Is Green Fluorescent Protein Toxic to the Living Cells?

Abstract

Green fluorescent protein (GFP) has become more popular to be used as a living marker for positively transfected clones in many studies. To establish stable cell lines constitutively expressing GFP, three GFPs expressed from plasmid pBIEGFP, pSG5GFP, and pRSGFP were introduced into NIH/3T3, BHK-21, Huh-7, and HepG2 cells. All the GFPs we used are the mutant forms of a common wild phenotype. The pBIEGFP expressed enhanced GFP (EGFP). The pRSGFP and pSG5GFP expressed red shift GFP (RSGFP). The RSGFP gene in pSG5GFP was driven by a strong SV40 promoter and showed at least 20-fold higher RSGFP expression by western blot analysis. Despite of the variation in the levels of GFP expression, many GFP expressing cells contracted, rounded-up, and died, which was confirmed by decreasing luciferase activity. CPP32 activity and flow cytometric analyses further demonstrate that cells expressing GFP underwent apoptosis. Our observation is contradictory to other reports that GFP is nontoxic to the cells. Most importantly, this paper shows for the first time the link between expression of GFP and induction of apoptosis. This finding should promote studies of GFP cytotoxicity and attempts to isolate new non-toxic mutants of GFP.