Hyperpolarized 13C NMR studies of glucose metabolism in living breast cancer cell cultures.

The recent development of dissolution dynamic nuclear polarization (DNP) gives NMR the sensitivity to follow metabolic processes in living systems with high temporal resolution. In this article, we apply dissolution DNP to study the metabolism of hyperpolarized U-(13)C,(2)H7-glucose in living, perfused human breast cancer cells. Spectrally selective pulses were used to maximize the signal of the main product, lactate, whilst preserving the glucose polarization; in this way, both C1-lactate and C3-lactate could be observed with high temporal resolution. The production of lactate by T47D breast cancer cells can be characterized by Michaelis-Menten-like kinetics, with K(m) = 3.5 ± 1.5 mM and V(max) = 34 ± 4 fmol/cell/min. The high sensitivity of this method also allowed us to observe and quantify the glycolytic intermediates dihydroxyacetone phosphate and 3-phosphoglycerate. Even with the enhanced DNP signal, many other glycolytic intermediates could not be detected directly. Nevertheless, by applying saturation transfer methods, the glycolytic intermediates glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, phosphoenolpyruvate and pyruvate could be observed indirectly. This method shows great promise for the elucidation of the distinctive metabolism and metabolic control of cancer cells, suggesting multiple ways whereby hyperpolarized U-(13)C,(2)H7-glucose NMR could aid in the diagnosis and characterization of cancer in vivo.

Mapping pathophysiological features of breast tumors by MRI at high spatial resolution.

Magnetic resonance imaging (MRI) is a noninvasive method that reveals anatomical details in vivo and detects lesions for diagnosis. Although standard breast MRI cannot clearly delineate breast cancer, contrast-enhanced MRI enables the detection of breast masses with high sensitivity. Dynamic studies demonstrated that malignant lesions were characterized by a faster signal enhancement rate than benign ones. Dynamic MRI of human breast cancer in mice revealed high heterogeneity in the distribution of contrast-enhanced curves and derived pathophysiological features, indicating the importance of high spatial resolution. With clinical MRI, it is difficult to achieve simultaneously high spatial and temporal resolution. In previous dynamic studies, the emphasis was on high temporal resolution and mainly empiric analyses. We describe here a new model-based method that optimizes spatial resolution by using only three time points, and yet characterizes tumor heterogeneity in terms of microvascular permeability and extracellular fraction. Mapping these pathophysiological features may aid diagnosis and prognosis assessment, while the high spatial resolution may improve the capacity to detect smaller lesions. The method was tested in human breast tumors implanted in mice and in a limited number of benign and malignant breast lesions of patients.

Metabolic studies of estrogen- and tamoxifen-treated human breast cancer cells by nuclear magnetic resonance spectroscopy.

The effects of 17 beta-estradiol treatment versus tamoxifen on the metabolism of human breast cancer T47D-clone 11 cells were studied by noninvasive 31P and 13C nuclear magnetic resonance techniques. 31P nuclear magnetic resonance spectra revealed differences between estrogen and tamoxifen treated cells. The steady state content of phosphorylcholine and of the nucleoside diphosphates was higher in the tamoxifen treated cells by 33 and 140%, respectively, relative to estrogen treated cells. The intracellular pH of 7.2 and the content of the nucleoside triphosphates, Pi, phosphocreatine, glycerolphosphorylcholine, and glycerolphosphorylethanolamine and uridine diphosphoglucose remained the same in both treatments. Glucose utilization and subsequent lactate, glutamate, alanine, and glycerol 3-phosphate synthesis were monitored on line following administration of specifically labeled [13C]glucose. In estrogen treated cells the rate of lactate production via glycolysis was 560 fmol/cell/h and the initial rate of 13C labeling of the glutamate pool via the Krebs cycle was 6.8 fmol/cell/h. In the tamoxifen treated cells these rates were 2-fold lower, at 250 and 2.9 fmol/cell/h for lactate and glutamate labeling, respectively. In estrogen treated cells, the calculated content of glutamate (19 fmol/cell), alanine (11 fmol/cell), and glycerol 3-phosphate (8 fmol/cell) was higher than in tamoxifen treated cells, where only glutamate labeling was detected (13 fmol/cell). The observed differences in the in vivo kinetics of glucose metabolism may provide a sensitive measure for detecting the response of human breast cancer cells to estrogen versus tamoxifen treatments.

