The weak rate of sphingolipid biosynthesis shown by basal keratinocytes isolated from aged vs. young donors is fully rejuvenated after treatment with peptides of a potato hydrolysate.

A new study was carried out to bring more information on the effect of the potato proteins ferment. Basal keratinocytes obtained from freshly excised skin samples of two groups of five donors, a young one (25-36-year-old) and an aged one (59-70-year-old) were established in culture. The results showed a downward trend in the content of all lipid fractions in untreated keratinocytes of aged donors when compared with young ones. We found major differences in the response of keratinocytes to potato proteins ferment treatment between young and old donors. Whereas the lipid content of cells from young donors increased either moderately or actually decreased in some cases in comparison with the untreated controls, the lipid biosynthesis was strongly stimulated in aged donors' keratinocytes whose lipid contents globally became close to those found in young donors. However, the changes elicited by potato proteins ferment treatment were not seen at the same extent for all lipid classes. Cholesterol content increased up to three-fold and alpha-hydroxy fatty acids were augmented up to seven-fold, whereas the increase in normal fatty acids was quite moderate. In sphingolipids labelled by incubation of keratinocytes in culture medium containing [(14)C]-serine, ceramides and glucosylceramides in cells from aged donors showed the highest uptake of radioactivity, with somewhat less incorporation in sphingomyelin and gangliosides. Therefore, it seems that potato proteins ferment has a much more potent stimulatory activity on the lipid biosynthesis of basal keratinocytes of aged donors, thereby normalizing the cellular lipid content that obviously decreases along with ageing. Although our results were obtained only with basal keratinocytes in this study, potato proteins ferment could be beneficial to maintain an efficient skin barrier in ageing people, provided that the peptides can get through to the basal membrane upon topical application.

A set of glycoproteins recognized by A2B5 antibody with major bands at 55 and 76 kDa is overexpressed in human head and neck squamous cell carcinomas.

A2B5 antibody was found to strongly label frozen sections of human head and neck squamous cell carcinomas. The low amount of glycolipids (c-series gangliosides and sulfatides) purified from the same tumors and reactive with A2B5 by immunostaining on thin-layer plates could not account for the high level of tissue labeling. Proteins were extracted from both normal tissues and squamous cell carcinomas and analyzed by Western blot with A2B5 antibody on PVDF membranes. The antibody was found to stain a set of glycoproteins with two major bands at 55 and 76 kDa present in normal tissues and overexpressed in carcinomas. Staining was abolished by prior treatment of the PVDF membranes either with Arthrobacter ureafaciens neuraminidase or with a solution of 10 mM periodate that is known to destroy carbohydrates. Our results show that the carbohydrate epitope recognized by A2B5 antibody can be displayed by both glycolipids and glycoproteins.

A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges.

Solid-phase extraction (SPE) methods are easy, rapid, and reliable. Their growing popularity is in part due to their operational simplicity and cost reduction in solvents, and partly because they are easier to automate. Sphingolipids are implicated in various cellular events such as growth, differentiation, and apoptosis. However, their separation by small SPE cartridges has attracted limited attention. Here we describe an SPE procedure on aminopropyl cartridges that by sequential elution allows the separation of a lipid mixture into free ceramides, neutral glycosphingolipids, neutral phospholipids (sphingomyelin), and a fraction containing the acidic phospholipids and phosphorylated sphingoid bases, phosphoceramides and sulfatides. Individual components are obtained in high yield and purity. We applied the procedure to obtain data on separation of [(3)H]myristic acid-labeled sphingolipids from fish gills, and from human melanoma tumor tissue. Individual lipids in the SPE fractions were identified by chromatography on several high-performance thin-layer chromatography (HPTLC) systems. The chromatographic behavior of free sphingoid bases is also reported.

Single-step gas chromatography-mass spectrometry analysis of glycolipid constituents as heptafluorobutyrate derivatives with a special reference to the lipid portion.

