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Receptor‐Mediated Targeting of Toxin Results in Selection of Genetically Protected Hepatocytes Without Bystander Toxicity
Author(s) -
Volarevic Martina,
Wu Catherine H,
Smolic Robert,
Andorfer John H,
Wu George Y
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1137-c
Subject(s) - microbiology and biotechnology , toxicity , hepatocyte , cell culture , receptor , conjugate , biology , transfection , in vitro , chemistry , biochemistry , organic chemistry , mathematical analysis , genetics , mathematics
AIM To target G418 specifically to liver cells via asialoglycoprotein receptors (AsGR) for selection of stably transfected neomycin resistant hepatocytes (NR+) without collateral damage. METHODS: A novel G418 conjugate was synthesized by peptide coupling to a galactose‐terminal asialoorosomucoid (AsOR). Uptake studies were done by labeled‐ligand assays, and toxicity by MTT assays on Huh7 (AsGR+, NR−), GFP‐Huh7 (AsGR+, NR+), and 3T3 (AsGR−, NR−) cell lines. RESULTS: Uptake of conjugate (7–8 moles) of G418 per AsOR was the same as for AsOR alone. Conjugate inhibited growth of Huh7 cells by 84.3%. This inhibition was blocked by excess free AsOR. Incubation of 3T3 cells with free G418, but not conjugated G418 resulted in toxicity. GFP‐Huh cells were not susceptible either to free or conjugated G418. Co‐cultures of GFP‐Huh7 and 3T3 in G418 alone resulted in only inhibition of 3T3 cells. In contrast, co‐cultures with AsOR‐G418 resulted in no significant toxicity to either cell type. CONCLUSIONS: G418‐protein conjugates retained inhibitory activity targeted against AsGR (+), NR (−) cells. G418 modification to form a conjugate did not block its activity. Lack of effect of G418 conjugate on ASGR (−) cells suggests that the effects were specific, and not due to release of free G418 in the medium. The presence of NR gene in AsGR (+) cells protected those cells, and the effect without collateral damage to co‐cultured AsGR (−) cells. Supported by Lopata Chair in Hepatitis Research and R01‐DK042182.