Metabolism of alternative substrates and the bioenergetic status of EMT6 tumor cell spheroids.

In order to evaluate the ability of EMT6/Ro multicellular spheroids to utilize various pathways of energy production, (13)C and (31)P MRS have been employed to monitor the metabolism of glucose, glutamine, acetate and propionate. EMT6/Ro spheroids perfused with culture medium containing 5.5 mM glucose maintain stable levels of nucleotide triphosphates (NTP) and phosphocreatine (PCr) for up to 48 h, even in the absence of glutamine. The metabolism of 1-(13)C-glucose was almost entirely to 3-(13)C-lactate (88 +/- 12%, n = 7), even though the perfusion medium was equilibrated with 95% O(2). Labeling was also observed in other glycolytic metabolites, primarily alanine and alpha-glycerolphosphate. A low level of (13)C labeling in glutamate, indicative of mitochondrial oxidative metabolism (TCA cycle), was consistently detected when spheroids were perfused with 1-(13)C-glucose, almost exclusively in the C4 position of glutamate. Labeling of glutamate C2 and C3 was always less than 20% of the labeling in C4 and was usually undetectable. No evidence of adjacent carbon labeling in individual glutamate molecules (indicative of multiple cycles of label incorporation) was found, even in high-resolution (13)C NMR spectra of extracts from cells or spheroids. Despite the predominantly glycolytic metabolism of glucose, the mitochondrial substrate glutamine (2 mM, in the presence of < or =0.5 mM glucose from fetal bovine serum), supported stable levels of NTP and PCr in the tumor cells for up to 12 h. In the presence of 2.5 mM acetate, the bioenergetic status of cells in EMT6 spheroids declined slowly but measurably, and no incorporation of label from 2-(13)C-acetate into other metabolites was detected either in intact perfused spheroids or in high-resolution spectra of extracts. In contrast, when the anaplerotic TCA cycle substrate 3-(13)C-propionate replaced acetate, the high-energy phosphate levels in EMT6/Ro spheroids were somewhat reduced, but stabilized at a new lower level. Incubation of spheroids with 3-(13)C-propionate (with natural abundance glucose and glutamine) resulted in label detectable in the C2 and C3 of glutamate, but the primary labeled compound was methylmalonate, an intermediate in propionate metabolism. Addition of vitamin B(12), a cofactor for methylmalonyl CoA reductase, to the growth medium 24 h prior to perfusion with propionate resulted in the elimination of the methylmalonate resonance. A variety of 2- and 3-labeled metabolites were detected, including succinate, malate and glutamate. Labeling of C2 and C3 of lactate implicated cytoplasmic malic enzyme activity.

Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes.

The cellular uptake of liposomes is generally believed to be mediated by adsorption of liposomes onto the cell surface and subsequent endocytosis. This report examines the effect of liposome surface charge on liposomal binding and endocytosis in two different cell lines: a human ovarian carcinoma cell line (HeLa) and a murine derived mononuclear macrophage cell line (J774). The large unilamellar liposomes were composed of 1, 2-dioleolyl-sn-glycero-3-phosphatidylcholine with and without the addition of either a positively charged lipid, 1, 2-dioleoyl-3-dimethylammonium propanediol (DODAP), or a negatively charged lipid, 1,2-dioleolyl-sn-glycero-3-phosphatidylserine. In some experiments 5 mol % of the anionic PEG2000-PE or a neutral PEG lipid of the same molecular weight was added. HeLa cells were found to endocytose positively charged liposomes to a greater extent than either neutral or negatively charged liposomes. This preference was not lipid-specific since inclusion of a cationic cyanine dye, DiIC18(3), to impart positive charge in place of DODAP resulted in a similar extent of endocytosis. In contrast the extent of liposome interaction with J774 cells was greater for both cationic and anionic liposomes than for neutral liposomes. The greater uptake of positively charged liposomes by HeLa cells was also observed with sterically stabilized liposomes (PEG liposomes). Although the overall amount of endocytosis for all the PEG liposomes examined was attenuated relative to conventional liposomes, the extent of endocytosis was greatest for positively charged PEG liposomes, whereas negatively charged PEG2000-PE liposomes were hardly endocytosed by the HeLa cells. Incorporation of a neutral PEG lipid into liposomes permits the independent variation of liposome steric and electrostatic effects in a manner that may allow interactions with cells of the reticuloendothelial system to be minimized, yet permit strong interactions between liposomes and proliferating cells.

