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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: A special feature of this tumor is the heterogeneity of its cells.These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling.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

Intracellular calcium response to four different agonists in MUG-Chor1 cells was measured.A) Histamine (His), acetylcholine (ACh), serotonin (5HT), and P2Y purinoceptor agonist mes-ATP were applied at a concentration of 10 µM for a period of 60 seconds as indicated by the grey bars; the ratio between 340 nm and 380 nm is given. After stimulation, the respective agonist was washed out for another 60 seconds. B) The first two peaks of ratio values of MUG-Chor1 cells indicate ACh-application in the presence of extracellular calcium (NT+[Ca2+]ex; upper, open bar); peak reduction was induced by removing external calcium (NT w/o [Ca2+]ex; upper, grey bar). Small bars represent the time of perfusion with ACh at the two different concentrations (10 µM: lower, grey bar; 100 µM: lower, black bar). C) Within the bar chart a summary of ACh-induced changes is given. The first two bars represent effects in [Ca2+]i induced by ACh (10 µM; 100 µM) given as average value (± standard deviation) of the change in intracellular calcium concentration [Ca2+]i (nM). The usage of calcium within the perfusion solution as well as the different concentrations of ACh is indicated below the x-axis. Significant changes evaluated by the students t-test are given (***, p<0.001; **, p<0.01).
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pone-0114251-g008: Intracellular calcium response to four different agonists in MUG-Chor1 cells was measured.A) Histamine (His), acetylcholine (ACh), serotonin (5HT), and P2Y purinoceptor agonist mes-ATP were applied at a concentration of 10 µM for a period of 60 seconds as indicated by the grey bars; the ratio between 340 nm and 380 nm is given. After stimulation, the respective agonist was washed out for another 60 seconds. B) The first two peaks of ratio values of MUG-Chor1 cells indicate ACh-application in the presence of extracellular calcium (NT+[Ca2+]ex; upper, open bar); peak reduction was induced by removing external calcium (NT w/o [Ca2+]ex; upper, grey bar). Small bars represent the time of perfusion with ACh at the two different concentrations (10 µM: lower, grey bar; 100 µM: lower, black bar). C) Within the bar chart a summary of ACh-induced changes is given. The first two bars represent effects in [Ca2+]i induced by ACh (10 µM; 100 µM) given as average value (± standard deviation) of the change in intracellular calcium concentration [Ca2+]i (nM). The usage of calcium within the perfusion solution as well as the different concentrations of ACh is indicated below the x-axis. Significant changes evaluated by the students t-test are given (***, p<0.001; **, p<0.01).

Mentions: Changes in intracellular calcium [laCa2+]i were analyzed by the application of the two neurotransmitters serotonin (5-HT) and ACh and furthermore by adding histamine or mes-ATP, respectively. As given within the representative original traces the addition of histamine and 5-HT did not induce any changes. On the other hand, we were able to demonstrate that particularly mes-ATP (less so at 130%), and ACh (to a greater extent with 220%), triggered a rise in [Ca2+]i (Figure 8A). The use of two different concentrations in ACh (10 and 100 µM) did not alter the magnitude of the effect but the removal of external Ca2+ led to a reduction of measurable [Ca2+]i by 58% (Figure 8B). Perfusion by ACh of MUG-Chor1 cells induced the intracellular Ca2+ within the nanomolar range by 131±50.9 nM, 100 µM ACh in comparison to 10 µM ACh did not further change its response (−7±31.2 nM). The observed reduction of [Ca2+]i to 30±14.6 nM by Ca2+-free perfusion significantly changed to 61.8±8.1 nM when changing back to Ca2+-rich extracellular medium (Figure 8C).


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)

Intracellular calcium response to four different agonists in MUG-Chor1 cells was measured.A) Histamine (His), acetylcholine (ACh), serotonin (5HT), and P2Y purinoceptor agonist mes-ATP were applied at a concentration of 10 µM for a period of 60 seconds as indicated by the grey bars; the ratio between 340 nm and 380 nm is given. After stimulation, the respective agonist was washed out for another 60 seconds. B) The first two peaks of ratio values of MUG-Chor1 cells indicate ACh-application in the presence of extracellular calcium (NT+[Ca2+]ex; upper, open bar); peak reduction was induced by removing external calcium (NT w/o [Ca2+]ex; upper, grey bar). Small bars represent the time of perfusion with ACh at the two different concentrations (10 µM: lower, grey bar; 100 µM: lower, black bar). C) Within the bar chart a summary of ACh-induced changes is given. The first two bars represent effects in [Ca2+]i induced by ACh (10 µM; 100 µM) given as average value (± standard deviation) of the change in intracellular calcium concentration [Ca2+]i (nM). The usage of calcium within the perfusion solution as well as the different concentrations of ACh is indicated below the x-axis. Significant changes evaluated by the students t-test are given (***, p<0.001; **, p<0.01).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114251-g008: Intracellular calcium response to four different agonists in MUG-Chor1 cells was measured.A) Histamine (His), acetylcholine (ACh), serotonin (5HT), and P2Y purinoceptor agonist mes-ATP were applied at a concentration of 10 µM for a period of 60 seconds as indicated by the grey bars; the ratio between 340 nm and 380 nm is given. After stimulation, the respective agonist was washed out for another 60 seconds. B) The first two peaks of ratio values of MUG-Chor1 cells indicate ACh-application in the presence of extracellular calcium (NT+[Ca2+]ex; upper, open bar); peak reduction was induced by removing external calcium (NT w/o [Ca2+]ex; upper, grey bar). Small bars represent the time of perfusion with ACh at the two different concentrations (10 µM: lower, grey bar; 100 µM: lower, black bar). C) Within the bar chart a summary of ACh-induced changes is given. The first two bars represent effects in [Ca2+]i induced by ACh (10 µM; 100 µM) given as average value (± standard deviation) of the change in intracellular calcium concentration [Ca2+]i (nM). The usage of calcium within the perfusion solution as well as the different concentrations of ACh is indicated below the x-axis. Significant changes evaluated by the students t-test are given (***, p<0.001; **, p<0.01).
Mentions: Changes in intracellular calcium [laCa2+]i were analyzed by the application of the two neurotransmitters serotonin (5-HT) and ACh and furthermore by adding histamine or mes-ATP, respectively. As given within the representative original traces the addition of histamine and 5-HT did not induce any changes. On the other hand, we were able to demonstrate that particularly mes-ATP (less so at 130%), and ACh (to a greater extent with 220%), triggered a rise in [Ca2+]i (Figure 8A). The use of two different concentrations in ACh (10 and 100 µM) did not alter the magnitude of the effect but the removal of external Ca2+ led to a reduction of measurable [Ca2+]i by 58% (Figure 8B). Perfusion by ACh of MUG-Chor1 cells induced the intracellular Ca2+ within the nanomolar range by 131±50.9 nM, 100 µM ACh in comparison to 10 µM ACh did not further change its response (−7±31.2 nM). The observed reduction of [Ca2+]i to 30±14.6 nM by Ca2+-free perfusion significantly changed to 61.8±8.1 nM when changing back to Ca2+-rich extracellular medium (Figure 8C).

Bottom Line: A special feature of this tumor is the heterogeneity of its cells.These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling.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