Tamoxifen enhances cell death in implanted MCF7 breast cancer by inhibiting endothelium growth.

Magnetic resonance imaging at high spatial resolution and histochemical staining were applied to monitor the influence of tamoxifen versus estrogen on the growth, endothelial density, and extent of necrosis in tumors of MCF7 human breast cancer cells implanted in nude mice. Concomitantly with tamoxifen growth arrest, a highly significant decrease, by more than 2-fold, in the endothelial density of viable tumor regions had occurred, together with a significant increase in the extent of necrosis. The results suggest that the antiestrogenic activity of tamoxifen in breast cancer, which results in enhanced necrosis and tumor regression, is due to the inhibition of angiogenesis and of endothelial growth, thus reducing vascularization and impairing tumor perfusion.

A permanently charged tamoxifen derivative displays anticancer activity and improved tissue selectivity in rodents.

A quaternized form of tamoxifen (TAM), tamoxifen methiodide (TMI), was shown to demonstrate very low brain uptake compared to TAM and, unexpectedly, was considerably less estrogenic than TAM in the uterus. The agonist activity of TMI in the bone was similar to that of TAM. TMI manifested significant dose-dependent tumoricidal activity with a rapid onset of action against MCF-7 human breast cancer implants in nude mice and a mean reduction in tumor size of 60% over six weeks.

NMR kinetic studies of the ionophore X-537A-mediated transport of manganous ions across phospholipid bilayers.

Nuclear magnetic resonance spectroscopy has been applied as a method for studying manganous ions transport across the membrane of phosphatidylcholine vesicles. The rates of the ionophore X-537A (lasalocid A)-mediated Mn2+ transport have been measured as a function of ionophore concentration, pH of the vesicle suspension, and temperature. The translocation was found to occur via a neutral complex composed of one manganous ion bound in two ionized X-537A molecules (Mn X2). The activation energy for the overall transport process was determined to be 22 +/- 5 kcal/mol. Also a pKa of 5.0 +/- 0.2 was determined for the ionophore acid dissociation equilibrium in the vesicle suspension.

The diffusional water permeability in the halotolerant alga dunaliella as measured by nuclear magnetic resonance.

T1 nuclear relaxation measurements of 1H and 17O of water have been applied to study the kinetics of the diffusional transport of water across the cytoplasmic cell membrane of Dunaliella salina and Dunaliella bardawil. The water permeability coefficients at 25 degree C were found to be 1.5.10-3 cm/s and 1.8.10-3 cm/s, respectively, with an activation energy of 3.7 kcal/mol. The results indicate that the cell membrane of Dunaliella exhibits high diffusional permeability to water, similar in magnitude to that found for other cells and model membranes, and a relatively low activation energy. This regularity is in contrast to the exceptionally low glycerol permeability of the membrane (Brown, F.F., Sussman, I., Avron, M. and Degani, H. (1982) Biochim. Biophys. Acta 690, 165-173.

Kinetics of water diffusion across phospholipid membranes. 1H- and 17O-NMR relaxation studies.

