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Journal of Lipid Research, Vol 36, 1866-1875, Copyright © 1995 by Lipid Research, Inc.

ARTICLES

Animal cell mutants unable to take up biologically active glycerophospholipids

RA Zoeller, MD Layne and EJ Modest
Department of Biophysics, Boston University School of Medicine, MA 02118, USA.

We have isolated two mutant strains from the murine, macrophage-like cell line, RAW 264.7, that are resistant to the cytotoxic effects of the antineoplastic, platelet activating factor analogue, 1-octadecyl-2- methyl-rac-glycero-3-phosphocholine (ET-18-OMe). The mutants were isolated using a single round of selection to ensure that resistance was due to a single gene defect. These mutants, RAW.R1 and RAW.R23, are approximately 20 times more resistant to ET-18-OMe (ID50 = 15-17 microM) than the parent strain (ID50 = 0.7-1.0 microM). Resistance to ET-18-OMe was due to a 90-95% reduction in the ability to take up and accumulate this compound. The uptake of other choline glycerophospholipids (e.g., platelet activating factor and 1-acyl-2- lyso-sn-glycero-3-phosphocholine) was also severely affected. This defect was not limited to choline glycerophospholipids; the uptake of an ethanolamine glycerophospholipid (1-alkyl-2-lyso-sn-glycero-3- phosphoethanolamine) was reduced by 80%. The uptake of palmitic acid, an amphipathic molecule bearing no phosphate-containing head group, was unaffected in the mutants. There was little metabolism of ET-18-OMe by either the wild-type or mutant cells. Binding of ET-18-OMe appeared to be normal in the mutants, but internalization of pre-bound ET-18-OMe was reduced. Uptake of non-lipid ligands such as horseradish peroxidase, lucifer yellow, and transferrin was normal in the mutants demonstrating that fluid-phase and receptor-mediated endocytosis is functional. The ability to generate mutants displaying a lesion that affects the uptake of both choline and ethanolamine phospholipids demonstrates that these species are internalized by RAW cells through one common primary route or through pathways that share a common factor. These mutants, and this approach to their isolation, offer a system with which to study and define the mechanisms of glycerophospholipid uptake into macrophages as well as other cell types.
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