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Extended ultrastructural characterization of chordoma cells: the link to new therapeutic options.

Kolb D, Pritz E, Steinecker-Frohnwieser B, Lohberger B, Deutsch A, Kroneis T, El-Heliebi A, Dohr G, Meditz K, Wagner K, Koefeler H, Leitinger G, Leithner A, Liegl-Atzwanger B, Zweytick D, Rinner B - PLoS ONE (2014)

Bottom Line: These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling.We quantified levels of ceramide and glycosylceramide species by the methyl tert-butyl ether extraction method and we assessed the intracellular calcium concentration with the ratiometric fluorescent dye Fura-2AM.Measurements of the changes in the intracellular calcium concentration revealed an increase in calcium due to the application of acetylcholine.

View Article: PubMed Central - PubMed

Affiliation: Center for Medical Research, Medical University of Graz, Graz, Austria; Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Graz, Austria.

ABSTRACT
Chordomas are rare bone tumors, developed from the notochord and largely resistant to chemotherapy. A special feature of this tumor is the heterogeneity of its cells. By combining high pressure freezing (HPF) with electron tomography we were able to illustrate the connections within the cells, the cell-cell interface, and the mitochondria-associated endoplasmic reticulum membrane complex that appears to play a special role among the characteristics of chordoma. These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling. Compared to other tumor cells, chordoma cells show a close connection of rough endoplasmic reticulum and mitochondria, which may influence the sphingolipid metabolism and calcium release. We quantified levels of ceramide and glycosylceramide species by the methyl tert-butyl ether extraction method and we assessed the intracellular calcium concentration with the ratiometric fluorescent dye Fura-2AM. Measurements of the changes in the intracellular calcium concentration revealed an increase in calcium due to the application of acetylcholine. With regard to lipid synthesis, glucosylceramide levels in the chordoma cell line were significantly higher than those in normal healthy cells. The accumulation of glycosylceramide in drug resistant cancer cells has been confirmed in many types of cancer and may also account for drug resistance in chordoma. This study aimed to provide a deep morphological description of chordoma cells, it demonstrated that HPF analysis is useful in elucidating detailed structural information. Furthermore we demonstrate how an accumulation of glycosylceramide in chordoma provides links to drug resistance and opens up the field for new research options.

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Related in: MedlinePlus

Lipid analyses.The GlyCer level (16:0; 24:0; 24:1) of chordoma cell lines (MUG-Chor1 and U-CH1) significantly increased compared to healthy human fibroblasts (fibro MUG-Chor1) and U2OS. The y-axis represents the amount of GlyCer (pg/mol). The x-axis represents the GlyCer with three different fatty acids attached to the amino group of sphingosine, namely palmitic acid (16: 0), lignoceric acid (24: 0), and nervonic acid (24: 1).
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pone-0114251-g009: Lipid analyses.The GlyCer level (16:0; 24:0; 24:1) of chordoma cell lines (MUG-Chor1 and U-CH1) significantly increased compared to healthy human fibroblasts (fibro MUG-Chor1) and U2OS. The y-axis represents the amount of GlyCer (pg/mol). The x-axis represents the GlyCer with three different fatty acids attached to the amino group of sphingosine, namely palmitic acid (16: 0), lignoceric acid (24: 0), and nervonic acid (24: 1).

Mentions: Since little is known about the lipid composition of chordomas, we performed a non-targeted differential lipidomic analysis. In this assay the level of GlyCer was strikingly and significantly different, while some triglyceride species (TG 52:1, TG 52:2, and TG 56:3) were marginally increased within chordoma cells (MUG-Chor1 and U-CH1) compared to osteosarcoma (U2OS) and human skin fibroblasts (data not shown). These lipid species were used for a concentrated targeted analysis, where the massive upregulation of GlyCer 16:0, GlyCer 24:0, and GlyCer 24:1 (Figure 9) could be corroborated. We discovered an accumulation of GlyCer in chordomas compared with fibroblasts and osteosarcoma. The slightly elevated triglyceride levels detected in the non-targeted lipidomic analysis could not be confirmed.


