Metabolic profile and quantification of deoxyribose synthesis pathways in HepG2 cells |
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Authors: | Josep J. Centelles Antonio Ramos-Montoya Shu Lim Sara Bassilian Laszlo G. Boros Silvia Marín Marta Cascante Wai-Nang Paul Lee |
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Affiliation: | (1) Department of Biochemistry and Molecular Biology, Faculty of Chemistry, and CeRQT at Barcelona Scientific Parc, University of Barcelona, Martí i Franquès, 1, 08028 Barcelona, Spain;(2) Department of Pediatrics, LABiomedical Research Institute, Harbor-UCLA Medical Center, 1124 West Casrson Street RB1, Torrance, CA 90502, USA |
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Abstract: | Traditional tracer studies of cell proliferation fail to distinguish between label enrichment due to increased DNA repair versus DNA replication. We used the emerging stable (non-radiating) isotope-based dynamic metabolic profiling technique on HepG2 cells to determine synthesis pathways of nucleic acids from glucose and rates of proliferation using CG-MS assay of RNA and DNA enrichment. Comparing the isotopic enrichment curve in DNA with the theoretical curve based on cell growth, we observed that the measured tracer enrichment was significantly higher, indicating that surplus label was acquired during DNA repair. In particular, after the first duplication (3 days), 80.13% of the total enrichment observed corresponds to duplication and 19.87% corresponds to DNA repair as calculated from the [1, 2-13C2]-glucose incorporation curve. Our data indicate contemporary measurements of cell proliferation rates relying on tracer incorporation may be overestimated. 13C label was distributed between m1 (m1/Σm = 80) and m2 (m2/Σm = 14) of deoxyribose, indicating that most of the glucose carbon was acquired via direct glucose oxidation in the pentose cycle. The stable isotope technique distinguishes rates of DNA synthesis and repair via the oxidative and non-oxidative pentose cycle, separately, in one test, without inhibition of either process. The contribution of DNA repair in malignant cells to isotope accumulation in deoxyribose remains to be investigated. |
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Keywords: | deoxyribose ribose GC-MS isotopomer distribution |
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