Functional validation of novel variants in B4GALNT1 associated with early-onset complex hereditary spastic paraplegia with impaired ganglioside synthesis.

Childhood-onset forms of hereditary spastic paraplegia are ultra-rare diseases and often present with complex features. Next-generation-sequencing allows for an accurate diagnosis in many cases but the interpretation of novel variants remains challenging, particularly for missense mutations. Where sufficient knowledge of the protein function and/or downstream pathways exists, functional studies in patient-derived cells can aid the interpretation of molecular findings. We here illustrate the case of a 13-year-old female who presented with global developmental delay and later mild intellectual disability, progressive spastic diplegia, spastic-ataxic gait, dysarthria, urinary urgency, and loss of deep tendon reflexes of the lower extremities. Exome sequencing showed a novel splice-site variant in trans with a novel missense variant in B4GALNT1 [NM_001478.5: c.532-1G>C/c.1556G>C (p.Arg519Pro)]. Functional studies in patient-derived fibroblasts and cell models of GM2 synthase deficiency confirmed a loss of B4GALNT1 function with no synthesis of GM2 and other downstream gangliosides. Collectively these results established the diagnosis of B4GALNT1-associated HSP (SPG26). Our approach illustrates the importance of careful phenotyping and functional characterization of novel gene variants, particularly in the setting of ultra-rare diseases, and expands the clinical and molecular spectrum of SPG26, a disorder of complex ganglioside biosynthesis.

Ganglioside GD2 Enhances the Malignant Phenotypes of Melanoma Cells by Cooperating with Integrins.

Gangliosides have been considered to modulate cell signals in the microdomain of the cell membrane, lipid/rafts, or glycolipid-enriched microdomain/rafts (GEM/rafts). In particular, cancer-associated gangliosides were reported to enhance the malignant properties of cancer cells. In fact, GD2-positive (GD2+) cells showed increased proliferation, invasion, and adhesion, compared with GD2-negative (GD2-) cells. However, the precise mechanisms by which gangliosides regulate cell signaling in GEM/rafts are not well understood. In order to analyze the roles of ganglioside GD2 in the malignant properties of melanoma cells, we searched for GD2-associating molecules on the cell membrane using the enzyme-mediated activation of radical sources combined with mass spectrometry, and integrin ß1 was identified as a representative GD2-associating molecule. Then, we showed the physical association of GD2 and integrin ß1 by immunoprecipitation/immunoblotting. Close localization was also shown by immuno-cytostaining and the proximity ligation assay. During cell adhesion, GD2+ cells showed multiple phospho-tyrosine bands, i.e., the epithelial growth factor receptor and focal adhesion kinase. The knockdown of integrin ß1 revealed that the increased malignant phenotypes in GD2+ cells were clearly cancelled. Furthermore, the phosphor-tyrosine bands detected during the adhesion of GD2+ cells almost completely disappeared after the knockdown of integrin ß1. Finally, immunoblotting to examine the intracellular distribution of integrins during cell adhesion revealed that large amounts of integrin ß1 were localized in GEM/raft fractions in GD2+ cells before and just after cell adhesion, with the majority being localized in the non-raft fractions in GD2- cells. All these results suggest that GD2 and integrin ß1 cooperate in GEM/rafts, leading to enhanced malignant phenotypes of melanomas.

Ganglioside GD3 induces convergence and synergism of adhesion and hepatocyte growth factor/Met signals in melanomas.

Ganglioside GD3 is highly expressed in human melanomas and enhances malignant properties of melanomas, such as cell proliferation and invasion activity. In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL-I). Although stimulation of melanoma N1 cells (GD3+ and GD3-) with either HGF or adhesion to CL-I did not show marked differences in the phosphorylation levels of Akt at Ser473 and Thr308 between two types of cells, simultaneous treatment resulted in definite and markedly increased activation of Akt in GD3+ cells. Similar increases were also shown in Erk1/2 phosphorylation levels with the costimulation in GD3+ cells. When resistance to induced apoptosis by H2O2 was examined, only GD3+ cells treated with both HGF and adhesion to CL-I showed clearly low percentages of dead cells compared with GD3- cells or GD3+ cells treated with either one of the stimulants. Cell growth measured by 5-ethynyl-2' deoxyuridine uptake also showed synergistic effects in GD3+ cells. These results suggested that GD3 plays a crucial role in the convergence of multiple signals, leading to the synergistic effects of those signals on malignant properties of melanomas.