In a previous work (Zanetta et al. Glycobiology 9, 255-266 (1999)), it was reported that all constituents of gangliosides could be obtained as heptafluorobutyrate derivatives after methanolysis in a single gas chromatography analysis. This report demonstrates that gas chromatography coupled with mass spectrometry in the electron impact mode allows identification and quantification of long-chain bases and fatty acids without interference from monosaccharides. On the basis of ions specific for families and for individual compounds, sphingosines, sphinganines, and phytosphingosines (including ramified, unsaturated, hydroxylated, and etherified compounds) can be identified. Fatty acid methyl esters, including linear, ramified, unsaturated, and hydroxylated species, are identified and quantified in the same way. Possible extensions of this method to the fatty moiety of other lipids (alkylacylglycerol and dimethyl acetal) are discussed.

Occurrence of monosialosyl pentahexaosylceramide GalNAc-GM1 as specific tumor-associated ganglioside of human head and neck squamous cell carcinomas.

In a recent study of the ganglioside profiles of human head and neck squamous cell carcinomas versus normal tissue, one unidentified GX ganglioside was found exclusively in tumor extracts, migrating between GM1 and GD3 by thin-layer chromatography. To determine the chemical structure of this ganglioside which accounted for 3-8% of the total gangliosides, the lipid samples were pooled and separated by high-pressure liquid chromatography to obtain individual ganglioside species purified to homogeneity. The tumor-associated GX ganglioside was analyzed by gas-liquid chromatography, mass spectrometry and immunostaining on thin-layer plates with mouse monoclonal antibodies after enzymatic cleavage. The data allowed the identification of GX ganglioside as GalNAc-GM1 that has been reported as a very minor brain ganglioside in humans. Thus, GalNAc-GM1 is a specific tumor-associated ganglioside in human head and neck squamous cell carcinomas that could be potentially valuable for clinicians.

Analysis of glycosphingolipids of human head and neck carcinomas with comparison to normal tissue.

Glycosphingolipids of head and neck carcinomas from six tumor-bearing patients were analyzed and compared to those of normal tissue from similar areas. The total glycosphingolipid content and the lipid-bound sialic acid were much higher in carcinomas than in normal tissue. Major neutral glycolipids were glucosylceramide, lactosylceramide, trihexosylceramide and paragloboside. Sulfatides were seen only in extracts from normal tissue which also showed a rather simple ganglioside pattern with #GM3 and GD3 as major species, whereas tumors showed additional species such as GM2 and GD2, along with a strong increase in LM1, GM1, GD1a and GT1b.

Very low density lipoproteins and interleukin 2 enhance the immunogenicity of 9-O-acetyl-GD3 ganglioside in BALB/c mice.

Gangliosides expressed by tumor cells constitute potential targets for immunotherapy. A major limitation of protocols aiming to immunize patients against tumor gangliosides is the weak immunogenicity of these molecules. We have previously shown that exogenous gangliosides are essentially bound to serum lipoproteins. In this study we have analyzed the influence of human serum lipoproteins on the immunogenicity of purified human ganglioside 9-O-acetyl-GD3 in BALB/c mice. Although expressed at very low levels in mice, this ganglioside was not immunogenic when administered in the form of micelles. However 9-O-acetyl-GD3 adsorbed onto Very Low Density Lipoproteins (VLDL) was strongly and reproducibly immunogenic, inducing both an IgM and an IgG response, with higher titers than those obtained with total serum. The IgM antibody response appeared after a single injection whereas the IgG response was observed after 3 weeks but was stronger and more durable. The antibody response to 9-O-acetyl-GD3 bound to other serum fractions was weak or absent. The addition of recombinant interleukin 2 (IL-2) enhanced weak antibody responses to 9-O-acetyl-GD3 thereby facilitating responses to ganglioside in micelles and in protein-free Very Low Density Particles. Using in vitro assays, we demonstrated that VLDL-bound ganglioside 14C-GM3 was more sensitive to the effect of neuraminidase than gangliosides bound to other lipoprotein fractions, suggesting greater accessibility of VLDL-bound gangliosides. These results indicate that VLDL-bound gangliosides are the most immunologically active fraction of serum gangliosides. VLDL or similar particles and recombinant IL-2 may be useful adjuvants for immunization with gangliosides.

