Optimal ligand binding by the recombinant human glucocorticoid receptor and assembly of the receptor complex with heat shock protein 90 correlate with high intracellular ATP levels in Spodoptera frugiperda cells.

The full-length human glucocorticoid receptor (hGR), overexpressed in Spodoptera frugiperda (Sf9) cells, associates with heat shock protein 90 (hsp90) and hsp70 and binds dexamethasone with high affinity. Baculovirus infection of Sf9 cells grown in TNM-FH medium results in the rapid depletion of glucose from the medium within 24 h. Noting a discrepancy between hGR protein levels and ligand binding capacity in such cultures, we hypothesized that the depletion of glucose from the medium could result in intracellular ATP depletion and consequently affect the ligand binding capacity of the recombinant hGR. Supplementation of the Sf9 culture medium with additional glucose resulted in a three-fold increase in intracellular ATP levels, and a three-fold increase in 3H-dexamethasone binding capacity, without altering the protein levels of hGR, hsp90 or hsp70. However, more hsp90 co-immunoprecipitated with hGR from cells grown in glucose supplemented medium. Our data support the hypothesis that high-affinity ligand binding by hGR requires the ATP-dependent formation of the hGR:hsp90 heterocomplex. Besides having practical consequences for the production of recombinant GR and other related proteins, our findings could ultimately have relevance in diseases such as diabetes mellitus.

Role of intracellular free Ca(II) and Zn(II) in dexamethasone-induced apoptosis and dexamethasone resistance in human leukemic CEM cell lines.

The levels of intracellular free Ca(II) and Zn(II) during dexamethasone (dex)-induced apoptosis in CEM cell lines were determined by 19F nuclear magnetic resonance (NMR), using the fluorinated intracellular chelator 1,2-bis-(2- amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5-FBAPTA). The effects of these divalent metal ions on growth rate and DNA degradation were evaluated. Measurements were done on one dex-sensitive (CEM-C7) and three different dex-resistant variants (CEM-C1, CEM-4R4, and CEM-ICR27). Dex caused a continuous increase in the Ca(II) level in dex-sensitive CEM-C7 cells, while in CEM-C1 cells dex caused an initial increase in the Ca(II) level which in approximately 36 h was restored to its normal value. The intracellular Ca(II) level in CEM-4R4 cells was not significantly affected by dex, while that of CEM-ICR27 cells decreased after dex incubation. Only the dex-sensitive CEM-C7 cells showed dex-induced DNA degradation. An intracellular free Zn(II) level of approximately 1 nM was measured for the dex-resistant CEM-C1 cells. No detectable level of intracellular Zn(II) was found in the other cell lines. Incubation with < 100 microM Zn(II) did not inhibit dex-induced apoptosis in CEM-C7 cells (e.g., DNA degradation). Treatment with approximately 250 microM Zn(II) caused significant decrease in growth rate in all cell lines and prevented dex-induced DNA degradation in CEM-C7 cells. A calibrated amount of Ca(II) ionophore (A23187), used to increase Ca(II) concentrations up to the dex-induced levels, did not induce DNA degradation in CEM-C7 or CEM-C1 cells. While elevation of intracellular Ca(II) by itself is not sufficient to initiate apoptosis in CEM-C7 cells, the results reported here suggest that Ca(II) is involved in the killing mechanism as a secondary factor. The combination of dex and ionophore caused significant DNA degradation in CEM-C1 cells, which normally showed resistance to each compound individually. The combination of dex and the Zn(II) chelator phenanthroline also caused extensive DNA degradation in the normally dex-resistant CEM-C1 cells, suggesting that Zn(II) plays a role in the dex resistance of these cells.

31P NMR characterization of cellular metabolism during dexamethasone induced apoptosis in human leukemic cell lines.

