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 competitive 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 incorporation of [14C]glucose into total cell material (acid-soluble plus - 20- acid-insoluble) and into acid-insoluble material (macromolecules) 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 incorporation 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 resuspension 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 immediately 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 incorporation 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. 5A, portions of a suspen- and immediately thereafter with 40,MM 2-deoxy-D-['4C]glucose sion of cells were supplemented with various concentrations (7 cpm/pmol). (A) At various times of incubation at 370 du- of CB, and after 90 and 240 min of incubation, samples of plicate 0.5-ml samples of each suspension were analyzed for radio- each portion were monitored for the incorporation of [14C]- activity in total cell material. All points represent averages of the duplicate samples. (B) At 120 min of incubation, acid-extracts deoxyglucose. The results indicate that deoxyglucose trans- were prepared from samples of 2 X 107 cells of each suspension port was inhibited by CB to about the same extent whether and a 50-,ul sample of each acid-extract was chromatographed on it was added at the same time as CB or 90-240 min later. The 3MM Whatman paper with solvent 28. The graph is a composite results indicate that the effective concentration of CB did of the radioactivity profiles of the acid-extracts from CB-treated not decrease during the 300 min of incubation with the cells. and untreated cells. In contrast, 12-15% of the 30 MM deoxyglucose added to the Downloaded by guest on September 29, 2021 1432 Ce!-Biology: Estensen and Piagein Proc. Nat. Acad. &i. USA 69 (187B) medium was accumulated by the control cells during the 60- min incubation periods. Thus the data indicate that CB was

tof 0 j Iz oa w WI A,a E 0 c 0 a rc- £ 0 MINUTES

3 D. PULSE-CHASE

x I, uIL 4I MINUTES WI Q ::CB (jtLM) 3 ;O,@fro I "I. ':I -a WI I A N WI ea b I **.II A A ifl, ,. J 0 9 w VA WI 60 0 60 0 DISTANCE FROM ORIGIN MINUTES 0 I-U (CM)

0 U FIG. 4. Effect of CB on n-[14C]glucosamine uptake and metabolism. (A-C) Samples of a suspension of 2 X 106 cells/ml of glucose-free basal medium 42 were supplemented, where indicated, with 4.1 AM CB and with 100 MM D-[14C]glucosamine MINUTES (1.9 cpm/pmol). At various times of incubation at 370, duplicate FIG. 3. Effect of CB on glucose metabolism at glucose con- 0.5-ml samples of each suspension were analyzed for radioactivity centrations of 100 MM (A-C) or 10 mM (D-F) in the medium. in total cell material (A) or acid-insoluble material (B). All points (A-C) and (D-F) represent separate experiments. In each, one represent averages of the duplicate samples. After 120 min of portion of a suspension of 2 X 106 cells/ml of HEPES-buffered, incubation, acid-extracts were prepared from 1.6 X 107 cells, and glucose-free basal medium 42 was supplemented with 4.1 MAM CB 50 Ml of each acid-extract were chromatographed on 3MM (*-_, A -), and another portion remained untreated Whatman paper with solvent 28 (C). (C) is a composite of the (0-0-, A). Then D-['4C]glucose was added to both radioactivity profiles of acid-extracts from the CB-treated portions: to (A-C), 100 M&M (5 Ci/mol); to (D-F) 10 mM (0.1 (- -) and untreated (O-O) cells. (D) Cells were sus- Ci/mol). A sample of each suspension was incubated in 14CO2 pended to 2 X 106 cells/ml in glucose-free basal medium 42 collection flasks. The scintillation vials were replaced at 30-min that had been equilibrated at 20° and contained 2.5 MM D- intervals and analyzed for radioactivity. The points in (C) and [14C]glucosamine (50 Ci/mol). The suspension was incubated at (F) represent accumulative values. The remainders of each sus- 200 and monitored for radioactivity in total cell material (Circles) pension were incubated separately, and at various times du- and acid-insoluble material (triangles). After 55 min of incubation, plicate 0.5-ml samples were analyzed for radioactivity in total the remaining cells were collected by centrifugation and washed cell material and acid-insoluble material (B) and (E). All points once in cold (40) glucose-free basal medium 42. Equal portions represent averages of the duplicate samples. Lactate production of the cells were then suspended (0 time) to the original cell (A) and (D) was estimated by chromatography of 2-,ul samples density in warm (37°) glucose-free basal medium 42 either with of the culture fluid on 3MM Whatman paper with solvent 36. or without CB. The suspensions were further incubated at 370 The line designated Total in A represents the total amounts of and monitored for radioactivity in total cell material and acid- glucose converted to lactate, total cell material, and C02. All insoluble material. All points represent averages of duplicate 0.5- values are expressed in glucose equivalents. ml samples. Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Transport Inhibition by Cytochalasin B 1433

