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Cytochalasin B: Inhibition of Glucose and Glucosamine Transport* (Deoxyglucose/Novikoff Rat Hepatoma Cells/Cell Membrane) RICHARD D

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Cytochalasin B: Inhibition of Glucose and Glucosamine Transport* (Deoxyglucose/Novikoff Rat Hepatoma Cells/Cell Membrane) RICHARD D Proc. Nat. Acad. Sci. USA Vol. 69, No. 6, pp. 1430-1434, June 1972 Cytochalasin B: Inhibition of Glucose and Glucosamine Transport* (deoxyglucose/Novikoff rat hepatoma cells/cell membrane) RICHARD D. ESTENSEN AND PETER G. W. PLAGEMANN Departments of Pathology and Microbiology, Medical School, University of Minnesota, Minneapolis, Minn. 55455 Communicated by Robert A.'Good, February 22, 1972 ABSTRACT Cytochalasin B has been shown to potently indicate that transport may be the rate-limiting step in the inhibit the transport of glucose, deoxyglucose, and glucos- metabolism of various substances. amine by Novikoff hepatoma cells in suspension culture without affecting their intracellular phosphorylation and metabolism. Deoxyglucose transport is inhibited by cyto- MATERIALS AND METHODS ehalasin B in a simple competitive manner. Although this Novikoff rat hepatoma cells (subline NlS1-67), propagated in inhibition is not sufficient to explain the biological action of the drug on cytokinesis, it does explain earlier observa- suspension culture (18, 19), were suspended to 2 X 106 cells/ tions on inhibition by cytochalasin B of the incorporation ml in basal medium 42 (18), or glucose-free basal medium 42 of glucose and glucosamine, and probably of other extra- (17), or HEPES-buffered, glucose-free basal medium 42 (17). cellular precursors, into macromolecules by various Suspensions of cells were supplemented with CB by addition types of cells. of the appropriate volume of an 8.2-mM stock solution of CB in Cytochalasin B (CB) was originally noted by Carter (1) to dimethyl sulfoxide or absolute ethanol. Addition of equivalent inhibit cytoplasmic division (cytokinesis) without inhibiting volumes of the solvents had no effect on the various processes nuclear division (karyokinesis), to inhibit cell movement, and investigated. to cause dramatic changes in cell shape. Subsequent reports The incorporation of uniformly labeled 2-deoxy-D-'14C]- have indicated a wide range of biological effects, including glucose or D-[14C]glucose (International Chemical and Nuclear inhibition of phagocytosis (2-4), pinocytosis (5), secretion of Corp.), or D-[1-14C]glucosamine (Amersham/Searle) into thyroid (6) and growth hormone (7), and the inhibition of total cell material (acid-soluble plus acid-insoluble) or into morphogenesis (8, 9). It has been suggested that the effects acid-insoluble material (macromolecules) was determined as of CB may be due to an inhibition of microfilament function described (16, 17, 20). The acid-soluble pools were extracted (10-12). While the effects on the morphology of microfila- from labeled cells with perchloric acid, and the acid-extracts ments is apparent, such effects may not account for the pri- were analyzed by ascending paper chromatography with mary action of the drug (13). Direct interaction of CB with solvent 28 (17, 20). The conversion of D-[14C]glucose to ex- the cell membrane, on the other hand, could account for all tracellular lactate was determined by chromatography of the biological effects of the chemical. samples of the culture fluid (17), and 14CO2 production was One of us (14) has previously observed that CB inhibits measured by incubation of the cell suspensions in 14CO2 the incorporation of uridine and thymidine, but not of choline, collector flasks (17). The phosphorylation of glucose by in into macromolecules by cultured Novikoff rat hepatoma cells. vitro preparations from NlS1-67 cells was measured as de- Preliminary experiments indicated that glucose incorporation scribed (17). is also inhibited. Similarly, the inhibition by CB of phagocy- RESULTS tosis is accompanied an inhibition of gly- by leukocytes by Effect of CB on glucose and deoxyglucose colysis and respiration (3, 4) as measured by the formation transport and metabolism of lactate and CO2, respectively, from glucose. Glucosamine incorporation into mucopolysaccharides by various types of The results in Fig. 1A illustrate that the incorporation of cells is also inhibited by CB, and it has therefore been sug- ["4C]deoxyglucose into total cell material was markedly in- gested that CB inhibits mucopolysaccharide synthesis (15). hibited by GB. Chromatographic analyses of acid-extracts However, the finding that nucleoside incorporation into nu- from labeled cells indicated that CB caused a uniform reduc- cleic acids by Novikoff cells is inhibited by CB without sig- tion in the amounts of intracellular radioactivity associated nificantly affecting the increase in cell mass (14), suggested with free deoxyglucose as well as of its phosphorylated de- that CB might inhibit the incorporation of extracellular metab- rivatives (Fig. 1B). On the other hand, CB, at a