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Rhabdomyosarcoma cells show an energy producing anabolic metabolic phenotype compared with primary myocytes.

Fan TW, Kucia M, Jankowski K, Higashi RM, Ratajczak J, Ratajczak MZ, Lane AN - Mol. Cancer (2008)

Bottom Line: Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool.The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Louisville, KY, USA. twmfan@gmail.com

ABSTRACT

Background: The functional status of a cell is expressed in its metabolic activity. We have applied stable isotope tracing methods to determine the differences in metabolic pathways in proliferating Rhabdomysarcoma cells (Rh30) and human primary myocytes in culture. Uniformly 13C-labeled glucose was used as a source molecule to follow the incorporation of 13C into more than 40 marker metabolites using NMR and GC-MS. These include metabolites that report on the activity of glycolysis, Krebs' cycle, pentose phosphate pathway and pyrimidine biosynthesis.

Results: The Rh30 cells proliferated faster than the myocytes. Major differences in flux through glycolysis were evident from incorporation of label into secreted lactate, which accounts for a substantial fraction of the glucose carbon utilized by the cells. Krebs' cycle activity as determined by 13C isotopomer distributions in glutamate, aspartate, malate and pyrimidine rings was considerably higher in the cancer cells than in the primary myocytes. Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool. Specific labeling patterns in these metabolites show the increased importance of anaplerotic reactions in the cancer cells to maintain the high demand for anabolic and energy metabolism compared with the slower growing primary myocytes. Serum-stimulated Rh30 cells showed higher degrees of labeling than serum starved cells, but they retained their characteristic anabolic metabolism profile. The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.

Conclusion: The specific 13C isotopomer patterns showed that the major difference between the transformed and the primary cells is the shift from energy and maintenance metabolism in the myocytes toward increased energy and anabolic metabolism for proliferation in the Rh30 cells. The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis. This may be a common adaptive strategy in cancer cells.

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HSQC TOCSY spectrum of myocyte cell extract. Sugar region of a high resolution HSQC-TOCSY spectrum of myocytes recorded with a mixing time of 50 ms. The resolution in F1 is adequate to show 13C-13C splittings in for example the beta form of G6P. upper: 1D projection, lower: 2D spectrum
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Figure 12: HSQC TOCSY spectrum of myocyte cell extract. Sugar region of a high resolution HSQC-TOCSY spectrum of myocytes recorded with a mixing time of 50 ms. The resolution in F1 is adequate to show 13C-13C splittings in for example the beta form of G6P. upper: 1D projection, lower: 2D spectrum

Mentions: To confirm further the assignment for G6P (as opposed to glucose), 2-D high-resolution 1H-13C HSQC-TOCSY spectra were analyzed and that for the myocytes is illustrated in Figure 12. In this spectrum, covalent connectivities from 13C1 to H1 then relayed to H2 and H3 as well as 13C3 to H3 then to H4 of βG6P were clearly observed. Also evident was the set of 13C6 to H6 connectivities of βG6P, thus providing unequivocal identification of βG6P in the crude extract of the myocytes. Moreover, the high-resolution spectrum revealed 13C coupling patterns of βG6P carbons, i.e. doublet for C1, triplet for C3-5, as well as doublet for C6, which indicate the presence of uniformly labeled βG6P and thus its origin from [U-13C]-glucose.


Rhabdomyosarcoma cells show an energy producing anabolic metabolic phenotype compared with primary myocytes.

Fan TW, Kucia M, Jankowski K, Higashi RM, Ratajczak J, Ratajczak MZ, Lane AN - Mol. Cancer (2008)

HSQC TOCSY spectrum of myocyte cell extract. Sugar region of a high resolution HSQC-TOCSY spectrum of myocytes recorded with a mixing time of 50 ms. The resolution in F1 is adequate to show 13C-13C splittings in for example the beta form of G6P. upper: 1D projection, lower: 2D spectrum
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2577687&req=5

Figure 12: HSQC TOCSY spectrum of myocyte cell extract. Sugar region of a high resolution HSQC-TOCSY spectrum of myocytes recorded with a mixing time of 50 ms. The resolution in F1 is adequate to show 13C-13C splittings in for example the beta form of G6P. upper: 1D projection, lower: 2D spectrum
Mentions: To confirm further the assignment for G6P (as opposed to glucose), 2-D high-resolution 1H-13C HSQC-TOCSY spectra were analyzed and that for the myocytes is illustrated in Figure 12. In this spectrum, covalent connectivities from 13C1 to H1 then relayed to H2 and H3 as well as 13C3 to H3 then to H4 of βG6P were clearly observed. Also evident was the set of 13C6 to H6 connectivities of βG6P, thus providing unequivocal identification of βG6P in the crude extract of the myocytes. Moreover, the high-resolution spectrum revealed 13C coupling patterns of βG6P carbons, i.e. doublet for C1, triplet for C3-5, as well as doublet for C6, which indicate the presence of uniformly labeled βG6P and thus its origin from [U-13C]-glucose.

Bottom Line: Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool.The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Louisville, KY, USA. twmfan@gmail.com

ABSTRACT

Background: The functional status of a cell is expressed in its metabolic activity. We have applied stable isotope tracing methods to determine the differences in metabolic pathways in proliferating Rhabdomysarcoma cells (Rh30) and human primary myocytes in culture. Uniformly 13C-labeled glucose was used as a source molecule to follow the incorporation of 13C into more than 40 marker metabolites using NMR and GC-MS. These include metabolites that report on the activity of glycolysis, Krebs' cycle, pentose phosphate pathway and pyrimidine biosynthesis.

Results: The Rh30 cells proliferated faster than the myocytes. Major differences in flux through glycolysis were evident from incorporation of label into secreted lactate, which accounts for a substantial fraction of the glucose carbon utilized by the cells. Krebs' cycle activity as determined by 13C isotopomer distributions in glutamate, aspartate, malate and pyrimidine rings was considerably higher in the cancer cells than in the primary myocytes. Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool. Specific labeling patterns in these metabolites show the increased importance of anaplerotic reactions in the cancer cells to maintain the high demand for anabolic and energy metabolism compared with the slower growing primary myocytes. Serum-stimulated Rh30 cells showed higher degrees of labeling than serum starved cells, but they retained their characteristic anabolic metabolism profile. The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.

Conclusion: The specific 13C isotopomer patterns showed that the major difference between the transformed and the primary cells is the shift from energy and maintenance metabolism in the myocytes toward increased energy and anabolic metabolism for proliferation in the Rh30 cells. The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis. This may be a common adaptive strategy in cancer cells.

Show MeSH
Related in: MedlinePlus