The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) possesses antiproliferative and apoptotic activity at pharmacological doses. In this study we show that addition of antioxidants to HL-60 cells cultured in the presence of 3 microM HPR, markedly suppresses the apoptopic effect of the retinoid and significantly prolongs cell survival (48-96 h). We also show, by the use of the oxidation-sensitive probe 2',7'-dichlorofluorescin diacetate (DCF-DA) and in combination with flow cytometric and spectrofluorimetric analysis, that treatment of cells with 3 microM HPR results in an immediate and sustained production of intracellular free radicals, most likely hydroperoxides. Interestingly, the formation of these HPR-induced free radicals is effectively blocked by the water soluble antioxidants L-ascorbic acid and N-acetyl-L-cysteine. Neither 3-15 microM N-(4-methoxyphenyl) retinamide (MPR), the structurally similar but biologically inert analog of HPR, nor 3 microM doses of the retinoids all-trans retinoic acid, 9-cis-retinoic acid, TTNPB and SR11237 induce intracellular free radicals, thus indicating that the specificity of this phenomenon is restricted to HPR. Altogether, we provide the first direct evidence that HPR stimulates the generation of intracellular free radicals, which appear to have a causative role in the induction of apoptosis in vitro. Our findings raise the possibility that the therapeutic efficacy of HPR may, at least in part, depend on these apoptosis-inducing oxidative phenomena.

Human cultured fibroblasts were fed with G,, ganglioside species isotopically labeled at C3 of C1 Ssphingosine, or at C3 of C18-sphinganine, or at the sialic acid acetyl group, and with C18-sphingosine and CIS-sphinganine, both labeled at C1. After a lipid pulse the cells were subjected until 7-day chase; measurements were then made of the radioactive products resulting from the administered long-chain base and ganglioside species catabolism and the salvage processes of catabolic fragments. From the data we drew the following conclusions.The G,, species differing in the long-chain base structure were taken up by the cells and metabolized. About 80% of the total catabolic CIS-sphingosine and C18-sphinganine were recycled for the biosynthesis of complex sphingolipids, the rest being degraded.Results obtained by administering ganglioside species of G,, containing radioactive sphingosine or the free radioactive sphingosine to fibroblasts suggested the existence in the cells of two quite separate pools of sphingosine. One pool was the direct result of either the catabolism of radioactive G,, highdensity microdomains or the diffusion of exogenous sphingosine into the cell; this pool was mainly used for the biosynthesis of the G,, species that contain palmitic and stearic acids. The other pool of sphingosine, the cell basal pool, came from the catabolism of radioactive sphingolipids in the recycling of sphingosine, and was used for the biosynthesis of the G,, species that mainly contain very long fatty acid chains, the main fibroblast endogenous species of GD3. Administration of the ganglioside species of G,, containing sphinganine or free sphinganine to fibroblasts yielded the G,, species containing mainly very long-chain fatty acids and sphingosine.These results show the possible existence of a pool of ganglioside-derived sphingosine, quite separate from the cell basal pool of sphingosine, suggesting that sphingosine derived from sphingolipid catabolism is reduced to sphinganine before entering the sphingolipid biosynthetic pathway.Keywords: ganglioside ; biosynthesis ; sphingosine ; sphinganine ; recycling.Gangliosides, sialic acid containing glycosphingolipids that are normal components of plasma membranes [I], are biosynthesized in the Golgi apparatus and degraded in the lysosomes [2] to fragments that are recycled for biosynthetic purposes [3-61. Among the ganglioside catabolic fragments, sphingosine has attracted scientific interest and increasing attention. It has been shown to act as an intracellular effector through the modulation of protein kinase C and other enzyme activities [7-91. Most of our present information arises from the administration of sphingosine to cells, but little is known about a potential role of a pool of sphingosine originated from ganglioside catabolism in modulating cell function.Catabolic sphingosine is partly recycled, but much of it is degraded [6, ; thus it is difficult to determine to what degree this recycling affects the overall sphingolipid turnover. Moreover, the de novo biosy...

Oxidn. of di-Et α-benzylmalonate (I) by Mn(III) acetate in acetic acid at 70° in the presence of alkynes RC≡CR1 (R = cyclohexyl, CO2Et, Me3Si, Ph, CH2OH, R1 = H; R = R1 = Ph, Et, CO2Et, Pr; R = CO2Me, R1 = Me) leads to dihydronaphthalene derivs. II in moderate to good yields. A homolytic mechanism, involving oxidn. of I to the corresponding malonyl radical, its addn. to a triple bond, and intramol. homolytic arom. substitution of the vinyl radical adducts, is suggested. Propargylic hydrogen abstraction and dimerization at the malonic position for less reactive alkynes are the main side reactions obsd. Relative and abs. rates of addn. of α-benzylmalonyl radicals to representative alkynes and alkenes, deduced from competitive expts., indicate a lower reactivity toward alkynes than toward the corresponding substituted alkenes. Both SOMO-LUMO and SOMO-HOMO interactions in the transition state lower the activation energy of these homolytic addns. with alkenes, but only the SOMO-HOMO interaction dominates with the examd. alkynes. 2-Naphthoic acid derivs. can be efficiently obtained by oxidative decarboxylation of II with NaI and ai

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Changes in the free long-chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total LCB mixtures, extracted from the cells maintained in culture up to 22 days, were derivatized with o-phthalaldehyde and fractionated by reversed-phase HPLC, and each LCB species was quantified. Four main LCBs were components of the total LCB mixtures of cultured cells: Cl 8sphingosine, C18-sphinganine, C20-sphingosine, and C20-sphinganine. They were found in all the cells analyzed, from 0 to 22 days of culture, with their contents being in the sequence C18-sphingosine > C18-sphinganine > C20-sphingosine > C20-sphinganine and varying from 0.02 ±0.015 pmol/mg of cell protein for C20sphinganine at day 0 to 223 ±22 pmol/mg of cell protein for C18-sphingosine at day 8. Sphinganines were found to be minor components of the total LCB mixture, with C20-sphinganine being particularly scarce in nondifferentiated cells. The cell content of C20-sphinganine progressively increased from day 0 to 22 of culture; that of C18sphinganine increased up to day 8, when cells are differentiated, and then remained quite constant. The changes of C18-and C20-sphingosine levels during cell culture were qualitatively similar to those of Cl 8-and C20-sphinganine, but the content of the sphingosines was much higher than that of the sphinganines.