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Open Full Page [CANCER RESEARCH 46, 3768-3774, August 1986] 31PNuclear Magnetic Resonance Study of a Human Colon Adenocarcinoma Cultured Cell Line1 Franck Desmoulin, Jean-Philippe Galons, Paul Cantoni, Jacques Marvaldi, and Patrick J. Cozzone2 Laboratoire de Biologie Physicochimique, Institut de Chimie Biologique, Universitéd'Aix-Marseille, Place Victor Hugo 13003 Marseille (France) ABSTRACT Saccharomyces cerevisiae (14-16). Investigation of mammalian cell lines has received only scant appraisal because of several •"Pnuclear magnetic resonance (NMR) spectroscopy has been used to technical difficulties. Work has been limited to a few cell types, monitor the energy metabolism in a human colon adenocarcinoma cell essentially HeLa cells (17, 18), Ehrlich ascites tumor cells (19), line (HT 29). NMR spectra were recorded at 80.9 MHz on approximately 2.5 x 10" cells continuously perfused with culture medium within a 20- and normal and transformed fibroblasts (20, 21). In addition, a detailed analysis of the metabolite content of radiation-induced iiini NMR sample tube. Typical NMR spectra display a series of well-resolved resonances fibrosarcoma cells by using multinuclear NMR has been re assigned to nucleoside triphosphates (mainly adenosine S'-triphosphate), cently published (22). uridine diphosphohexose derivatives (uridine 5'-diphosphate-A/-acetyl- A major difficulty to overcome in the study of perfused cells glucosamine, uridine 5/-diphosphate-Ar-acetylgalactosamine, uridine 5'- is the preservation of physiological conditions in the NMR diphosphate-glucose), intra- and extracellular inorganic phosphate, and sample tube where the accumulation of metabolite by-products phosphomonoesters (mainly phosphorylcholine and glucose 6-phos- and the required bubbling of gas may affect the viability of the phate). Measurement of phosphorylated metabolite concentrations from cells. Various perfusion techniques have been developed to the intensity of NMR signals is in good agreement with the results ensure the protection of cell metabolic integrity (23-26). These provided by conventional biochemical assays. techniques have been discussed in the recent review by Evan- 31P NMR allows to follow noninvasively the effect of anoxia on HT 29 cells. The results indicate that the cells are able to maintain about ochko et al. (27) on the application of NMR to the study of isolated tumor cells, excised tumors, and in vivo tumors. 60% of their initial nucleoside triphosphate level after 2 h of anaerobic In this report, we present results of the 3IP NMR study of a perfusion. Cells accumulate inorganic phosphate during anoxia and the intracellular-extracellular pH gradient increases from 0.5 in well-oxygen human colon adenocarcinoma cell line (HT 29 line) established ated cells to more than l pH unit under anoxic conditions. The value of in 1964 by J. Fogh (28). All NMR measurements were carried intracellular pH of well-oxygenated HT 29 cells is 7.1. out on cell preparations constantly perfused with the culture The effect of glucose starvation upon energy metabolism has also been medium while in the NMR sample tube. The perfusion medium examined in real time by NMR: a rapid decline of adenosine 5'-triphos- provided the cells with various substrates and oxygen while phate down to 10% of the initial value is observed over a period of 2 h. removing damaging metabolite by-products. The perfusion sys In contrast, the level in uridine diphosphohexoses reaches a new steady tem was kept at constant temperature in order to maintain the state value representing 60% of the initial one. Refeeding the cells with 25 HIMglucose leads to a dramatic drop of internal pH reflecting the cells under the steady state conditions required for NMR signal activation of the glycolytic pathway. averaging. Intracellular levels of phosphate metabolites and external and cytosolic pH values were simultaneously measured from the NMR spectra of well-oxygenated cells or hypoxic INTRODUCTION cells. The effect of glucose starvation which has been shown to alter the energetic metabolism of HT 29 (29, 30) has also been Over the past few years, development of instrumental tech nology in NMR3 has permitted the observation of phosphorus investigated in this NMR study. The results illustrate the pos sibility offered by NMR to analyze in a noninvasive manner containing metabolites in living systems. The technique has precise metabolic variations of cultured cell lines as a valuable been extensively applied to the study of either perfused organs contribution to the understanding of specific metabolic events or suspensions of whole cells. More recently, it has been ex related to cell growth and cell differentiation. tended to whole animals and humans because of the availability of wide-bore horizontal magnets and the development of meth ods which provide a spatial localization of the observed metab MATERIALS AND METHODS olites (1-5). 