A versatile oxygenator and perfusion system for magnetic resonance studies.

A compact, reusable membrane oxygenator has been constructed for the perfusion of cultured cells and isolated organs. While the oxygenator was designed to be compatible with nuclear magnetic resonance (NMR) spectroscopy studies, it can also be used for any experiment which requires warming and oxygenation of perfusates. For the NMR studies, the oxygenator can be positioned at the opening of the magnet bore which allows oxygenation and warming of the perfusate immediately prior to delivery to the tissue, therefore eliminating problems with heat or oxygen loss which may occur with the long perfusion lines. (c) 1996 John Wiley & Sons, Inc.

31P NMR spectroscopic studies of the effects of cyclophosphamide on perfused RIF-1 tumor cells.

To determine whether direct cellular effects of chemotherapy are responsible for 31P NMR spectral changes observed in treated tumors in vivo, RIF-1 fibrosarcoma cells were examined in vitro before, during, and after treatment with 4-hydroperoxycyclophosphamide (4-HC), an activated form of cyclophosphamide. When RIF-1 cells were treated with 4-HC in a metabolically stable but nonproliferating state, the 31P NMR spectra were identical with those of untreated cells for up to 70 h. When actively proliferating RIF-1 cells were treated with 4-HC, the intensities of the nucleotide triphosphate resonances, which increased linearly during control cell growth, remained constant for 50 h or longer. These studies demonstrate that the bioenergetic improvement observed following treatment of RIF-1 tumors in vivo [S.-J. Li, J.P. Wehrle, S.S. Rajan, R.G. Steen, J.D. Glickson, and J. Hilton, Cancer Res. 48, 4736 (1988)] does not result from direct effects of cyclophosphamide metabolites on RIF-1 cell metabolism, but rather from indirect effects of treatment on tumor or host physiology.

Dimethyl methylphosphonate (DMMP): a 31P nuclear magnetic resonance spectroscopic probe of intracellular volume in mammalian cell cultures.

Dimethyl methylphosphonate (DMMP), when added to a suspension of erythrocytes, has been reported to have a lower frequency chemical shift inside of cells than outside. This work further investigates the same phenomenon in hollow-fiber bioreactor cultures of six mammalian cell lines and describes the application of DMMP as a measure of intra- versus extracellular volumes in mammalian cell cultures. No toxic effects of the DMMP were observed at the concentrations used here. The dependence of the shift of intracellular DMMP on intracellular protein content was shown to be similar for cultured mammalian and red blood cells. Also consistent with shifts in erythrocytes, an increase in the intracellular protein concentration due to a reduction in cultured cell volume increased the magnitude of the shift to lower frequency. Longitudinal relaxation (T1) values for intra- and extracellular DMMP were measured so that partially saturated DMMP peaks in 31P NMR spectra of mammalian cell cultures can be corrected to give the relative volumes of the intra- and extracellular compartments; this information provides a relative measure of culture growth. Intracellular volume measured by this method can also be used to quantify intracellular metabolites such as ATP during the growth of the culture. To explore the mechanism behind the intracellular shift, we have also addressed the three possible contributions to the chemical shift of DMMP: hydrogen-bonding interactions, magnetic susceptibility, and ionic strength. Data is presented which eliminates the latter two mechanisms and strongly supports the hypothesis that the observed intracellular shift is due to a reduction in hydrogen bonding between water and DMMP in the cytoplasm.

