Apoptosis is associated with triacylglycerol accumulation in Jurkat T-cells.

Magnetic resonance spectroscopy is increasingly used as a non-invasive method to investigate apoptosis. Apoptosis was induced in Jurkat T-cells by Fas mAb. (1)H magnetic resonance spectra of live cells showed an increase in methylene signal as well as methylene/methyl ratio of fatty acid side chains at 5 and 24 h following induction of apoptosis. To explain this observation, (1)H magnetic resonance spectra of cell extracts were investigated. These demonstrated a 70.0+/-7.0%, 114.0+/-8.0% and 90.0+/-5.0% increase in the concentration of triacylglycerols following 3, 5 and 7 h of Fas mAb treatment (P<0.05). Confocal microscopy images of cells stained with the lipophilic dye Nile Red demonstrated the presence of lipid droplets in the cell cytoplasm. Quantification of the stained lipids by flow cytometry showed a good correlation with the magnetic resonance results (P > or =0.05 at 3, 5 and 7 h). (31)P magnetic resonance spectra showed a drop in phosphatidylcholine content of apoptosing cells, indicating that alteration in phosphatidylcholine metabolism could be the source of triacylglycerol accumulation during apoptosis. In summary, apoptosis is associated with an early accumulation of mobile triacylglycerols mostly in the form of cytoplasmic lipid droplets. This is reflected in an increase in the methylene/methyl ratio which could be detected by magnetic resonance spectroscopy.

Imaging biochemistry: applications to breast cancer.

The use of magnetic resonance spectroscopy (MRS) to investigate breast tumour biochemistry in vivo is reviewed. To this end, results obtained both from patients in vivo and from tumour extracts and model systems are discussed. An association has been observed between transformation and an increase in phosphomonoesters (PMEs) detected in the 31P MRS spectrum, as well as an increase in choline-containing metabolites detected in the 1H spectrum. A decrease in PME content after treatment is associated with response to treatment as assessed by tumour volume. Experiments in model systems aimed at understanding the underlying biochemical processes are presented, as well as data indicating the usefulness of MRS in monitoring the uptake and metabolism of some chemotherapeutic agents.

Magnetic resonance detects changes in phosphocholine associated with Ras activation and inhibition in NIH 3T3 cells.

Ras is frequently mutated in cancer, and novel therapies are being developed to target Ras signalling. To identify non-invasive surrogate markers of Ras activation and inhibition, we used(31)P magnetic resonance spectroscopy (MRS) and investigated NIH 3T3 cells compared to a mutant ras transfected counterpart. The MR spectra indicated that phosphocholine (PC) levels increased significantly from 3 +/- 2 fmol cell(-1)in NIH 3T3 cells to 13 +/- 4 fmol cell(-1)in the transfected cells. The PC/NTP ratio increased significantly from 0.3 +/- 0.1 to 0.7 +/- 0.3. This could not be explained by either a faster proliferation rate or by alterations in cell cycle distribution. Both cell lines were treated with simvastatin, 17-AAG and R115777, agents which inhibit Ras signalling. Cell proliferation was inhibited in both cell lines. The spectrum of NIH 3T3 cells was not affected by treatment. In contrast, in the ras transfected cells growth inhibition was associated with an average 35 +/- 5% drop in PC levels and a comparable drop in PC/NTP. Thus the MRS visible increase in phosphocholine is associated with Ras activation, and response to treatment is associated with partial reversal of phosphocholine increase in ras transfected cells. MRS might therefore be a useful tool in detecting Ras activation and its inhibition following targeted therapies.

Treatment of SW620 cells with Tomudex and oxaliplatin induces changes in 2-deoxy-D-glucose incorporation associated with modifications in glucose transport.

