neurodegeneration and suggest a novel mechanism of lipofuscin formation, a common phenomenon that occurs during normal aging and in some neurodegenerative diseases.

Lag1 (longevity assurance gene 1) homologues, a family of transmembrane proteins found in all eukaryotes, have been shown to be necessary for (dihydro)ceramide synthesis. All Lag1 homologues contain a highly conserved stretch of 52 amino acids known as the Lag1p motif. However, the functional significance of the conserved Lag1p motif for (dihydro)ceramide synthesis is currently unknown. In this work, we have investigated the function of the motif by introducing eight point mutations in the Lag1p motif of the mouse LASS1 (longevity assurance homologue 1 of yeast Lag1). The (dihydro)ceramide synthase activity of the mutants was tested using microsomes in HeLa cells and in vitro. Six of the mutations resulted in loss of activity in cells and in vitro. In addition, our results showed that C18:0 fatty acid CoA (but not cis-C18:1 fatty acid CoAs) are substrates for LASS1 and that LASS1 in HeLa cells is sensitive to fumonisin B 1 , an in vitro inhibitor of (dihydro)-ceramide synthase. Moreover, we mutated the Lag1p motif of another Lag homologue, human LASS5. The amino acid substitutions in the human LASS5 were the same as in mouse LASS1, and had the same effect on the in vitro activity of LASS5, suggesting the Lag1p motif appears to be essential for the enzyme activity of all Lag1 homologues.