concentra- olites into macromolecules by inhibiting their uptake into the tion of 160 ,M, had no significant effect on the phosphoryla- cells. This conclusion is supported by the present results and tion of deoxyglucose or glucose by an in vitro preparation is in agreement with results of previous studies (16, 17) that (not shown). Evidence has been presented elsewhere (17) that indicates that deoxyglucose is taken up by NlS1-67 cells by facilitated diffusion with, a Km between 1 and 2 mM, Abbreviation: CB, cytochalasin B. and that the rate of incorporation of deoxyglucose into total * This is no. V in a series of papers. No. IV of the series is Becker, cell material is a valid measure of the transport rate. The E. L., Davis, A. T., Estensen, R. D. & Quie, P. G., J. Immunol., Lineweaver-Burk plots in Fig. 2 of the initial rates of deoxy- 108, 396. glucose transport in the presence and absence of CB indicate 1430 Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Transport Inhibition by Cytochalasin B 1431 that CB inhibited deoxyglucose transport in a simple com- petitive manner. The Ki for the inhibition (about 1 MAM) was over 1000-fold lower than the Km for deoxyglucose transport. At a concentration of 100 MAM in the medium, the incorpora- tion of [14C]glucose into total cell material (acid-soluble plus - 20- acid-insoluble) and into acid-insoluble material (macro- molecules) and its conversion to CO2 was inhibited about 85% U 15- by 4.1 MAM CB, and its conversion to lactate about 95% (Fig. 01 3, A-C). In view of the results with deoxyglucose described already and the fact that glucose and deoxyglucose appear to be taken up by the same transport system (17), it seems -I 2 3 S likely that CB inhibited glucose metabolism by inhibiting its 5- transport into the cells. At a concentration of 10 mM, at which most glucose uptake is by simple diffusion, the in- corporation of [14C]glucose into cell material and CO2 was only slightly affected, while lactate production was reduced about 65% (Fig. 3, D-F). The apparent greater effect of CB V/DEOXYGLUCOSE (mM) on lactate production than oln the incorporation of glucose FIG. 2. Lineweaver-Burk plots of the initial rates of deoxyglu- into cell material or CO2 mimicked the effect of lowering the cose transport in the absence and presence of CB. Portions of a glucose concentration in the medium (17) and simply re- suspension of 2 X 106 cells/ml of glucose-free basal medium 42 flected the reduced uptake of glucose by the cells. were mixed with CB to the indicated concentrations and im- mediately thereafter, 10-ml samples of each suspension were Effect of CB on glucosamine incorporation into supplemented with 0.2, 0.33, 0.5, or 1 mM 2-deoxy-D-[14C]glucose acid-soluble pool and macromolecules (257 cpm/nmol) or 4 mM 2-deoxy-D-[14C]glucose (60 cpm/nmol). The results in Fig. 4 A and B show that CB at a concentration The suspensions were incubated on a gyrotory shaker at 370, of 4.1 MAM also markedly inhibited the incorporation of [14CJ- and after 5 min, duplicate 1-ml samples of each suspension were glucosamirle (at a concentration of 100 AMM) into total cell analyzed for radioactivity in total cell material. These values material and were considered estimates of the initial transport rates (17). The acid-insoluble material (macromolecules). apparent Km and Ki values were estimated from the slopes of the Glucosamine was incorporated into macromolecules only Lineweaver-Burk lines. 0, 0 MM CB, Km 1.8 mM; 0, 4.1 relatively slowly, and most of the AM glucosamine taken up by CB, Ki - luM; A, 8.2,uM CB, Ki-1MM. Vb x the cells accumulated intracellularly as glucosamine-6- 2 f. phosphate (Fig. 4C). The incorporation of glucosamine into 0D both total cell material and acid-insoluble material and into -J w the various intracellular acid-soluble components was in- 0 hibited by CB to about the same extent (compare Fig. 4, A, a- B, and C). This finding suggests that the incorporation of glu- Sw 4!- cosamine into macromolecules was also solely a consequence 0 of an inhibition of glucosamine uptake by CB. The results of 0 IL' the pulse-chase experiment in Fig. 4D support this conclusion. 0 0 2 Cells were first incubated with [4GC]glucosamine at 200. U 0I- Macromolecular synthesis is largely inhibited at this tem- 0 a.E K perature. The cells were collected by centrifugation, washed 0 free of residual [14C]glucosamine, and further incubated in 0'a fresh medium at 370 with and without CB. The results in Fig. 4D show that CB had no significant effect on the in- corporation of the labeled intracellular acid-soluble glucos- MINUTES DISTANCE FROM ORIGIN (CM) amine derivates into macromolecules. FIG. 1. Effect of CB on deoxyglucose transport. Samples of a suspension of 2 X 106 cells/ml of glucose-free basal medium 42 Persistence of inhibition of deoxyglucose transport by CB and reversibility of effect were supplemented with O(O-O), 0.41 (0 0) or 4.1 juM (A A) CB or with 0.05% (v/v) dimethylsulfoxide (/-A), In the experiment illustrated in Fig.
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