2nd Meeting

Sale Marasino, Italy, June 2/4, 2003

Sphingolipids as regulators of cell death: facts and uncertainties.

T. Levade , N. Andrieu-Abadie, E. Bonhoure, S. Carpentier, O. Cuvillier, V. Garcia, V. Gouazé, S. Malagarie, I. Popa# , J. Portoukalian# , B. Ségui, C. Tardy, R.J. Veldman*
INSERM U.466, Lab. Biochimie, CHU Rangueil, Toulouse, France ; # INSERM U.346, Lyon, France ; * (present address) The Netherlands Cancer Institute, Amsterdam

Programmed cell death is a regulated process that is required for normal development and tissue homeostasis. The major biochemical pathways involved in apoptosis include the activation of caspases and mitochondrial events. Since the pioneering work of Obeid and Hannun on ceramide and TNF-induced apoptosis one decade ago, a wealth of reports have been published supporting a role for sphingolipids in the initiation and/or regulation of the apoptotic program [1]. Different sphingolipids have been implicated, including ceramide, sphingosine, sphingosine-1-phosphate as well as some glycolipids, which could trigger or modulate either programmed cell death or cell survival [2]. Despite intensive search, the molecular mechanisms that underlie the production of action of (bioactive) sphingolipids, and more generally the functions of sphingolipids in cell death/growth regulation remain to be clarified. Several instances of still unsolved issues will be presented. These include i) the contribution of acid and neutral sphingomyelinases to the generation of ceramide as a potential proapoptotic second messenger, both in cultured cell systems and in animals, ii) the subcellular location of this bioactive ceramide, and especially its localization to membrane microdomains, iii) the nature of ceramide targets (for example, the role of cathepsin D as a ceramide-controlled protease in apoptosis will be discussed), iv) the role of enzymatic conversion of ceramide to glycolipids in cell survival/resistance, and v) the role of ganglioside production in apoptosis signalling. These various issues will be critically discussed in the light of recent observations .

1 – Hannun YA, Luberto C. (2000) Trends Cell Biol . 10 : 73-80
2 – Levade T, Hannun YA, Spiegel S. (2002) Biochim. Biophys. Acta 1585 : 51-226

Design and synthesis of ceramide analogues as a new strategy for anticancer drug development.

Marco Macchia
Dipartimento di Scienze Farmaceutiche, Università di Pisa

Sphingomyelins play a primary role in the regulation of cellular responses to exogenous stimuli as well as cell growth, differentiation, transformation, and cell-cell contact. In particular, membrane sphingomyelin is hydrolyzed in response to extracellular stimuli, such as tumour necrosis factor-alpha (TNF- a ) and interleukin 1, generating the putative second messenger ceramide. Ceramide, in turn, is thought to propagate the signal into the cell interior by the activation of phosphatases and mitogen-activated protein kinases. The mode of action of ceramide and the regulation of its production have recently attracted great attention due to the emerging role of this lipid messenger as an intracellular effector molecule in apoptosis. On the other hand, growth factors, such as platelet-derived growth factor, trigger further hydrolysis of ceramide to sphingosine and activate sphingosine kinase to form sphingosine 1-phosphate with promotion of cell growth. For these reasons, the dynamic balance between levels of ceramide and sphingosine 1-phosphate, is an important factor that determines whether a cell survives or dies .

In view of the emerging role of ceramide as an intracellular effector molecule, possible therapeutic targets of new pharmacological interventions directed at regulating abnormal tumour growth include the development of analogues that mimic the effects of endogenous ceramide, are metabolically stable to prevent further metabolism to sphingosine, and possess an inhibitory activity on ceramidase to prevent generation of sphingosine from endogenous sources of ceramide: on this basis, a series of conformationally restrained and metabolically stable ceramide analogues have been developed in our laboratory; these compounds proved to induce cytotoxicity and apoptosis in human cancer cells.

Sphingomyelin metabolism in response to the effect of the UV cosmic (stratospheric) radiation.

Perrella G. °, Cataldi S.*, Albi E.*, Toller M., Meli A., Del Terra E., Casani S.,
°Department of Experimental and Clinical Pathology and Medicine University of Udine
*Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Perugia

Classic radiation dosimetry, performed with traditional physical methods, does not evaluate the kinetics of radiation absorbance or the biological response(s) to radiation. Both are key aspects in biological systems and cells, which possess elaborate radiation response mechanisms (DNA repair, cellular components turnover, apoptosis, etc.).

Biological dosimetry on eukaryotic cell cultures provides nowadays elaborate experimental targets very close to animal and human organs or organisms, which can be used as radiation target systems.

In this study, the available technologies allow us to use FRTL5 cells to evaluatey the biological radiation effects under the following different physiological conditions: 1) in proliferative state, in Thyroid Stimulating Hormone (TSH) additioned growth medium; 2) in non proliferative state, in TSH-deprived culture medium. For both conditions the FRTL5 cells have been exposed to ionizing radiation (11,23 m Sv/h) by mean of stratospheric balloons or UVC (20 J/m 2 ). Shielded cells have been used as non irradiated control in the stratospheric balloons.

The FRTL5 cells are a strain of normal and differentiated follicular thyroid cells, obtained, characterized and also patented by us some years ago, presently an international standard for the clinical or autoimmune thyroid diseases. The FRTL5 cell line permanently express in vitro most of the in vivo tissue-specific thyroid characteristics, such as Thyroglobulin synthesis and secretion, Iodide active transport, Peroxidase production and Thyrotropin (TSH) sensitivity, etc. Other cell types (in particular of human origins) now under test in our laboratory may possibly be used in addition.

