We generated red blood cells (RBC) from cord blood (CB) CD34+ cells using a four-phase culture system. We first cultured CB CD34+ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34+ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34+ cells from a different donor using the same coculture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 x 10(12) RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 x 10(13) RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.

Gangliosides are glycosphingolipids found on the cell surface. They act as recognition molecules or signal modulators and regulate cell proliferation and differentiation. N-glycolylneuraminic acid (NeuGc)-containing gangliosides have been detected in some neoplasms in humans, although they are usually absent in normal human tissues. Our aim was to evaluate the presence of NeuGc-containing gangliosides including GM3 (NeuGc) and assess their relationship with the prognosis of non-small-cell lung cancer (NSCLC). NeuGc-containing ganglioside expression in NSCLC tissues was analyzed immunohistochemically using the mouse monoclonal antibody GMR8, which is specific for gangliosides with NeuGc alpha 2,3Gal-terminal structures. On the basis of NeuGc-containing ganglioside expression, we performed survival analysis. We also investigated the differences in the effects of GM3 (N-acetylneuraminic acid [NeuAc]) and GM3 (NeuGc) on inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase in A431 cells. As a result, the presence of NeuGc-containing gan-gliosides was evident in 86 of 93 (93.5%) NSCLC samples. The NSCLC patients with high NeuGc-containing ganglioside expression had a low overall survival rate and a significantly low progression free survival rate. In the in vitro study, the inhibitory effect of GM3 on EGFR tyrosine kinase in A431 cells after exposure to GM3 (NeuGc) was lower than that after exposure to GM3 (NeuAc). In conclusion, NeuGc-containing gangliosides including GM3 (NeuGc) are widely expressed in NSCLC, and NeuGc-containing ganglioside expression is associated with patient survival. The difference in the effects of GM3 (NeuGc) and GM3 (NeuAc) on the inhibition of EGFR tyrosine kinase might contribute to improvement in the prognosis of NSCLC patients. (Cancer Sci 2013; 104: 43-47) L ung cancer is the most frequently diagnosed major cancer worldwide. (1) The World Health Organization classification of lung cancer identifies squamous cell carcinoma, adenocarci-noma, large cell carcinoma and small cell carcinoma as its four major types. These tumors are commonly divided into small-cell lung cancer and non-small-cell lung cancer (NSCLC) depending on differences in their biology and treatment. The low rate of cure for NSCLC can be attributed to the high metastasis rate at diagnosis and the lack of effective treatments to cure such a metastatic disease. Gangliosides are ubiquitous membrane-associated glycos-phingolipids containing at least one sialic acid residue. They act as recognition molecules or signal modulators and regulate cell proliferation and differentiation. (2,3) Delay in cell growth of the human epidermoid carcinoma cell line A431 is caused by GM3 (N-acetylneuraminic acid [NeuAc])-mediated inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase. (4) GM3 is a ganglioside that binds to the extracellular domain of EGFR and inhibits its dimerization without inhibiting ligand binding. (5) Kawashima et al. described the significance of carbohydrate-carbohydrate interactions betw...

Summary Extracts from sea urchin intestine were screened for new anti-tumour drugs. Four glycolipids, 3'-sulphonoquinovosyl-1', 2'-diacylglyceride (A-4), 3'-sulphonoquinovosyl-1 '-monoacylglyceride (2'-Iyso A-4, A-5), NeuGca2-6GIcP1-1 ceramide (A-6) and HSO3-8NeuGca2-6Glcp1-1ceramide (A-7), were isolated from the intestine of sea urchin, Strongylocentrotus intermedius, and characterized by means of proton nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. When tested for cytotoxic activity against tumour cells in vitro, A-5 showed significant activity, but A-4, -6 and -7 did not. In addition, the hydrophilic derivatives of A-4 or -5 had no cytotoxicity. Furthermore, the anti-tumour effects on nude mice bearing solid tumours of a human lung adenocarcinoma cell line A-549 were evaluated in vivo using A-4 and -5. As a result, A-5 was found to be significantly effective in suppressing the growth of solid tumours, whereas A-4 had no effect. Pathologically, the solid tumours showed haemorrhagic necrosis areas after treatment with A-5. In this study, we have demonstrated the anti-tumour effect of sulphonoquinovosyl-lysoglyceride (A-5), which provides important information that this sulpholipid could be a useful drug for cancer chemotherapy.

Glycolipids were extracted from primary bladder tumors of 14 patients and 2 normal counterparts. Their expression pattern was assessed by thin-layer chromatography (TLC). The most remarkable change was massive accumulation of GM3 in superficial bladder tumors compared with invasive tumors. This change was also confirmed by immunohistochemistry using anti-GM3 monoclonal antibody. The activities of glycosyltransferases responsible for GM3 synthesis (GM3 synthase, Gb3 synthase and GD3 synthase) were consistent with upregulated expression of GM3 in superficial tumors. It was suggested that the marked GM3 accumulation in superficial tumors was caused not only by upregulated Key words: GM3; bladder tumor; invasionGlycolipids are major components of the cell membrane and they change dramatically in quantity and quality during differentiation and malignant transformation. 1,2 In our previous studies on testicular seminoma, globotriaosyl ceramide (Gb3) was markedly accumulated, 3 and galactosylgloboside was inversely correlated with metastatic potential. 4 Gb3 was very useful for differentiation between testicular malignant lymphoma and seminoma, which were sometimes difficult to differentiate histopathologically. 5 In renal cell carcinoma, increased expression of the longer chain glycolipids is closely associated with metastatic potential and predicts early postoperative development of metastasis. 6 Bladder tumors are classified into 2 main categories, superficial and invasive, by their distinctive growth patterns and biologic behavior. 7,8 Most superficial tumors are papillary, often multifocal and only occasionally progress to invasive disease; they also have a good prognosis. Most invasive tumors are nodular, metastasize in early phase and have a poor prognosis. Although these differences are considered to be due to the difference in carcinogenesis, the biochemical background has not yet been documented. There have been no intensive studies on glycolipid expression in human bladder tumors in regard to distinctive growth patterns and biologic behavior.In the present study, the glycolipid composition of human bladder tumors was assessed and massive accumulation of GM3 ganglioside in superficial papillary tumors was revealed. Furthermore, exogeneously given GM3 inhibited bladder tumor cell invasion. MATERIAL AND METHODS Human tissuesFourteen samples of bladder tumor tissue and 2 normal counterparts were subjected to glycolipid extraction. All patients were treated at the Department of Urology, Tohoku University Hospital. No radiotherapy or chemotherapy were given before tumor removal. Table I shows the clinicopathological feature. The local ethics committee approved the study and written consent was obtained from all patients. The tumor staging system was based on the UICC TNM classification. 8a Ta, T1 and T2 tumors are categorized as superficial and T2, T3 and T4 tumors are invasive. Cell linesBoth T-24 9 and KK-47 10 are human bladder tumor cell lines. They were maintained in RPMI-1640 supplemented with 10% FCS. Extr...

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