Gel-entrapment of perfluorocarbons: a fluorine-19 NMR spectroscopic method for monitoring oxygen concentration in cell perfusion systems.

Oxygenation is a major determinant of the physiological state of cultured cells. 19F NMR can be used to determine the oxygen concentration available to cells immobilized in a gel matrix by measuring the relaxation rate (1/T1) of perfluorocarbons (PFC) incorporated into the gel matrix. In calcium alginate gel beads without cells the relaxation rate (1/T1) of the trifluoromethyl group of perfluorotripropylamine (FTPA) varies linearly with oxygen concentration, with a slope of 1.26 +/- 0.15 x 10(-3) s-1 microM-1 and an intercept of 0.50 +/- 0.04 s-1. During perfusion with medium equilibrated with 95%/5% O2/CO2, changes in PFC T1s indicate that the average oxygen concentration was reduced from 894 +/- 102 microM in the absence of cells to 476 +/- 65 microM and 475 +/- 50 microM in the presence of 0.7 x 10(8) EMT6/Ro and RIF-1 murine tumor cells per milliliter of gel, respectively. The presence of 0.2 microliters of FTPA/ml of gel had no effect on the energy status of the cells as indicated by 31P NMR spectra. To calculate oxygen gradients within the beads from the average PFC T1 of the sample, a mathematical model was used assuming that oxygen is the limiting nutrient for cell metabolism and that the cellular oxygen consumption rate is independent of oxygen concentration. Data for EMT6/Ro cells were fit using experimentally determined perfusion parameters together with literature values for cell volume and oxygen consumption rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Design and application of NMR-compatible bioreactor circuits for extended perfusion of high-density mammalian cell cultures.

MR spectroscopy of cultured cells allows non-invasive analyses of the metabolism of cells with specific phenotypes under defined conditions. This technique can be used to investigate the intracellular metabolism of cells or extended to critically evaluate phenomena observed by in vivo MRS. In this paper, a cell maintenance system is described which allows MR analyses with unparalleled spectral resolution, S/N and stability. This system consists of a 25 mm diameter hollow fiber bioreactor and a supporting circuit. The hollow fiber reactor was chosen because it yields a large filling factor which can be perfused through defined volumes. The fibers were 300 microns diameter microporous (0.2 micron) cellulose acetate/cellulose nitrate membranes with high porosity, which allow bulk convective flow throughout the extracapillary space. This flow (Starling flow) is necessary to disrupt steady-state gradients in substrates and waste products. In many respects, the design of the supporting circuit is more important than the bioreactor itself, since it provides the reactor with the proper chemical and physical environment. Hence, this circuit can be applied to a variety of bioreactor configurations. The circuit consists of a hollow fiber oxygenator and a bleed-and-feed system housed in a temperature-controlled cabinet. Culture of mammalian cells in this reactor yields 31P spectra which have excellent spectral and temporal resolution. At confluence, endogenous 31P line widths were typically < 10 Hz (at 162 MHz) and well resolved spectra were obtained in < 30 s.

31P NMR spectroscopic study of the effects of gamma-irradiation on RIF-1 tumor cells perfused in vitro.

In order to examine the mechanisms underlying radiation-induced changes in phosphorus metabolite levels observed in RIF-1 tumors in vivo, RIF-1 cells in culture were perfused for up to 70 h following gamma-irradiation with 0-25 Gy and monitored continuously by 31P NMR spectroscopy at 8.5 T. Cells immobilized in the sample volume by incorporation into calcium alginate beads were bioenergetically stable, but did not replicate at the cell density used. Following an initial increase in PCr and NTP, which occurred in both control and irradiated cells, a dramatic decline in high-energy phosphates was detected beginning 24-30 h after irradiation with 15 or 25 Gy. In contrast, unirradiated cells or cells treated with 10 Gy remained metabolically stable for up to 72 h. The metabolic changes induced by irradiation of the cultured cells, which reflected cell death and lysis, were distinctly different from those observed in RIF-1 tumors in vivo during the same postirradiation time interval--an increase in high-energy relative to low-energy phosphates. This suggests that the spectral changes in vivo do not result from direct modification of cellular energy metabolism by radiation injury.