We studied sphingomyelin metabolism modifications in response to the effect of UV and stratospheric radiation related to the apoptosis process.

The results: exposure of FRTL5 cell lines to U.V. radiation appears to inhibit neutral sphingomyelinase activity and to increase sphingomyelin synthase activity in the nuclear cellular compartment. This effect is comparable to the enzymatic activities registered during the G2/M cell cycle phase and apoptosis of hepatocytes.

Both inhibition of neutral sphingomyelinase and enhancing of sphingomyelin synthase activities by high altitude (stratospheric) radiations occur in all FRTL5 samples, including controls.

Previous studies have already demonstrated that neutral sphingomyelinase stimulates cell proliferation; thus, it might be assumed that the reduced activity of this enzyme by radiations could determine a decreased cell growth rate. Moreover, the radiation-induced increase of sphingomyelin synthase activity could indicate that FRTL5 cells stop their cell cycle at G2/M, thus avoiding to proceed to mitosis. In other words, exposure of cells to high altitude radiations could determine alteration of metabolic pathways, so that cells are with the time be induced to enter the apoptosis process.

The increased sphingomyelin intracellular concentration due to the radiation-induced enhanced enzymatic activity could modify membrane fluidity, which will be reduced, and thus interfering with nucleus-cytoplasm exchange processes. Moreover, as G2/M nuclear transition step should slow down, mitotic fuse formation could be affected.

Serum deprivation activates the sphingomyelin metabolism in normal neuronal nuclei.

Elisabetta Albi1 , Samuela Cataldi1 , Laura Colombaioni2 , Francesca Mazzoni3 , Mariapia Viola Magni1 , Vladimir Voccoli3 , and Mercedes Garcia-Gil3 .
Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100, Perugia; 2) Institute of Neuroscience CNR, Pisa; 3) Dpt. of Physiology and Biochemistry, University of Pisa.

Sphingolipids are known as molecules involved in cell proliferation, differentiation and apoptosis processes. Recent evidences suggest that the subcellular localization of ceramide may have a critical role in determining the cellular response. Since enzymes involved in ceramide metabolism such as sphingomyelinase, sphingomyelin synthase, sphingosine kinase and ceramidase have been found in the nucleus (Albi and Viola Magni, 1997, 1999; Tsugane et al., 1999, Jaffrezou et al., 2001; Kleuser et al., 2001), we have studied whether nuclear sphingomyelin metabolism varies during apoptosis induced by serum deprivation in the embryonic hippocampal cell line (HN9.10e), by measuring the activites of sphingomyelinase and sphingomyelin-synthase. We have previously shown, during the apoptosis induced by serum deprivation, an increase of cell ceramide levels, a translocation of Bax, a release of cytochrome c, and a maintenance of mitochondrial functionality followed by calcium deregulation and caspase-3 activation. We report now that nuclear sphingomyelinase increases biphasically, with an earlier peak one hour after deprivation, and a second at 15 h until 48h after deprivation. In contrast, nuclear sphingomyelin-synthase decreases after serum deprivation with a minimun after 1 hour. These changes in nuclear metabolism of sphingomyelin occur before the nuclear translocation of the NFkB transcription factor, and the nuclear raise of Ca ++ . Our results suggest that the formation of ceramide through nuclear sphingomyelinase activation may have a role in serum deprivation-induced apoptosis.

Albi E. and Viola-Magni M. (1997) Biochem. Biophys. Res. Commun. 236 , 29-33.
Albi E. and Viola-Magni M. (1999) FEBS Lett. 460 , 369-372.
Jaffrezou J.P., Bruno A.P., Moisand A., Levade T. and Laurent G. (2001) FASEB J. 15 , 123-133.
Kleuser B., Maceyka M., Milstien S., and Spiegel S. (2001) FEBS Lett. 503 , 85-90.
Tsugane K., Tamiya-Koizumi K., Nagino M., Nimura Y. and Yoshida S. (1999) J. Hepatol. 31 , 8-17.

Lysosomes, cell death and drug resistance in cancer: when sphingolipids and cathepsins get together.

Roberta Castino, Marzia Mirabelli, Giuseppina Nicotra, Anna Aspesi, Marina Démoz, and Ciro Isidoro
Università del Piemonte Orientale “A. Avogadro”, Dipartimento di Scienze Mediche di Novara

Altered regulation of cell survival and cell death is considered an important factor contributing to tumour development and progression, as well as to the resistance to anti-cancer therapy. Recent data emphasise the role of lysosomes in the induction of cell death, not only in necrosis and autophagic type cell death but also in apoptosis. At least two classes of lysosomal hydrolases contribute to the regulation of the cellular response toward cytotoxic cytokines and antiblastic drugs: (1) those involved in sphingolipid metabolism, such as acid sphingomyelinase and acid ceramidase, and (2) acid endopeptidases such as Cathepsins B and D.

