coelenterazine and Glioma

Secreted Gaussia luciferase (Gluc) has been shown to be a useful tool for ex vivo monitoring of in vivo biological processes. The Gluc level in the blood was used to detect tumor growth, metastasis and response to therapy, gene transfer, and circulating cells viability, as well as transcription factors activation, complementing in vivo bioluminescence imaging. The sensitivity of the Gluc blood assay is limited due to the absorption of blue light by pigmented molecules such as hemoglobin, resulting in quenching of the signal and therefore lower sensitivity. To overcome this problem, we designed an alternative microtiter well-based binding assay in which Gluc is captured first from blood using a specific antibody followed by the addition of coelenterazine and signal acquisition using a luminometer. This assay showed to be over 1 order of magnitude more sensitive in detecting Gluc in the blood as compared to the direct Gluc blood assay enhancing ex vivo monitoring of biological processes.

Topics: Animals; Copepoda; Genes, Reporter; Glioma; Humans; Imidazoles; Luciferases; Luminescent Measurements; Mice; Mice, Nude; Pyrazines; Tumor Cells, Cultured

In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which analog is most suitable for activity measurements in live cells and living animals, we characterized 10 CLZN analogs using Renilla luciferase (Rluc) as the reporter enzyme. For each analog, we monitored enzyme activity, auto-oxidation, and efficiency of cellular uptake. All CLZN analogs tested showed higher auto-oxidation signals in serum than was observed in phosphate buffer or medium, mainly as a result of auto-oxidation by binding to albumin. CLZN-f, -h, and -e analogs showed 4- to 8-fold greater Rluc activity, relative to CLZN-native, in cells expressing the enzyme from a stable integrant. In studies using living mice expressing Rluc in hepatocytes, administration of CLZN-e and -native produced the highest signal. Furthermore, distinct temporal differences in signal for each analog were revealed following intravenous or intraperitoneal delivery. We conclude that the CLZN analogs that are presently available vary with respect to hRluc utilization in culture and in vivo, and that the effective use of CLZN-utilizing enzymes in living animals depends on the selection of an appropriate substrate.

Topics: Animals; Anthozoa; Cell Line, Tumor; Dose-Response Relationship, Drug; Genes, Reporter; Glioma; HeLa Cells; Humans; Imidazoles; Injections, Intravenous; Kinetics; Luciferases; Luminescent Measurements; Male; Mice; Mice, Inbred Strains; Molecular Structure; Protein Binding; Pyrazines; Radionuclide Imaging; Rats; Substrate Specificity; Time Factors; Transfection