Randomized phase II trial comparing carboplatin plus weekly paclitaxel and docetaxel alone in elderly patients with advanced non-small cell lung cancer: north japan lung cancer group trial 0801.

BACKGROUND: Standard first-line chemotherapy for elderly non-small cell lung cancer (NSCLC) patients has been monotherapy with vinorelbine or gemcitabine. Docetaxel has also been considered as an alternative option for the elderly population in Japan. We have previously demonstrated the high efficacy of carboplatin plus weekly paclitaxel for elderly NSCLC patients. Consequently, we conducted a randomized phase II study to select the proper regimen for a future phase III trial. METHODS: Eligible patients were aged 70 years or older with newly diagnosed advanced NSCLC. Patients were randomly assigned either to a combination of carboplatin (area under the curve: 6 mg/mL per minute) with weekly paclitaxel (70 mg/m²) (CP regimen) or to single-agent docetaxel (60 mg/m²). The primary endpoint of this study was objective response rate. Secondary endpoints were progression-free survival, overall survival, and toxicity profile. RESULTS: Among 83 eligible patients (41 to CP, 42 to docetaxel), the objective response rates were 54% (95% confidence interval: 39%-69%) and 24% (95% confidence interval: 11%-37%) and median progression-free survival was 6.6 months and 3.5 months in the CP arm and the docetaxel arm, respectively. Severe neutropenia, febrile neutropenia, and nausea were significantly frequent in the docetaxel arm, whereas toxicities in the CP arm were generally moderate. One treatment-related death was observed in the docetaxel arm. CONCLUSION: The CP regimen achieved higher activity with less toxicity than single-agent docetaxel. Considering the results of this phase II trial and the IFCT-0501 trial, we have selected the CP regimen for a future phase III trial in elderly patients with advanced NSCLC.

UVB-irradiated keratinocytes induce melanoma-associated ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6.

Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.

Crucial roles of exosomes secreted from ganglioside GD3/GD2-positive glioma cells in enhancement of the malignant phenotypes and signals of GD3/GD2-negative glioma cells.

Neuroectoderm-derived tumors characteristically express gangliosides such as GD3 and GD2. Many studies have reported that gangliosides GD3/GD2 enhance malignant phenotypes of cancers. Recently, we reported that human gliomas expressing GD3/GD2 exhibited enhanced malignant phenotypes. Here, we investigated the function of GD3/GD2 in glioma cells and GD3/GD2-expressing glioma-derived exosomes. As reported previously, transfectant cells of human glioma U251 MG expressing GD3/GD2 showed enhanced cancer phenotypes compared with GD3/GD2-negative controls. When GD3/GD2-negative cells were treated with exosomes secreted from GD3/GD2-positive cells, clearly increased malignant properties were observed. Furthermore, increased phosphorylation of signaling molecules was detected after 5-15 min of exosome treatment, ie, higher tyrosine phosphorylation of platelet-derived growth factor receptor, focal adhesion kinase, and paxillin was found in treated cells than in controls. Phosphorylation of extracellular signal-regulated kinase-1/2 was also enhanced. Consequently, it is suggested that exosomes secreted from GD3/GD2-positive gliomas play important roles in enhancement of the malignant properties of glioma cells, leading to total aggravation of heterogenous cancer tissues, and also in the regulation of tumor microenvironments.

Extracellular vesicles released from ganglioside GD2-expressing melanoma cells enhance the malignant properties of GD2-negative melanomas.

