Samuela Cataldi

Nuclear sphingomyelin pathway in serum deprivation-induced apoptosis of embryonic hippocampal cells

Sphingomyelin (SM) cycle has been involved in the regulation of proliferation, differentiation, and apoptosis. Increases in ceramide have been found after a larger number of apoptotic stimuli including cytokines, cytotoxic drugs, and environmental stresses. Accumulating evidence suggest that the subcellular localization of ceramide generation is a critical factor in determining the cellular behavior. Since recently enzymes involved in ceramide metabolism such as sphingomyelinase, SM synthase, sphingosine kinase and ceramidase have been found in the nucleus of hepatocyte cells, we have studied first the presence and the physicochemical characteristics of SM metabolism enzymes in nuclei isolated from embryonic hippocampal cells (cell line HN9.10e). The activities of sphingomyelinase and SM‐synthase have been assayed and the ceramide production evaluated at different times after serum deprivation in these neurones cultivated in serum‐deficient medium. We report that both enzymes are present in the nucleus of embryonic hippocampal cells and differ from those present in the homogenate in optimum pH. After serum deprivation, that induces a time‐dependent decrease in cell viability and increase of the cell percentage in G1 phase of the cell cycle, a nuclear sphingomyelinase activation together with SM‐synthase inhibition and a consequent increase of nuclear ceramide pool have been demonstrated. No similar enzyme activity modifications in homogenate have been identified. The possible role of nuclear sphingomyelinase/sphingomyelin‐synthase balance in serum deprivation‐induced apoptosis in the embryonic hippocampal cell is discussed.

A possible role of cholesterol-sphingomyelin/phosphatidylcholine in nuclear matrix during rat liver regeneration

BACKGROUND/AIMS Phospholipids and cholesterol in chromatin have been previously demonstrated. The lipid fraction changes during cell proliferation in relation to activation of enzymes of phospholipid metabolism. The aim of the present work is to clarify if chromatin lipids may derive or not from nuclear matrix and if they have different roles. METHODS The subnuclear fractions were isolated from rat hepatocyte nuclei and the lipid fraction was extracted and analysed by chromatography in normal and regenerating liver. The phosphatidylcholine-sphingomyelin metabolism enzymes activity was assayed, by using radioactive substrates. RESULTS In nuclear matrix, cholesterol and sphingomyelin are respectively five and three times higher than those present in chromatin; the amount of phosphatidylcholine, which it is enriched in saturated fatty acids, is lower, thus indicating a less fluid structure. The lower content in phosphatidylcholine may be justified by the phosphatidylcholine-dependent phospholipase C activity, which increases during liver regeneration, reaching a peak at the beginning of S-phase, when also cholesterol and sphingomyelin increase. CONCLUSIONS The nuclear matrix lipids are independent from chromatin lipids; the ratio cholesterol-sphingomyelin/phosphatidylcholine is higher and, as a consequence, nuclear matrix is less fluid in relation to DNA synthesis, suggesting a specific role of nuclear matrix as a structure involved in DNA duplication.

Involvement of nuclear phosphatidylinositol-dependent phospholipases C in cell cycle progression during rat liver regeneration.

Nuclear lipid metabolism is involved in the regulation of cell proliferation. Modulation of the expression and activity of nuclear PI‐phospholipase C (PI‐PLC) has been reported during liver regeneration after partial hepatectomy, although it has not been determined whether different PLC isoforms play specific roles in the regulation of cell cycle progression. Here, we report evidence that the increased activity of nuclear PLCs in regenerating rat liver occurs before the peak of DNA replication and involves the enzyme activity associated to the chromatin and not that associated to the nuclear membrane. Immunocytochemical analyses indicate that PI‐PLC β1 isoform is exclusively localized at the chromatin level, PI‐PLC β1 co‐localizes with DNA replication sites much more than PI‐PLC γ1, which is also present at the nuclear envelope. These findings and the increased amount of PI‐PLC γ1 occurring after the peak of DNA replication suggest that PI‐PLC β1 and γ1 play different roles in cell cycle progression during regenerating liver. The increased activity of PI‐PLC β1 constitutively present within the hepatocyte nucleus, should trigger DNA replication, whereas PI‐PLC γ1 should be involved in G2/M phase transition through lamin phosphorylation. J. Cell. Physiol. 197: 181–188, 2003.

