ANIMAL-CELL MUTANTS UNABLE TO TAKE UP BIOLOGICALLY-ACTIVE GLYCEROPHOSPHOLIPIDS

We have isolated two mutant strains from the murine, macrophage-like c ell line, RAM 264.7, that are resistant to the cytotoxic effects of th e antineoplastic, platelet activating factor analogue, 1-octadecyl-2-m ethyl-rac-glycero-3-phosphocholine (ET-18-OMe). The mutants were isola ted using a single round of selection to ensure that resistance was du e to a single gene defect. These mutants, RAW.R1 and RAW.R23, are appr oximately 20 times more resistant to ET-18-OMe (ID50 = 15-17 mu M) tha n the parent strain (ID50 0.7-1.0 mu M). Resistance to ET-18-OMe was d ue to a 90-95% reduction in the ability to take up and accumulate this compound. The uptake of other choline glycerophospholipids (e.g., pla telet activating factor and 1-acyl-2-lyso-sn-glycero-3-phosphocholine) was also severely affected. This defect was not limited to choline gl ycerophospholipids; 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 phosph ate-containing head group, was unaffected in the mutants. There was li ttle metabolism of ET-18-OMe by either the wildtype or mutant cells. B inding of ET-18-OMe appeared to be normal in the mutants, but internal ization of pre-bound ET-18-OMe was reduced. Uptake of non-lipid ligand s such as horseradish peroxidase, lucifer yellow, and transferrin was normal in the mutants demonstrating that fluid-phase and receptor-medi ated endocytosis is functional. The ability to generate mutants displa ying 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 glyce rophospholipid uptake into macrophages as well as other cell types.