The gangliosides as a possible molecular coupling factor between the proportion of radiosensitive cells in vitro and the metastatic potential in vivo within a human melanoma cell line.

With an experimental model of spontaneous lung metastases in immunosuppressed newborn rats, seven clones and variants with different metastatic potential and gangliosides expression were derived from a single parental human melanoma cell line M4Be. The cellular radiosensitivity of M4Be and its seven sublines was estimated using an in vitro colony assay. The total amount of gangliosides in M4Be and its seven sublines was determined by cell extraction and thin-layer chromatography, while the expression of GD3 gangliosides was estimated by flow cytometry with a monoclonal antibody. The radiation-cell survival curves of most clones and variants derived from M4Be showed a zero dose extrapolation clearly lower than 100%, suggesting that two populations of cells of very different radiosensitivity coexist within each of these clones and variants. Although the proportion of radiosensitive cells could be estimated from the shape of the survival curve, its radiosensitivity is too high to be properly evaluated by the colony assay. The eight survival curves differ essentially in the proportion of radiosensitive cells--which varied from 0% to 40% among M4Be and its seven sublines--whereas the cellular radiosensitivity of the radioresistant population was similar among them. The metastatic potential in vivo of M4Be and its seven sublines was not significantly related to the cellular radiosensitivity of their corresponding radioresistant population, but significantly increased with the fraction of radiosensitive cells. This relationship is valid only when the highly metastatic cells are cultured for no more than five passages in vitro as the fraction of radiosensitive cells is rapidly lost during subcultures. The relationship remains valid in vivo as metastatic melanoma-bearing newborn rats whole body irradiated with 20 cGy show no lung metastasis compared with controls. The radiosensitive cell fraction is inversely correlated with both the total ganglioside content (r = 0.84, P < 0.02) and the number of cells positively labelled with the monoclonal antibody directed to GD3 (r = 0.92, P < 0.001). The incubation of a radiosensitive clone with the exogenous bovine brain ganglioside GM1 significantly increases the proportion of radioresistant cells and suppresses its metastatic potential, while the inhibition of the endogenous gangliosides synthesis in the radioresistant cell line M4Be increases the proportion of radiosensitive cells. This study provides a possible explanation for the correlation between the metastatic potential and the proportion of radiosensitive cells within the seven sublines derived from a single parental human melanoma cell line.

Gangliosides protect human melanoma cells from ionizing radiation-induced clonogenic cell death.

With an experimental model of spontaneous lung metastases of melanoma developed in this laboratory, a range of sublines (variants and clones) with different metastatic potential and ganglioside expression was established from a single human melanoma cell line M4Be. Using an in vitro clonogenic assay and provided that cells were cultured for no more than five passages, variations in cellular radioresistance of M4Be and seven sublines derived from M4Be were detected. This study shows a positive correlation between the cell intrinsic radioresistance of M4Be and its seven sublines and their total ganglioside content. More precisely, the proportion of radioresistant cells in M4Be and the seven sublines correlated with the number of cells determined by flow cytometry that were positively labelled with a monoclonal antibody directed to GD3 disialoganglioside. Blocking the cellular biosynthesis of gangliosides with the inhibitor Fumonisin B1 or cleaving with Vibrio cholerae neuraminidase the cell surface ganglioside-bound sialic acid in a radioresistant poorly metastatic subline increased its radiosensitivity in vitro. In contrast, enrichment of a radiosensitive metastatic subline with exogenous bovine brain GM1 increased its radioresistance in vitro. These results suggest that, in the radiation dose range important for radioprotection (0-1 Gy), membrane gangliosides radioprotect human melanoma cells in vitro.