A central molecule that mediates the death signal triggered by cytotoxic cytokines and chemotherapeutic drugs is ceramide. Hyper-expression of acid ceramidase has been shown to confer resistance to cytotoxic treatments with TNF a , whereas pharmacological inhibition of acid ceramidase renders the cells highly susceptible to the cytokine. In accord with the above observation, the induction of apoptosis in cancer cells by ionising radiation, cytotoxic cytokines and various chemotherapeutic drugs has been shown to require the activation of Acid Sphingomyelinase, the enzyme that leads to ceramide production within lysosomes. Finally, sphingosine, that is formed by hydrolysis of ceramide by acid ceramidase, exerts a cytotoxic effect via destabilisation of the lysosomal membrane and consequent leakage of lysosomal hydrolases. Another class of lysosomal hydrolases that has been shown to play a role in drug resistance comprises the proteases Cathepsins B and D. Both this acid endopeptidases have been shown to mediate the death signal triggered by cytotoxic cytokines and various chemotherapeutic drugs. Of note, the expression of Cathepsin D is regulated also by the levels of p53. In addition, the maturation and activation of pro-cathepsin D is catalysed by lysosomal ceramide.

These data suggest that defective functioning of the lysosomal metabolism (of certain lipids and proteins) in cancer cells may constitute a novel mechanism of chemo-resistance to antineoplastic drugs as well as of immune-resistance against cytotoxic cytokines.

We will review the literature on this subject and will present some original data recently obtained in our laboratory.

Funded by MIUR-Cofin2001, AIRC, Regione Piemonte and CNR-target project on Biotechnology

The impact of ceramide on lysosomal targeting: a study with wild-type and glycosylation knock-out mutated cathepsin D-green fluorescent protein chimeras.

Camilo Gianinazzi , Nicol Trincheri, Carlo Follo, Anna Aspesi, Marina Démoz, Roberta Castino and Ciro Isidoro
Università del Piemonte Orientale “A. Avogadro”, Dipartimento di Scienze Mediche di Novara

Ceramide is a sphingosine-based lipid naturally occurring within the cell that acts as a pleiotropic second messenger in a number of cellular functions ranging from proliferation and differentiation to senescence and apoptosis. Onset of cell death is associated with morphological changes that in an ordered, as yet not known, sequence involve the various organelles and the cytoskeleton. This implies that location of organelles has to change in order to provide a topological organisation of membranes that favours the biochemical cross-talk among the organelles involved in the cell death process. Also, molecules must exit from one compartment and enter to another in order to propagate the death signal. Leakage of molecules from an organelle imply (a transient) loss of stability of membranes. The aim of the present work was to assess the impact of ceramide on protein transport and membrane traffic, with a particular focus on lysosomes and lysosomal targeting of cathepsin D, a lysosomal protease involved in the cell death process. To this end we have followed by confocal immunofluorescence the localisation of the fusion protein Cathepsin D-Green Fluorescent Protein (CD-GFP) in cells treated with N-acetyl sphingosine (NAS), a synthetic analogue of ceramide. Cathepsin D (CD) is synthesised in the rough endoplasmic reticulum as a di-glycosylated precursor that is transported to the Golgi apparatus and eventually to the endosomal-lysosomal compartments via Mannose-6-Phosphate (M6P)-dependent and M6P-independent pathways. The latter pathway can be studied by expressing mutagenized cDNA coding for CD in which either one or both N-glycosylation sites have been knocked-out. We and other have already shown that ceramide influences the transport and proteolytic maturation of Cathepsins D. In the present work we describe the effects of NAS on the M6P-dependent and M6P-independent targeting and subcellular localisation of CD in cells expressing the wild-type or the N-Glycosylation mutated CD-GFP constructs.

Funded by MIUR-Cofin2001 and CNR-target project on Biotechnology

Role of ceramide in the control of autophagy in colon cancer cells.

Francesca Scarlatti, Chantal Bauvy*, Giusy Sala, Daniel Meley*, Patrice Codogno*, Riccardo Ghidoni
Lab. Biochemistry and Molecular Biology, San Paolo Hospital medical School, University of Milan; * INSERM U504, Villejuif, France

Autophagy is a degradative pathway that culminates in the lysosomal compartment after the formation of a cytoplasmic vacuole engulfing macromolecules and organelles. The autophagic mechanism triggers a non-apoptotic programmed cell death which inhibits the tumor development. Consequently, the autophagic pathway is an important player in the elimination of cancer cells

A regulatory signaling mechanism of autophagy involves the class I PI3K/PKB pathway, that is known to inhibit autophagy in human cancer cells.

Our study aims to investigate the relationship between the autophagic signaling pathway and the increase of endogenous ceramide levels.

The literature suggests ceramide as an important mediator of apoptosis. However, very little is known on its ability to interfere with the autophagic cellular death. We tested this possibility in HT-29 colorectal cancer cells, that are known to undergo autophagy, either spontaneously and after serum deprival stimulus. In this cell line, C2-ceramide induces apoptosis and the morphological signs, along with DNA fragmentation, occur after 24-48 h of incubation, while PARP occurs not earlier than 8 h from incubation.

We found that a 3-hours treatment of HT-29 cells with C2-ceramide induces autophagy in a dose-dependend manner, as suggested by increase in protein degradation. Moreover, we found that HT-29 cells treated with C2-ceramide reduce expression levels of protein kinase B/Akt by activating Serine 473 dephosphorilylation. C2-ceramide-induced dephosphorylation promotes autophagy by reverting PI3kinase/PKB inhibitory pathway.

In addition, we found that in HT-29 cells the treatment with C2-ceramide induces a dramatic increase (up to 8-fold) of the endogenous long-chain ceramide, which is inhibited by FB 1 , a ceramide synthase inhibitor, but not by myriocin, an SPT inhibitor. Consequently our results show that in HT-29 cancer cells, after treatment with C2-ceramide, endogenous ceramide controls autophagy by interfering with the class I PI3K signaling pathway.

