Oxygen sensitivity of reporter genes: implications for preclinical imaging of tumor hypoxia

Mol Imaging. Jul-Aug 2007;6(4):219-28.

Abstract

Reporter gene techniques have been applied toward studying the physiologic phenomena associated with tumor hypoxia, a negative prognostic indicator. The purpose of this study was to assess the potential adverse effects of hypoxic conditions on the effectiveness of four commonly used reporter genes: Renilla luciferase, monomeric red fluorescent protein, thymidine kinase, and lacZ. Tumor-forming A375 cells expressing a trifusion reporter consisting of Renilla luciferase, monomeric red fluorescent protein, and thymidine kinase were subjected to decreasing oxygen tensions and assayed for reporter expression and activity. A375 cells expressing beta-galactosidase were similarly exposed to hypoxia, with activity of the reporter monitored by cleavage of the fluorescent substrate 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-beta-galactoside (DDAOG). Generation of signal in in vivo tumor models expressing bioluminescent or beta-galactosidase reporters were also examined over the course of hypoxic stresses, either by tumor clamping or the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Our findings indicate that bioluminescent and fluorescent reporter activity are decreased under hypoxia despite minimal variations in protein production, whereas beta-galactosidase reporter activity per unit protein was unchanged. These results demonstrate that combining beta-galactosidase with the DDAOG optical probe may be a robust reporter system for the in vivo study of tumor hypoxia.

MeSH terms

  • Animals
  • Cell Hypoxia / physiology*
  • Cell Line, Tumor
  • Fluorescent Dyes / analysis
  • Gene Expression Regulation, Neoplastic / drug effects*
  • Genes, Reporter*
  • Humans
  • Imaging, Three-Dimensional*
  • Luciferases, Renilla / metabolism
  • Luminescent Measurements
  • Luminescent Proteins / metabolism
  • Male
  • Mice
  • Mice, Nude
  • Neoplasm Proteins / biosynthesis
  • Neoplasms / metabolism*
  • Neoplasms / pathology*
  • Oxygen / metabolism
  • Oxygen / pharmacology*
  • Reproducibility of Results
  • Thymidine Kinase / metabolism
  • beta-Galactosidase / metabolism

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • Neoplasm Proteins
  • red fluorescent protein
  • Luciferases, Renilla
  • Thymidine Kinase
  • beta-Galactosidase
  • Oxygen