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Stable isotope resolved metabolomics analysis of ribonucleotide and RNA metabolism in human lung cancer cells

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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 by de novo synthesized 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-detected 15N NMR. The incorporation of 13C 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.

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Abbreviations

DSS:

2,2′-Dimethylsilapentane-5-sulfonate

GSH:

Reduced glutathione

HSQC:

Heteronuclear single quantum coherence

MSA:

Methyl seleninic acid

NSCLC:

Non small cell lung cancer

SIRM:

Stable isotope resolved metabolomics

TOCSY:

Total correlation spectroscopy

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Acknowledgments

The authors thank Dr. S. Arumugam for assistance in the NMR measurements. This study was supported in part by National Science Foundation EPSCoR grant # EPS-0447479; NIH NCRR Grant 5P20RR018733, 1R01CA118434-01A2 (to TWMF), 1RO1 CA101199 (to TWMF), R21CA133668-01 (to ANL) from the National Cancer Institute; the Kentucky Challenge for Excellence, and the Brown Foundation.

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Authors and Affiliations

  1. Department of Chemistry, University of Louisville, 2210 S. Brook St, Rm 348 John W. Shumaker Research Building, Louisville, KY, 40292, USA

    Teresa W-M. Fan & Andrew N. Lane

  2. Center for Regulatory Environmental Analytical Metabolomics, 2210 S. Brook St., Louisville, KY, 40292, USA

    Teresa W-M. Fan & Andrew N. Lane

  3. JG Brown Cancer Center, Clinical Translational Research Building, Room 217, 505 S. Hancock St., Louisville, KY, 40202, USA

    Teresa W-M. Fan, Jinlian Tan & Andrew N. Lane

  4. Department of Medicine, Clinical Translational Research Building, 505 S. Hancock St., Louisville, KY, 40202, USA

    Martin M. McKinney & Andrew N. Lane

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  1. Teresa W-M. Fan
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Correspondence to Andrew N. Lane.

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11306_2011_337_MOESM1_ESM.doc

Figure S1. Effect of MSA on 13C incorporation from [U-13C]-glucose into the ribose subunit of A549 RNA. A549 cells were grown in [U-13C]-glucose in the absence or presence of MSA. Total RNA was extracted and digested for 2-D 1H TOCSY NMR analysis in terms of fractional (%) 13C enrichment in C1’ of AMP ribose as described in the methods. Each data point represents an average of two replicates. Red: control, Blue: +5 μM MSA. (DOC 85 kb)

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Fan, T.WM., Tan, J., McKinney, M.M. et al. Stable isotope resolved metabolomics analysis of ribonucleotide and RNA metabolism in human lung cancer cells. Metabolomics 8, 517–527 (2012). https://doi.org/10.1007/s11306-011-0337-9

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  • DOI: https://doi.org/10.1007/s11306-011-0337-9

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