Nuclear Lipid Microdomain as Place of Interaction between Sphingomyelin and DNA during Liver Regeneration

Nuclear sphingomyelin is a key molecule for cell proliferation. This molecule is organized with cholesterol and proteins to form specific lipid microdomains bound to the inner nuclear membrane where RNA is synthesized. Here, we have reported the ability of the sphingomyelin present in the nuclear microdomain to bind DNA and regulate its synthesis, and to highlight its role in cell proliferation induced by partial hepatectomy. During G1/S transition of the cell cycle, sphingomyelin and DNA content is very high and it is strongly reduced after exogenous sphingomyelinase treatment. During the S-phase of the cell cycle, the stimulation of sphingomyelinase and inhibition of sphingomyelin–synthase are accompanied by the DNA synthesis start. To assess the specificity of the results, experiments were repeated with trifluoperazine, a drug known to affect the synthesis of lipids and DNA and to stimulate sphingomyelinase activity. The activity of sphingomyelinase is stimulated in the first hour after hepatectomy and sphingomyelin–DNA synthesis is strongly attenuated. It may be hypothesized that the nuclear microdomain represents a specific area of the inner nuclear membrane that acts as an active site of chromatin anchorage thanks to the stabilizing action of sphingomyelin. Thus, sphingomyelin metabolism in nuclear lipid microdomains is suggested to regulate cell proliferation.

Nuclear lipid microdomains regulate nuclear vitamin D3 uptake and influence embryonic hippocampal cell differentiation.

In the cell nucleus, the 1,25-(OH)2 vitamin D3 (1,25-(OH)2D3) receptor (VDR) is localized in specialized microdomains enriched in sphingomyelin and cholesterol. The integrity of these microdomains is necessary for 1,25-(OH)2D3–induced differentiation of embryonic hippocampal cells. Serum deprivation alters nuclear microdomains, which lose the VDR.

The impact of long-term exposure to space environment on adult mammalian organisms: a study on mouse thyroid and testis.

Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis. In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10−7M and 10−8M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains. In testes, immunofluorescent reaction for 3β- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1β were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3β and 17β steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 β expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. −90% vs. laboratory and ground controls), indicating that the space environment may lead to degenerative changes in seminiferous tubules. Space-induced changes of structure and function of thyroid and testis/epididymis could be responsible for variations of hormone levels in human during space missions. More research, hopefully a reflight of MDS, would be needed to establish whether the space environment acts directly on the peripheral glands or induces changes in the hypotalamus-pituitary-glandular axis.

Plasmalogens in rat liver chromatin: new molecules involved in cell proliferation.

A minor component of chromatin, the phospholipid fraction, changes during cell cycle as result of the activation of intranuclear lipid metabolism enzymes including phosphatidylcholine‐dependent phospholipase C activity. It is known that this enzyme may be activated by phosphatidylcholine plasmalogen (Plg). Until now, there has been little evidences for the presence of Plgs inside the nucleus. The aim of our study is to ascertain if they are present in the nucleus and are responsible of the activation of phosphatidylcholine‐dependent phospholipase C during cell proliferation and apoptosis. Therefore, we have analysed the Plg composition of the whole homogenate, cytosol, nuclei and chromatin of hepatocytes. The phosphatidylcholine‐dependent phospholipase C activity was assayed using both phosphatidylcholine and plasmalogenyl‐phosphatidylcholine as substrates. Our results show, for the first time, that Plgs are present in chromatin and the plasmalogenyl‐phosphatidylcholine stimulates the phosphatidylcholine‐dependent phospholipase C activity more than phosphatidylcholine. Finally, in order to verify the possible role of these molecules during cell proliferation and apoptosis, we used liver of rats fed with ciprofibrate which stimulates hepatocytes proliferation during the treatment and, after withdrawal, apoptosis. After 3 days of ciprofibrate treatment, the chromatin plasmalogenyl‐phosphatidylcholine increases as well as the phosphatidylcholine‐dependent phospholipase C activity. After drug withdrawal, when the hepatocytes undergo to apoptosis, the plasmalogenyl‐phosphatidylcholine content together with phosphatidylcholine‐dependent phospholipase C activity decreases. Therefore, it can be concluded that plamalogens are present in the chromatin, and probably may have a function both in regulating phosphatidylcholine dependent phospholipase C and cell cycle.

