Cell and membrane lipid analysis by proton magnetic resonance spectroscopy in five breast cancer cell lines

Abstract

Summary The lipid composition of five human breast cancer cell lines T47D, was assessed by proton magnetic resonance spectroscopy (MRS) in whole cells and membraneenriched fractions. The proportions of the three main lipid resonances in 1D spectra were different for each cell line. These resonances included mobile methyl and methylene functions from fatty acids of triglycerides and phospholipids and N-trimethyl from choline of phospholipids. T47D and Materials and methods Cell cultureFive human breast carcinoma cell lines were used: MCF-7, MDA-MB 231, T47D, SKBR3 and ZR-75-l (Engel & Young, 1978). All these cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% foetal calf serum, as adherent cells.The cells were mass cultured in the same conditions, in 150 cm2 flasks. The cells from three flasks (107 cells) were harvested at confluency with a rubber policeman scraper and centrifuged at 1,000 r.p.m. for 10 min. For whole cell MRS experiments, the cells were washed three times with 2 ml of phosphate-buffered saline (PBS: KH2PO4 0.2 g 1-, KCI 0.2 g 1-', NaCl 8 g 1-', Na2HPO41.15 g 1-') in deuterium oxide (D20). The final pellet was suspended in PBS/D20 to obtain a final volume of 0.5 ml and placed in a 5 mm MRS tube. For cell membrane preparation, the same number of cells were washed in cool PBS and disrupted with a hypotonic Tris (tris[hydroxymethyl]aminoethane) buffer (0.01 M), followed by a freeze-thaw cycle at -20C; finally, polytron was used three times for 5 s. The resulting homogenate was centrifuged at 1,000 g for 10 min and the supernatant was centrifuged at 47,000 g for 30 min (Beckmann TL 100, rotor TL 100-2). For MRS experiments, the final pellet was suspended in PBS/D20 and 0.5 ml of the suspension was placed in a 5 mm MRS tube. The specific Na+/K+ ATPase activity was determined in the presence or absence of digitoxigenin using a coupled assay method previously described (Noel & Godfraind, 1984

Radiation effects on soluble metabolites in cultured HeLa cells examined by 1H MRS: Changes in concentration of glutathione and of lipid catabolites induced by gamma rays and proton beams

“…2b). Most of these signals are rather usual in PCA extracts of different origin [1][2][3] and have been assigned in other tumor cells with a similar methodology [43,44].…”

Pimonidazole binding in C6 rat brain glioma: relation with lipid droplet detection

“…The presence of mobile lipids has been detected by proton magnetic resonance spectroscopy ( 1 H MRS) in animal and human tumours: in cultured cells (Mountford and Wright, 1988;Le Moyec et al, 1992), in biopsies (Lean et al, 1993;Kuesel et al, 1994a), and in vivo (Ott et al, 1993;Negendank et al, 1996). The location of these lipids has been widely discussed during the past years.…”

Noninvasive estimation of tumour viability in a xenograft model of human neuroblastoma with proton magnetic resonance spectroscopy (1H MRS)

“…Metabolites with chemical shifts between 0.9 and 1.4 ppm are frequently observed in 1 H MRS spectra of human tumours (Guidoni et al, 1987;Le Moyec et al, 1992;. These signals principally correspond to fatty acyl chains with methylene (CH 2 ) and methyl (CH 3 ) groups, and are commonly designated mobile lipids (MLs).…”