Chemotherapeutic Agents Up-regulate the Cytomegalovirus Promoter: Implications for Bioluminescence Imaging of Tumor Response to Therapy
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Bioluminescence imaging is widely used to evaluate tumor growth and response to therapy in living animals. In cells expressing luciferase under the control of a constitutive promoter, light output in part depends on viable cell number, so that changes in bioluminescence intensity may be correlated with changes in viable tumor mass over time. We have found that treatment of cancer cell lines expressing luciferase under control of the cytomegalovirus (CMV) promoter with staurosporine, doxorubicin, and paclitaxel results in a transient increase in bioluminescence, which is positively correlated with apoptosis and inversely correlated with cell viability. In contrast, similar treatment of cell lines expressing luciferase under control of the SV40 promoter did not exhibit this result. We found that low doses of staurosporine induced bioluminescence in CMV- but not SV40-driven luciferase cell lines, whereas high doses elicited induction in both, indicating promoter-dependent and promoter-independent mechanisms of bioluminescence induction. The promoter-dependent increase in bioluminescence intensity from CMV-driven luciferase is a result of induction of luciferase mRNA and protein expression. We extended these findings in vivo; doxorubicin treatment resulted in a transient induction in bioluminescence when normalized to tumor volume in CMV- but not SV40-driven luciferase-expressing xenografts. We found that inhibition of the p38 mitogen-activated protein kinase pathway blocked bioluminescence induction by doxorubicin, paclitaxel, and staurosporine in CMV-driven luciferase-expressing cells. These findings have important implications when using bioluminescence to monitor the efficacy of anticancer therapy and underscore the complex regulation of the CMV promoter, which is widely used for high-level protein expression in mammalian cells.Keywords:
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Abstract Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small‐molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Herein, we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. When using adeno‐associated viral (AAV) vectors to express luciferases in the brain, we found that mutant luciferases that are inactive or weakly active with d ‐luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH‐sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.
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Bioluminescence refers to the emission of light from a living system in which photoproteins such as luciferase enzymes oxidize their substrates to produce light. Because of its high-sensitivity and low-toxicity, bioluminescence imaging (BLI) is particularly useful for in vitro assays and in vivo small animal imaging. It provides a powerful tool to study various important biological questions and processes including gene and protein expression, protein-protein interactions, protein-nucleic acid interactions, and cell signaling pathway functions. This review highlights some of the latest developments in the design and applications of molecular probes for BLI. Keywords: Bioluminescence imaging, Molecular Imaging, Probe, Luciferase, Bioluminescence Resonance Energy Transfer, MRI, Luciferase Mutugenesis, D-luciferin Analogs, Luciferase Fused Proteins, Intramolecular Enzyme Complementation
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Bioluminescence of the medusa Periphylla is based on the oxidation of coelenterazine catalyzed by luciferase. Periphylla has two types of luciferase: the soluble form luciferase L, which causes the exumbrellar bioluminescence display of the medusa, and the insoluble aggregated form, which is stored as particulate material in the ovary, in an amount over 100 times that of luciferase L. The eggs are especially rich in the insoluble luciferase, which drastically decreases upon fertilization. The insoluble form could be solubilized by 2-mercaptoethanol, yielding a mixture of luciferase oligomers with molecular masses in multiples of approximately 20 kDa. Those having the molecular masses of 20 kDa, 40 kDa, and 80 kDa were isolated and designated, respectively, as luciferase A, luciferase B, and luciferase C. The luminescence activities of Periphylla luciferases A, B, and C were 1.2∼4.1 × 1016 photon/mg · s, significantly higher than any coelenterazine luciferase known, and the quantum yields of coelenterazine catalyzed by these luciferases (about 0.30 at 24 °C) are comparable to that catalyzed by Oplophorus luciferase (0.34 at 22 °C), which has been considered the most efficient coelenterazine luciferase until now. Luciferase L (32 kDa) could also be split by 2-mercaptoethanol into luciferase A and an accessory protein (approx. 12 kDa), as yet uncharacterized. Luciferases A, B, and C are highly resistant to inactivation: their luminescence activities are only slightly diminished at pH 1 and pH 11 and are enhanced in the presence of 1∼2 M guanidine hydrochloride; but they are less stable to heating than luciferase L, which is practically unaffected by boiling.