VERY LONG CHAIN FATTY ACID SPHINGOMYELIN IN NUCLEAR LIPID MICRODOMAINS OF HEPATOCYTES AND HEPATOMA CELLS: CAN THE EXCHANGE FROM C24:0 TO C16:0 AFFECT SIGNAL PROTEINS AND VITAMIN D RECEPTOR?

The 24:0 sphingomyelin of nuclear lipid microdomains from normal cells shifts to 16:0 sphingomyelin in nuclear lipid microdomains from cancer cells. The narrower microdomains in the nucleus are associated with the changes to proteins involved in hepatocarcinogenesis.

Severe hypocholesterolaemia is often neglected in haematological malignancies

AIM OF THE STUDY It is generally believed that high levels of cholesterol (hypercholesterolaemia) are life-threatening, while low levels seem to be positive. Unfortunately this assumption is far from true, and can be indicative of an underlying serious medical condition in most of the cases (i.e. cancer). However, the biological role of severe hypocholesterolaemia is poorly understood. Here, the possible biological process is being investigated. Cholesterol plays a key role in cell proliferation, hence it has been suggested that low cholesterol levels are probably linked to the high cellular cholesterol demands from neoplastic cells. SUMMARY OF THE METHODS We used serum and isolated T-lymphocytes from patients with acute lymphoblast leukaemia and human lymphoblast cell line to test this hypothesis. RESULTS We found that patients with low serum cholesterol levels have instead high levels of cholesterol in lymphocytes. These data were supported with in vitro studies. In fact we have demonstrated that low cholesterol level in the culture medium was related to the neoplastic cellular growth, suggesting a greater use by lymphoma cells for their proliferation. Therefore by inhibiting cholesterol synthesis by mevastatin, in vitro, we showed that cholesterol levels did not change significantly in culture medium and the cellular growth was inhibited. CONCLUDING STATEMENT Following these preliminary results, blood cholesterol levels could be potentially considered a good biological marker to follow up the neoplastic process.

Gentamicin Arrests Cancer Cell Growth: The Intriguing Involvement of Nuclear Sphingomyelin Metabolism

The use of gentamicin for the treatment of bacterial infection has always been an interesting and highly speculated issue for the scientific community. Conversely, its effect on cancer cells has been very little investigated. We studied the effect of high doses of gentamicin on non-Hodgkin’s T-cell human lymphoblastic lymphoma (SUP-T1). We showed that gentamicin delayed cell growth and induced cell death in lymphoma cells with a rather mild effect on lymphocytes. In SUP-T1 cells, GAPDH, B2M, CDKN1A and CDKN1B were down-expressed in comparison with lymphocytes. Gentamicin treatment in SUP-T1 cells restored the expression of GAPDH, B2M and CDKN1A to values similar to those of lymphocytes and caused overexpression of CDKN1B. The drug acted via sphingomyelin metabolism; in whole cells, sphingomyelinase activity was stimulated, whereas in purified nuclei, sphingomyelinase activity was inhibited and that of sphingomyelin-synthase was stimulated, with a consequent high level of nuclear sphingomyelin content. We suggest that the increase of nuclear sphingomyelin might enrich the nucleus of lipid microdomains that act as a platform for active chromatin and, thus, might be responsible for gene expression. It is possible that in lymphoblastic lymphoma, high doses of gentamicin induce a beneficial therapeutic outcome.