Extended ultrastructural characterization of chordoma cells: the link to new therapeutic options.

Kolb D, Pritz E, Steinecker-Frohnwieser B, Lohberger B, Deutsch A, Kroneis T, El-Heliebi A, Dohr G, Meditz K, Wagner K, Koefeler H, Leitinger G, Leithner A, Liegl-Atzwanger B, Zweytick D, Rinner B - PLoS ONE (2014)

Lipid analyses.The GlyCer level (16:0; 24:0; 24:1) of chordoma cell lines (MUG-Chor1 and U-CH1) significantly increased compared to healthy human fibroblasts (fibro MUG-Chor1) and U2OS. The y-axis represents the amount of GlyCer (pg/mol). The x-axis represents the GlyCer with three different fatty acids attached to the amino group of sphingosine, namely palmitic acid (16: 0), lignoceric acid (24: 0), and nervonic acid (24: 1).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257693&req=5

pone-0114251-g009: Lipid analyses.The GlyCer level (16:0; 24:0; 24:1) of chordoma cell lines (MUG-Chor1 and U-CH1) significantly increased compared to healthy human fibroblasts (fibro MUG-Chor1) and U2OS. The y-axis represents the amount of GlyCer (pg/mol). The x-axis represents the GlyCer with three different fatty acids attached to the amino group of sphingosine, namely palmitic acid (16: 0), lignoceric acid (24: 0), and nervonic acid (24: 1).
Mentions: Since little is known about the lipid composition of chordomas, we performed a non-targeted differential lipidomic analysis. In this assay the level of GlyCer was strikingly and significantly different, while some triglyceride species (TG 52:1, TG 52:2, and TG 56:3) were marginally increased within chordoma cells (MUG-Chor1 and U-CH1) compared to osteosarcoma (U2OS) and human skin fibroblasts (data not shown). These lipid species were used for a concentrated targeted analysis, where the massive upregulation of GlyCer 16:0, GlyCer 24:0, and GlyCer 24:1 (Figure 9) could be corroborated. We discovered an accumulation of GlyCer in chordomas compared with fibroblasts and osteosarcoma. The slightly elevated triglyceride levels detected in the non-targeted lipidomic analysis could not be confirmed.

Bottom Line: These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling.We quantified levels of ceramide and glycosylceramide species by the methyl tert-butyl ether extraction method and we assessed the intracellular calcium concentration with the ratiometric fluorescent dye Fura-2AM.Measurements of the changes in the intracellular calcium concentration revealed an increase in calcium due to the application of acetylcholine.

View Article: PubMed Central - PubMed

Affiliation: Center for Medical Research, Medical University of Graz, Graz, Austria; Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Graz, Austria.

ABSTRACT
Chordomas are rare bone tumors, developed from the notochord and largely resistant to chemotherapy. A special feature of this tumor is the heterogeneity of its cells. By combining high pressure freezing (HPF) with electron tomography we were able to illustrate the connections within the cells, the cell-cell interface, and the mitochondria-associated endoplasmic reticulum membrane complex that appears to play a special role among the characteristics of chordoma. These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling. Compared to other tumor cells, chordoma cells show a close connection of rough endoplasmic reticulum and mitochondria, which may influence the sphingolipid metabolism and calcium release. We quantified levels of ceramide and glycosylceramide species by the methyl tert-butyl ether extraction method and we assessed the intracellular calcium concentration with the ratiometric fluorescent dye Fura-2AM. Measurements of the changes in the intracellular calcium concentration revealed an increase in calcium due to the application of acetylcholine. With regard to lipid synthesis, glucosylceramide levels in the chordoma cell line were significantly higher than those in normal healthy cells. The accumulation of glycosylceramide in drug resistant cancer cells has been confirmed in many types of cancer and may also account for drug resistance in chordoma. This study aimed to provide a deep morphological description of chordoma cells, it demonstrated that HPF analysis is useful in elucidating detailed structural information. Furthermore we demonstrate how an accumulation of glycosylceramide in chordoma provides links to drug resistance and opens up the field for new research options.

Show MeSH
Related in: MedlinePlus