fumonisin-b1 and dihydroceramide

A new variant of a group of pediatric neurodegenerative diseases known as neuronal ceroid lipofuscinosis (NCL) or Batten disease has been identified. It is termed CLN9-deficient. CLN9-deficient fibroblasts have a distinctive phenotype of rapid growth and increased apoptosis and diminished levels of ceramide, dihydroceramide, and sphingomyelin. Transfection with CLN8 but not other NCL genes corrected growth and apoptosis in CLN9-deficient cells, although the entire CLN8 sequence was normal. CLN8 is one of the TRAM-Lag1-CLN8 proteins containing a Lag1 motif. The latter imparts (dihydro)ceramide synthase activity to yeast cells. Transfection with the yeast gene Lag1 Sc and the human homolog LASS1 increased ceramide levels and partially corrected growth and apoptosis in CLN9-deficient cells. LASS2,-4,,-5, and -6 also corrected growth and apoptosis. Dihydroceramide levels and dihydroceramide synthase activity were markedly diminished in CLN9-deficient cells. Sequencing of LASS1, LASS2, LASS4, LASS5, and LASS6 genes was normal, and expression levels were increased or normal in CLN9-deficient cells by reverse transcription-PCR. N-(4-Hydroxyphenyl)retinamide (4-HPR), a dihydroceramide synthase activator, corrected growth and apoptosis and increased dihydroceramide synthase activity. Ceramide levels dropped further, and there was no increase in de novo ceramide synthesis, probably due to the effects of 4-HPR as activator of dihydroceramide synthase and inhibitor of dihydroceramide desaturase. Fumonisin B1, a dihydroceramide synthase inhibitor, exaggerated the CLN9-deficient phenotype of accelerated growth, decreased ceramide and increased apoptosis. This was neutralized by 4-HPR. We conclude that the CLN9 protein may be a regulator of dihydroceramide synthase and that 4-HPR could be developed as a treatment for CLN9-deficient patients.

Topics: Apoptosis; Cell Line; Cell Proliferation; Ceramides; Fenretinide; Fibroblasts; Fumonisins; Humans; Membrane Proteins; Neuronal Ceroid-Lipofuscinoses; Oxidoreductases; Saccharomyces cerevisiae Proteins; Sphingosine N-Acyltransferase

Ceramide is formed through sphingomyelin hydrolysis or de novo synthesis and may play a key role in cell growth, differentiation and apoptosis. To clarify which pathway tumor cells use to form ceramide and how its formation is regulated, we determined the levels of dihydroceramide and ceramide in mice inoculated with Sarcoma 180, B16 melanoma or Lewis lung carcinoma cells. The levels in these tumor masses were very high compared to those in other healthy tissues. The high levels were significantly reduced by a single administration of the dihydroceramide synthase inhibitor fumonisin B(1), but not by a sphingomyelinase inhibitor, sphingomyelin analog-1 (SMA-1), suggesting that the tumor cells have a very effective means of synthesizing dihydroceramide and ceramide. To investigate the characteristics of dihydroceramide synthase, we prepared microsomes from Sarcoma 180 tumor masses and healthy mouse liver cells, and compared their catalytic activities on dihydroceramide formation. A kinetic analysis using sphinganine and palmitoyl CoA as substrates revealed that the enzyme present in the tumor formed dihydroceramide 3 times more efficiently than that in healthy liver cells. Partial purification of dihydroceramide synthase from bovine liver microsomes revealed that the enzyme was present in healthy tissues as a 333 kDa form constructed of 47 kDa subunit proteins. However, gel filtration of the enzyme solubilized from the Sarcoma 180 tumor masses demonstrated that its molecular weight was 1300 kDa. These results suggest that malignant transformation causes the cell to produce a form of dihydroceramide synthase with a larger than normal molecular mass; the increased molecular mass may account for the enzyme's increased catalytic efficiency.

Topics: Amides; Animals; Apoptosis; Catalysis; Cattle; Ceramides; Chromatography, Gel; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fumonisins; Gene Expression Regulation, Enzymologic; Kinetics; Liver; Liver Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Microsomes; Microsomes, Liver; Organophosphonates; Oxidoreductases; Tumor Cells, Cultured

Overexpression of upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene (LAG1), selectively induces the synthesis of stearoyl-containing sphingolipids in mammalian cells (Venkataraman, K., Riebeling, C., Bodennec, J., Riezman, H., Allegood, J. C., Sullards, M. C., Merrill, A. H. Jr., and Futerman, A. H. (2002) J. Biol. Chem. 277, 35642-35649). Gene data base analysis subsequently revealed a new subfamily of proteins containing the Lag1p motif, previously characterized as translocating chain-associating membrane (TRAM) protein homologs (TRH). We now report that two additional members of this family regulate the synthesis of (dihydro)ceramides with specific fatty acid(s) when overexpressed in human embryonic kidney 293T cells. TRH1 or TRH4-overexpression elevated [3H](dihydro)ceramide synthesis from l-[3-3H]serine and the increase was not blocked by the (dihydro)ceramide synthase inhibitor, fumonisin B1 (FB1). Analysis of sphingolipids by liquid chromatography-electrospray tandem mass spectrometry revealed that TRH4 overexpression elevated mainly palmitic acid-containing sphingolipids whereas TRH1 overexpression increased mainly stearic acid and arachidic acid, which in both cases were further elevated upon incubation with FB1. A similar fatty acid specificity was obtained upon analysis of (dihydro)ceramide synthase activity in vitro using various fatty acyl-CoA substrates, although in a FB1-sensitive manner. Moreover, in homogenates from TRH4-overexpressing cells, sphinganine, rather than sphingosine was the preferred substrate, whereas no preference was seen in homogenates from TRH1-overexpressing cells. These findings lend support to our hypothesis (Venkataraman, K., and Futerman, A. H. (2002) FEBS Lett. 528, 3-4) that Lag1p family members regulate (dihydro)ceramide synthases responsible for production of sphingolipids containing different fatty acids.

Topics: Acyl Coenzyme A; Animals; Cell Line; Ceramides; Chromatography, Liquid; COS Cells; DNA, Complementary; Enzyme Inhibitors; Fatty Acids; Fumonisins; Gene Expression Regulation; Humans; Kinetics; Membrane Proteins; Mice; Microscopy, Fluorescence; Multigene Family; Palmitic Acids; Protein Structure, Tertiary; Sphingolipids; Sphingosine N-Acyltransferase; Time Factors; Tissue Distribution; Transfection; Tumor Suppressor Proteins