Oxygen sensitivity of reporter genes: implications for preclinical imaging of tumor hypoxia.
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.
Cecic
Ivana
I
Department of Radiation Oncology and Molecular Imaging Program, Stanford, Stanford University School of Medicine, Stanford, CA 94305-5847, USA.
Chan
Denise A
DA
Sutphin
Patrick D
PD
Ray
Pritha
P
Gambhir
Sanjiv Sam
SS
Giaccia
Amato J
AJ
Graves
Edward E
EE
D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology*; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000032:analysis; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D021621:Imaging, Three-Dimensional; D049410:Luciferases, Renilla / Q000378:metabolism; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000096:biosynthesis; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology*; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology*; D015203:Reproducibility of Results; D013937:Thymidine Kinase / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism; D000097573:Red Fluorescent Protein
D005456:Fluorescent Dyes; D049410:Luciferases, Renilla; D008164:Luminescent Proteins; D009363:Neoplasm Proteins; D010100:Oxygen; D013937:Thymidine Kinase; D001616:beta-Galactosidase; D000097573:Red Fluorescent Protein