31P NMR has been used to study the effects of dexamethasone on phosphorus metabolism in one dexamethasone (dex)-sensitive (CEM-C7) and three different dex-resistant (CEM-C1, CEM-4R4, and CEM-ICR27) human leukemic cell lines. The use of these cell lines, containing widely varying amounts of glucocorticoid receptors, made it possible to evaluate the receptor-mediated contributions to the modes of action of dexamethasone in these cells. To evaluate the effects of dexamethasone without any significant contribution from experimental conditions, all the experiments were done with parallel controls. Results obtained showed: 1) significantly different levels of phosphorylethanolamine (PE) and phosphorylcholine (PC) among cell lines, suggesting significant differences in phospholipid metabolism; 2) the dexamethasone induced reduction of phosphomonoester (PE+PC), ATP, and metabolic rates probably through glucocorticoid receptor mediated mechanisms; 3) the dexamethasone induced stimulation of cellular metabolism in a process which seems to be independent of glucocorticoid receptors; and 4) the dexamethasone induced alkaline shift of intracellular pH in all the cell lines except ICR27. The reduction in PME levels seems to be an earlier step in dexamethasone-induced apoptosis than the reduction in ATP. The degree of alkaline shift was found to correlate with the number of glucocorticoid receptors present. The possible involvement of phospholipid metabolites as second messengers in dexamethasone-induced apoptosis is discussed.

Inhibitory effects of ascorbic acid on growth of leukemic and lymphoma cell lines.

Vitamin C has been suggested and disputed as an anti-cancer agent. Previous in vitro studies using either primary cell cultures from cancer patients or tumor cell lines have suggested that tumor cells with different lineages may have different sensitivities to ascorbic acid. In this study we report characterization of the effects of ascorbic acid on growth of two ascorbic acid sensitive and one ascorbic acid resistant lymphocyte tumor cell lines. The cytotoxic effects of ascorbic acid on the sensitive cell lines were time and dosage dependent. Furthermore, the energy state of the ascorbic acid sensitive cells was affected by the presence of ascorbic acid before the cells became apparently non-viable, as demonstrated by 31P nuclear magnetic resonance spectroscopy. The existence of these lymphocyte cell lines with varying sensitivities to ascorbic acid may provide a useful model system for further understanding of vitamin C action on cancer cells.

Bulk magnetic susceptibility induced broadening in the 19F NMR of suspended leukemic cells.

The relevance of bulk magnetic susceptibility (BMS) induced broadening to in vivo NMR studies of intact cells has been examined and the significance of the contribution of BMS difference to the resolution of intra- and extracellular resonances was demonstrated. BMS difference between intra- and extracellular compartments was found to limit the resolution of intra- and extracellular 19F resonances of fluoro compounds in leukemic cells.

19F NMR studies of changes in membrane potential and intracellular volume during dexamethasone-induced apoptosis in human leukemic cell lines.

The induction of apoptosis in leukemic cells by dexamethasone is well known, but the mechanism of this type of cell death and of dexamethasone resistance by some variants is still poorly understood. Apoptotic cell death is preceded by many changes in cellular properties, such as glucose metabolism, cell size, cell density, and others. In this study, 19F-NMR has been used to characterize changes in cell membrane potential and intracellular accessible volume during dexamethasone induced apoptosis. One dex-sensitive (CEM-C7) and three dex-resistant variants (CEM-C1, CEM-ICR27, and CEM-4R4) were examined. We have observed separate intracellular and extracellular resonances for trifluoroacetate and trifluoroacetamide added to suspended leukemic cells. From the equilibrium distribution of these fluoro-compounds between intra and extracellular spaces, the changes in membrane potential and intracellular accessible volume were calculated. The membrane potential for CEM-C7 cells was found to significantly decrease in the presence of dexamethasone (9-mV decrease within 18 h of dexamethasone treatment), while that of CEM-ICR27 was found in some samples to increase on dexamethasone incubation. The membrane potential for CEM-C1 decreased slightly, while that of CEM-4R4 was not appreciably affected by dexamethasone. The reduction of membrane potential seems to be an early step in the mechanism of dexamethasone induced apoptosis. Although the intracellular volume varied with cell type and dexamethasone incubation (for CEM-C7), the fractional intracellular volume (alpha = Vin/Vcell) was found to be the same (0.82 +/- 0.06) for all the cell lines in the presence and absence of dexamethasone.

Growth inhibitory effects of bioflavonoids and related compounds on human leukemic CEM-C1 and CEM-C7 cells.