Surface expression of GD3 disialogangliosides in human melanoma cells is correlated to both metastatic potential in vivo and radiosensitivity in vitro.

With an experimental model of spontaneous lung metastases of melanoma developed in this laboratory, 7 sublines (variants and clones) with different metastatic potential and ganglioside expression were established from a single human melanoma cell line M4Be. Clones and variants derived from M4Be have been characterized at their surface by their gangliosides expression that were determined by flow cytometry with monoclonal antibodies. Gangliosides are membrane glycolipids containing sialic acid. Using an in vitro clonogenic assay and provided that cells were cultured for no more than 5 passages, variations in the cellular radiosensitivity of M4Be and of the 7 sublines were detected. This study shows that the lower the expression of GD3 disialoganglioside at the cell surface, both the higher their radiosensitivity in vitro and their metastatic potential in vivo. These results suggest that highly metastatic human melanoma cells are radiosensitive and deficient in surface gangliosides. Strengthening of this hypothesis arise from experiments showing that the incubation of radiosensitive cells with exogenous ganglioside significantly increases their radioresistance in vitro and reduces their metastatic potential in vivo.

Optimal conditions to radiolabel (3H or 14C) aminosugar-containing glycosphingolipids by de-N-acetylation and re-N-acetylation.

The optimal conditions were examined for selective re-N-acetylation with 14C or 3H.acetic anhydride of de-N-acetylated aminosugar-containing glycosphingolipids. Re-N-acetylation, which is nearly quantitative within 10 minutes in methanol, occurs selectively up to a maximal 100% yield when using a molar ratio of 5 mol of acetic anhydride per mole of aminosugar present in the glycosphingolipid. Above this molar ratio, it was observed some O-acetylation of carbohydrates which could be removed by mild alkali treatment. The method allows the choice of 14C- or 3H-labeling of glycosphingolipids with a final specific radioactivity which depends solely on the one of acetic anhydride. The binding of specific antibodies to glycosphingolipids, which was abolished upon de-N-acetylation, was again detectable after re-N-acetylation with radioactive acetic anhydride, suggesting that the native structures were recovered. This procedure of radiolabeling offers safety, rapidity and broad applicability to alkali-stable aminosugar-containing glycosphingolipids.

Deficiency of ganglioside biosynthesis in metastatic human melanoma cells: relevance of CMP-NeuAc:LacCer alpha 2-3 sialyltransferase (GM3 synthase).

The glycosphingolipid patterns were analyzed on two clones derived from a human melanoma cell line and selected for their respectively high and low metastatic ability in immunosuppressed newborn rats. Conversely to the weakly metastatic cells which exhibited a pattern similar to that of the parental cell line, highly metastatic human melanoma cells appeared to be deficient in ganglioside biosynthesis. An accumulation of lactosylceramide was found in the latter cells, with low amounts of GM3 as the only ganglioside detected and a fourfold decreased activity of GM3 synthase (EC 2.4.99.9). After subcutaneous injection of metastatic cells in newborn rats, the cells proliferating in the tumor induced at the injection site re-expressed the four common gangliosides of melanoma: GM3, GM2, GD3 and GD2, whereas the cells growing in the lungs as metastatic nodules were deficient in ganglioside synthesis and showed an accumulation of lactosylceramide. Taken together, our results suggest that the human melanoma cells which are able to escape from the primary tumor and invade the lungs have an impaired ganglioside biosynthesis with a deficient GM3 synthase.

Antigen-specific primary immune response of human B-lymphocytes after in vitro immunization with GM3 ganglioside.