Exosomes (small extracellular vesicles: EVs) have attracted increasing attention from basic scientists and clinicians since they play important roles in cell-to-cell communication in various biological processes. Various features of EVs have been elucidated regarding their contents, generation and secretion mechanisms, and functions in inflammation, regeneration, and cancers. These vesicles are reported to contain proteins, RNAs, microRNAs, DNAs, and lipids. Although the roles of individual components have been rigorously studied, the presence and roles of glycans in EVs have rarely been reported. In particular, glycosphingolipids in EVs have not been investigated to date. In this study, the expression and function of a representative cancer-associated ganglioside, GD2, in malignant melanomas was investigated. Generally, cancer-associated gangliosides have been shown to enhance malignant properties and signals in cancers. Notably, EVs derived from GD2-expressing melanomas enhanced the malignant phenotypes of GD2-negative melanomas, such as cell growth, invasion, and cell adhesion, in a dose-dependent manner. The EVs also induced increased phosphorylation of signaling molecules such as EGF receptor and focal adhesion kinase. These results suggest that EVs released from cancer-associated ganglioside-expressing cells exert many functions that have been reported as a function of these gangliosides and regulate microenvironments, including total aggravation of heterogeneous cancer tissues, leading to more malignant and advanced cancer types.

Phase I study of irinotecan by 24-h intravenous infusion in combination with 5-fluorouracil in metastatic colorectal cancer.

BACKGROUND: This study was intended to ascertain the feasibility of a combination therapy with irinotecan by 24-h intravenous infusion (24-h CPT-11) and 5-fluorouracil (5-FU) for patients with metastatic colorectal cancer, to estimate the dose-limiting toxicity (DLT) and the maximum tolerated dose (MTD), to determine the recommended dose (RD) for the Phase II study, and to evaluate the efficacy of the combination therapy. METHODS: The dosage regimen was as follows: CPT-11 was given by 24-h CPT-11 on day 1, followed by 24-h intravenous infusion of 5-FU on day 2. This regimen was repeated every 2 weeks. The dose of CPT-11 was escalated in five steps from 50 to 75, 100, 125, or 150 mg/m(2) (levels 1-5), whereas the dose of 5-FU was fixed at 800 mg/m(2). RESULTS: Twenty-six patients were recruited for this study, and 25 of the 26 patients were eligible for the assessment. The DLTs of 24-h CPT-11/5-FU therapy included grade 3 diarrhea in 1 patient treated at level 1, and grade 3 neutropenia in 1 patient and grade 4 neutropenia in 1 patient at level 4. In level 5, in 3 cases the next administration could not be done for 22 days or more as a consequence of anorexia. Thus, the level 5 was made a MTD and the level 4 was made a RD. The main side effects of grade 3 or higher, although nausea/vomiting occurred, were mild and tolerable in severity overall. The overall response rate was 24.0% (6PR/25). CONCLUSION: This study suggests that 24-h CPT-11/5-FU therapy is feasible and effective for treatment of metastatic colorectal cancer.

Ganglioside GD3 enhances adhesion signals and augments malignant properties of melanoma cells by recruiting integrins to glycolipid-enriched microdomains.

Ganglioside GD3 is widely expressed in human malignant melanoma cell lines and tumors. Previously, we reported that GD3+ cells show stronger tyrosine phosphorylation of focal adhesion kinase (FAK), p130(Cas), and paxillin when treated with fetal calf serum than GD3- cells. In this study, we analyzed the changes in the signals mediated by the interaction between integrins and extracellular matrices (ECM) to clarify how GD3 enhances cell signals in the vicinity of the cell membrane. An adhesion assay with a real time cell electronic sensing system revealed that GD3+ cells had stronger adhesion to all extracellular matrices examined. In particular, GD3+ cells attached more strongly to collagen type I and type IV than controls. Correspondingly, they showed stronger tyrosine phosphorylation of FAK and paxillin during adhesion to collagen type I. In the floating pattern of detergent extracts, a high level of integrin beta1 was found in glycolipid-enriched microdomain (GEM)/rafts in GD3+ cells before adhesion, whereas a smaller amount of integrin beta1 was detected in the GEM/rafts of controls. Some phosphorylated forms of FAK as well as total FAK were found in GEM/rafts during cell adhesion only in GD3+ cells. Another signal consisting of integrin-linked kinase/Akt was also activated during adhesion more strongly in GD3+ cells than in controls. In double stained GD3+ cells, GD3 and integrin beta1 co-localized at the focal adhesion with a punctate pattern. All these results suggested that integrins assembled and formed a cluster in GEM/rafts, leading to the enhanced signaling and malignant properties under GD3 expression.