not accumulated extensively by the cells nor was it degraded during incubation with the cells. The fact that deoxyglucose transport is inhibited by CB in a competitive manner suggests that CB binds reversibly to the transport sites. This was also indicated by the results of removing cells from the CB-containing media and sus- pending them in fresh medium. When cells exposed for 10 or 55 min to 4.1 MM CB were collected by centrifugation and either washed once in basal medium 42 or resuspended in basal medium 42 without washing, they were able to incor- porate ['4C]deoxyglucose at almost the rates exhibited by the FIG. 5. Persistence of CB effect on deoxyglucose transport (A) untreated cells (Fig. 5B). and reversibility of inhibition (B). (A) Portions of suspension of 2 X 106 cells/ml of glucose-free basal medium 42 were supple- DISCUSSION mented with the indicated concentrations of CB (0 time) and incubated at 37°. At 0, 90, and 240 min, samples of each suspen- The present results demonstrate that CB competitively in- sion were supplemented with 30 ,uM 2-deoxy-D-['4C]glucose hibits, with high efficiency, the transport of glucose and de- ([14C] DG; 6 cpm/pmol), and further incubated at 37°. At various oxyglucose and probably of glucosamine. The affinity of CB times thereafter, duplicate 0.5-ml samples were analyzed for radio- for the glucose transport system is at least three orders of activity in total cell material. 0, 0uM CB; *, 0.41 ,uM CB; A, 4.1 magnitude higher than that of the substrate itself. Other ex- ,uM CB. (B) Two samples of an exponential phase culture in periments (Estensen and Plagemann, in preparation) have medium 67 (2.4 X 106 cells/ml) were supplemented with 4.1 ,uM shown that CB also competitively inhibits uridine and thy- CB and incubated at 370 for 10 min. The cells were collected by midine transport but has no effect on choline Since centrifugation. The cells of one sample were directly suspended transport. (0 time) to 2 X 106 cells/ml in glucose-free basal medium 42 glucose, uridine, and thymidine are transported by different (A A\) containing 40MM 2-deoxy-D-[14C]glucose (8 cpm/pmol), systems (refs. 17 and 21; and Plagemann, in preparation), CB and those of the other sample were suspended after an additional interacts with at least three transport systems. washing in glucose-free basal medium 42 (A A). At the same The results of the pulse-chase experiment clearly demon- time, other samples of untreated cells were suspended to 2 X strate that CB has no effect on the incorporation of glu- 106 cells/ml in glucose-free basal medium 42 with ( - 0) or cosamine into macromolecules, at least for the first 30-60 without (0-O) 4.1 ,uM CB and containing [14C]deoxyglucose. min after addition of the chemical to the cells. Results from The suspensions were incubated at 370 and at various times, du- similar pulse-chase experiments with uridine and thymidine plicate 0.5-ml samples of each suspension were analyzed for radio- have shown that CB has no effect on the incorporation of activity in total cell material. After 55 min of incubation, samples ribonucleotides or deoxyribonucleotides into RNA or DNA, were removed from the suspension incubated with ["4C]deoxyglucose plus CB (0 *). The cells were collected by centrifuga- respectively. Thus the inhibitions by CB of the incorporation tion and suspended to the original cell density directly in basal of extracellular precursors into macromolecules by NlSl-67 medium 42 containing ['4CGdeoxyglucose (V-V) or after a cells seem to be solely attributable to an inhibition of their single washing in glucose-free basal medium 42 (V V). These transport into the cell. Other transport inhibitors, like Persan- suspensions were also incubated at 370 and monitored for radio- tine (16, 17) or phenethyl alcohol (22), have a similar effect. activity in total cell material. All points represent averages of In view of this observation with NlS1-67 cells, it seems likely duplicate samples. that the inhibition by CB of glucose metabolism by leukocytes (3, 4) and of the incorporation of glucosamine into mucopolysaccharides by various types of cells (15) is also binding of CB to various membrane transport sites may in- simply a consequence of the effect of CB on the transport of terfere with other biological functions of the cell membrane. the glucose and glucosamine into the cell. It seems unlikely, In the absence of specific knowledge of the molecular processes however, that the various other biological effects of CB, such involved in