Abstract
Luciferase transfected cell lines are used extensively for cancer models, revealing valuable biological information about disease mechanisms. However, these genetically encoded reporters, while useful for monitoring tumor response in cancer models, can impact cell metabolism. Indeed firefly luciferase and fatty acyl-CoA synthetases differ by a single amino acid, raising the possibility that luciferase activity might alter metabolism and introduce experimental artifacts. Therefore knowledge of the metabolic response to luciferase transfection is of significant importance, especially given the thousands of research studies using luciferase as an in vivo bioluminescence imaging reporter. Untargeted metabolomics experiments were performed to examine three different types of lymphoblastic leukemia cell lines (Ramos, Raji and SUP-T1) commonly used in cancer research, each were analyzed with and without vector transduction. The Raji model was also tested under perturbed starvation conditions to examine potential luciferase-mediated stress responses. The results showed that no significant metabolic differences were observed between parental and luciferase transduced cells for each cell line, and that luciferase overexpression does not alter cell metabolism under basal or perturbed conditions.
Similar content being viewed by others
Abbreviations
- ACN:
-
Acetonitrile
- BLI:
-
Bioluminescence imaging
- dPBS:
-
Dulbecco’s phosphate buffered saline
- ESI:
-
Electrospray ionization
- EV:
-
Empty vector
- FA:
-
Formic acid
- FACS:
-
Fatty Acyl CoA synthetase
- FLuc:
-
Photinus pyralis luciferase
- HILIC:
-
Hydrophilic interaction liquid chromatography
- HPLC:
-
High performance liquid chromatography
- IPA:
-
Isopropanol
- LC/MS:
-
Liquid Chromatography/Mass Spectrometry
- MeOH:
-
Methanol
- MS:
-
Mass Spectrometry
- NH4Ac:
-
Ammonium acetate
- NH4OH:
-
Ammonium hydroxide
- Q-TOF/MS:
-
Quadrupole Time-Of-Flight Mass Spectrometer
- RPLC:
-
Reversed-phase liquid chromatography
References
Brutkiewicz, S., Mendonca, M., Stantz, K., Comerford, K., Bigsby, R., Hutchins, G., et al. (2007). The expression level of luciferase within tumour cells can alter tumour growth upon in vivo bioluminescence imaging. Luminescence, 22, 221–228.
Czupryna, J., & Tsourkas, A. (2011). Firefly luciferase and RLuc8 exhibit differential sensitivity to oxidative stress in apoptotic cells. PLoS ONE, 6, e20073.
Ellis, J. M., Frahm, J. L., & Li, L. O. (2010). Acyl-coenzyme a synthetases in metabolic control. Current Opinion Lipidol, 21, 212–217.
Gatignol, A., Durand, H., & Tiraby, G. (1988). Bleomycin resistance conferred by a drug-binding protein. FEBS Lett, 230, 171–175.
Gould, S. G., Keller, G. A., & Subramani, S. (1987). Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J Cell Biol, 105, 2921–2923.
Greer, L. F, 3rd, & Szalay, A. A. (2002). Imaging of light emission from the expression of luciferases in living cells and organisms: A review. Luminescence, 17, 43–74.
Gulick, A. M. (2009). Conformational dynamics in the Acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase. ACS Chem Biol, 4, 811–827.
Guranowski, A., Sillero, M. A. G., & Sillero, A. (1990). Firefly luciferase synthesizes P1, P 4-bis (5′-adenosyl) tetraphosphate (Ap4A) and other dinucleoside polyphosphates. FEBS Lett, 271, 215–218.
Ivanisevic, J., Zhu, Z., Plate, L., Tautenhahn, R., Chen, S., O’Brien, P. J., et al. (2013). Toward ‘Omic’ scale metabolite profiling: a dual separation—mass spectrometry approach for coverage of lipids and central carbon metabolism. Anal Chem, 85, 6876–6884.
Keller, G. A., Gould, S., Deluca, M., & Subramani, S. (1987). Firefly luciferase is targeted to peroxisomes in mammalian cells. Proc Natl Acad Sci USA, 84, 3264–3268.
Keyaerts, M., Remory, I., Caveliers, V., Breckpot, K., Bos, T. J., Poelaert, J., et al. (2012). Inhibition of firefly luciferase by general anesthetics: effect on in vitro and in vivo bioluminescence imaging. PLoS ONE, 7, e30061.
McElroy, W. D., DeLuca, M., & Travis, J. (1967). Molecular uniformity in biological catalyses. The enzymes concerned with firefly luciferin, amino acid, and fatty acid utilization are compared. Science, 157, 150–160.
