**2. Bioluminescence**

Bioluminescence (BL) is defined as the production of light by a living organism. Many organisms such as bacteria, fungi, fish, marine invertebrates, and insects use BL for the purpose of mating, camouflage, repulsion, communication and illumination. The chemical reaction that produces BL requires a pigment known generally as luciferin and enzymes called luciferase (see Reaction 1). The reaction between luciferase and its substrate is an oxidation reaction which is sometimes mediated by cofactors such as calcium and may require energy in the form of ATP.

Bioluminescence Applications in Preclinical Oncology Research 141

luciferins that are currently used in biological research. Luciferase, is the general term for a class of enzymes which catalzye oxidation reactions involving luciferins. The structure of luciferase seems to have an impact on the wavelength of photons emitted and color of light produced so that reactions may exhibit yellow-green to red light. Although many of the luciferase enzymes discussed in Table 1 have been applied to in vitro and in vivo work, firefly luciferase (F-Luc) is most commonly employed in oncology research. In the chemical reaction between F-Luc and its substrate D-luciferin, ATP and oxygen promote the formation of oxy-luciferin species. The emission spectrum of the F-Luc catalyzed reaction is broad with an emission peak at approximately 560 nm and a large component above 600 nm. Yellow-green to yellow-orange light is emitted following the relaxation of oxy-luciferin

D-Luciferin + ATP (reversible uses luciferase and Mg2+) luciferase luciferyl–AMP + PPi Luciferyl–AMP + oxygen (uses luciferase and Mg2+) luciferase + oxyluciferin + AMP +

In the following sections the application of biological models expressing luciferase are described in detail. Mammalian systems do not naturally express luciferase and therefore these systems must be engineered to expresse the enzyme. A vector capable of constitutive expression of luciferase or vectors designed to achieve controlled expression of the enzyme can be used Fusion genes can be used as reporter constructs to track gene delivery and integration. Expression of fusion proteins can be used to follow protein localization and protein-protein interactions. Cells with stable expression of luciferase can be used for in vitro assays or be injected into animals to examine metabolism, immunity, angiogenesis, or to establish disease that can be monitored using BLI. Additionally, transgenic animals may also be developed where reporter gene expression is introduced

Luciferase positive tumor cells used for generating animal models in cancer research are widely available in almost all histological types such as breast, cervical, colorectal, lung, prostate, ovarian cancer and melanoma. Developing a luciferase expressing mammary cell line can be accomplished in house by standard transfection or transduction methodology using reagents, such as plasmid vectors carrying the luciferase genes, which are

Tang et al use a typical transfection procedure to create a luciferase positive neuroprogenitor cell line where the pGL3Basic plasmid (Promega, Madison, WI) carrying F-Luc was digested with HindIII and BamHI and the 1.9-kb cDNA fragment encoding F-Luc was isolated and cloned into a second vector (the pHGCX). The resulting pHGCX vector contained the F-Luc gene driven by the cytomegalovirus (CMV) promoter which enables constitutive expression of luciferase. The pHGCX vector also contained the gene for enhanced green fluorescent protein (eGFP) under the control of the viral immediate early

**3. Constructing bioluminescent mammalian cells and animal models** 

to its ground state (see reaction 2).

CO2 + hv (de Wet, Wood et al. 1987)

through the germline.

commercially available.

**3.1 Engineering Luciferse expressing cells** 

Reaction 2

#### Reaction 1 Luciferin + O2 → oxyluciferin + light

Luciferins are a family of light emitting proteins that act as substrates to luciferase. Luciferins evolved many times in various organisms, hence there are a variety of luciferins that are structurally and chemically distinct (Shimomura 2006). Table 1 provides a list of

Table 1. The structure, luminescence wavelength and cofactors required by the luciferases and luciferins used in biological and chemical research (Shimomura 2006)

luciferins that are currently used in biological research. Luciferase, is the general term for a class of enzymes which catalzye oxidation reactions involving luciferins. The structure of luciferase seems to have an impact on the wavelength of photons emitted and color of light produced so that reactions may exhibit yellow-green to red light. Although many of the luciferase enzymes discussed in Table 1 have been applied to in vitro and in vivo work, firefly luciferase (F-Luc) is most commonly employed in oncology research. In the chemical reaction between F-Luc and its substrate D-luciferin, ATP and oxygen promote the formation of oxy-luciferin species. The emission spectrum of the F-Luc catalyzed reaction is broad with an emission peak at approximately 560 nm and a large component above 600 nm. Yellow-green to yellow-orange light is emitted following the relaxation of oxy-luciferin to its ground state (see reaction 2).

Reaction 2

140 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

Luciferins are a family of light emitting proteins that act as substrates to luciferase. Luciferins evolved many times in various organisms, hence there are a variety of luciferins that are structurally and chemically distinct (Shimomura 2006). Table 1 provides a list of

**Organism Luciferin structure Co-factors Luminescece** 

D-Luciferin ATP Mg2+ 560-615nm

coelenterazine Ca2+ 480nm

Latia O2 536

Dinoflagellates Tetrapyrrole O2 474nm

Table 1. The structure, luminescence wavelength and cofactors required by the luciferases

and luciferins used in biological and chemical research (Shimomura 2006)

Cypridina O2 450-460nm

Reaction 1 Luciferin + O2 → oxyluciferin + light

Firefly

Sea Pansy

Snail

(Photinus pyralis) Clickbeetle (Pyrophorus plagiophthalamus)

(Renilla reniformis) Marine copepod (Gaussia princeps)

(Latia neritoides)

Marine Ostracod (Vargula Hilgendorfi) D-Luciferin + ATP (reversible uses luciferase and Mg2+) luciferase luciferyl–AMP + PPi

Luciferyl–AMP + oxygen (uses luciferase and Mg2+) luciferase + oxyluciferin + AMP + CO2 + hv (de Wet, Wood et al. 1987)
