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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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Alanine and Lactate isotopomer patterns in Rh30 cell extract. 1H NMR spectrum expansion of the Rh30 cell extract spectrum from Figure 5 showing the satellite peaks of lactate and Ala.
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Figure 6: Alanine and Lactate isotopomer patterns in Rh30 cell extract. 1H NMR spectrum expansion of the Rh30 cell extract spectrum from Figure 5 showing the satellite peaks of lactate and Ala.

Mentions: Thirty four metabolites in the TCA extracts of the cell biomass were analyzed using GC-MS (Figures 2, 3), twenty of which were also analyzed by 1H NMR (Figure 5, 6). An additional eight compounds were determined by 1H NMR alone for a total of forty two identified metabolites. The profiles of selected metabolites in Rh30 and myocytes determined by GC-MS are compared in Figures 2 and 3. Lactate, Gly, Thr, Glu, glutathione (GSH), Arg, and phosphocholine (P-choline), taurine and myo-inositol were among the more abundant metabolites (cf. Figures 2, 3, 4, 5). There was good agreement between GC-MS and NMR determinations, indicating high analytical reliability. Figs 2, 3, 4, 5 also illustrate that there are significant differences in levels of numerous metabolites between the myocytes and Rh30 cells. Notably there was a much higher content of amino acids, GSH, taurine, P-choline, myo-inositol, adenine nucleotides (5'-AXP), and uracil nucleotides in the Rh30 cells. The high abundance of P-choline in Rh30 compared with the myocytes (Figure 5) appears to be a distinct feature of proliferating cells [35,37,41-46].


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)

Alanine and Lactate isotopomer patterns in Rh30 cell extract. 1H NMR spectrum expansion of the Rh30 cell extract spectrum from Figure 5 showing the satellite peaks of lactate and Ala.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Alanine and Lactate isotopomer patterns in Rh30 cell extract. 1H NMR spectrum expansion of the Rh30 cell extract spectrum from Figure 5 showing the satellite peaks of lactate and Ala.
Mentions: Thirty four metabolites in the TCA extracts of the cell biomass were analyzed using GC-MS (Figures 2, 3), twenty of which were also analyzed by 1H NMR (Figure 5, 6). An additional eight compounds were determined by 1H NMR alone for a total of forty two identified metabolites. The profiles of selected metabolites in Rh30 and myocytes determined by GC-MS are compared in Figures 2 and 3. Lactate, Gly, Thr, Glu, glutathione (GSH), Arg, and phosphocholine (P-choline), taurine and myo-inositol were among the more abundant metabolites (cf. Figures 2, 3, 4, 5). There was good agreement between GC-MS and NMR determinations, indicating high analytical reliability. Figs 2, 3, 4, 5 also illustrate that there are significant differences in levels of numerous metabolites between the myocytes and Rh30 cells. Notably there was a much higher content of amino acids, GSH, taurine, P-choline, myo-inositol, adenine nucleotides (5'-AXP), and uracil nucleotides in the Rh30 cells. The high abundance of P-choline in Rh30 compared with the myocytes (Figure 5) appears to be a distinct feature of proliferating cells [35,37,41-46].

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