Seven compounds, which included some naturally occurring dietary substances, were tested for their inhibitory effects on growth and metabolism of human leukemic CEM-C1 and CEM-C7 cell lines. Among the active compounds the naturally occurring dietary constituents were found to be the most active. The strongest inhibitory effects were observed with 3',4',5,7-tetrahydroxy-flavone (luteolin) and 4,4'-dihydroxychalcone. 31P-NMR spectra of cells incubated for 24 h with 30 microM of either of these compounds show complete ATP depletion. Also glucose uptake by the cells as measured by 13C-NMR is completely inhibited by these compounds. These results may be relevant to the tumor suppressing activity of bioflavonoids and the role of these compounds in chemoprevention.

Nerve growth factor effects on pyridine nucleotides after oxidant injury of rat pheochromocytoma cells.

Neurotrophic factors regulate neuronal survival and neurite growth in development and following injury. Oxidative stress produced in neurons as a consequence of primary injury, or during reperfusion following ischemia, may contribute to cell death. Here, the effects of nerve growth factor (NGF) on the response to H2O2 injury were examined in the PC12 rat pheochromocytoma cell line. Specifically, the effect of NGF on cell viability after H2O2 injury was measured. Pretreatment with NGF enhanced survival after H2O2 treatment, as measured by Trypan blue dye exclusion, radiolabeled amino acid incorporation, tetrazolium salt reduction, or cytoplasmic enzyme release. One early event associated with H2O2 treatment was a rapid decrease in NAD+. Although initial decreases in NAD+ levels were similar in control and NGF-treated cells, the latter recovered more rapidly and extensively. The decline in total NAD observed after NGF treatment was almost equal in magnitude to the measured increase in NADP. Inhibition of poly(ADP-ribose) polymerase also enhanced viability following H2O2 injury. Treatment with both NGF and an inhibitor of this enzyme resulted in a greater reduction of H2O2 toxicity than was observed with either agent alone. These data suggest that NGF protection is multifactorial and that a significant component of the NGF effect is due to its regulatory role in the metabolism of pyridine nucleotides.

13C NMR studies of glucose metabolism in human leukemic CEM-C7 and CEM-C1 cells.

Glucose metabolism of human leukemic cell lines CEM-C7 and CEM-C1 was investigated in vivo by 13C NMR using 13C-labeled glucose. Exact knowledge of glucose concentration, cell count, and cell viability of the cell suspensions made it possible to analyze glucose metabolism in detail. In both cell lines aerobic glycolysis accounts for virtually all glucose consumption. The use of D-[13C2]glucose provided a simple method to measure the glucose flux through the pentose phosphate pathway as 9% (CEM-C1) and 11% (CEM-C7) of glucose channeled into glycolysis. The dexamethasone-sensitive CEM-C7 cells consume glucose at a rate about 50% higher than the dexamethasone-resistant CEM-C1 cells. It is shown that this higher consumption correlates with a larger size of the CEM-C7 cells. Therefore in CEM cells the development of drug resistance does not seem to involve related changes in cell energetics.

31P nuclear magnetic resonance studies of growth inhibition and dexamethasone resistance in human leukemic cells.

31P Nuclear magnetic resonance spectra of perchloric acid extracts from wild-type human leukemic CEM-C7 cells and the dexamethasone-resistant CEM-C1 mutant reveal significant differences in concentrations of phospholipid precursors and ATP+, which indicate metabolic differences between these two cell lines. At high cell concentrations the CEM-C7 cells are growth inhibited, which is reflected by low phospholipid precursor levels, indicative of low phospholipid turnover. The CEM-C1 mutant does not exhibit this growth inhibition and has constant phospholipid precursor levels over the same cell concentration range. Dexamethasone causes phospholipid precursor and ATP levels in CEM-C7 to drop after 48 h, but spectra obtained for CEM-C1 cells continue to show high cell viability up to 72 h.

Cation nuclear magnetic resonance (NMR). 7Li- and 23Na-NMR results obtained with human erythrocytes.