The sphingomyelinase activity regulates the cholesterol level in the nucleus.

Albi E ., Cataldi S., Lazzarini R, Rossi G., Tringali A.R., Tringali S., Viola Magni,MP.
Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Perugia, Italy.

Cholesterol (CHO) exchange between membranes containing sphingomyelin (SM) occurs at a much slower rate than between membranes lacking SM (1). Numerous studies have shown that the strong interaction between unesterified CHO and SM arises from the van der Waals interactions between CHO and the saturated lipid acyl chains, whereas the hydroxy groups of SM are less important (2, 3). The cellular concentrations of SM and CHO are positively correlated in several pathological and experimental conditions (4). In atherosclerosis, abnormal deposition of CHO, cholesteryl esters, and SM has been demonstrated (5 ); SM and CHO accumulate in the intima as a function of ageing (6). Co-localization of SM and CHO has been demonstrated using bacterial sphingomyelinase; hydrolysis of SM results in a movement of CHO to the interior of the cell where it is esterified and reduces sterol synthesis, repressing 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity (7). On the other hand, the 25-hydroxycholesterol (25 OH-CHO), an inhibitor of HMG-CoA reductase, reduces the CHO synthesis and stimulates SM synthesis (4). The CHO-SM relation suggests a mechanism for sterol distribution in which sterol levels are determined by the intrinsic characteristics of each membrane (8). The aim of this work is to establish the relationship between CHO and SM in a intranuclear complex formed by a small amount of DNA, proteins, double-strand RNA, SM and phosphatidylcholine (PC). The results show that in the in intranuclear complex, the amount of CHO is similar to that of SM, and it increases after digestion with exogenous sphingomyelinase. After this treatment the double strand RNA which is RNase-resistent becames RNAse-sensitive. Moreover the cholesterol inhibits the chromatin sphingomyelin-synthase activity. It may be concluded that in intranuclear complex the CHO play a role on SM metabolism modifying the bridge between two RNA strands and consequently regulating transcription.

• Kan CC, Ruan Z, and Bittman R. Biochemistry 1991; 30: 7759-7766; 2) Lund-Katz S, Laboda HM, McLean LR, and Phillipis MC. Biochemistry 1988; 27: 3416-3423; 3) McIntosh TJ, Simon SA, Needham D, and Huang C. Biochemistry 1992; 31: 2020-2024; 4) Ridgway ND. J Lipid Res 1995; 36: 1345-1358; 5) Smith EB, and Cantab BA. Lancet 1960; 1: 799-803; 6) Eisenberg S, Stein Y, and Stein O. Biochim Biophys Acta 1969; 176: 557-569; 7) Slotte JP, and Bierman EL. Biochem J 1988; 250: 653-658; 8) Wattenberg BW, and Silbert DF. J Biol Chem 1983; 258: 2284-2289.

Possible mechanism of inhibition of human PBL proliferation by DMS an inhibitor of sphingosine kinase.

Lucia Cavallini, Nada Sonda, Francesca Pistollato and Adolfo Alexandre
Dipartimento di Chimica Biologica-Università di Padova

The activation of T lymphocytes is the central event in the development of immunity. IL-2 is produced early upon stimulation, together with the high affinity IL-2 receptor, (CD25). They mediate proliferation and differentiation responses, as well as apoptosis at the end of the process of clonal expansion. We performed experiments on the role of the sphingolipid intermediates on lymphocyte proliferation induced by phytohemoagglutinin (PHA) by using some inhibitors of the sphingolipid pathway.

Previous studies in our lab showed the inhibition of proliferation of activated T cell by by fumonisin B1, an inhibitor of ceramide synthase and perhaps of a neutral sphingomyelinase, and by N,N-dimethylsphingosine (DMS) and threo-dihydrosphingosine (DHS), both inhibitors of Sphingosine kinase (SPHK). Alltogether these experiments suggest the involvement of some sphingolipid compound as obligatory intermediate of lymphocyte proliferation. Among the inhibitors tested DMS is the most efficient, inducing an almost complete inhibition at 2-3 uM, followed by DHS at 5-10 mM. IL2 expression is slightly decreased by DMS and more affected by DHS. The expression of CD25 expression is strongly inhibited by DMS, but not by DHS. The DMS inhibition (75-100%) is also observed in a model of IL-2-dependent proliferation where PBL are pre-stimulated with PHA for 74 hours to induce a high expression of CD25 , and after washing proliferation is induced by IL2. In this system DHS is again a poor inhibitor. In all cases the effects of DMS are evidenced by inhibition of the increase of cyclin D2 and D3, pRb phosphorylation and an almost complete absence of expression of cyclin A and cyclin E; the data are consistent with a block in the G1-S transition of the cell cycle. SPHK activity measured in extracts of PHA and IL2 restimulated PBL showed an increase. The increment of the activity was completely abrogated by PBL traetment with DMS but not with DHS at similar concentration, suggesting a positive correlation between the enzyme activity and proliferation.

