Publications

1989

1. Studies of the metabolism of human breast cancer spheroids by NMR

   Ronen S. & Degani H. (1989) Magnetic Resonance in Medicine. 12, 2, p. 274-281  Abstract

   Spheroids present a model for tumor behavior in vivo. In NMR studies, a problem of spheroid adhesion occurs. Here, we present a method which overcomes this problem and show the variations in phosphate metabolite concentrations as well as the rate of glucose metabolism as a function of spheroid age. © 1989 Academic Press, Inc.
2. ³¹P NMR studies of the thermodynamics and kinetics of the creatine kinase reaction

   Eldar H. & Degani H. (1989) Magnetic Resonance in Medicine. 11, 1, p. 121-126  Abstract

   The thermodynamic and kinetic parameters of the reaction catalyzed by creatine kinase (CK) were measured in vitro in the temperature range 13 to 35°C, using ³¹P NMR spectroscopy, including magnetization transfer methods. The apparent equilibrium constant of the reaction and the associated enthalpy for the formation of ATP at 35°C, pH 8.2, and excess [Mg²⁺] were 3.5 × 10⁹ M⁻¹ and −2.4 ± 0.5 kcal/mol, respectively. The rates at equilibrium at 35°C catalyzed by 1 unit/ml CK were 12.4 and 10.7 μM /s at pH 7 and 8, respectively. The rate constants per 1 unit CK/ 1 ml at 35°C, pH 7, were 1.3 × 10⁸ s⁻¹M⁻² and 9.9 × s⁻¹ M⁻¹ in the direction of ATP and PCr formation, respectively. The activation energies in both directions were similar and corresponded to 15 ± 2 kcal/mol at pH 7 and 17.5 ± 1.5 kcal/mol at pH 8. Comparison of in vivo results with the above in vitro data may provide information regarding the activity and kinetics of the CK reaction.
3. Metabolic Studies of Estrogen- and Tamoxifen-treated Human Breast Cancer Cells by Nuclear Magnetic Resonance Spectroscopy

   Neeman M. & Degani H. (1989) Cancer Research. 49, 3, p. 589-594  Abstract

   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.

4. Early estrogen-induced metabolic changes and their inhibition by actinomycin D and cycloheximide in human breast cancer cells: 31P and 13C NMR studies: ³¹P and ¹³C NMR studies

   Neeman M. & Degani H. (1989) Proceedings of the National Academy of Sciences of the United States of America. 86, 14, p. 5585-5589  Abstract

   Metabolic changes following estrogen stimulation and the inhibition of these changes in the presence of actinomycin D and cycloheximide were monitored continuously in perfused human breast cancer T47D clone 11 cells with ³¹P and ¹³C NMR techniques. The experiments were performed by estrogen rescue of tamoxifen-treated cells. Immediately after perfusion with estrogen-containing medium, a continuous enhancement in the rates of glucose consumption, lactate production by glycolysis, and glutamate synthesis by the Krebs cycle occurred with a persistent 2-fold increase at 4 hr. The content of phosphocholine had increased by 10% to 30% within the first hour of estrogen stimulation, but the content of the other observed phosphate metabolites as well as the pH remained unchanged. Pretreatment with either actinomycin D or cycloheximide, at concentrations known to inhibit mRNA and protein synthesis, respectively, and simultaneous treatment with estrogen and each inhibitor prevented the estrogen-induced changes in glucose metabolism. This suggested that the observed estrogen stimulation required synthesis of mRNA and protein. These inhibitors also modulated several metabolic activities that were not related to estrogen stimulation. The observed changes in the in vivo kinetics of glucose metabolism may provide a means for the early detection of the response of human breast cancer cells to estrogen versus tamoxifen treatment.
5. Estradiol induction of accelerated energy metabolism in prepuberal rat uteri in vitro: mRNA hybridization and [¹³C]NMR studies

   KAYE A., SHINKARENKO L., WAISMAN A., VICTOR T. & Degani H. (1989) Journal of Steroid Biochemistry. 34, 1-6, p. 289-292  Abstract

   In vitro treatment with 30 nM 17β-estradiol stimulated the induction of mRNA for the brain type isozyme of creatine kinase BB (CKBB) and stimulated glucose metabolism in perifused uteri from 27-29-day-old rats. The perifusion conditions maintained the normal NMR spectrum of high energy phosphates for at least 24 h. This technique permitted the demonstration that perifused rat uteri stimulated by 17β-estradiol show increased mRNA for creatine kinase BB, 1 h after estrogen addition. The time-course of increase, measured by Northern blot hybridization, parallels that seen in mRNA extracted from uteri after in vivo induction by i.p. injection of 5μg 17β-estradiol; the maximal increase is seen at 2-4 h. Experiments utilizing actinomycin D (4 μg/ml) for inhibition of RNA synthesis showed that CKB mRNA from both untreated and estradiol stimulated uteri had a similar half-life, of approximately 2 h, indicating that CKB mRNA is transcriptionally regulated. In the same system, the rate of glycolysis was measured by NMR spectroscopy using [1-¹³C]glucose. Following in vitro stimulation with 30 nM estradiol, glycolysis increased within 3 h, in parallel to increases previously found in uteri from in vivo stimulated rats.