Milsom, C. C., Lee, C. R., Hackl, C., Man, S., & Kerbel, R. S. (2013). Differential post-surgical metastasis and survival in SCID, NOD-SCID and NOD-SCID-IL-2Rgamma(null) mice with parental and subline variants of human breast cancer: implications for host defense mechanisms regulating metastasis. PLoS ONE, 8, e71270.
Oba, Y., Iida, K., & Inouye, S. (2009). Functional conversion of fatty acyl-CoA synthetase to firefly luciferase by site-directed mutagenesis: a key substitution responsible for luminescence activity. FEBS Lett, 583, 2004–2008.
Oba, Y., Ojika, M., & Inouye, S. (2003). Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase. FEBS Lett, 540, 251–254.
Oliva-Trastoy, M., Defais, M., & Larminat, F. (2005). Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells. Mutagenesis, 20, 111–114.
Patti, G. J., Tautenhahn, R., Rinehart, D., Cho, K., Shriver, L. P., Manchester, M., et al. (2013). A view from above: cloud plots to visualize global metabolomic data. Anal Chem, 85, 798–804.
Patti, G. J., Tautenhahn, R., & Siuzdak, G. (2012). Meta-analysis of untargeted metabolomic data from multiple profiling experiments. Nat Prot, 7, 508–516.
Schipper, M. L., Patel, M. R., & Gambhir, S. S. (2006). Evaluation of firefly luciferase bioluminescence mediated photodynamic toxicity in cancer cells. Mol Imaging Biol, 8, 218–225.
Sim, H., Bibee, K., Wickline, S., & Sept, D. (2011). Pharmacokinetic modeling of tumor bioluminescence implicates efflux, and not influx, as the bigger hurdle in cancer drug therapy. Cancer Res, 71, 686–692.
Tautenhahn, R., Patti, G. J., Rinehart, D., & Siuzdak, G. (2012). XCMS Online: a web-based platform to process untargeted metabolomic data. Anal Chem, 84, 5035–5039.
Theodossiou, T., Hothersall, J. S., Woods, E. A., Okkenhaug, K., Jacobson, J., & MacRobert, A. J. (2003). Firefly luciferin-activated rose bengal: in vitro photodynamic therapy by intracellular chemiluminescence in transgenic NIH 3T3 cells. Cancer Res, 63, 1818–1821.
Thorne, N., Shen, M., Lea, W. A., Simeonov, A., Lovell, S., Auld, D. S., et al. (2012). Firefly luciferase in chemical biology: a compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter. Chem Bol, 19, 1060–1072.
Tiffen, J. C., Bailey, C. G., Ng, C., Rasko, J. E., & Holst, J. (2010). Luciferase expression and bioluminescence does not affect tumor cell growth in vitro or in vivo. MolCancer, 9, 299.
van Staveren, W. C., Solis, D. Y., Hebrant, A., Detours, V., Dumont, J. E., & Maenhaut, C. (2009). Human cancer cell lines: Experimental models for cancer cells in situ? For cancer stem cells? Biochim et Biophys Acta, 1795, 92–103.
Watkins, P. A., & Ellis, J. M. (1822). Peroxisomal acyl-CoA synthetases. Biochim et Biophys Acta, 2012, 1411–1420.
Zhang, C. C., Yan, Z., Li, W., Kuszpit, K., Painter, C. L., Zhang, Q., et al. (2012). [(18)F]FLT-PET imaging does not always “light up” proliferating tumor cells. Clin Cancer Res, 18, 1303–1312.
Zhu, Z. J., Schultz, A. W., Wang, J., Johnson, C. H., Yannone, S. M., Patti, G. J., et al. (2013). Liquid chromatography quadrupole time-of-flight mass spectrometry characterization of metabolites guided by the METLIN database. Nat Prot, 8, 451–460.
Acknowledgments
The authors thank Cathy Zhang and Max Hallin (Pfizer Oncology Research) for insightful discussions. These studies were funded by Pfizer and National Institutes of Health grants 5R01CA170737-02 (GS and BHF), P30 MH062261-13 (GS), 1R21CA170492-01 (GS) and W81XWH-13-1-0402 (GS).
Conflict of interests
The authors declare no Conflict of interests.
Author information
Authors and Affiliations
Corresponding authors
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Johnson, C.H., Fisher, T.S., Hoang, L.T. et al. Luciferase does not alter metabolism in cancer cells. Metabolomics 10, 354–360 (2014). https://doi.org/10.1007/s11306-014-0622-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11306-014-0622-5