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To enhance the efficiency of firefly luciferase/luciferin bioluminescence imaging, a series of N-cycloalkylaminoluciferins (cyaLucs) were developed by introducing lipophilic N-cycloalkylated substitutions. The experimental results demonstrate that these cyaLucs are effective substrates for native firefly luciferase (Fluc) and can produce elevated bioluminescent signals in vitro, in cellulo, and in vivo. It should be noted that, in animal studies, N-cyclobutylaminoluciferin (cybLuc) at 10 μM (0.1 mL), which is 0.01% of the standard dose of d-luciferin (dLuc) used in mouse imaging, can radiate 20-fold more bioluminescent light than d-luciferin (dLuc) or aminoluciferin (aLuc) at the same concentration. Longer in vivo emission imaging using cybLuc suggests that it can be used for long-time observation. Regarding the mechanism of cybLuc, our cocrystal structure data from firefly luciferase with oxidized cybLuc suggested that oxidized cybLuc fits into the same pocket as oxyluciferin. Most interestingly, our results demonstrate that the sensitivity of cybLuc in brain tumor imaging contributes to its extended application in deep tissues.
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Abstract In vivo bioluminescence imaging is becoming a very important tool for the study of a variety of cellular and molecular events or disease processes in living systems. In vivo bioluminescence imaging is based on the detection of light emitted from within an animal. The light is generated as a product of the luciferase–luciferin reaction taking place in a cell. In this study, we implanted mice with tumour cells expressing either a high or a low level of luciferase. In vivo bioluminescence imaging was used to follow tumour progression. Repeated luciferin injection and imaging of high and low luciferase‐expressing tumours was performed. While low luciferase‐expressing tumours grew similarly to vector controls, growth of the high luciferase‐expressing tumours was severely inhibited. The observation that a high level of luciferase expression will inhibit tumour cell growth when an animal is subjected to serial in vivo bioluminescence imaging is potentially an important factor in designing these types of studies. Copyright © 2007 John Wiley & Sons, Ltd.
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1 Abstract Odontosyllis undecimdonta is a marine syllid polychaete that produces bright internal and exuded bioluminescence. Despite over fifty years of biochemical investigation into Odontosyllis bioluminescence, the light-emitting small molecule substrate and catalyzing luciferase protein have remained a mystery. Here we describe the discovery of a bioluminescent protein fraction from O. undecimdonta, the identification of the luciferase using peptide and RNA sequencing, and the in vitro reconstruction of the bioluminescence reaction using highly purified O. undecimdonta luciferin and recombinant luciferase. Lastly, we found no identifiably homologous proteins in publicly available datasets. This suggests that the syllid polychaetes contain an evolutionarily unique luciferase among all characterized luminous taxa. 3 Highlights The polychaete O. undecimdonta uses a luciferin-luciferase bioluminescence system O. undecimdonta bioluminescence does not require additional cofactors The luciferase of the Japanese fireworm is 329 amino acids long Recombinant luciferase is not secreted when expressed in human cells Exogenous luciferin does not seem to penetrate cell membranes-only lysate luminesces The luciferase transcript is supported by full-length cDNA reads with 5’ and 3’ UTR
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Firefly luciferase is ubiquitously used as a genetic reporter for the non-invasive bioluminescence imaging of small animal models. This widespread use of Firefly luciferase in vivo has been facilitated by genetic engineering producing mutants which are extremely stable at physiological conditions. In addition, the red-shifting of bioluminescence has resulted in the enhanced penetration of light emission through biological tissue. However, the use of bioluminescence in vivo is still largely limited to the tracking of single events within a model. This is due to the differential attenuation of light <600nm, making the spectral unmixing of bioluminescent signals extremely challenging. Consequently, there is a real need to move bioluminescence into the near-infrared for dual-colour imaging. As we seem to have reached the limits of mutational based red-shifting, research has more recently focused upon chemical modification of the D-luciferin substrate. But any modification of the DLuciferin substrate is inevitably going to require subsequent mutagenesis of Firefly luciferase to optimise the light emitting reaction. The first part of this project describes the development and validation of a high throughput screening platform for bioluminescent proteins, to advance the identification of mutants with enhanced characteristics. Focus then turns to the use of genetically engineered Firefly luciferase colour mutants for in vivo bioluminescence imaging. Small animal tumour models, representing increasing tissue depth, were engrafted with Firefly luciferase colour mutants to explore the feasibility of dual colour imaging and establish the true benefit of red-shifting bioluminescence. Finally, bioluminescence in the near infra-red is used for dual bioluminescence imaging, tracking two tumour populations in a B-cell lymphoma mouse model through the spectral unmixing of Firefly luciferase colour mutants with the novel substrate infra-luciferin.
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