Membrane sialidase NEU3 is highly expressed in human melanoma cells promoting cell growth with minimal changes in the composition of gangliosides.

NEU3 is a membrane sialidase specific for gangliosides. Its increased expression and implication in some cancers have been reported. Here, we analyzed NEU3 expression in malignant melanoma cell lines and its roles in the cancer phenotypes. Quantitative RT-PCR revealed that high levels of the NEU3 gene were expressed at almost equivalent levels with those in colon cancers. To examine the effects of overexpression of NEU3, NEU3 cDNA-transfectant cells were established using a melanoma cell line SK-MEL-28 and its mutant N1 lacking GD3. SK-MEL-28 sublines overexpressing both the NEU3 gene and NEU3 enzyme activity showed no changes in both cell growth and ganglioside expression, while N1 cells showed a mild increase in cell proliferation with increased phosphorylation of the EGF receptor and neo-synthesis of Gb3 after NEU3 transfection. In contrast, NEU3 silencing resulted in a definite reduction in cell growth in a melanoma line MeWo, while ganglioside patterns underwent minimal changes. Phosphorylation levels of ERK1/2 with serum stimulation decreased in the NEU3-silenced cells. All these results suggest that NEU3 is highly expressed to enhance malignant phenotypes including apoptosis inhibition in malignant melanomas.

Phase II trial of S-1 plus low-dose cisplatin for unresectable and recurrent gastric cancer (JFMC27-9902 Step2).

AIMS: The efficacy and the toxicity of oral fluorouracil derivative S-1 plus low-dose cisplatin in unresectable or recurrent gastric cancer were evaluated by a phase II study. METHODS: S-1 was administered orally for 28 days following 14 days' rest at 80-120 mg/body/day, depending on body surface area. During administration of S-1, cisplatin was given twice a week at the recommended dose (10 mg/m(2)), which was determined by a phase I study. Data from 34 patients in phase II and 8 patients treated with the recommended dose of cisplatin in phase I were analyzed. The primary endpoint was objective response. RESULTS: The response rate was 47.1%. The median survival time was 11.0 months and the median progression-free survival was 6.9 months. The grade 3/4 toxicities observed in 10% or more of the treated patients were neutropenia (16.7%), anemia (16.7%) and anorexia (11.9%). The serum concentration of cisplatin was 794 ± 341 ng/ml at day 25 of the first course. CONCLUSIONS: S-1 plus low-dose cisplatin may be a clinically useful regimen for unresectable or recurrent gastric cancer because of its infrequent adverse events in spite of considerable efficacy and its convenience of no hydration and no hospitalization.

TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes.

Analyses of expression and regulation of ganglioside synthases in melanocytes are important to understand roles of gangliosides in melanomagenesis. In this study, we analyzed the expression and regulatory mechanisms of glycosyltransferase genes responsible for ganglioside synthesis in normal melanocytes. We reported previously that culture supernatants of UVB-irradiated keratinocytes induced upregulation of ganglioside GD3 synthase gene in melanocytes, and mainly TNFα was responsible for it. Then, we found that elimination of dibutyryl cyclic AMP and IBMX from the medium also resulted in upregulation of the GD3 synthase gene. The addition of α-melanocyte-stimulating hormone which increases cAMP, to the medium led to a significant reduction in the GD3 synthase gene expression level, and a PKA inhibitor enhanced the GD3 synthase gene level. These results suggest that signals mediated via TNFα and cAMP oppositely regulate GD3 synthase gene expression in melanocytes. The results of an IKK inhibitor indicate the possibility that TNFα induces GD3 synthase gene expression via NF-κB signaling in melanocytes. When melanoma cells were treated by these factors, no fluctuation in the GD3 synthase gene expression level was observed, although an IKK inhibitor significantly suppressed it, suggesting that ganglioside synthase genes are regulated in distinct manners between melanocytes and melanomas.

