Alessandro Floridi

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.

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.

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.

Nuclear Lipid Microdomain as Resting Place of Dexamethasone to Impair Cell Proliferation

The action of dexamethasone is initiated by, and strictly dependent upon, the interaction of the drug with its receptor followed by its translocation into the nucleus where modulates gene expression. Where the drug localizes at the intranuclear level is not yet known. We aimed to study the localization of the drug in nuclear lipid microdomains rich in sphingomyelin content that anchor active chromatin and act as platform for transcription modulation. The study was performed in non-Hodgkin’s T cell human lymphoblastic lymphoma (SUP-T1 cell line). We found that when dexamethasone enters into the nucleus it localizes in nuclear lipid microdomains where influences sphingomyelin metabolism. This is followed after 24 h by a cell cycle block accompanied by the up-regulation of cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin-dependent kinase inhibitor 1B (CDKN1B), growth arrest and DNA-damage 45A (GADD45A), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes and by the reduction of signal transducer and activator of transcription 3 (STAT3) and phospho signal transducer and activator of transcription 3 (phoshoSTAT3) proteins. After 48 h some cells show morphological changes characteristic of apoptosis while the number of the cells that undergo cell division and express B-cell lymphoma-2 (Bcl-2) is very low. We suggest that the integrity of nuclear lipid microdomains is important for the response to glucocorticoids of cancer cells.

Analysis of Serum Sphingomyelin Species by Uflc-Ms/Ms in Patients Affected with Monoclonal Gammopathy

Cancer cells are hungry of cholesterol incorporated from serum with avidity and used to favour the expressions of proteins involved in cell proliferation such as RNA polymerase II, STAT3, PKCz and cyclin D1. Numerous studies have shown that exists a strong interaction between unesterified cholesterol and saturated fatty acid sphingomyelin which arises from the Van der Waals interaction. Since sphingomyelin and cholesterol association is responsible for the formation of membrane lipid raft involved in cell signalling, we studied the possible hyposphingomyelinemia associated to hypocholesterolemia in the patients with cancer. The blood of 23 patients with monoclonal gammopathy were analyzed for cholesterol, 12:0 sphingomyelin, 16:0 sphingomyelin and 18:1sphingomyelin content. The results demonstrated that only the patients with very low level of cholesterol (65-99 mg/dl) had low amount of sphingomyelin and, in particular, of saturated sphingomyelin specie (16:0 sphingomyelin). The possibility that the hypocholesterolemia in cancer was secondary to hyposphingomyelinemia was discussed.

A Firmer Understanding of the Effect of Hypergravity on Thyroid Tissue: Cholesterol and Thyrotropin Receptor

Maintaining a good health requires the maintenance of a body homeostasis which largely depends on correct functioning of thyroid gland. The cells of the thyroid tissue are strongly sensitive to hypogravity, as already proven in mice after returning to the earth from long-term space missions. Here we studied whether hypergravity may be used to counteract the physiological deconditioning of long-duration spaceflight. We investigated the influence of hypergravity on key lipids and proteins involved in thyroid tissue function. We quantified cholesterol (CHO) and different species of sphingomyelin (SM) and ceramide, analysed thyrotropin (TSH) related molecules such as thyrotropin-receptor (TSHR), cAMP, Caveolin-1 and molecule signalling such as Signal transducer and activator of transcription-3 (STAT3). The hypergravity treatment resulted in the upregulation of the TSHR and Caveolin-1 and downregulation of STAT3 without changes of cAMP. TSHR lost its specific localization and spread throughout the cell membrane; TSH treatment facilitated the shedding of α subunit of TSHR and its releasing into the extracellular space. No specific variations were observed for each species of SM and ceramide. Importantly, the level of CHO was strongly reduced. In conclusion, hypergravity conditions induce change in CHO and TSHR of thyroid gland. The possibility that lipid rafts are strongly perturbed by hypergravity-induced CHO depletion by influencing TSH-TSHR interaction was discussed.

