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Bayesian analysis used MrBayes version Ronquist and Huelsenbeck
Fig. 4. Correlation of acute MβCD-induced apoptosis and high level cell surface expression of GM2. (A) MβCD-induced cell death monitored with a CytoTox-Glo cytotoxicity assay kit using IMR-32, K562, GOTO, Lu-135, Schwannian GOTO, and HeLa cells. Cells were treated with 10 mM MβCD and subjected to the viability assay at the indicated time points. Lethality of the cells was calculated as described in Supplementary Materials and Methods. Bars represent means ± SEM of four experiments. (B) FACS analysis to measure the cell surface GM2 of the indicated cells. Cells were processed in the presence and absence of mAb I-13 and subjected to FACS analysis. GOTO cells exhibited a striking reduction in the reactivity toward I-13 upon differentiation into Schwann cells, suggesting differentiation-dependent alteration in GM2 contents.Figure optionsDownload full-size imageDownload as PowerPoint slideDiscussionBy using a newly generated monoclonal antibody against ganglioside GM2, a major component of lipid rafts, we characterized the neuroblastoma GOTO cell and found that GM2 is exceptionally highly enriched on the surface of GOTO cells compared to BrdU-differentiated Schwann cells (Fig. 1) and other cells including THP-1, HL60, MOLT-3, MEG-01, U937, TIG-1, G-361, and HeLa cells (Fig. 1A). Raft localization of GM2 was confirmed by its colocalization with flotillin-1 in the GEM (raft) fractions separated by sucrose density gradient fractionation of GOTO cells homogenized in sodium carbonate (Fig. 1F). The flotillin family consists of two homologous members: flotillin-1 and flotillin-2. Both are ubiquitously expressed and associated with membrane microdomains (lipid rafts) by means of fatty Nutlin-3 modifications and hydrophobic amino acid stretches [20], [21] and [22]. The results reported here strongly suggest that the neuroblastoma GOTO cell contains lipid rafts rich in GM2. This property of the GOTO cell would make it useful for studying lipid rafts since (i) lipid rafts are attracting much attention as platforms that are crucial for certain types of cell signaling [23] and [24] and endocytosis [25], [26] and [27] and (ii) GM2-rich rafts have not been characterized in detail yet while GM1-rich rafts are relatively well characterized using cholera toxin subunit B (CTB) as a probe for ganglioside GM1 [28].The marked alterations in the abundance of GM2-rich rafts, observed here on differentiation of GOTO cells into Schwann cells, is not surprising but rather expected, but our demonstration is the first experimental evidence for such an alteration (i.e. differentiation-dependent marked difference in the physicochemical properties of lipid rafts). Another interesting point is that the Schwannian differentiation was accompanied by a formation of a large number of focal adhesions (Fig. 1B) and by a striking resistance to MβCD (Fig. 4A), an observation that is somehow consistent with previous reports that (i) overexpression of focal adhesion kinase (FAK) renders the host cell resistant to MβCD-induced apoptosis and (ii) cholesterol depletion by MβCD treatment induces apoptosis of A431 cells through downregulation of FAK and internalization of lipid rafts/caveolae [29].Methylated β-cyclodextrin (MβCD) is a water soluble derivative of β-cyclodextrin (β-CD), a cyclic oligosaccharide formed by seven units of α-d-(+)-glucopyranose, and possesses the ability to encapsulate a variety of compounds in its hydrophobic cavity [30]. In the field of membrane lipid research, this property of MβCD has been used to deplete cholesterol from the plasma membrane. For example, it has been successfully used to perturb the function of lipid rafts by decreasing their cholesterol content, which led to retardation of cellular functions and to the implication that lipid rafts act as an organization center of proteins for signal transduction [23] and [24], endocytosis [25], [26] and [27], apoptosis [31] and [32] and also a docking site for the entry of viruses, bacteria, and toxins [33] and [34]. In the present study, we demonstrated that GOTO cells rapidly undergo apoptotic cell death when treated with MβCD (Fig. 2, Fig. 3 and Fig. 4). It has been reported that MβCD has the potential to induce relatively slow apoptosis of several human cell lines including human keratinocyte, epidermoid carcinoma, prostate cancer, and breast cancer cells [35] and [36]. Additionally, our results indicated that human neuroblastoma, chronic myelogenous leukemia, and small cell lung carcinoma cell lines are extremely hypersensitive to MβCD-mediate cholesterol depletion (Fig. 4). One possible explanation for this hypersensitivity is that GM2/cholesterol-rich rafts are associated with the apoptosis signaling cascade and destabilization of the rafts by cholesterol depletion leads to activation of apoptosis. Since the GM2/cholesterol-rich nature is a property common to most neuroblastoma cells, the MβCD-induced cell death may have clinical implications. Neuroblastoma is one of the most malignant tumors of childhood, which frequently shows spontaneous regression. The high rate of spontaneous regression in infants is currently explained in relation to delayed activation of normal apoptotic pathways resulting from the absence of nerve growth factor in their microenvironments [37]. The unique property of the lipid rafts on GOTO cells may also contribute to the regression.AcknowledgmentsWe thank Setsuko Sato and Tomoko Okada for secretarial assistance. This work was supported by the 21st Century and Global COE Programs of the Ministry of Education, Culture, Sport, Science and Technology of Japan (MEXT).Appendix A. Supplementary dataSupplementary data. Supplementary materials and methods.Help with DOC filesOptionsDownload file ( K)





 
 
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