The effect of inhibition of SPHK could be due to an increase of ceramide. Ceramide has been reported as involved in the CD28 costimulation of lymphocytes, but also in numerous different effects (e.g. activation of PP-1 and -2 phosphatases, activation of caspases). Direct measurements of ceramide in PHA stimulated and IL-2 restimulated PBL revealed a slight decrease in the ceramide content which was marginally and not significant prevented by DMS. Sphingosine and SPP levels in human PBL are so low to be nearly undetectable. Besides they did not show extensive variation in the activated and DMS inhibited cells. So a significant mass variation of the different metabolites seems not correlated with the massive effect of DMS. A direct effect of DMS seems a more probable explanation for its effect, and the target could be a discrete pattern of enzymes. Parallel measurements of SPHK and SPP-Lyase and Diacyl-glycerol kinase activities show inhibition by DMS, but also the increase of retinoblastoma protein is impaired and that of cyclins A and E whose synthesis depend on Rb transcription/ activation. Lactate dehydrogenase variations (used as marker of protein content) induced by DMS are however far less evident than those on SPHK. Preliminary data comparing the effects of DMS and sphingosine on the inhibition of PBL proliferation show that DMS has the same efficacy as > 10 mM sphingosine, reported to be a lysosomotropic agent. An increase of Sphingosine or DMS could be the physiological cause of apoptosis at the end of the process of clonal expansion. This aspect is under investigation.

Composition in polyunsatured fatty acids of membrane phospholipid is involved in the regulation of cell proliferation in tumor human cell lines.

Marina Maggiora, Antonella Trombetta, Germana Martinasso, Rosa Angela Canuto and Giuliana Muzio
Dipartimento di Medicina ed Oncologia Sperimentale, Università di Torino

The length and the number of unsaturation of phospholipid fatty acids determine not only modification in the physic behaviour of membranes, but also influence its function, such as interaction ligand-receptor, hormone and growth factor receptors, signalling transduction, enzyme metabolising xenobiotics. These interactions can affect cell proliferation and death for apoptosis. Our previous results demonstrated that enriching rat hepatoma cells with arachidonic acid inhibits cell proliferation and reverts tumor-associated phenotype of class 2 and 3 aldehyde dehydrogenases (1). These effects due to arachidonic acid enrichment are consequent to the restoration of normal content of this fatty acid in membrane phospholipids, being lower in rat hepatoma cells in comparison with normal hepatocytes. In the light of these observations we extended our study on human tumor cell lines by using different polyunsaturated fatty acids (linoleic, conjugated linoleic and arachidonic acids).

Different types of human tumor cells are used: two hepatoma cell lines with different tumorigenicity, non-tumorigenic HepG2 cells and tumorigenic SK-HEP-1 cells, and a lung cell line, A549 cells.

Preliminary results show that polyunsaturated fatty acids inhibit cell proliferation and induce apoptosis in a dose-dependent manner. Moreover, the entity of the cytostatic and cytotoxic effect differs in relation with the cell type and, in the hepatoma cell lines it is directly correlated with the degree of malignancy. Flow cytometry analysis of DNA distribution evidences that in cells undergoing growth inhibition an increased G0/G1 population occurs; whereas in cells undergoing apoptosis an increased percentage of cells in S phase is present. About the mechanisms underlying the inhibition of cell proliferation induction MAPK pathway is under investigation; about apoptosis induction ceramide involvement and mitochondria pathway are investigated.

This research is supported by a grant from Compagnia di San Paolo, Turin .

Involvement of the de novo ceramide synthesis in IGFBP-3-mediated endothelial cell apoptosis.

Riccarda Granataa, Marzia De Petrinia, Marina Talianoa, Ada Castellia, G. Garbarinoa, Giulia Somenzib, Riccardo Ghidonib, Ezio Ghigoa.
a Division Endocrinology and Metabolism, Department Internal Medicine; University of Turin, Italy; b Laboratory of Genetics and Biochemistry, San Paolo University Hospital; University of Milan, Italy.

IGFBP-3 modulates IGFs action and exerts direct IGF-independent actions inducing apoptosis or promoting cell growth in different cell types. IGFBP-3 expression has been shown in both cardiomyocytes and endothelial cells. Doxorubicin exerts its apoptotic effect through the ceramide death pathway and we have found that IGFBP-3 mediates apoptosis in cardiomyocytes. In this study we aimed to investigate the role of IGFBP-3 in endothelial cell apoptosis induced by doxorubicin and its relation with ceramide in the induction of the apoptotic response. We evaluated 1) the expression of IGFBP-3 and IGF-I in human endothelial cells (HUVEC) exposed to doxorubicin (0.5 m M) by Western blot and radio-immunoassay; 2) the effect of IGFBP-3 (1000 ng/ml) on cell survival and apoptosis of HUVEC exposed to doxorubicin and to the ceramide synthase inhibitor fumonisin B1 (FB1) by MTT, and FACS analysis; 3) IGF-I secretion in cells exposed to IGFBP-3 with or without doxorubicin and/or FB1; 4) the effect of IGFBP-3 antisense oligonucleotides on cell survival and apoptosis (by HOECHST staining) in all experimental conditions; 5) the variations in ceramide levels; 6) cell motility of cells treated with IGFBP-3, doxorubicin and/or FB1. An inhibitor of the first step in the de novo synthesis of ceramide, myriocin (50 nM), was also tested for the apoptotic effect of IGFBP-3 in association with doxorubicin. We found that IGFBP-3 was up-regulated upon doxorubicin-induced cell death, with increased ceramide production and no change in IGF-I secretion. Exogenous IGFBP-3 reduced apoptosis in serum deprived cells also decreasing ceramide levels. When associated to doxorubicin, IGFBP-3 enhanced apoptosis and increased ceramide levels. The protective effect of FB1 over doxorubicin-induced cell death and apoptosis was unexpectedly enhanced by IGFBP-3 with reduced ceramide levels and dramatically increased IGF-I secretion. IGFBP-3 antisense oligonucleotides counteracted apoptosis induced by doxorubicin but also reduced the protective effect of both FB1 and FB1+IGFBP-3 over doxorubicin-induced cell death. Finally, IGFBP-3 increased cell motility of HUVEC exposed to doxorubicin and FB1. Preliminary results indicate that IGFBP-3 even attenuates doxorubicin-induced apoptosis in the presence of myriocin. In conclusion, IGFBP-3 modulates endothelial cell fate, protecting or enhancing serum starvation- and doxorubicin-induced apoptosis and potentiating the protective effect of fumonisin B1 over doxorubicin. These actions appear to be paralleled by ceramide variations and increase in IGF-I secretion. Thus, IGFBP-3 likely exerts complex modulation of the ceramide signaling pathway directly or indirectly through the de novo ceramide synthesis, regulating apoptosis in endothelial cells and even IGF-I release that, in turn, would per se exert antiapoptotic action.