A short discussion of practical results and theoretical aspects of nuclear magnetic resonance (NMR) on intracellular monovalent cations is presented. 7Li- and 23Na-NMR relaxation studies on human erythrocytes are described which indicate that lithium and sodium ions are essentially free inside the cell. However, there is a contribution to transverse relaxation originating from cations diffusing through electric field gradients imposed by the cytoskeleton, which is reflected in the NMR line shape. 39K-NMR results obtained by other authors for intracellular potassium are compared with the 7Li- and 23Na-NMR results presented here. The few 39K-NMR results obtained so far do not suggest that significant differences in dynamic behavior exist between intracellular potassium and sodium. 39K-NMR studies will, in the future, certainly enhance our understanding of the intracellular state of potassium ions.

Considerations for brain pH assessment by 31P NMR.

Recently in vivo NMR spectroscopy has been used to measure brain pH non-invasively. Both the inorganic orthophosphate (Pi) chemical shift (delta) and the difference between the chemical shifts of phosphocreatine (PCr) and Pi(delta delta PCr-Pi) have been proposed as indicators of brain pH. However, the precise delta of Pi may be difficult to determine under normoxic conditions as is the delta of PCr under hypoxic/ischemic conditions. Ideally one needs a NMR delta parameter that: (1) linearly changes between pH 6.0-8.0, (2) is either relatively unaffected or predictably affected by cations (e.g., Mg2+) other than H+, and that (3) comes from readily observable 31P NMR resonances whose delta's can be accurately assessed under all physiological conditions. Therefore, we undertook a systematic 31P NMR study of the pH and Mg2+ titration curves for 16 phosphorus-containing metabolites observed in brain by 31P NMR. On the basis of the titration curves, the delta delta's for PCr-Pi, phosphoethanolamine (PE)-Pi, and PCr-PE fulfill criteria (1) and (2), but not criterion (3). However, the delta delta of ATP gamma-alpha fulfills all three criteria and potentially provides information on the intracellular Mg2+ concentration.

31P nuclear magnetic resonance (NMR) spectroscopy of brain in aging and Alzheimer's disease.

Phosphorus-31 (31P) NMR is proving to be a powerful analytical method for investigating molecular/metabolic issues in neural tissues. Recent studies have demonstrated high levels of phosphomonoesters and phosphodiesters in mammalian brain, and revealed the influence of brain maturation, development, and aging on these levels. Preliminary studies in Alzheimer's disease have demonstrated elevated levels of phosphomonoesters and phosphodiesters in the areas of Alzheimer's brain which exhibit neuropathological changes. Moreover, phosphomonoester levels were also elevated in areas of Alzheimer's brain that were devoid of neuropathological changes. These findings suggest that the phosphomonoester elevations in Alzheimer's brain antedate changes in cellular morphology and structure. Abnormalities in protein kinase function could potentially explain these findings, as well as the reported hyperphosphorylation of tau protein in Alzheimer's brain. Recent studies from this laboratory suggest that aluminum could also be involved in the changes in phosphomonoesters and phosphodiesters.

Fluorine-19 nuclear magnetic resonance investigation of fluorine-19-labeled phospholipids. 1. A multiple-pulse study.

A multiple-pulse nuclear magnetic resonance technique has been used to measure the order parameter, SFF, at 40 MHz for dimyristoylphosphatidylcholine labeled with a difluoromethylene group at the 4-, 8-, or 12-position of the sn-2-acyl chain dispersed in water in the liquid-crystalline phase. The Carr-Purcell-Meiboom-Gill multiple-pulse sequence can resolve the homonuclear dipolar coupling between the two fluorine nuclei, thus making a direct determination of the order parameter, SFF, for the F-F internuclear vector possible. Other interactions, such as the 19F chemical shift anisotropy, heteronuclear dipolar couplings, and field inhomogeneity, which normally obscure the dipolar splitting, are effectively canceled. The order parameters obtained in this work compare well with those obtained by 19F nuclear magnetic resonance line-shape analysis of the 19F-labeled phospholipids reported in the following paper [Dowd, S. R., Simplaceanu, V., & Ho, C. (1984) Biochemistry (following paper in this issue)] as well as comparable SCD order parameters, determined for the deuterium-carbon internuclear vector of deuterium-labeled phospholipids [Oldfield, E., Meadows, M., Rice, D., & Jacobs, R. (1978) Biochemistry 17, 2727-2740]. The present results clearly show the usefulness of using nuclear magnetic resonance spectroscopy to investigate lipid-lipid and protein-lipid interactions, especially for those systems containing a difluoromethylene group in the acyl chain of a phospholipid molecule.

