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Sphingolipids are a complex family of molecules that participate in many aspects of cell structure and function, including an essential cellular process known as autophagy. Autophagy is a degradation and recycling pathway whereby intracellular components are sequestered into double-membrane vesicles, known as autophagosomes, for subsequent fusion with lysosomes and degradation. Autophagy takes part in cell survival, host immune defense against pathogens, and other biological processes, but is also sometimes lethal. Ceramide, sphingosine 1-phosphate, and more recently dihydroceramide have been shown to induce autophagy, which opens an interesting new field of cell regulation by sphingolipids. This dissertation describes two new cases in which sphingolipids participate in the induction of autophagy: a) RAW264.7 cells treated with Kdo2-Lipid A, a lipopolysaccharide sub-structure with endotoxin activity equal to LPS; and b) MCF7 cells treated with fenretinde, a chemotherapeutic agent which has shown success in clinical trials. It also analyzes the structural properties of fenretinide that contribute to its ability to modulate sphingolipid metabolism through inhibition of dihydroceramide desaturase, thereby elevating dihydroceramide and induction of autophagy. Autophagy was monitored by following the redistribution of GFP-LC3 into discrete punctate vesicles in response to the agents and by Western blotting; in parallel, the sphingolipid composition of the cells was monitored by liquid chromatography, electrospray ionization tandem mass spectrometry. These analyses revealed that Kdo2-Lipid A and fenretinide induce profound changes in sphingolipid metabolism in RAW264.7 and MCF7 cells, respectively, and that one of the purposes for increased de novo biosynthesis is to enable the production of autophagosomes, as the autophagic response was inhibited by myriocin. These studies have uncovered a direct link between sphingolipid metabolism and autophagy, which could pave the way for new therapeutic interventions for the treatment of pathogenic infection and be clinically useful in enhancing the efficacy of current cancer treatment strategies.

N-(4-Hydroxyphenyl) retinamide (4-HPR) is a derivative of all-trans-retinoic acid that induces apoptosis in cancer cell lines and is being tested in clinical trials as a relatively non-toxic anti-cancer agent. 4-HPR induces de novo sphingolipid biosynthesis and production of ceramide has been suggested to contribute to the growth arrest and apoptosis. To characterize the types of ceramides that might be involved, we used liquid chromatography, electrospray ionization tandem mass spectrometry (LC ESI-MS/MS) to analyze the sphingolipids, and found that 4-HPR increased total sphingolipid amounts, but unexpectedly, ceramides (i.e., N-acylsphingosines) changed very little, and in some cases decreased. Instead, dihydroceramides (i.e., N-acylsphinganines) increased as much as 10-fold, both as the free species and as the backbones of dihydrosphingomyelins and dihydrohexosylceramides. To determine if 4-HPR inhibits dihydroceramide desaturase, we synthesized NBD-dihydroceramide and treated Hek293 cells with 4-HPR and analyzed the metabolites by HPLC. These analyses showed that NBD-dihydroceramide was taken up by the cells and converted to NBD-ceramides and more complex NBD-sphingolipids in control cells, however, within one hour of treatment with 10 ~{ and L~}M 4-HPR, the production of NBD-ceramide was blocked. In vitro assays of the desaturase using NBD-dihydroceramide also showed rapid and complete inhibition by 4-HPR. Interestingly, when Hek cells were treated with 4-HPR for one hour then the medium was changed, the recovery of dihydroceramide desaturase activity was very slow (i.e., t1/2 > 66 h); therefore, either 4-HPR is difficult to remove from cells or the inhibition is essentially irreversible. These findings establish that 4-HPR not only induces de novo sphingolipid biosynthesis but also inhibits dihydroceramide desaturase, resulting in production of abnormally high proportions of sphingolipids with dihydroceramide as the backbone. This raises the possibility that some of the effects of 4-HPR on cell behavior may be due to the presence of these abnormal species.

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No description available.