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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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HCCH TOCSY spectrum of myocyte cell extract. 48 h myoctye cells and 24 h Rh30 cells grown in the presence of [U-13C]-glucose were extracted as described in the methods. 1D NMR spectra were recorded at 18.8 T as described in the methods and elsewhere[35]. The HCCH TOCSY spectrum of the myocyte extract was recorded with a mixing time of 12 ms showing cross peak patterns of consecutively labeled carbons of glycogen resonances, G6P and fully labeled ribose moieties of nucleotides (e.g. 5'AXP), lactate, Ala, and Glu.
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Figure 11: HCCH TOCSY spectrum of myocyte cell extract. 48 h myoctye cells and 24 h Rh30 cells grown in the presence of [U-13C]-glucose were extracted as described in the methods. 1D NMR spectra were recorded at 18.8 T as described in the methods and elsewhere[35]. The HCCH TOCSY spectrum of the myocyte extract was recorded with a mixing time of 12 ms showing cross peak patterns of consecutively labeled carbons of glycogen resonances, G6P and fully labeled ribose moieties of nucleotides (e.g. 5'AXP), lactate, Ala, and Glu.

Mentions: Additional 2-D 1H NMR experiments helped identify 13C-labeled glucose-6-phosphate (G6P) and/or glycogen in the Rh30 and myocyte extracts. These included 1H HCCH-TOCSY and 1H-13C HSQC-TOCSY. The HCCH-TOCSY spectrum of the myocyte extract (Figure 11) displayed covalent connectivities of protons attached to consecutively 13C-labeled carbons of various metabolites such as lactate, Ala, Glu, β-glucose-6-phosphate (βG6P), glycogen, and AXP. The Rh30 spectrum (not shown) also exhibited similar covalent networks except for the absence of connectivities for glycogen, lower abundance connectivities for βG6P, and more prominent connectivities for 5'-UXP and 5'-GXP. The connectivity patterns for these metabolites is consistent with the presence of [13C-1-5]-βG6P and [U-13C-glucose]-glycogen, in addition to [U-13C]-lactate, [U-13C]-Ala, and [13C-2,3,4]-Glu.


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)

HCCH TOCSY spectrum of myocyte cell extract. 48 h myoctye cells and 24 h Rh30 cells grown in the presence of [U-13C]-glucose were extracted as described in the methods. 1D NMR spectra were recorded at 18.8 T as described in the methods and elsewhere[35]. The HCCH TOCSY spectrum of the myocyte extract was recorded with a mixing time of 12 ms showing cross peak patterns of consecutively labeled carbons of glycogen resonances, G6P and fully labeled ribose moieties of nucleotides (e.g. 5'AXP), lactate, Ala, and Glu.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 11: HCCH TOCSY spectrum of myocyte cell extract. 48 h myoctye cells and 24 h Rh30 cells grown in the presence of [U-13C]-glucose were extracted as described in the methods. 1D NMR spectra were recorded at 18.8 T as described in the methods and elsewhere[35]. The HCCH TOCSY spectrum of the myocyte extract was recorded with a mixing time of 12 ms showing cross peak patterns of consecutively labeled carbons of glycogen resonances, G6P and fully labeled ribose moieties of nucleotides (e.g. 5'AXP), lactate, Ala, and Glu.
Mentions: Additional 2-D 1H NMR experiments helped identify 13C-labeled glucose-6-phosphate (G6P) and/or glycogen in the Rh30 and myocyte extracts. These included 1H HCCH-TOCSY and 1H-13C HSQC-TOCSY. The HCCH-TOCSY spectrum of the myocyte extract (Figure 11) displayed covalent connectivities of protons attached to consecutively 13C-labeled carbons of various metabolites such as lactate, Ala, Glu, β-glucose-6-phosphate (βG6P), glycogen, and AXP. The Rh30 spectrum (not shown) also exhibited similar covalent networks except for the absence of connectivities for glycogen, lower abundance connectivities for βG6P, and more prominent connectivities for 5'-UXP and 5'-GXP. The connectivity patterns for these metabolites is consistent with the presence of [13C-1-5]-βG6P and [U-13C-glucose]-glycogen, in addition to [U-13C]-lactate, [U-13C]-Ala, and [13C-2,3,4]-Glu.

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