title: Stable Isotope Resolved Metabolomics Analysis of Ribonucleotide and RNA Metabolism in Human Lung Cancer Cells.
abstract: We have developed a simple NMR-based method to determine the turnover of nucleotides and incorporation into RNA by stable isotope resolved metabolomics (SIRM) in A549 lung cancer cells. This method requires no chemical degradation of the nucleotides or chromatography. During cell growth, the free ribonucleotide pool is rapidly replaced byde novosynthesized nucleotides. Using [U-13C]-glucose and [U-13C,15N]-glutamine as tracers, we showed that virtually all of the carbons in the nucleotide riboses were derived from glucose, whereas glutamine was preferentially utilized over glucose for pyrimidine ring biosynthesis, via the synthesis of Asp through the Krebs cycle. Incorporation of the glutamine amido nitrogen into the N3 and N9 positions of the purine rings was also demonstrated by proton-detected15N NMR. The incorporation of13C from glucose into total RNA was measured and shown to be a major sink for the nucleotides during cell proliferation. This method was applied to determine the metabolic action of an anti-cancer selenium agent (methylseleninic acid or MSA) on A549 cells. We found that MSA inhibited nucleotide turnover and incorporation into RNA, implicating an important role of nucleotide metabolism in the toxic action of MSA on cancer cells.
authors.affiliation: Department of Chemistry, University of Louisville, 2210 S. Brook St, Rm 348 John W. Shumaker Research, Building, Louisville, KY 40292, USA ; Center for Regulatory Environmental Analytical Metabolomics, 2210 S. Brook St., Louisville, KY 40292, USA ; JG Brown Cancer Center, Clinical Translational Research Building, 505 S. Hancock St., Louisville, KY 40202, USA.
authors.forename: Teresa W-M
authors.initials: TW
authors.lastname: Fan
authors.affiliation: JG Brown Cancer Center, Clinical Translational Research Building, 505 S. Hancock St., Louisville, KY 40202, USA.
authors.forename: Jinlian
authors.initials: J
authors.lastname: Tan
authors.affiliation: Department of Medicine, Clinical Translational Research Building, 505 S. Hancock St., Louisville, KY 40202, USA.
authors.forename: Martin M
authors.initials: MM
authors.lastname: McKinney
authors.affiliation: Department of Chemistry, University of Louisville, 2210 S. Brook St, Rm 348 John W. Shumaker Research, Building, Louisville, KY 40292, USA ; Center for Regulatory Environmental Analytical Metabolomics, 2210 S. Brook St., Louisville, KY 40292, USA ; JG Brown Cancer Center, Clinical Translational Research Building, 505 S. Hancock St., Louisville, KY 40202, USA ; Department of Medicine, Clinical Translational Research Building, 505 S. Hancock St., Louisville, KY 40202, USA.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Lane
keywords: RNA biosynthesis; SIRM; Selenium; nucleotide pools