Study supported by MURST, University of Turin and SMEM Foundation

Modifying the cellular response to ceramide-mediated stress by gene manipulation of lysosomal cathepsin D expression.

Anna Aspesi , Roberta Castino, Camilo Gianinazzi, Marzia Mirabelli, Carlo Follo, Marina Démoz, and Ciro Isidoro
Università del Piemonte Orientale “A. Avogadro”, Dipartimento di Scienze Mediche di Novara

Ceramide is generated as a second messenger in response to various environmental stimuli such as cytokines, serum deprivation and cytotoxic chemotherapeutic drugs, and it is believed to play a major role in mediating their cellular effects. Ectopic administration of ceramide analogues (for instance, C2-ceramide) can mimic these cellular effects. We have assessed culture conditions to induce apoptotic cell death in L929 fibrosarcoma cells by treatments with C2-ceramide or chemotherapeutic drugs known to rise the intracellular levels of ceramide. Cathepsin D is a lysosomal protease that has been shown to play an active role in the onset of cytokine- and antiblastic drug-triggered apoptosis in tumour cells. Cathepsin D is also a target of endosomal-lysosomal ceramide generated by activation of acid sphingomyelinase under cytotoxic conditions. In the present work we describe the consequences of the transgenic hyperexpression of Cathepsin D in L929 cells subjected to increased levels of ceramide. We show that sensitivity of transfected cells to ceramide is proportional to the rate of Cathepsin D expression. The molecular mechanism underlying ceramide-induced cytotoxicity include the destabilisation of lysosomal membranes, likely associated with leakage of lysosomal cathepsins.

Funded by MIUR-Cofin2001, AIRC, Regione Piemonte and CNR-target project on Biotechnology

Differentiation and cell signaling: ceramide-dependent modulation of neuroblastoma cell viability.

Deborah Pace , Raffaele Picco , F abrizio Condorelli , Pier Luigi Canonico
DISCAFF Dept. Università del Piemonte Orientale, Novara, Italy

Ceramide, either produced by sphingomyelin hydrolysis or by neosynthesis, has been proposed as a possible second messenger for different extracellular signals. The increase in the intracellular ceramide levels may lead to induction of apoptotic death or to cell differentiation depending on experimental models including neuronal-derived cell lines. Since retinoic acid (RA) is known as the most effective inducer of differentiation (mainly in hematopoyetic and neuronal models) we decided to study the possible interactions of the ceramide-dependent pathways with RA-induced neuronal differentiation. Two different human neuroblastoma cell lines (SK-N-BE, SH-SY5Y) were exposed for increasing lengths of time (4 h minimal, 1 week maximal) to different concentrations of RA (10 nM-1 mM) and/or cell-permeable C2 ceramide (C2 10 nM-10 mM) to evaluate their response in terms of neuronal phenotype acquisition and viability. In similar conditions high C2 concentrations (1-10 mM) showed an addictive effect on RA (10 mM) induced differentiation evaluated both morphologically and by western-blot analysis of neuronal protein marker expression. Paradoxically, lower concentrations of C2 were cytotoxic according to trypan-blue exclusion assay (10 nM = 45 ± 8% and 100 nM = 35 ± 6% blue positive), an effect antagonized by RA (10 mM) co-treatment. This effect was probably due to induction of apoptosis as shown by DAPI-stained cells nuclei. Moreover we evaluated by western-blot analysis the subcellular localization of some member of Bcl-2 family and cytochrome c in SK-N-BE cells. These results set the basis to investigate further ceramide involvement in RA-induced differentiation.

Preliminary data on glyco(sphingo)lipid amount and distribution in food.

Bruno Berra , Gigliola Montorfano, Attilio Giacosa *
Institute of General Physiology and Biochemistry “G. Esposito” University of Milano, Italy; Gastroenterology and Nutrition Department, National Cancer Institute, Genova, Italy

Sphingolipids are commonly assumed to be present mainly in neuronal tissue; however, they also appear in lipoproteins, milk fat, globule membranes, the lamellar barrier of skin, the Golgi apparatus and the lisosomes. The amounts vary considerably, with the highest proportions being in membrane-rich tissues. They are also present in relatively large amounts in some foods, such as dairy products and soybeans, which contain more gangliosides and glucosylcerebrosides, respectively than brain.

