Limitations of the reporter green fluorescent protein under simulated tumor conditions.
This paper reports a detailed analysis of the effect of low oxygen conditions (hypoxia) on the reporter green fluorescent protein (GFP). It questions the feasibility of using GFP for gene expression studies under tumor conditions. Hypoxia is a characteristic of both experimental and clinical tumors. Several important factors are pointed out which need to be considered when using GFP as reporter gene. GFP fluorescence is the final product of a long and complex pathway involving transcription, translation, and posttranslational modifications. All of these steps may be affected by the availability of oxygen. We show specifically that cellular GFP fluorescence decreased with reduced oxygenation, anoxia virtually eliminated fluorescence and protein levels, and fluorescence recovery after anoxia required 5-10 h of reoxygenation. In conclusion, GFP appears to be a good marker gene to study location or movement of proteins or cells but should be used with great caution as a reporter of gene expression under tumor conditions.
Coralli
C
C
Tumour Microcirculation Group, Gray Laboratory Cancer Research Trust, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, United Kingdom.
Cemazar
M
M
Kanthou
C
C
Tozer
G M
GM
Dachs
G U
GU
D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology; D005260:Female; D005453:Fluorescence; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D010100:Oxygen / Q000378:metabolism* / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D010100:Oxygen