UNLABELLED: Many studies suggest that changes in the uptake of the glucose analog FDG after therapy, compared with pretreatment uptake, predicts tumor response to therapy. However, clinical interpretation is compromised by a limited understanding of the effect of therapy on FDG and 2-deoxy-D-glucose (DG) uptake at the tumor cell level. METHODS: Uptake of 2-deoxy-D-[1-(3)H]glucose (3H-DG) by SW620 colonic tumor cells was measured before and 8, 16, 24, and 48 h after treatment with the novel platinum drug oxaliplatin and the novel thymidylate synthase inhibitor Tomudex. Glucose transport was determined by measuring the initial rate of uptake of the nearly nonmetabolized glucose analog 3-O-methyl-D-[1-(3)H]glucose (3H-OMG). The effect of these drugs on cell cycle kinetics was determined using flow cytometry. RESULTS: Treatment of SW620 cells with oxaliplatin was found to decrease uptake of 3H-DG after up to 24 h, but uptake returned to control levels after longer treatment. The initial decrease in 3H-DG incorporation was associated with a lower rate of glucose transport. Treatment of cells with Tomudex induced an increase in 3H-DG uptake that depended on treatment duration. Both glucose transport and the volume of distribution of 3H-OMG were higher in Tomudex-treated cells than in control cells. Flow cytometry showed that oxaliplatin induced a G2 and M arrest, whereas a buildup of cells in the S phase was associated with Tomudex treatment. Both treatments induced apoptosis in SW620 cells. CONCLUSION: Changes in uptake of DG by SW620 colonic tumor cells responding to therapy is specific to the drug type. Modulation of glucose transport was associated with changes in 3H-DG uptake.

Applications of magnetic resonance in model systems: cancer therapeutics.

The lack of information regarding the metabolism and pathophysiology of individual tumors limits, in part, both the development of new anti-cancer therapies and the optimal implementation of currently available treatments. Magnetic resonance [MR, including magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), and electron paramagnetic resonance (EPR)] provides a powerful tool to assess many aspects of tumor metabolism and pathophysiology. Moreover, since this information can be obtained nondestructively, pre-clinical results from cellular or animal models are often easily translated into the clinic. This review presents selected examples of how MR has been used to identify metabolic changes associated with apoptosis, detect therapeutic response prior to a change in tumor volume, optimize the combination of metabolic inhibitors with chemotherapy and/or radiation, characterize and exploit the influence of tumor pH on the effectiveness of chemotherapy, characterize tumor reoxygenation and the effects of modifiers of tumor oxygenation in individual tumors, image transgene expression and assess the efficacy of gene therapy. These examples provide an overview of several of the areas in which cellular and animal model studies using MR have contributed to our understanding of the effects of treatment on tumor metabolism and pathophysiology and the importance of tumor metabolism and pathophysiology as determinants of therapeutic response.

Magnetic resonance detects metabolic changes associated with chemotherapy-induced apoptosis.

Apoptosis was induced by treating L1210 leukaemia cells with mechlorethamine, and SW620 colorectal cells with doxorubicin. The onset and progression of apoptosis were monitored by assessing caspase activation, mitochondrial transmembrane potential, phosphatidylserine externalization, DNA fragmentation and cell morphology. In parallel, 31P magnetic resonance (MR) spectra of cell extracts were recorded. In L1210 cells, caspase activation was detected at 4 h. By 3 h, the MR spectra showed a steady decrease in NTP and NAD, and a significant build-up of fructose 1,6-bisphosphate (F-1,6-P) dihydroxyacetonephosphate and glycerol-3-phosphate, indicating modulation of glycolysis. Treatment with iodoacetate also induced a build-up of F-1,6-P, while preincubation with two poly(ADP-ribose) polymerase inhibitors, 3-aminobenzamide and nicotinamide, prevented the drop in NAD and the build-up of glycolytic intermediates. This suggested that our results were due to inhibition of glyceraldehyde-3-phosphate dehydrogenase, possibly as a consequence of NAD depletion following poly(ADP-ribose) polymerase activation. Doxorubicin treatment of the adherent SW620 cells caused cells committed to apoptosis to detach. F-1,6-P was observed in detached cells, but not in treated cells that remained attached. This indicated that our observations were not cell line- or treatment-specific, but were correlated with the appearance of apoptotic cells following drug treatment. The 31P MR spectrum of tumours responding to chemotherapy could be modulated by similar effects.

