A Novel Thermostable Beetle Luciferase Based Cytotoxicity Assay

Overview

Journal Sci Rep

Specialty Science

Date 2021 May 12

PMID 33976304

Citations 1

Authors

Sunju Choi

Hittu Matta

Ramakrishnan Gopalakrishnan

Venkatesh Natarajan

Songjie Gong

Alberto Jeronimo

Wei-Ying Kuo

Bryant Bravo

Preet M Chaudhary

Affiliations

Soon will be listed here.

Abstract

Cytotoxicity assays are essential for the testing and development of novel immunotherapies for the treatment of cancer. We recently described a novel cytotoxicity assay, termed the Matador assay, which was based on marine luciferases and their engineered derivatives. In this study, we describe the development of a new cytotoxicity assay termed 'Matador-Glo assay' which takes advantage of a thermostable variant of Click Beetle Luciferase (Luc146-1H2). Matador-Glo assay utilizes Luc146-1H2 and D-luciferin as the luciferase-substrate pair for luminescence detection. The assay involves ectopic over-expression of Luc146-1H2 in the cytosol of target cells of interest. Upon damage to the membrane integrity, the Luc146-1H2 is either released from the dead and dying cells or its activity is preferentially measured in dead and dying cells. We demonstrate that this assay is simple, fast, specific, sensitive, cost-efficient, and not labor-intensive. We further demonstrate that the Matador-Glo assay can be combined with the marine luciferase-based Matador assay to develop a dual luciferase assay for cell death detection. Finally, we demonstrate that the Luc146-1H2 expressing target cells can also be used for in vivo bioluminescence imaging applications.

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References

Tolani B, Matta H, Gopalakrishnan R, Punj V, Chaudhary P . NEMO is essential for Kaposi's sarcoma-associated herpesvirus-encoded vFLIP K13-induced gene expression and protection against death receptor-induced cell death, and its N-terminal 251 residues are sufficient for this process. J Virol. 2014; 88(11):6345-54. PMC: 4093867. DOI: 10.1128/JVI.00028-14. View

BRUNNER K, Mauel J, Cerottini J, Chapuis B . Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology. 1968; 14(2):181-96. PMC: 1409286. View

Kim J, Kalimuthu S, Ahn B . In vivo cell tracking with bioluminescence imaging. Nucl Med Mol Imaging. 2015; 49(1):3-10. PMC: 4354780. DOI: 10.1007/s13139-014-0309-x. View

El-Amouri S, Cao P, Miao C, Pan D . Secreted luciferase for in vivo evaluation of systemic protein delivery in mice. Mol Biotechnol. 2012; 53(1):63-73. PMC: 4040271. DOI: 10.1007/s12033-012-9519-6. View

Decker T, Lohmann-Matthes M . A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods. 1988; 115(1):61-9. DOI: 10.1016/0022-1759(88)90310-9. View

Auld D, Thorne N, Maguire W, Inglese J . Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression. Proc Natl Acad Sci U S A. 2009; 106(9):3585-90. PMC: 2638738. DOI: 10.1073/pnas.0813345106. View

Gopalakrishnan R, Matta H, Chaudhary P . A purine scaffold HSP90 inhibitor BIIB021 has selective activity against KSHV-associated primary effusion lymphoma and blocks vFLIP K13-induced NF-κB. Clin Cancer Res. 2013; 19(18):5016-26. PMC: 3804723. DOI: 10.1158/1078-0432.CCR-12-3510. View

Yang Y, Groshong J, Matta H, Gopalakrishnan R, Yi H, Chaudhary P . Constitutive NF-kappaB activation confers interleukin 6 (IL6) independence and resistance to dexamethasone and Janus kinase inhibitor INCB018424 in murine plasmacytoma cells. J Biol Chem. 2011; 286(32):27988-97. PMC: 3151044. DOI: 10.1074/jbc.M110.213363. View

Tafreshi N, Hosseinkhani S, Sadeghizadeh M, Sadeghi M, Ranjbar B, Naderi-Manesh H . The influence of insertion of a critical residue (Arg356) in structure and bioluminescence spectra of firefly luciferase. J Biol Chem. 2007; 282(12):8641-7. DOI: 10.1074/jbc.M609271200. View

10.

Rabinovich P, Zhang J, Kerr S, Cheng B, Komarovskaya M, Bersenev A . A versatile flow-based assay for immunocyte-mediated cytotoxicity. J Immunol Methods. 2019; 474:112668. PMC: 6891822. DOI: 10.1016/j.jim.2019.112668. View

11.

Olsson T, Gulliksson H, Palmeborn M, Bergstrom K, Thore A . Leakage of adenylate kinase from stored blood cells. J Appl Biochem. 1983; 5(6):437-45. View

12.

Matta H, Gopalakrishnan R, Choi S, Prakash R, Natarajan V, Prins R . Development and characterization of a novel luciferase based cytotoxicity assay. Sci Rep. 2018; 8(1):199. PMC: 5760659. DOI: 10.1038/s41598-017-18606-1. View

13.

Fu X, Tao L, Rivera A, Williamson S, Song X, Ahmed N . A simple and sensitive method for measuring tumor-specific T cell cytotoxicity. PLoS One. 2010; 5(7):e11867. PMC: 2912327. DOI: 10.1371/journal.pone.0011867. View

14.

Corey M, Kinders R, Brown L, Vessella R . A very sensitive coupled luminescent assay for cytotoxicity and complement-mediated lysis. J Immunol Methods. 1997; 207(1):43-51. DOI: 10.1016/s0022-1759(97)00098-7. View

15.

Auld D, Zhang Y, Southall N, Rai G, Landsman M, MacLure J . A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution. J Med Chem. 2009; 52(5):1450-8. PMC: 3430137. DOI: 10.1021/jm8014525. View

16.

Zambito G, Hall M, Wood M, Gaspar N, Ridwan Y, Stellari F . Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging. iScience. 2021; 24(1):101986. PMC: 7811125. DOI: 10.1016/j.isci.2020.101986. View

17.

Hosseinkhani S . Molecular enigma of multicolor bioluminescence of firefly luciferase. Cell Mol Life Sci. 2010; 68(7):1167-82. PMC: 11114832. DOI: 10.1007/s00018-010-0607-0. View

18.

Natarajan V, Gopalakrishnan R, Matta H, Choi S, Gong S, Jeronimo A . A Fast and Sensitive Luciferase-based Assay for Antibody Engineering and Design of Chimeric Antigen Receptors. Sci Rep. 2020; 10(1):2318. PMC: 7012821. DOI: 10.1038/s41598-020-59099-9. View

19.

Zaritskaya L, Shurin M, Sayers T, Malyguine A . New flow cytometric assays for monitoring cell-mediated cytotoxicity. Expert Rev Vaccines. 2010; 9(6):601-16. PMC: 2911950. DOI: 10.1586/erv.10.49. View

20.

Bakhtiarova A, Taslimi P, Elliman S, Kosinski P, Hubbard B, Kavana M . Resveratrol inhibits firefly luciferase. Biochem Biophys Res Commun. 2006; 351(2):481-4. DOI: 10.1016/j.bbrc.2006.10.057. View