3IP NMR spectroscopy provides a unique way of Cell Culture and Growth Conditions. HT 29 cells were routinely simultaneously measuring extra- and intra-cellular pH on the grown as monolayers in ISO-cm2 sterile plastic flasks (Corning) in basis of the chemical shift, and determining stationary intracel Dulbecco's modified Eagle's medium containing 25 HIMglucose and lular concentrations of phosphate metabolites and their fluctua supplemented with 10% fetal calf serum. The culture medium was tions under a variety of metabolic events. So far, the majority replaced every 2 days. Cells were cultured for 2 weeks in an incubator of the studies on cells by 3IP NMR has been dedicated to at 37°Cin a humidified atmosphere containing 95% air and 5% COz prokaryotic cells including Escherichia coli (6-8), Staphylococ- until they reached confluency. They were harvested at confluency after cus aureus (9), Tetrahymena (10), Rhodopseudomonas sphae- a 5-min treatment at 20°Cwith 0.05% Trypsin-0.5 HIM EDTA in a Ca2+- and Mg2+-free 150 mM phosphate buffer, pH 7.2. For NMR roides (11), and yeast, Acanthamoeba castellani (12, 13), and experiments, trypsinized cells were pelleted by centrifugation. The Received 12/4/84; revised 1/21/86; accepted 4/17/86. pellet was washed twice with fresh culture medium and the cells were 1This work was supported by the Centre National de la Recherche Scientifique resuspended in 1 ml of the same medium to give a final count of 2.5 x (UÀ202) and grants from the Ministère de la Recherche et de la Technologie 10*cells confined into a 0.7- x 10-cm flat dialysis membrane (Spectra- (GBM 83-M-0802, 84-M-0804 and 85-M-0564), the CNAMTS (Contrat de Recherche Externe 1983-1986), théMinistèrede l'Education, and théFondation por). Viability of cells was tested by their ability to exclude trypan blue. Typically, viability values of 90-95 and 70-90% were measured before pour•'Towhome la Recherche requests Médicale. for reprints should be addressed. and after the NMR experiment. Similar results on cell viability were 'The abbreviations used are: NMR, nuclear magnetic resonance; DPDE, obtained by the 51Crrelease technique. diphosphodiesters (mainly uridine diphosphohexose or diphosphohexosamine derivatives); G-6-P, glucose 6-phosphate; NTP. nucleoside triphosphates; P¡™, Cell Extracts. After removal of the medium from the flasks, the cell intracellular Pâ;P", extracellular P¡;NDP, nucleoside diphosphates. layers were rinsed once with ice-cold 0.9% NaCl solution and then were 3768 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1986 American Association for Cancer Research. 31P NMR OF HUMAN COLON ADENOCARCINOMA CELLS frozen by flotation of the flask on liquid nitrogen. The frozen cell layer MDPA Pi was scraped with 2.4 ml of ice-cold 0.9 M HC1O4. The mixture was kept 10 min at 0°Cand then centrifuged for 5 min at 5000 x g and 4°C.The clear supernatant was neutralized with 5 N KOH and the NTPa NAD solution centrifuged for 10 min at 8000 x g and 4°C.Extracts were + + stored at -70°C. NDPa DPDE NMR Spectroscopy and Cell Perfusion. 31PNMR spectra of perfused HT 29 cells were recorded on a Nicolet NT200 wide-bore spectrometer operating at 80.9 MHz. Each spectrum corresponds to the Fourier NTP/3 transform of the sum of 600 free induction decays (10-min accumula tion). Flip angle of 60°with repetition time of 1.2 s was selected in order to optimize the signal-to-noise ratio and to minimize the satura tion of the signals arising from intracellular metabolites and I',". Under those conditions, a partial saturation (15%) of external P¡wasobserved. All chemical shifts were expressed as ppm relative to 85% phosphoric acid, using as secondary reference the signal at 20.8 ppm of hexachlo- rocyclotriphosphazene contained in a capillary (31) for in vivo experi ments. In some cases, the glycerophosphorylcholine signal at 0.50 ppm was used as internal reference at pH 7.1 and 37°C. The dialysis membrane containing the cell suspension as described above was folded 4-5 times in order to fit into a 20-mm NMR sample r ' tube. The 1-ml solution bathing the cells was continuously renewed by 20 10 -10 -20 PPM Fig. 1. Proton-coupled 31PNMR spectrum (80.9 MHz) of the perchloric acid perfusing the system with 180 ml of culture medium. The volume of extract of HT 29 cells. The solution contains 1 mM methylenediphosphonic acid fresh medium inside the NMR tube was around 10 ml. The perfusate (MDPA) as internal reference. The spectrum corresponds to the average of 10 was maintained at pH 7.5 and oxygenated with a 5% CO2-95% air blocks of 624 scans each. The signal-to-noise ratio was improved by multiplying mixture using a membrane oxygenator (Scimed). the free induction decay with an exponential (5 Hz line broadening). The extract 31P NMR spectra of the cell perchloric acid extracts were recorded (8 ml total volume) was prepared from 400 x IO6 cells and corresponds to a protein concentration of 20 mg/ml.
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