In vivo 31P nuclear magnetic resonance spectroscopy of subcutaneous 9L gliosarcoma: effects of tumor growth and treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea on tumor bioenergetics and histology.

In vivo 31P nuclear magnetic resonance spectroscopy was used to examine the bioenergetics of the rat 9L gliosarcoma during untreated growth and in response to chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea. Tumor growth was associated with a decline in the phosphocreatine and nucleoside triphosphate resonances, consistent with an increase in tumor hypoxia during untreated growth. Following chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea (10 mg/kg), tumor levels of phosphocreatine and nucleoside triphosphate rebounded while the level of inorganic phosphate in the tumor declined. Histological comparison of treated and untreated tumor sections 4 days posttreatment showed that the treated tumor had a lower proportion of necrotic cells, a higher proportion of viable cells, and a 5-fold higher level of interstitial space than the control tumor.

Decarboxylation of alpha-difluoromethylornithine by ornithine decarboxylase.

The mechanism of inactivation of rodent ornithine decarboxylase by alpha-difluoromethylornithine (DFMO) was studied using the inhibitor labelled with 14C in both the 1 and the 5 positions. [1-14C]DFMO was a substrate and was decarboxylated by the enzyme yielding 14CO2. A radioactive metabolite derived from [5-14C]DFMO was bound to the enzyme, and the extent of binding paralleled the irreversible inactivation of ornithine decarboxylase. The partition ratio of decarboxylation to binding was approx. 3.3. These results provide support for the postulated mechanism of action of DFMO [Metcalf, Bey, Danzin, Jung, Casera & Vevert (1978) J. Am. Chem. Soc. 100, 2551-2553], in which enzymic decarboxylation of the inhibitor leads to the generation of a conjugated imine, which then alkylates a nucleophilic residue on the enzyme.

Effect of 1,3,6-triaminohexane and 1,4,7-triaminoheptane on growth and polyamine metabolism in SV-3T3 cells treated with 2-difluoromethylornithine.

The possibility that one or both of the synthetic triamines, 1,3,6-triaminohexane and 1,4,7-triaminoheptane, could substitute for the naturally occurring polyamines in the growth of SV-3T3 cells was investigated. It was found that these triamines did lead to a restoration of growth in cells in which spermidine content had been depleted by exposure to the ornithine decarboxylase inhibitor 2-difluoromethylornithine. This resumption of a normal growth rate occurred prior to the reduction in the content of cellular decarboxylated S-adenosylmethionine, suggesting that this nucleoside (which increases in concentration several hundred-fold in cells treated with 2-difluoromethylornithine) does not cause the reduction of cell growth. However, unlike the increase in cell growth brought about by spermidine, which continued indefinitely, the increase produced by 1,3,6-triaminohexane or 1,4,7-triaminoheptane was transient. Cell growth in the presence of 2-difluoromethylornithine and these triamines stopped after about three or four population doublings. This corresponded to the time at which the intracellular spermine content of the cells was reduced to values less than 20% of normal. It is suggested that the increased growth rate of spermidine-depleted cells in response to these triamines is due to their uptake into the cell and ability to displace spermine from intracellular sites, thus making spermine available to fulfill the polyamine function(s) essential for growth. These results indicate that the naturally occurring polyamines spermidine or spermine are essential for continued cell growth and cannot be replaced by analogues containing only primary amino groups.

Synthesis, absorption, and toxicity of N1,N8-bis(2,3-dihydroxybenzoyl)spermidine, a potent iron chelator.

A synthesis of N1,N8-bis(2,3-dihydroxybenzoyl)spermidine, a potent iron chelator, is developed employing N4-benzylspermidine as the starting material. Both the toxicity and the absorption properties of this compound are evaluated, further supporting its potential as a clinical iron chelator.