In vitro immunization of human B-lymphocytes was performed with liposomes containing the monosialoganglioside GM3, with or without either complete tetanus toxoid or a synthetic T helper epitope derived from tetanus toxin (determinant 830-843). The immunized B-cells were Epstein-Barr virus transformed and the human anti-ganglioside antibody response was evaluated using an indirect ELISA against different mono- and disialogangliosides. Clones producing antigen-specific human antibodies of the IgM isotype against the ganglioside GM3 used as the immunogen were selected and one clone, IM-11, was further characterized. In addition, a method of positive selection using GM3-coated magnetic beads has been developed which allowed us to rescue unstable clones. The binding of the human antibody IM-11 to a large panel of glycosphingolipids separated on thin-layer plates was studied. The human MAb IM-11 was found to bind strongly to NeuAcGM3, IV3 NeuAcnLc4 and sulfate containing glycosphingolipids and weakly to NeuGcGM3. Immunohistological staining of melanoma and breast cancer biopsy sections showed a selective reactivity of IM-11 with tumor cells which varied among different tumors.

Correlation between the radiosensitivity in vitro of clones and variants derived from a human melanoma cell line and their spontaneous metastatic potential in vivo.

With an experimental model of spontaneous lung metastases of human melanoma in immunosuppressed newborn rats, a large panel of clones and variants with different metastatic potential were derived from a single human melanoma parental cell line (M4Be). Seven clones and variants from M4Be were selected, respectively, for their low (parental, clone 1), intermediate (clones 2 and 3, subvariant 1-) and high (variant 1, subvariant 1+, clone 4) metastatic potential. This paper investigates the relationship between the in vivo metastatic potential of the eight cell lines and their sensitivity to ionizing radiation in vitro (range 0.05-7 Gy). The radiosensitivity was estimated from the mean inactivation dose, a parameter equal to the area under the survival curve plotted in linear coordinates. Examination of the eight survival curves, obtained with cells cultured for no more than five passages after defrost, shows that clone 1, subvariant 1- and the M4be parental line are the most radioresistant cells, clone 4 and subvariant 1+ are the most radiosensitive cells, while clones 2 and 3 and variant 1 showed an intermediate response to radiation. The metastatic potential in vivo of the parental line and the seven sublines is significantly correlated to their radiosensitivity in vitro: the higher the metastatic potential, the higher the radiosensitivity.

Induction of IgG antibodies directed to a M(r) 31,000 melanoma antigen in patients immunized with vaccinia virus melanoma oncolysates.

Pre- and postimmunization sera from eight tumor-free melanoma patients undergoing vaccinia melanoma oncolysate (VMO) therapy were used to investigate the humoral response to antigens from infected and uninfected melanoma cells and from vaccinia virus. Immunodetection on Western blots showed that all patients, in addition to reacting to several other proteins, developed IgG antibodies to a M(r) 31,000 protein antigen within 1 month of immunization. This M(r) 31,000 antigen is expressed both on VMO and on melanoma metastases in situ, disappears in primary cultures of these metastases, and is absent in extracts from vaccinia virus, from human melanoma cell lines, and from normal melanocytes, suggesting that this M(r) 31,000 protein is reexpressed following vaccinia virus infection of human melanoma cells. Periodate treatment of the blotted antigens abolished reactivity of patients' postimmunization sera with the M(r) 31,000 antigen, thus showing that this antigen is a glycoprotein and that the relevant epitope is likely to reside on its carbohydrate moiety. These anti-M(r) 31,000 IgG antibodies were absent in the sera of VMO-treated patients before immunization, absent in the serum of a normal donor hyperimmunized with vaccinia virus, and absent in normal human sera. In addition, these anti-M(r) 31,000 antibodies appeared 1 week after the first VMO injection, remained stable during the treatment, and decreased when the treatment was stopped. Such antibodies can also be demonstrated in sera of melanoma patients bearing metastases but disappeared following resection of their metastases. Thus, in melanoma patients, immunization with VMO induces an antibody response directed against a M(r) 31,000 glycoprotein likely to represent a new melanoma antigen. Further identification of this antigen could be of utmost interest for the further development of melanoma vaccines.