How Microgravity Changes Galectin-3 in Thyroid Follicles

After long-term exposure to real microgravity thyroid gland in vivo undergoes specific changes, follicles are made up of larger thyrocytes that produce more cAMP and express more thyrotropin-receptor, caveolin-1, and sphingomyelinase and sphingomyelin-synthase; parafollicular spaces lose C cells with consequent reduction of calcitonin production. Here we studied four immunohistochemical tumor markers (HBME-1, MIB-1, CK19, and Galectin-3) in thyroid of mice housed in the Mouse Drawer System and maintained for 90 days in the International Space Station. Results showed that MIB-1 proliferative index and CK19 are negative whereas HBME-1 and Galectin-3 are overexpressed. The positivity of Galectin-3 deserves attention not only for its expression but also and especially for its localization. Our results highlighted that, in microgravity conditions, Galectin-3 leaves thyrocytes and diffuses in colloid. It is possible that the gravity force contributes to the maintenance of the distribution of the molecules in both basal membrane side and apical membrane side and that the microgravity facilitates slippage of Galectin-3 in colloid probably due to membrane remodelling-microgravity induced.

Why high cholesterol levels help hematological malignancies: role of nuclear lipid microdomains

BackgroundDiet and obesity are recognized in the scientific literature as important risk factors for cancer development and progression. Hypercholesterolemia facilitates lymphoma lymphoblastic cell growth and in time turns in hypocholesterolemia that is a sign of tumour progression. The present study examined how and where the cholesterol acts in cancer cells when you reproduce in vitro an in vivo hypercholesterolemia condition.MethodsWe used non-Hodgkin’s T cell human lymphoblastic lymphoma (SUP-T1 cell line) and we studied cell morphology, aggressiveness, gene expression for antioxidant proteins, polynucleotide kinase/phosphatase and actin, cholesterol and sphingomyelin content and finally sphingomyelinase activity in whole cells, nuclei and nuclear lipid microdomains.ResultsWe found that cholesterol changes cancer cell morphology with the appearance of protrusions together to the down expression of β-actin gene and reduction of β-actin protein. The lipid influences SUP-T1 cell aggressiveness since stimulates DNA and RNA synthesis for cell proliferation and increases raf1 and E-cadherin, molecules involved in invasion and migration of cancer cells. Cholesterol does not change GRX2 expression but it overexpresses SOD1, SOD2, CCS, PRDX1, GSR, GSS, CAT and PNKP. We suggest that cholesterol reaches the nucleus and increases the nuclear lipid microdomains known to act as platform for chromatin anchoring and gene expression.ConclusionThe results imply that, in hypercholesterolemia conditions, cholesterol reaches the nuclear lipid microdomains where activates gene expression coding for antioxidant proteins. We propose the cholesterolemia as useful parameter to monitor in patients with cancer.

Critical role for the protons in FRTL-5 thyroid cells: nuclear sphingomyelinase induced-damage.

Proliferating thyroid cells are more sensitive to UV-C radiations than quiescent cells. The effect is mediated by nuclear phosphatidylcholine and sphingomyelin metabolism. It was demonstrated that proton beams arrest cell growth and stimulate apoptosis but until now there have been no indications in the literature about their possible mechanism of action. Here we studied the effect of protons on FRTL-5 cells in culture. We showed that proton beams stimulate slightly nuclear neutral sphingomyelinase activity and inhibit nuclear sphingomyelin-synthase activity in quiescent cells whereas stimulate strongly nuclear neutral sphingomyelinase activity and do not change nuclear sphingomyelin-synthase activity in proliferating cells. The study of neutral sphingomyelinase/sphingomyelin-synthase ratio, a marker of functional state of the cells, indicated that proton beams induce FRTL-5 cells in a proapoptotic state if the cells are quiescent and in an initial apoptotic state if the cells are proliferating. The changes of cell life are accompanied by a decrease of nuclear sphingomyelin and increase of bax protein.