TET1 is a tumour suppressor that inhibits colon cancer growth by derepressing inhibitors of the WNT pathway.

Ten eleven translocation (TET) enzymes catalyse the oxidative reactions of 5-methylcytosine (5mC) to promote the demethylation process. The reaction intermediate 5-hydroxymethylcytosine (5hmC) has been shown to be abundant in embryonic stem cells and tissues but strongly depleted in human cancers. Genetic mutations of TET2 gene were associated with leukaemia, whereas TET1 downregulation has been shown to promote malignancy in breast cancer. Here we report that TET1 is downregulated in colon tumours from the initial stage. TET1 silencing in primary epithelial colon cells increase their cellular proliferation while its re-expression in colon cancer cells inhibits their proliferation and the growth of tumour xenografts even at later stages. We found that TET1 binds to the promoter of the DKK gene inhibitors of the WNT signalling to maintain them hypomethylated. Downregulation of TET1 during colon cancer initiation leads to repression, by DNA methylation, the promoters of the inhibitors of the WNT pathway resulting in a constitutive activation of the WNT pathway. Thus the DNA hydroxymethylation mediated by TET1 controlling the WNT signalling is a key player of tumour growth. These results provide new insights for understanding how tumours escape cellular controls.

Comparison between extra virgin olive oil and oleic acid rich sunflower oil: effects on postprandial lipemia and LDL susceptibility to oxidation.

The aim of our study was to determine whether the minor polar components of virgin olive oil could have favorable effects (1) on fasting and postprandial lipid profile and (2) on low-density lipoprotein (LDL) composition and susceptibility to oxidation in vitro. Ten normolipidic subjects were included in a crossover study (two diet periods of 3 weeks) and received either virgin olive oil (OO diet) or oleic acid rich sunflower oil. An oral fat load was performed at the end of each period. The plasma lipid levels were not significantly different after both diets in the fasting and postprandial states. A few minor variations of the LDL composition were observed only in the postprandial lipemia, and they were different after both diets. The LDL oxidation susceptibility was evaluated by the formation of conjugated dienes. With LDL isolated in the fasting state, the diene production decreased (p = 0.0573) only after the OO diet. The dienes determined at time 0 and the maximal dienes obtained during the oxidation reaction decreased (p = 0.0145 and p = 0.0184, respectively) only after the OO fat load. Nevertheless, the diene production decrease was not significant (p = 0.0848). Our results suggest a mild effect of minor components of virgin olive oil related to a decrease of LDL susceptibility to oxidation; further analyses are necessary to give clear conclusions about their role.

Vitamin D metabolites activate the sphingomyelin pathway and induce death of glioblastoma cells.

1 alpha, 25-dihydroxyvitamin D3 was previously shown to induce cell death in brain tumour cell lines when added to the medium at micromolar concentration. In this paper we show that Cholecalciferol, a poor ligand of the vitamin D receptor, also induces cell death of HU197 human glioblastoma cell line and early passages cultures derived from a recurrent human glioblastoma. This finding suggests that the effects of vitamin D metabolites on brain tumour cells are at least partially independent from the activation of the classic nuclear receptor pathway. Vitamin D metabolites have been shown to activate the sphingomyelin pathway inducing an increase in cellular ceramide concentration. We determined the levels of sphingomyelin ceramide and ganglioside GD3 in Hu197 cells after treatment with cholecalciferol. A significant increase in ceramide concentration and a proportional decrease in sphingomyelin was already present after 6 hours of cholecalciferol treatment when no morphological changes were visible in the cultures. Treatment with ceramides (N-acetylsphingosine or natural ceramide from bovine brain) of the same cells also induces cell death. Similarly, treatment of the same cells with bacterial Sphingomyelinase also results in cell death. The demonstration of an increase in intracellular ceramide after cholecalciferol treatment and the ability of ceramide to induce cell death suggest that the sphingomyelin pathway may be implicated in the effect of vitamin D metabolites on human glioblastoma cells. Inhibition of ceramide biosynthesis by fumonisin B1 treatment did not alter the dose response curve of HU197 cells to cholecalciferol. Insensitivity to fumonisin B1 together with a decrease in sphingomyelin content after cholecalciferol treatment indicate that activation of sphingomyelinase should be responsible for the increase in intracellular ceramide concentration.

Prolonged in vitro culture modifies the surface lipid composition of murine melanoma cell lines.