Measurements of human breast cancer using magnetic resonance spectroscopy: a review of clinical measurements and a report of localized 31P measurements of response to treatment.

A review of the literature has shown that in human breast tumours, large signals from phosphomonoesters (PME) and phosphodiesters (PDE) are evident. In serial measurements in 19 patients with breast cancer, a decrease in PME was significantly associated with a stable or responding disease (p = 0.017), and an increase in PME was associated with disease progression. Extract studies have shown PME to comprise of phosphoethanolamine (PEth) and phosphocholine (PCho), with the PEth to PCho ratio ranging from 1.3 to 12. The PCho content of high grade tumours was found to be higher than low grade tumours. In some animal models, changes in PCho have been shown to correlate with indices of cellular proliferation, and spheroid studies have shown a decrease in PCho content in spheroids with smaller growth fractions. A serial study of 25 patients with advanced primary breast tumours undergoing hormone, chemotherapy or radiotherapy treatments, showed that in this heterogenous group there were significant changes in metabolites that were seen during the first 3 weeks (range 2-4 weeks) of treatment, that correlated with volume change over this period, employed here as a measure of response. Changes in PME (p = 0.003), total phosphate (TP) (p = 0.008) and total nucleoside tri-phosphate (TNTP) (p = 0.02) over 3 (+/-1) weeks were significantly associated with response, as were the levels of PME (p<0.001), PDE (p = 0.01), TP (p = 0.001) and TNTP (p = 0.007) at week 3 (+/-1). PME at week 3 (+/-1) was also significantly associated with the best volume response to treatment (p = 0.03). A reproducibility analysis of results from the observation of normal breast metabolism in four volunteers showed a mean coefficient of variation of 25%, after correcting for changes resulting from the menstrual cycle. Reproducibility studies in four patients with breast cancer showed a mean coefficient of variation of 33%, with the reproducibility being better in patients measured on different days (difference in TP was -6%) compared with those measured on the same day (difference in TP was -29%).

Comparative NMR study of a differentiated rat hepatoma and its dedifferentiated subclone cultured as spheroids and as implanted tumors.

H4IIEC3 (H4), a differentiated rat hepatoma line and H5, its dedifferentiated subclone, were investigated as proliferating spheroids and as implanted subcutaneous tumors in juvenile rats. H4 cells formed tight, round spheroids whereas H5 cells formed loose, grape-like structures. 31P MR spectra showed that phosphocreatine was present in H5 spheroids but not in H4 spheroids or tumors. [13C]Lactate production from [13C]glucose, with no detectable uptake of [13C]alanine, indicated that energy production in H5 spheroids was primarily via glycolysis. No [13C]glucose utilization was detected in H4 spheroids, but uptake of alanine and accumulation of labeled lactate, glutamate and glutamine indicated oxidation via the tricarboxylic acid (TCA) cycle. Tumors of H4 cells were well perfused, unlike tumors of H5 cells which were highly necrotic. Following i.v. infusion with [13C]alanine, [13C]lactate and glutamate, evidence of oxidation via the TCA cycle, were observed in H4 tumors. Thus the results obtained by 31P and 13C MRS correlated with the differentiation state of H4 and H5 spheroids and tumors.

The application of 13C NMR to the characterization of phospholipid metabolism in cells.