Nuclear relaxation measurements of 1H and 17O of water have been applied to study the kinetics of water diffusion across vesicular lipid membranes. Differentiation between the intra- and extravesicular media was achieved by entrapping Mn2+ inside the vesicles. The water permeability of egg phosphatidylcholine vesicles was found to be 2.9 x 10(-3) cm/s at 25 degrees C, with an activation energy of 10.5 kcal/mol which remains constant through the temperature range 0-65 degrees C. The water permeability across vesicular bilayers of L-alpha-dipalmitoyl phosphatidylcholine exhibited a sharp change through the lipid phase transition. The permeability in the lipid crystalline phase (45 degrees C) was found to be 7.2 x 10(-3) cm/s with an activation energy of 7.2 kcal/mol. Below the transition at the gel phase (35 degrees C) a permeability of 1.0 x 10(-3) cm/s was determined. The results indicate that water diffuses through lipid membranes in the liquid crystalline phase in a similar fashion to its diffusion in hydrocarbon liquids. However, when the lipids undergo a phase transition to the gel state, this similarity does not hold any more and water diffusion becomes much more restricted than in hydrocarbon liquids. The change in water permeability through the phase transition was correlated with the changes observed in the lipid segmental motion determined from 13C T1 measurements.

Permeability of alkylamines across phosphatidylcholine vesicles as studied by 1H-NMR.

The exchange rate and enthalpy and entropy of activation of the diffusion of the first five n-alkylamines across egg phosphatidylcholine vesicles has been measured by 1H-NMR spectroscopy employing the 1:2 Gd3+-EDTA complex as a relaxation reagent. The permeability determined from the exchange rate of the ethyl through the pentyl derivatives increased sequentially with increasing chain length from 7.10(-7) to 4.10(-4) cm/s, respectively, at 25 degrees C. The permeability of methylamine was similar to that of ethylamine (1.10(-6) cm/s at 25 degrees C) and exhibited a relatively smaller entropy increase. The enthalpy of activation for the transfer reaction was high for all amine derivatives (20 kcal/mol). The entropy of activation increased with increasing chain length. The results indicate that the rate of diffusion is dominated by the partition into the membrane. Methylamine, being the smallest molecule in this series, can probably diffuse also through vacancies formed by the internal motions of the lipid chains.

The kinetics of ionophore X-537A-mediated transport of manganese through dipalmitoylphosphatidylcholine vesicles. A 1H-NMR study.

We have studied the kinetics of ionophore X-537A-mediated transport of manganese ions into small unilamellar vesicles formed from dipalmitoylphosphatidylcholine. To follow the transport we used the paramagnetic effect of manganese on the 1H-NMR signal from choline trimethylammonium groups on the inner phospholipid monolayer. The transport of only one manganese ion produces an intravesicular concentration which is high enough (approx. 1 mM) to substantially broaden this signal. The observed signal thus arises predominantly from those vesicles which contain no manganese. Therefore, as manganese is transported into the vesicles the observed signal decreases in intensity, but does not broaden. The initial time-dependence of the intensity of the signal, S(t), can be approximated by the simple first-order rate law: S(t) = S(0) exp(-k't), where k' is the probability per unit time for the transport of a manganese ion from the external medium to the intravesicular space. From the dependence of k' on the ionophore X-537A concentration we conclude that manganese is transported into the vesicles via both 1 : 1 and 2 : 1 complexes with ionophore X-537A. At low ratios of ionophore X-537A to vesicles transport via the 1 : 1 complex predominates; at high ratios transport via the 2 : 1 complex predominates. From the dependence of k' on manganese concentration we determined that under our conditions the equilibration of ionophore X-537A between vesicles is much faster than the transport of manganese through the vesicles. Lastly, from the dependence of k' on temperature, we conclude that the ionophore X-537A-mediated transport of manganese into the dipalmitoylphosphatidylcholine vesicles is very sensitive to the gel-liquid crystalline phase transition.

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.

Chemotherapy-induced changes in the energetics of human breast cancer cells; 31P- and 13C-NMR studies.