Lipid composition of two murine melanoma cell variants (B16, without malignant properties and B16-F10, with high metastatic activity), has been examined at different stages of growth. The aim of the work was to identify cell surface modifications due to the time length of in vitro culture, that could be one variable to consider when metastatic potential is studied. Some of the analyzed parameters (ganglioside- and glycoprotein-bound neuraminic acid, cholesterol, neutral glycolipids, phospholipids, triacylglycerols) undergo statistically significant variations at the various passages in B16-F10 line. Fatty acids composition of the phospholipidic fraction was changed only at the last observed passage (100) in B16 line. No one of the examined parameters justifies the ability of B16-F10 cells to invade distant districts and to originate new tumors. Probably detailed lipid analysis on cellular subfractions, as already performed in this study on total lipid extract of the whole cell, could be a valuable tool to identify differences related with metastatic potential.

Oncogene transgenic mice: an useful model to study in vivo the relationships between gangliosides and oncogenes.

Several studies have demonstrated that transfer of oncogenes in cultured cells reproducibly induces transmissible alterations in their ganglioside profile; the transfection of the same oncogene into different cell lines and the different localization of the oncogene product result in a different ganglioside expression. In the present study the modifications of the ganglioside pattern in mammary carcinomas induced in transgenic mice by the activated form of the rat neu oncogene have been investigated. Whereas control mammary tissues contain quite exclusively GM3, all neoplastic samples show a substantial decrease of this ganglioside, an accumulation in variable amount of GM3-derived species (GM1, GD3, GD1a, GD1b, GT and GQ) and the appearance of new, not yet identified, sialic acid containing molecules. Interestingly, three out of 10 tumors analyzed, even if histologically comparable to the others but with a larger dimension, show a significative difference as regard to the GM1, GD3 and GD1a content. Our data suggest that an activated oncogene may induce also in vivo a specific and transmissible alteration in the ganglioside pattern, but this distribution could be susceptible to further modifications during the tumor progression.

Glycosphingolipid expression in solid tumours and transformed cell lines.

Glycosphingolipids are assumed to play a crucial role in cell-cell and cell-substrate interactions, including cell adhesion, proliferation, differentiation and apoptosis. Furthermore, cell surface glycolipid profile changes in the so called "social disorders", such as malignant transformation. To better investigate these modifications, the ganglioside composition in different solid tumours and in two transformed cell lines was analyzed. In some of these models we also tried to correlate the pattern of gangliosides to the key enzymes involved in their metabolism. The results we obtained can be summarized as follows:(1), meningiomas with or without chromosome 22 deletion: predominance of ganglioside GD3 in the former and of ganglioside GM3 in the latter. Correlation between GM3/GD3 ratio and SAT-2 activity; (2), mammary carcinomas developed in MMTV/c-neu transgenic mice: accumulation of GM3-derived species. The different ganglioside distribution seems to correlate with the tumour size; (3), Sarcoma Galliera-strain cells SGS/3A and normal syngenic murine fibroblasts FG: transformed cells exhibit a lower activity of sialyltransferases (SAT-1, SAT-2, SAT-4) compared to normal fibroblasts, suggesting a possible correlation with the ganglioside pattern. The neuraminidase activity seems to correlate to the glycoprotein sialic acid content; (4), 3T3 normal murine fibroblasts and SVT2 transformed cells: GM3 is absent in 3T3, while it accounts for the main ganglioside species in SVT2. On the contrary, GM2 present in a large amount in normal fibroblasts, is practically absent in transformed cells. No correlation has been observed between ganglioside profile and glycosyltransferase activities so far examined.

Prolonged in vitro culture modifies the surface lipid composition of murine melanoma cell lines.

Lipid composition of two murine melanoma cell variants (B16, without malignant properties and B16-F10, with high metastatic activity), has been examined at different stages of growth. The aim of the work was to identify cell surface modifications due to the time length of in vitro culture, that could be one variable to consider when metastatic potential is studied. Some of the analyzed parameters (ganglioside- and glycoprotein-bound neuraminic acid, cholesterol, neutral glycolipids, phospholipids, triacylglycerols) undergo statistically significant variations at the various passages in B16-F10 line. Fatty acids composition of the phospholipidic fraction was changed only at the last observed passage (100) in B16 line. No one of the examined parameters justifies the ability of B16-F10 cells to invade distant districts and to originate new tumors. Probably detailed lipid analysis on cellular subfractions, as already performed in this study on total lipid extract of the whole cell, could be a valuable tool to identify differences related with metastatic potential.

Infantile sialic acid storage disease: biochemical studies.