Differential roles of gangliosides in malignant properties of melanomas.

Ganglioside GD3 is widely expressed in human malignant melanomas, and has been reported to be involved in the increased cell proliferation and invasion. In this study, we established GM3-, GM2-, GM1-, GD3-, or GD2-expressing melanoma cell lines by transfecting cDNAs of glyscosyltransferases, and effects of individual gangliosides on the cell phenotypes and signals were examined. The phenotypes of established ganglioside-expressing cells were quite different, i.e. cell growth increased as following order; GD2+, GD3+ > GM1+, GM2+, GM3+ cells. Cell invasion activity increased as GD3+ ≧ GM2+ > GM1+, GM3+, GD2+ cells. Intensity of cell adhesion to collagen I (CL-I) and spreading increased as GD2+ >> GD3+, GM1+ > GM2+, GM3+ cells. In particular, cell adhesion of GD2+ cells was markedly strong. As for cell migration velocity, GD2+ cells were slower than all other cells. The immunocytostaining revealed close localization of gangliosides and F-actin in lamellipodia. Immunoblotting of phosphorylated p130Cas and paxillin by serum treatment reveled that these phosphorylations were more increased in GD3+ cells than in GD2+ or GM3+ cells, while phosphorylation of Akt underwent similarly increased phosphorylation between GD3+ and GD2+ cells compared with GM3+ cells. While GD2 and GD3 enhanced cell growth, GD3 might also contribute in cell invasion. On the other hand, GD2 might contribute in the solid fixation of melanoma cells at metastasized sites. These results suggested that individual gangliosides exert distinct roles in the different aspects of melanomas by differentially regulating cytoskeletons and signaling molecules.

Application of ion-trap LC/MS/MS for determination of acrylamide in processed foods.

Ion-trap LC/MS/MS was evaluated for use in the determination of acrylamide (AA) in processed foods. Multiple reaction monitoring (MRM) analysis of a series of AA standard solutions containing deuterium-labeled acrylamide (AA-d3) as an internal standard was performed. A linear relationship between the concentration of AA and the ratio of peak area (AA/AA-d3) in the extracted ion chromatogram (m/z 55, 58 derived from m/z 72, 75, respectively) was obtained over a wide range of 2-20,000 ng/mL. The quantification limit of AA was 2 ng/mL. In analyses of 37 commercial foods, AA was detected in a potato snack at the maximum value of 3,570 ng/g and found in 23 foods prepared or cooked at high temperature. The samples were analyzed in triplicate and the relative standard deviations (RSD) were less than 15% in many processed foods.

Formation of acrylamide from glucans and asparagine.

Model foods consisting of carbohydrates, asparagine (Asn), albumin, and sodium chloride were heated at 180°C for various times, and the levels of acrylamide (AA) in these foods were determined by LC/MS/MS. When glucans such as ß-cyclodextrin (ß-CD), starch and cellulose were used as carbohydrates in the above model, the levels of AA formed were approximately the same as or much higher than those observed in the glucose model. Glucans were heated in the absence of Asn for one hour, and their degradation products were analyzed for sugar components by HPAEC-PAD and for volatile compounds by GC/MS. The amounts of glucose detected in the glucan models, however, were too low to consider that AA was formed from the glucans in these model foods via the intermediate production of glucose. By contrast, several carbonyl compounds such as acetaldehyde and acetone were detected in the glucan degradation products. Furthermore, AA was formed when acetaldehyde and Asn were heated together in sealed vials at 180°C. These results showed that AA was formed from glucans and Asn, not via glucose produced by glucan hydrolysis, but via volatile carbonyl compounds such as acetaldehyde produced by glucan pyrolysis.