The early changes in the energetics of T47D-clone 11 human breast cancer cells, following treatment with adriamycin and several other anti-cancer drugs were characterized by 31P- and 13C-NMR spectroscopy. Treatment of the cells with cytotoxic doses of either adriamycin (10(-5) M), daunomycin (10(-5) M) or actinomycin-D (2 x 10(-6) M) induced an immediate increase in the content of the nucleoside triphosphate (NTP) pool. A maximum increase of 30 to 50% was reached 6 to 8 h after treatment, and was followed by a gradual decrease, in accord with the decline in cell number due to cell death. High-performance liquid chromatography measurements indicated that the adriamycin-induced build-up of the NTP pool was mainly due to a specific increase in ATP and GTP. Treatment with cytotoxic doses of cytosine arabinofuranoside (10(-4) M) and cis-platin (10(-4) M) and with the antiestrogen tamoxifen at a dose which inhibited growth (2 x 10(-6) M) did not induce an early increase in the NTP content. Adriamycin and actinomycin-D did not alter significantly the rates of glucose consumption and lactate production via glycolysis during the first 4 to 8 h of treatment. Both drug, however, caused during this time interval a 50% inhibition in the rate of glutamate synthesis via the Krebs cycle. Complementary flow cytometry studies have indicated that within 4 h of treatment with either adriamycin or actinomycin-D there is no detectable change in cell cycle distribution. Treatment for longer time periods indicated that each drug affects the cell cycle distribution in a different manner. Thus, the early increase in NTP can not be associated with a specific cell cycle distribution. The results suggest therefore that drugs of the anthracycline and actinomycin type exert a similar specific and early metabolic induction which may affect the energy state of the cells. This induction may relate to the cytotoxic mechanism and could potentially serve as an early marker for response to treatment.

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.

Polyphosphate metabolism in the alga Dunaliella salina studied by 31P-NMR.

Polyphosphate synthesis and the state of the intracellular polyphosphates in the unicellular green alga Dunaliella salina were studied using in vivo 31P-NMR spectroscopy. By perfusing phosphate-depleted algal cells trapped inside agarose beads with orthophosphate (Pi) containing medium, we were able to follow the process of polyphosphate synthesis in whole, living cells. The results suggest that, in Dunaliella, low molecular weight, probably cyclic, polyphosphate intermediates are synthesized from Pi, and are then condensed to high molecular weight polymers. Studies of the intracellular organization of the polyphosphates by electron microscopy and solid-state NMR techniques indicate that most of these polymers are stored in the cell in a soluble form, and not in solid-like structures.

31P-NMR studies of phosphate transfer rates in T47D human breast cancer cells.

The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31P-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (Pi), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC----gamma ATP (0.2 mM/s), Pi----gamma ATP (0.2 mM/s) and the conversion beta ATP----beta ADP (1.3 mM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.1 mumol/min per mg protein) and adenylate kinase (approx. 0.4 mumol/min per mg protein) suggest that the beta ATP----beta ADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from Pi and the rate of consumption of oxygen atoms (4 X 10(-3) mM/s) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.

[NMR studies of glycerol permeability in lipid vesicles, erythrocytes and the alga Dunaliella].

Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T1 relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (delta H not equal to ) were determined for glycerol at 25 degree C: for phosphatidylcholine vesicles 5 . 10-6 cm/s and 11 +/- 2 kcal/mol; for pig erythrocytes 7 . 10-8 cm/s and 18 +/- 3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17 degrees C was found to be exceptionally low and only a lower limit (P less than 5 . 10-11 cm/s) could be calculated. At temperatures above 50 degree C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.

TNF-induced modulations of phospholipid metabolism in human breast cancer cells.

Tumor necrosis factor alpha (TNF) is a cytokine that is cytocidal for certain tumor cells and induces necrotic and apoptotic forms of cell death. Flow cytometry and transmission electron microscopy analysis demonstrated that in human breast cancer cells (MCF7) TNF induces cell cycle arrest in G0+G1/S, accompanied by apoptosis. 31P and 13C NMR spectroscopy was applied to study cellular metabolism of MCF7 cells during TNF-induced signal to apoptosis. Deuterated choline and 2H NMR spectroscopy were utilized to monitor the kinetics of the rate limiting reactions in phosphocholine metabolism. The NMR measurements revealed that immediately after administration of TNF, choline transport was inhibited by 52+/-6%. Later (approximately 15 h), the activity of phosphocholine:cytidine triphosphate cytidylyltransferase, a key enzyme in the biosynthesis of phosphatidylcholine, was enhanced two-fold. These two opposing changes led to a decrease in the level of phosphocholine. Throughout these changes the energetic state of the cells, determined by the level of nucleoside triphosphates and the rate of glucose metabolism via glycolysis, remained constant. The results indicate that TNF specifically modulates the kinetics of membrane-bound enzymes of the rate determining steps in phosphatidylcholine biosynthesis, possibly as part of early events involved in apoptosis.