Inhibition of immune cell proliferation and cytokine production by lipoprotein-bound gangliosides.

We have analyzed the immunomodulatory effect of human melanoma gangliosides bound to serum lipoprotein fractions on normal human immune-competent cells in vitro. Total melanoma gangliosides in micelles inhibited proliferation of peripheral blood mononuclear cells stimulated by various mitogens, modulated lymphocyte surface molecules CD2, CD3, CD4, CD5 and CD8 and inhibited the production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha) and IL-6 by stimulated adherent cells. Most of these effects were abrogated in the presence of serum. Purified serum lipoprotein fractions were tested for their ability to allow or inhibit the immunomodulatory effects of gangliosides. Melanoma gangliosides bound to very-low-density lipoproteins (VLDL) were shown to be as potent modulators of the immune response in vitro as when they were presented to cells in the form of micelles. Gangliosides bound to low-density lipoproteins were less active and gangliosides bound to high-density lipoproteins or the lipoprotein-free fraction had no immunomodulatory effects. Given the fact that gangliosides are predominantly bound to lipoproteins in serum, we conclude that lipoproteins are important determinants of the immunomodulating potential of tumor gangliosides, and that the immunomodulatory effects of melanoma gangliosides observed in vitro may also occur in vivo.

A monoclonal antibody directed to sulfatide inhibits the binding of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein to macrophages but not their infection by the virus.

We show here that human immunodeficiency virus (HIV) envelope glycoproteins (gp160/gp120) bind to sulfatide and galactosyl ceramide. By immunofluorescence labeling with monoclonal antibody (mAb) A2B5, specific for ganglioside/sulfatide, we detect negatively charged glycolipids on CD4+ cells of the macrophage lineage and lymphocytes. Labeling of monocyte-derived macrophages (MDM) with mAb A2B5 was reproducibly found in 29 healthy donors, independently of the culture method and duration up to 11 days. The binding of the mAb to neuraminidase-treated MDM was unchanged relative to control cells, but mAb binding decreased after arylsulfatase treatment, which indicates that MDM membrane sulfatide is its major ligand. Preincubating MDM with the mAb partially (40-60%) but significantly inhibited the binding of HIV-1LAI radiolabeled recombinant gp160 to the cells. Similarly, the mAb entailed limited (32%) but significant inhibition of gp160 binding to cells of the monocytic U937 line but not to lymphoid CEM cells. However, mAb A2B5 did not inhibit the infection of CEM nor of U937 cells by HIV-1LAI strain, nor of MDM by monocytotropic HIV-1BaL. Thus, although sulfatide may be involved in the binding of HIV env glycoprotein to MDM or monocytic U937 cells, this does not play a significant role in HIV infection of these CD4+ cells.

Shedding of GD2 ganglioside in patients with retinoblastoma.

Retinoblastoma is a rare tumor of the young child with an intraocular localization that leads to certain problems of diagnosis. With the aim of defining a biochemical marker--which is still lacking for this disease--the gangliosides of a pool of fresh retinoblastoma tumors were analyzed. The ganglioside pattern was shown to have GM3, GM2, GM1, GD3, GD2, GD1b and GT1b as the major components. The occurrence of a high concentration of GD2 in the tumors led us to investigate the possibility of changes in the level of GD2 in the sera of retinoblastoma patients, using quantitative immunostaining with GD2-specific mouse monoclonal antibodies (MAbs). In 9 out of 10 tumor-bearing patients, the serum level of GD2 ganglioside was significantly higher than the average value found in normal individuals. A 2-year follow-up of patients showed that successful treatment resulted in a rapid decrease in the serum level of GD2 down to the normal range, from which a subsequent elevation was seen only in relapsing patients. Although the clinical study needs further development, the results obtained to date suggest that GD2 is shed in the serum of tumor-bearing patients and that the level of GD2 could be a potential serum marker of human retinoblastoma.