31P and 13C NMR spectroscopy of lipid extracts of T47D human breast cancer spheroids and the use of 13C-labeled lipid precursors [3-13C]serine,[1,2-13C]ethanolamine, and [1,2-13C]choline enabled us to determine the rate of 13C incorporation into the major phospholipids and to show that the synthesis of phosphatidylethanolamine in T47D cells is via both the CDP-ethanolamine pathway and serine decarboxylation, with the extent of each depending on the concentration of ethanolamine in the medium. In the presence of low ethanolamine (3.4 microM), both pathways contribute in equal proportions, while in the presence of high ethanolamine, the CDP-ethanolamine pathway predominates.

Lipid metabolism in large T47D human breast cancer spheroids: 31P- and 13C-NMR studies of choline and ethanolamine uptake.

31P- and 13C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as large (300 microns) spheroids. Spheroids were perfused inside the spectrometer with 1,2-13C-labeled choline or ethanolamine (0.028 mM) and the buildup of labeled phosphorylcholine (PC) or phosphorylethanolamine (PE) was monitored. To analyze the NMR kinetic data, it was assumed that each signal represents a weighted average of signal from the proliferating and non-proliferating compartments of the large spheroid. The average ATP pool size was 4 +/- 1 fmol/cell compared to 8 +/- 1 fmol/cell in small (150 microns) proliferating spheroids (P less than 0.0002). The average PC pool size at steady state was reduced to 11 +/- 6 fmol/cell compared to 22 +/- 8 (P less than 0.007). This could be correlated with an overall reduction of choline uptake in the non-proliferating spheroid fraction. The rate of the enzyme choline kinase was 0.3 fmol/(cell h) compared to 1.0 fmol/(cell h) (P less than 0.0001) for proliferating cells. The rate constant of CTP:phosphocholine cytidyltransferase (0.05 h-1) was not significantly altered, but the rate of the enzyme was reduced from 1.3 to 0.2-0.5 fmol/(cell h). The pool size of PE in medium containing serum ethanolamine (1.7 microM) was approximately the same (15 fmol/cell) in small and large spheroids. In the presence of high ethanolamine (0.028 mM) the average PE level decreased slightly (11 fmol/cell) and the rate of the enzyme ethanolamine kinase in the non-proliferating fraction was 0.7 fmol/(cell h) versus 1.0 fmol/(cell h) in the proliferating cells (P less than 0.07). The rate constant of CTP:phosphoethanolamine cytidyltransferase (0.07 h-1) was not significantly altered but the corresponding reaction rate was reduced from 1.4 to 0.2-0.8 fmol/(cell h). The kinetics of choline incorporation did not alter in the presence of 0.028 mM ethanolamine.

Lipid metabolism in T47D human breast cancer cells: 31P and 13C-NMR studies of choline and ethanolamine uptake.

31P and 13C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as small (150 microns) spheroids. Spheroids were perfused inside the spectrometer with 1,2-13C-labeled choline or 1,2-13C-labeled ethanolamine (0.028 mM) and the buildup of labeled phosphoryl-choline (PC) or phosphorylethanolamine (PE) was monitored. Alternatively the PC and GPC pools were prelabeled with 13C and the reduction of label was monitored. 31P 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 31P and 13C 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.

NMR studies of the lipid metabolism of T47D human breast cancer spheroids.

The in vivo 31P NMR spectrum of T47D human breast cancer cells grown as spheroids shows changes in the phosphomonoester lipid precursors as a function of spheroid size. The ratio of phosphorylethanolamine (PE) to phosphorylcholine (PC) was 1.0 +/- 0.3 for 3-day-old, 150 microns spheroids. This ratio increased to 2.4 +/- 0.4 for spheroids 7 days and older and which were at least 300 microns in diameter. To investigate the phosphatidylethanolamine to phosphatidylcholine (PdylE/PdylC) ratio in the membranes, chloroform/methanol extracts of spheroids were performed. The 31P spectrum of these extracts showed no change with spheroid size, namely the PdylE/PdylC ratio was 0.5 +/- 0.06 for spheroids of all ages suggesting that membrane composition is strongly regulated at the precursor level.