Abstract
Reporter proteins find increasing application in biomedical studies in vitro and in vivo. However, to correctly interpret the results based on their use, it is important to understand whether reporter protein production is modulated in model cells and in what conditions such modulation may occur. Reporter activity was studied in Mel IL melanoma cells transiently transfected with a pCpG vector-based plasmid construct expressing firefly luciferase. Luciferase expression quickly dropped during the first two culture passages, which were followed by a quasi-stable period, when luciferase expression relatively slightly decreased with time. Phases of maximal and minimal luciferase production, which corresponded to the exponential and stationary growth phases, respectively, were observed during batch culture. When the medium was changed, luciferase production was stimulated in the stationary, but not exponential, cell growth phase. Severe hypoxia (0.1% O2) decreased the luciferase amount, suggesting substantial modulation of cell metabolism in total and luciferase production in particular. The targeted drug vemurafenib suppressed the luciferase production in Mel IL cells, whereas DMSO, which is often used as a drug solvent in experiments with cells, stimulated the luciferase production. Based on the results, it was hypothesized that modulation of reporter protein production in mammalian cells reflects the adaptation of intracellular metabolism to external conditions and may be a source of incorrect interpretations of experiments using reporter proteins.
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REFERENCES
Ginn S.L., Alexander I.E., Edelstein M.L., Abedi M.R., Wixon J. 2013. Gene therapy clinical trials worldwide to 2012: An update. J. Gene Med. 15, 65–77.
Yin H., Kanasty R.L., Eltoukhy A.A., Vegas A.J., Dorkin J.R., Anderson D.G. 2014. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541–555.
Gill D.R., Pringle I.A., Hyde S.C. 2009. Progress and prospects: The design and production of plasmid vectors. Gene Ther. 16, 165–171.
Bruter A.V., Kandarakov O.F., Belyavsky A.V. 2018. Persistence of plasmid-mediated expression of transgenes in human mesenchymal stem cells depends primarily on CpG levels of both vector and transgene. J. Gene Med. 20, e3009. doi 10.1002/jgm.3009
Okita K., Nakagawa M., Hyenjong H., Ichisaka T., Yamanaka S. 2008. Generation of mouse induced pluripotent stem cells without viral vectors. Science. 322, 949–953.
Leitậ̣̣̣̣̣o J.M.M., Esteves da Silva J.C.G. 2010. Firefly luciferase inhibition. J. Photochem. Photobiol. B. 101, 1–8. doi 10.1016/j.jphotobiol.2010.06.015
Doran D.M., Kulkarni-Datar K., Cool D.R., Brown T.L. 2011. Hypoxia activates constitutive luciferase reporter constructs. Biochimie. 93, 361–368.
Keyaerts M., Remory I., Caveliers V., Breckpot K., Bos T.J., Poelaert J., Bossuyt A., Lahoutte T. 2012. Inhibition of firefly luciferase by general anesthetics: effect on in vitro and in vivo bioluminescence imaging. PLoS One. 7, e30061.
Khalil A.A., Jameson M.J., Broaddus W.C., Lin P.S., Dever S.M., Golding S.E., Rosenberg E., Valerie K., Chung Th.D. 2013. The influence of hypoxia and pH on bioluminescence imaging of luciferase-transfected tumor cells and xenografts. Int. J. Mol. Imaging. 2013, ID 287697. http://dx.doi.org/.10.1155/2013/287697.
Pirt S.J. 1975. Principles of Microbe and Cell Cultivation. London: Blackwell.
Kandarakov O.F., Kopantseva E.E., Belyavsky A.V. 2016. Analysis of proliferation of melanoma cells and mesenchymal stem cells in co-culture and contribution of experimental conditions into interpretation of the results. Bull. Exp. Biol. Med. 162, 127–133.
Fallahi-Sichani M., Honarnejad S., Heiser L.M., Gray J.W., Sorger P.K. 2013. Metrics other than potency reveal systematic variation in responses to cancer drugs. Nat. Chem. Biol. 9, 708–714.
Swaika A., Crozier J.A., Joseph R.W. 2014. Vemurafenib: An evidence-based review of its clinical utility in the treatment of metastatic melanoma. Drug Des. Devel. Ther. 16, 775–787.
Thaler R., Spitzer S., Karlic H., Klaushofer K., Varga F. 2012. DMSO is a strong inducer of DNA hydroxymethylation in pre-osteoblastic MC3T3-E1 cells. Epigenetics. 7, 635–651.
Kaskova Z.M., Tsarkova A.S., Yampolsky I.V. 2016. 1001 lights: Luciferins, luciferases, their mechanisms of action and applications in chemical analysis, biology and medicine. Chem. Soc. Rev. 45, 6048–6077.
Jenke A.C., Stehle I.M., Herrmann F., Eisenberger T., Baiker A., Bode J., Fackelmayer F.O., Lipps H.J. 2004. Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome. Proc. Natl. Acad. Sci. U. S. A. 101, 11 322–11 327.
Würtele H., Little K.C., Chartrand P. 2003. Illegitimate DNA integration in mammalian cells. Gene Ther. 10, 1791–1799.
Izsvák Z., Chuah M.K., Vandendriessche T., Ivics Z. 2009. Efficient stable gene transfer into human cells by the Sleeping Beauty transposon vectors. Methods. 49, 287–297.
Neuhuber B., Swanger S.A., Howard L., Mackay A., Fischer I. 2008. Effects of plating density and culture time on bone marrow stromal cell characteristics. Exp. Hematol. 36, 1176–1185.
Moriyama E.H., Niedre M.J., Jarvi M.T., Mocanu J.D., Moriyama Y., Subarsky P., Li B., Lilge L.D., Wilson B.C. 2008. The influence of hypoxia on bioluminescence in luciferase-transfected gliosarcoma tumor cells in vitro. Photochem. Photobiol. Sci. 7, 675–680.
Brutkiewicz S., Mendonca M., Stantz K., Comerford K., Bigsby R., Hutchins G., Goebl M., Harrington M. 2007. The expression level of luciferase within tumour cells can alter tumour growth upon in vivo bioluminescence imaging. Luminescence. 22, 221–228.
Beazley-Long N., Gaston K., Harper S.J., Orlando A., Bates D.O. 2015. Novel mechanisms of resistance to vemurafenib in melanoma: V600E B-Raf reversion and switching VEGF-A splice isoform expression. Am. J. Cancer Res. 5, 433–441.
Kalra J., Anantha M., Warburton C., Waterhouse D., Yan H., Yang Y., Strut D., Osooly M., Masin D., Bally M.B. 2011. Validating the use of a luciferase labeled breast cancer cell line, MDA435LCC6, as a means to monitor tumor progression and to assess the therapeutic activity of an established anticancer drug, docetaxel (Dt) alone or in combination with the ILK inhibitor, QLT02. Cancer Biol. Ther. 11, 826–838.
ACKNOWLEDGMENTS
We are grateful to researchers of the Blokhin Cancer Research Center (Russian Academy of Medical Sciences) for kindly providing the Mel IL cell line and vemurafenib.
This work was supported by the Russian Science Foundation (project no. 14-35-00107-P). The results shown in Fig. 1 were obtained with support from the Program of Basic Research at State Academies of Sciences from 2013 to 2020 (project no. 01201363823).
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Translated by T. Tkacheva
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Kandarakov, O.F., Bruter, A.V. & Belyavsky, A.V. Modulation of Luciferase Production in Melanoma Cells in vitro. Mol Biol 52, 715–722 (2018). https://doi.org/10.1134/S0026893318050084
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DOI: https://doi.org/10.1134/S0026893318050084