Infantile free sialic acid storage disease (ISSD), is an inherited metabolic disorder characterized by hyperexcretion of free sialic acid in the urine and by its storage in the lysosomes of different tissues. In order to obtain more reliable data on the amount of total and free sialic acid, we analyzed the urine, brain, cerebellum, liver, spleen, and kidneys from a 3-month-old baby who died with a diagnosis of ISSD. The lysosomal nature of the disease was confirmed by an electron microscopic study of cells in culture. No significant abnormalities were found involving cholesterol, total phospholipids, glycolipids, and gangliosides in the tissues examined. However, differences in the tissue distribution of individual glycolipids and gangliosides were observed. The amount of free and total sialic acid was markedly increased, due to the storage of free sialic acid accompanied by its hyperexcretion in the urine. These results demonstrate and confirm that only acid monosaccharide transport from the lysosome compartment is involved in the pathogenesis of ISSD.

Altered membrane lipid composition in a human meningosarcoma.

In a sample of meningosarcoma, obtained at the time of surgery, the amount of total gangliosides and phospholipids was examined, together with the cholesterol content and the distribution of different ganglioside and phospholipid species. The phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4, 5-bisphosphate and phosphatidylcholine fatty acid composition was also analyzed. The ganglioside pattern in the meningosarcoma was different from the previously reported pattern in meningiomas of different histological origin, showing a higher concentration of GD3, indicating that the so-called b pathway of ganglioside biosynthesis was the preferred one in this type of tumor; moreover the percentage content of polysialylated gangliosides was very low. Cholesterol and phospholipid content was lower than in meningiomas; the phosphatidylcholine increase and the sphingomyelin decrease would indicate a lower membrane microviscosity, a characteristic of tumor cells. Phosphoinositide and phosphatidylcholine fatty acid analysis revealed a considerable amount of docosahexaenoic acid. This abnormal presence of this fatty acid could lead to the production, after receptor stimulation, of a diacylglycerol containing docosahexaenoic acid, which, in turn, could be responsible for an altered activation pattern of protein kinase C, in this way promoting carcinogenesis.

Treatment of sphingomyelinase deficiency by repeated implantations of amniotic epithelial cells.

Five young patients with Niemann-Pick disease type B were treated with repeated implantations of amniotic epithelial cells, as a source of exogenous sphingomyelinase. This treatment abolished the recurrent infections, mainly of the respiratory tract, and led to other improvements of the general conditions of the patients. In particular, we noticed a disappearance of vomiting, a recovery from muscular hypotrophy, and significantly reduced pulmonary distress. In four subjects, who were in a prepuberal state, there was a puberal spurt with a concomitant burst of growth. In two cases, characterized by a greater than normal content of sphingomyelin in urinary sediments, a single implantation caused a sustained normalization of sphingomyelin and total phospholipids in the urine. Finally, sphingomyelinase activity of peripheral leukocytes, when assayed 0.5 to 4 months after some of the implantations, showed a rise to heterozygous values in 30-40% of the assays.

Fatty acid pattern of the different phosphoinositide fractions in human meningiomas.

Previous studies showed no differences in the phospholipid content of human meningiomas compared to normal leptomeninges, but only a higher unsaturation degree in the individual phospholipid fractions of tumors. Inasmuch as phosphoinositides play a role in the membrane responsiveness to numerous effectors, we studied the fatty acid pattern of the different phosphoinositide fractions of 14 human meningiomas of different histological origin. The fatty acid analysis revealed remarkable differences among the histological types, and, above all, among the different phosphoinositide fractions of a single tumor class. The phosphoinositides derived from transitional meningiomas appeared to be the most saturated ones, because of their low arachidonic acid content. Furthermore, in all the meningiomas, long chain polyunsaturated fatty acids were present only in the phosphatidylinositol fractions and the polyphosphorylated compounds appeared to be significantly different from the corresponding monophosphorylated ones. The possible significance of the different fatty acid distribution in the three phosphoinositide classes is discussed.

A new procedure for gangliosidic N-acetylneuraminic acid analysis in serum.

In this paper a method is presented which is suitable for the extraction, purification and analysis of serum gangliosides. The advantage in comparison with other previously published procedures is the complete extraction of sialoglycolipids without contamination of sialoglycoproteins and/or sialoglycopeptides. The method could be used as a second-level test for the diagnosis and follow-up of cancer patients, and also could be potentially used for pharmaco-kinetic studies after ganglioside treatment.

Correlation between cytogenetic data and ganglioside pattern in human meningiomas.