Fluorine-19 nuclear magnetic resonance study of 5-fluorotryptophan-labeled histidine-binding protein J of Salmonella typhimurium.

Fluorine-19 nuclear magnetic resonance has been used to investigate the histidine-binding protein J from Salmonella typhimurium. The protein has been labeled with fluorine-19 by growing the bacterial cells of a tryptophan auxotroph in the presence of 5-fluorotryptophan. Incorporation of up to 70% was achieved. The binding of L-histidine to the 19F-labeled protein is not affected by the isotopic labeling. The protein contains one tryptophan residue, giving rise to a single 19F resonance. Upon binding L-histidine to 19F-labeled histidine-binding protein J, the observed 19F resonance is shifted downfield by about 0.6 parts per million, indicating a conformational change of the protein molecule and a more hydrophobic environment for the 19F nucleus. Additional fluorescence experiments confirm that the tryptophan residue is located inside the hydrophobic core of the protein. 19F spin-lattice relaxation times of the 19F-labeled protein as a function of temperature show no difference between the free protein and the protein-histidine complex. However, the linewidth for the free protein is much larger than that of the protein-substrate complex. This can be explained by slow fluctuations between different conformations of the free protein molecule having slightly different 19F chemical shifts. Both with and without the substrate, the tryptophan residue is immobile inside the protein molecule as shown by the total disappearance of the 19F signal upon broadband irradiation at the 1H frequency. Also, the 19F spin-lattice relaxation times indicate that the protein is a rather rigid structure, in which rapid motions of the tryptophan residue on the time scale of 10(-8) second are not prominent.

An electron spin resonance spin-label study of lipophilin in oriented phospholipid bilayers.

Model membranes consisting of dimyristoyl phosphatidylcholine and a hydrophobic protein from bovine myelin, lipophilin, were studied using the cholesterol-resembling cholestane ESR spin label. Orientation of the membranes made it possible to deconvolute the spectra into two fractions, one of oriented spin labels reflecting phospholipid bilayer of high order, and one of isotropically tumbling spin labels ascribed to the lipid fraction surrounding the protein molecule (boundary lipid). This isotropic tumbling is different from the behavior of phospholipid molecules near the protein, which retain some degree of order, and indicates that the boundary lipid fraction in our model system forms a rather fluid environment for the protein. A nonlinear relation was found between protein concentration and amount of boundary spin labels. Addition of cholesterol decreases the amount of boundary spin labels. Both findings form evidence for a preferential binding of cholesterol by the membrane protein.

Lipid-protein interactions in model membranes from bovine brain white matter. An ESR spin label and electron microscopy study.

Lipid-protein model membranes, prepared from bovine brain white matter and containing all the lipids and Folch-Lees proteolipids, have been studied in macroscopically oriented multibilayers. To examine the lipid environment the membranes were spin labeled with the cholestane spin label (3'-spiro(2'=(N-oxyl-4',4'-dimethyl-oxazolidine))5alpha-cholestane) and a fatty acid spin label (4',4'-dimethyloxazolidine-N-oxyl derivative of 5-ketostearic acid). The ESR spectra exhibit two components arising from fairly well oriented and completely unoriented lipids. Up to a temperature of 55 degrees C the amount of oriented lipids is almost constant, being about 35%. At higher temperatures this percentage drops rapidly to zero. It is shown that the presence of unoriented lipids arises mainly from disrupted areas in the lipid bilayer structure. This is confirmed by electron miccroscopy and from an analysis of the temperature dependence of the order parameters of the spin labels. The presence of locally disrupted lipid parts in the bilayer is discussed in relation to the interaction of the brain white matter lipids with Folch-Lees protein.