Response of MCF7 human breast cancer to tamoxifen: evaluation by the three-time-point, contrast-enhanced magnetic resonance imaging method.

Variations in the cellular volume fraction and in the microvascular permeability of MCF7 human breast tumors were used to assess response to tamoxifen. These pathophysiological features were mapped by applying the three-time-point, contrast-enhanced, high resolution magnetic resonance imaging method (H. Degani et al, Nat. Med., 3: 780-782, 1997). Short-term treatment with tamoxifen caused a highly significant increase in the fraction of pixels displaying intermediate contrast agent clearance pattern and a significant increase in the fraction of pixels displaying high rate of contrast agent entrance. These changes resulted from a marked rise in the extracellular volume fraction, indicating increased necrosis, and from an augmentation in the microvascular permeability, predominantly in the vicinity of the high extracellular volume fraction areas, as a result of stress-induced angiogenesis.

Effects of 17 beta-estradiol on high energy phosphate concentrations and the flux catalyzed by creatine kinase in immature rat uteri: 31P nuclear magnetic resonance studies.

31P nuclear magnetic resonance (NMR) was used to study the effects of 17 beta-estradiol on the content of phosphates and on the flux catalyzed by creatine kinase in immature rat uteri. Perifusion with oxygenated medium at 36 C maintained the uteri in a viable state for at least 10 h in vitro during 31P NMR measurements. In vitro administration of 17 beta-estradiol to the perifused uteri induced changes in the concentration of the high energy phosphates similar to those found after in vivo stimulation: a rapid fall in the concentrations of ATP, phospho-creatine, and the phosphomonoesters during the first 2 h, followed by a slower return to initial concentrations by approximately 6 h. Analysis of the time course of this modulation indicated that after estrogen stimulation, the energy utilization rate was about twice the production rate. The flux through the creatine kinase (CK) reaction was measured independently using 31P magnetization transfer techniques; it was found to increase in uteri 24 h after estradiol injection by the same extent (65%) as the specific activity of CK measured by a spectrophotometric assay. The congruence between the results of these two techniques (in the absence of increased substrate concentrations) provides evidence that the early stimulation of brain-type CK synthesis by estrogen results in a net increase in the concentration of this enzyme.

31P and 23Na nuclear magnetic resonance studies of resting and stimulated mast cells.

Exocytosis induced by crosslinking the type I receptor for Fc epsilon domains present on rat mucosal mast cells (RBL-2H3-line) requires the influx of Ca2+ ions and is markedly influenced by the concentration of monovalent cations (K+, Na+ and protons) in their medium. We investigated the role of these ions in coupling the immunological stimulus to secretion using NMR spectroscopy to monitor simultaneously intracellular pH, ATP and Na+ concentrations and the secretory response of living adherent mast cells. Using this methodology we observed that: (i) ATP concentration and intracellular pH are highly regulated and no changes could be resolved in them upon stimulation and during exocytosis. (ii) In the absence of potassium ions in the cells' medium, a decrease is observed in the intracellular pH and ATP concentration and an increase in the Na+ concentration. (iii) From the influx of extracellular Na+ following inhibition of the Na+, K(+)-ATPase by ouabain, we estimated the inward Na+ current of resting cells to 5 x 10(7) ions/(cell.s). This value does not vary by more than 10% during exocytosis.