LIPID-METABOLISM IN T47D HUMAN BREAST-CANCER CELLS - P-31 AND C-13-NMR STUDIES OF CHOLINE AND ETHANOLAMINE UPTAKE

P-31 and C-13-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as small (150-mu-m) spheroids. Spheroids were perfused inside the spectrometer with 1,2-C-13-labeled choline or 1,2-C-13-labeled ethanolamine (0.028 mM) and the buildup of labeled phosphorylcholine (PC) or phosphorylethanolamine (PE) was monitored. Alternatively the PC and GPC pools were prelabeled with C-13 and the reduction of label was monitored. P-31 spectra were recorded from which the overall energetic status as well as total pool sizes could be determined. The ATP content was 8 +/- 1 fmol/cell, and the total PC and PE pool sizes were 16 and 14 fmol/cell, respectively. PC either increased by 50% over 24 h or remained constant, while PE remained constant in medium without added ethanolamine but increased 2-fold within 30 h in medium containing ethanolamine, indicating a dependence on precursor concentration in the medium. The P-31 and C-13 data yielded similar kinetic results: the rate of the enzymes phosphocholine kinase and phosphoethanolamine kinase were both on the order of 1.0 fmol/cell per h, and the rate constants for CTP:phosphocholine cytidyltransferase and CTP:phosphoethanolamine kinase were 0.06 h-1 for both enzymes. The kinetics of choline incorporation did not alter in the presence of 0.028 mM ethanolamine indicating that they have non-competing pathways.