Characterization of nonexchangeable radioactivity in L1210 cells incubated with [14C]thiotepa: labeling of phosphatidylethanolamine.

Abstract

N,N',N''-Triethylenethiophosphoramide ([14C]thiotepa) accumulation by L1210 cells is a biphasic process. A very rapid initial phase is followed by a much slower second phase that reflects accumulation of radioactivity in a form that is not lost or exchanged when cells are resuspended and incubated in drug-free medium for up to 8 h. In this study we attempted to characterize this nonexchangeable radioactivity. Nuclei (10(7)) isolated from L1210 cells and incubated with [14C]thiotepa did not accumulate 14C during incubations of up to 5 h. Similarly, nuclei isolated from 10(7) L1210 cells that had been shown to accumulate nonexchangeable 14C after incubation with [14C]thiotepa did not show an increase in nuclear-associated 14C. Eighty to 85% of nonexchangeable 14C in L1210 cells incubated with [14C]thiotepa was soluble in ethanol or chloroform:methanol (2:1, v/v), and although most of this cell-associated nonexchangeable 14C was precipitated by trichloroacetic acid, subsequent treatment of that precipitate with methanol solubilized most of the 14C so that only 15 to 20% remained with the final precipitate. When chloroform:methanol-soluble nonexchangeable 14C was analyzed with thin-layer chromatography systems suitable for thiotepa or simple lipids, all radioactivity remained at the origin. In contrast, when analyzed with one- and two-dimensional thin-layer chromatographic systems suitable for complex lipids, all chloroform:methanol-soluble radioactivity was associated with a single lipid spot. This lipid cochromatographed with phosphatidylethanolamine, reacted with ninhydrin but not with 4-(p-nitrobenzyl)pyridine or the Dragendorff choline reagent, and was digested by phospholipases C and D, all of which lead to its identification as phosphatidylethanolamine. This extensive labeling of phosphatidylethanolamine in L1210 cells incubated with [14C]thiotepa can be explained by liberation of [14C]aziridine from [14C]thiotepa, hydrolysis of the [14C]aziridine to [14C]ethanolamine, and incorporation of that radiolabeled material into phosphatidylethanolamine via the normal cellular synthetic pathways for that lipid. This information implies that thiotepa serves, at least in part, as a prodrug for aziridine and has implications as to the mechanism of thiotepa-induced cytotoxicity in that aziridine is a monofunctional alkylating agent incapable of producing interstrand, intrastrand, or protein-DNA cross-links.