cytokinesis, cell movement, or phagocytosis, this as the inhibition of cytokinesis, cell movement, or phagocy- question cannot be answered at present. It might also be tosis, can be attributed to the observed interference with the possible, however, that the effects of CB may be due to the various transport systems. For instance, incubation of NiS1- inhibition of the transport of other essential substances, such 67 cells in glucose-free medium rapidly inhibits both nuclear as ions, into or out of the cells. This question is under further and cytoplasmic division, and incubation of the cells in com- investigation. The finding that the inhibitions of cytokinesis plete basal medium 42 in the presence of high concentrations (1, 14), of cell movement (1), and of phagocytosis (2-4), as of Persantine has little or no effect on cytokinesis (unpub- well as of the transport reactions, are readily reversed by lished data). Similarly, phagocytosis by leukocytes is only resuspension of the cells in fresh medium is consistent with slightly affected by omission of glucose from the incubation the concept that these effects may all be due to a superficial medium (4). Further, the effect of CB at a concentration of binding of CB to the cell membrane. The finding that the 4.1 MAM on glucose transport is largely overcome by glucose effectiveness of CB in inhibiting deoxyglucose transport does concentrations commonly present in cell-culture media or not diminish during 4 hr of incubation with NlS1-67 cells is body fluids (5-10 mM), whereas cytokinesis of NlS1-67 also in agreement with this view. These findings, however, cells is still inhibited in these media (14). Preliminary results do not rule out the possibility that small amounts of CB are suggest that the entry of substances by simple diffusion is taken up by the cells. Further work is required to elucidate only slightly inhibited, if at all, by CB (unpublished data). the mechanism by which an interaction of CB with the plasma Most biological effects of CB concern functions involving membrane may interfere with other membrane processes or the plasma membrane. It seems possible, therefore, that affect microfilament structure. Downloaded by guest on September 29, 2021 1434 Cell Biology: Estensen and Plagemann Proc. Nat. Acad. Sci. USA 69 (1972) NOTE ADDED IN PROOF 9. Wrenn, J. T. & Wessells, N. K. (1970) Proc. Nat. Acad. Sci. USA 66, 904-908. One of us has recently demonstrated that CB is a competi- 10. Schroeder, T. E. (1969) Biol. Bull. 137, 443. tive inhibitor of glucosamine transport (P. G. W. Plage- 11. Schroeder, T. E. (1970) Z. Zellforsch. 109, 431-449. mann, in preparation). 12. Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. O., We thank John Erbe, Eugene C. Durkin, Mary Kay Robbins, Luduena, M. A., Taylor, E. L., Wrenn, J. T. & Yamada, Anthony Chu, and Mary Reusch for excellent technical assist- K. M. (1971) Science 171, 135-143. ance, and Dr. S. B. Carter for the gift of cytochalasin B. This 13. Estensen, R. D., Rosenberg, M. & Sheridan, J. D. (1971) work was supported by the Minnesota Medical Foundation, The Science 173, 356-358. Elsa A. Pardee Foundation, and by Public Health Service re- 14. Estensen, R. D. (1971) Proc. Soc. Exp. Biol. Med. 136, 1256- search Grants AI 07250 and 1-ROl-CA-12607-01. 1260. 15. Sanger, J. W. & Holtzer, H. (1972) Proc. Nat. Acad. Sci. 1. Carter, S. B. (1967) Nature 213, 261-264. USA 69, 253-257. 2. Davis, A. T., Estensen, R. & Quie, P. G. (1971) Proc. Soc. 16. Plagemann, P. G. W. & Roth, M. F. (1969) Biochemistry 8, Exp. Biol. Med. 137, 161-164. 4782-4789. 3. Malawista, S. E. (1971) in Progress in Immunology, ed. 17. Renner, E. D., Plagemann, P. G. W. & Bernlohr, W. (1972) Amos, B. (Academic Press, New York and London), pp. J. Biol. Chem., in press. 187-192. 4. Zigmond, S. H. & Hirsch, J. G., Exp. Cell Res., in press. 18. Plagemann, P. G. W. & Swim, H. E. (1966) J. Bacteriol. 91, 5. Wagner, R. Rosenberg, M. & Estensen, R. (1971) J. Cell. 2317-2326. Biol. 50, 804-817. 19. Ward, G. A. & Plagemann, P. G. W. (1969) J. Cell. Physiol. 6. Williams, J. A. & Wolff, J. (1971) Biochem. Biophys. Res. 73, 213-231. Commun. 44, 422-425. 20. Plagemann, P. G. W. (1971) J. Cell. Physiol. 77, 213-240. 7. Schofield, J. G. (1971) Nature New Biol. 236, 215-216. 21. Plagemann, P. G. W. (1971) Biochim. Biophys. Acta 233, 8. Spooner, B. S. & Wessells, N. K. (1970) Proc. Nat. Acad. 688-701. Sci. USA 66, 360-364. 22. Plagemann, P. G. W. (1970) J. Cell. Physiol. 75, 315-328. Downloaded by guest on September 29, 2021