Partial or total loss of chromosome 22 is often associated with tumors of the central nervous system and in particular with meningiomas. As in the case of other tumors, the ganglioside pattern is modified in transformed tissues. Cytogenetic analysis of 30 human meningiomas has been performed and the results compared to biochemical analysis of ganglioside distribution on the membrane surface. The meningiomas were divided into 2 groups on the basis of the presence or absence of chromosome 22. Thirteen tumors exhibited partial or total monosomy of the chromosome, whereas 17 were normal or showed other chromosomal anomalies. The GM3 and GD3 content of the meningiomas belonging to the 2 groups revealed a significant correlation between amount and reciprocal ratio of these 2 gangliosides and cytogenetic data. Tumors with monosomy 22 had a higher content of ganglioside GD3 than samples without monosomy 22, where the main ganglioside was GM3. Other gangliosides such as GM1, GD1a, GD1b and GT were present in various amounts in the 2 groups. Considering the biosynthetic pathway of gangliosides, we hypothesize the involvement of a gene located on chromosome 22 in the regulation of the enzymes which catalyze either GD3 synthesis (sialyltransferase 2, SAT-2) or its degradation to GM3 (neuraminidase).

Gas chromatographic evaluation of the antimycotic democonazole for pharmacokinetic and bioavailability studies in man and animals.

A new gas chromatographic method for quantitative determination of 1-[2,4-dichloro-beta-[2-(p-chlorophenoxy)ethoxy] styryl]imidazole (demonoconazole), employing an electron capture detector, was developed. All analytical parameters were satisfactory within the range between 5 ng and 1 micrograms per ml of plasma, which means 20 pg-2 ng as absolute sensitivity. This method therefore is specific and sensitive enough for the quantitative determination of the drug in biological samples for pharmacokinetic and bioavailability studies to be performed in animals and human beings.

Presence of glycoproteins containing the polylactosamine structure in brain and liver of GM1 gangliosidosis patients. Comparative study between clinical types I and II, using endo-beta-galactosidase enzyme.

The material derived from defective degradation of glycoproteins, which accumulates in brain and liver of a patient with GM1 gangliosidosis type I, was investigated, and the structure of the main storage compounds determined. For comparison, brain and liver of a patient with GM1 gangliosidosis type II were also analyzed. Analysis of the glycopeptides obtained after pronase digestion of the defatted residue indicates the storage of glycoprotein-like material in type I, but not in type II. Treatment with endo-beta-galactosidase showed that the stored material contained N-acetyllactosamine repeating units. Two major oligosaccharides, OS I and OS II, were isolated after the enzyme treatment, whose structures are: GlcNAc beta 1----3 Gal (OS I) and Gal beta l----4GlcNAc beta 1----3 Gal (OS II). Treatment with exo-beta-galactosidase transformed the trisaccharide OS II into the disaccharide OS I, indicating that the deficiency of beta-galactosidase in GM1 gangliosidosis type I, but not in type II, also affects glycoprotein catabolism, leading to the accumulation of glycopeptides containing terminal beta-galactosyl residues and N-acetyllactosamine repeating units. These results indicate the severe impairment in the catabolism of glycoconjugates with beta-linked galactose in type I, although this impairment is not as pronounced in type II.

Usefulness and potential pitfalls of sialic acid determination in sera of patients with ovarian tumors.

Increasing evidence in the literature indicates that serum sialic acid is increased in cancer patients suggesting a possible usefulness of its determination as a tumor marker. However there are many discrepancies in the data reported, probably due to methodological errors, mainly in lipid bound sialic measurement. In this paper we illustrate the results obtained when we applied a method worked out in our laboratory for the determination of total and fractionated sialic acid (lipid and protein bound) to the analysis of sera from patients with ovarian tumors and the preliminary data on the follow up of selected cases. The potential pitfalls in using this relatively new tumor marker will be critically evaluated.

Gas-liquid chromatographic evaluation of bencyclane in biological samples for pharmacokinetic and bioavailability investigations: comparison of two analytical methods.

Analytical conditions that allow bencyclane, a vasodilator, to be evaluated in biological samples for pharmacokinetic and bioavailability investigations are reported. Two gas chromatographic methods were developed, one employing a flame-ionization detector, reaching a sensitivity of 0.5-1 micrograms/ml, and the other employing a thermionic specific detector and reaching a sensitivity of 10 ng/ml. The extraction recovery, reproducibility and specificity were all satisfactory with both methods. The former method is suitable for chemical quality controls and the latter has a sufficient sensitivity and reproducibility for determination of the drug in biological samples as required in pharmacokinetic investigations.