There is no known nutritional requirement for sphingolipids. Nonetheless studies with experimental animals have shown that consumption of shingolipids inhibits colon carcinogenesis, reduces serum low-density lipoprotein cholesterol and elevates high-density lipoproteins, which suggest that they are “functional” components of food.

No human clinical trials have yet evaluated sphingolipids for the prevention or treatment of colon cancer, nor any epidemiological studies of possible associations between sphingolipid consumption and cancer risk was made. Nonetheless, there is indirect evidence to suggest that sphingolipids can inhibit colon cancer in humans: (1) shingosine and ceramide induce apoptosis in a human adenocarcinoma cell line (HT29); and (2) feeding sphingolipids to Min mice (Min mice have a genetic defect similar to that found in human familial adenomatous polyposis and which is also found in a large percentage of sporadic human colon cancers) reduced the number of intestinal tumours by 55%.

An epidemiologic programme is ongoing in Italy to evaluate the association between sphingolipids content of diet and colorectal cancer risk. This is part of case-control study that has been performed in six Italian areas.

In this view detailed programme for the analysis of the sphingolipid content in different foods has been developed in our Institute.

Fresh Cheese
Soy Bean
Neutral glycolipids
mg/g of fresh weight 21.24±2.88 10.17±0.58 84.4±8.63 358.94 – 551.49
mg/g of fresh weight 71.91±3.16 62.32±1.73 390.81±2.87 67.14 – 247.95
mg/g of fresh weight 8.22±0.67 4.36±0.22 36.57±1.19 291.35 – 496.57
mg/g of fresh weight 10.76±0.67 4.91±0.22 45.27±1.72 n.p.
Other minor glycolipids
mg/g of fresh weight 2.26±0.04 0.90±0.02 2.57±0.47 25.73 – 91.75

Table show the preliminary data we have found when we analyzed foods normally consumed in our population (like milk and fresh cheese) or less popular foods as different kinds of soy beans (were large variations were observed). In this study we included also a variety of bacterial strains used in fermented milks, due to the therapeutical potential of these food in the treatment of colon cancer.

Overexpression of cytosolic sialidase neu2 induces myoblast differentiation in C2C12 cells.

A. Fanzani, R. Giuliani, F. Colombo, D. Zizioli, *M. Presta, A. Preti, and S. Marchesini
Unit of Biochemistry and *Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia

Cytosolic sialidase Neu2 has been implicated in myoblast differentiation. Here we observed a significant upregulation of Neu2 expression during differentiation of murine C2C12 myoblasts. This was evidenced both as an increase in Neu2 mRNA steady-state levels and in the cytosolic sialidase enzymatic activity.

To understand the biological significance of Neu2 upregulation in myoblast differentiation, C2C12 cells were stably transfected with the rat cytosolic sialidase Neu2 cDNA. Neu2 overexpressing clones were characterized by a marked decrement of cell proliferation and by the capacity to undergo spontaneous myoblast differentiation also when maintained under standard growth conditions. This was evidenced by the formation of myogenin-positive myotubes and by a significant decrease in the nuclear levels of cyclin D1 protein. No differentiation was instead observed in parental and mock transfected cells under the same experimental conditions.

The results indicate that Neu2 upregulation is per sè sufficient to trigger myoblast differentiation in C2C12 cells.

Calnexin suppresses GD3 synthase – induced apoptosis.

Barbara Tomassini*, Florence Malisan*, Luigi Franchi*, Chiara Nicolo’*, Gloria Brea Calvo* and Roberto Testi*
*Laboratory of Immunology and Signal Transduction, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Roma

The accelerated activity of the GD3 synthase, with consequent GD3 accumulation, is part of the response to environmental stressors in different cell types. Depending on specific, yet largely undefined, cellular settings, this can be followed by cellular adaptation or apoptosis, in large part due to GD3-induced mitochondrial damage. Subcellular localization of the GD3 synthase could significantly affect the biological outcome of GD3 accumulation. We found that binding to the molecular chaperone calnexin causes the retention of the GD3 synthase within the endoplasmic reticulum (ER) and prevents its relocalization to the Golgi. Calnexin-dependent ER retention does not affect the activity of the GD3 synthase, yet the de novo synthesized GD3 largely fails to reach the mitochondria. Accordingly, overexpression of calnexin suppresses the pro-apoptotic activity of the GD3 synthase, and the loss of calnexin sensitizes cells to stress-induced apoptosis. Consequently, we conclude that calnexin has the ability to affect the subcellular distribution of the GD3 synthase and the biological outcome of GD3 accumulation during the stress response process.

Isolation and characterization of the GM3 synthase cDNA from human placenta.

E. Sottocornola , P. Berselli, S. Zava, , B. Berra and I. Colombo
Inst. of General Physiology and Biochemistry, University of Milan

It is known that gangliosides have various important biological functions, and their functions as well as their biosynthesis are currently clarified (1, 2). In vertebrates, almost all the ganglio-series gangliosides are synthesized from a common precursor, ganglioside GM 3 , which has the simplest structure among the major gangliosides. GM 3 itself is known to participate in induction of differentiation, modulation of proliferation, signal transduction and integrin-mediated cell adhesion. GM 3 synthase (EC, ST3Gal V) is the enzyme involved in the last step of GM 3 biosynthesis: it catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto lactosylceramide, forming an a2-3 linkage. Whereas GM 3 is ubiquitously distributed in the plasma membranes of all eukaryotic cells, GM 3 synthase results expressed in a tissue specific manner, especially in brain, placenta, muscle and testis (3). Many important issues, such as human cDNA identification and characterization, genomic structure and regulation of gene expression, are still open.

To isolate the coding sequence of the gene of GM 3 synthase from human placenta we used the 5′- and 3′-Rapid Amplification of cDNA Ends technology (SMART RACE cDNA Amplification Kit, Clontech) using, as specific primers, oligonucleotides derived from the human GM 3 synthase cDNA sequence from differentiated HL60 cells (3). The different PCR products were cloned into the pCR2.1 vector (TA Cloning Kit, InVitrogen) and the nucleotide sequence was determined.

A cDNA, showing high sequence homology with that encoding the human GM 3 synthase from TPA-differentiated HL60 cells (3), has been successfully isolated and cloned from human placenta. The major difference between these two cDNAs is in the 5′-end, according to the existence of different promoter regions, responsible for tissue-specific expression of the gene. Furthermore, the cDNA from the human placenta contains, upstream and in frame with the ATG indicated as translation initiation site for the GM 3 synthase of HL60 cells, another ATG codon inserted in a sequence compatible with Kozak’s rule, suggesting that the protein of the human placenta has an additional portion in NH 2 -terminus. The complete coding region of the human placenta cDNA is going to be cloned in an expression vector, under the control of the CMV promoter, in order to evaluate its activity. On the other hand, in vitro translation experiments are going to be carried out to define the first start codon.

1) Hakomori S.I. (2000): Glycoconj. J. 17, 627-647
2) Kolter T. et al. (2002): J.Biol.Chem . 277, 25859-25862
3) Ishii A. et al. (1998): J.B.C . 273, 31652-31655

Cancer-associated suppression of β3Gal-T5 reduces sialy-lewis a and enhances sialyl-lewis X and poly-N-acetyllactosamines.

Lidia Mare and Marco Trinchera
Dipartimento di Scienze Biomediche Sperimentali e Cliniche, Università dell’Insubria, Varese

Glycosylation of glycoproteins and glycolipids is one of many molecular changes that accompany malignant transformation. Poly-N-acetyllactosamine (Gal β 1-4GlcNAc β 1-3) n sequences frequently terminated by the sialyl-Lewis x antigenic determinant (NeuAc α 2-3Gal β 1-4[Fuc a 1-3]GlcNAc, have been observed to increase in some cancer, and to correlate with poor prognosis. We reported that the expression of a specific β 1,3 galactosyltransferase (β 3Gal-T5) prevents poly-N-acetyllactosamine and sialyl-Lewis x expression on N-glycans in CHO cells. We also found that β 3Gal-T5 transcript is down-regulated in colon adenocarcinomas and presumably responsible for the differential glycosylation of CEA in cancer, where it acquires N-linked poly-N-acetyllactosamine chains, absent in the normal counterpart. The very low levels of β 3Gal-T5 transcript detectable in colon cancer specimens also open the question as to whether the CA19.9 antigen, that is sialyl-Lewis a epitope carried by a mucin backbone and circulating in cancer patient serum, is synthesized by β 3Gal-T5. Our working hypothesis is that β 3Gal-T5 is an enzyme associated with the normal phenotype and potentially able to opposite the malignant phenotype by preventing poly-N-acetyllactosamine extension, and thus synthesis of sialyl-Lewis x and related antigens.

To assess this hypothesis, we first try to prove that β 3Gal-T5 is actually the enzyme responsible for β 1,3Gal-T activity in gastrointestinal tissues. To this aim, we measured β 1,3Gal-T activity and transcript levels in cancer, normal colon mucosa, and various cancer cell lines and recombinant clones, and compared the kinetic properties of the activity detected. We also determined the amount of sialyl-Lewis a synthesized and secreted by such sources. We found that one β 1,3Gal-T activity, corresponding to β 3Gal-T5, is expressed in tissues and cells and responsible for Lewis antigen synthesis and secretion on O-glycans. Moreover, almost no sialyl-Lewis a was detectable in colon cancer specimens by dot-blot. We then studied the effect of β 3Gal-T5 suppression in a cancer cell line. To this purpose we transfected BxPc3 cells with a β 3Gal-T5 fragment placed in the antisense orientation under the control of a very strong promoter, and isolated a recombinant clone that stably expresses high levels of the antisense transcript. We found that the clone expresses much less sialyl-Lewis a on the cell surface than BxPc3 cells, and β 1,3Gal-T activity is faintly detectable. These data strongly confirm that β 3Gal-T5 is the gene responsible for β 1,3Gal-T activity and sialyl-Lewis a antigen in these cells.

Moreover the clone became positive to sialyl-Lewis x, that is undetectable in BxPc3 cells. A relevant amount of sialyl-Lewis x is also detected in the culture medium of the clone, while sialyl-Lewis a, that is secreted by BxPc3 cells, is not detectable. Sialyl-Lewis x expression in the antisense clone is reduced by treatment with b -benzyl-GalNAc and not affected by swainsonine. Similar results are obtained with sialyl-Lewis a in BxPc3 cells. Analysis of radioactive glycoconiugates in the antisense clone upon metabolic labeling with tritiated Gal indicated increased synthesis and secretion of poly-N-actetyllactosamine chains with respect to parental BxPc3 cells. Altogether the results suggest that sialyl-Lewis a synthesis is controlled by β 3Gal-T5 and reduced in cancer.