Increased Membrane Transport of 2-Deoxyglucose and 3-0-Methylglucose Is an Early Event in the Transformation of Chick Embryo Fibroblasts by Rous Sarcoma Virus DENNIS R. LANG AND MICHAEL J. WEBER Department ofMicrobiology, University ofCincinnati College ofMedicine, Cincinnati, Ohio 45267and Department ofMicrobiology, University oflllinois, Urbana, Illinois 61801

ABSTRACT Transformation of chicken embryo fibroblasts with Rous sarcoma virus results in cells with an enhanced rate of hexose uptake. We have examined transport of the glucose analogs 2-deoxyglucoseand 3-0-methylglucosein cells infected with a temperature sensitive variant of the virus. In cells shifted from restrictive to permissive conditions for transformation, increased transport of the non-phosphorylatable analog 3-0-methylglucoseoccurs a t the same time as that of 2-deoxyglucose, a phosphorylatable analog. This enhanced rate of transport can be observed within three hours of the temperature shift. There is a corresponding decrease in the transport rate of both analogs following shift to the restrictive temperature. These results suggest that increased transport is likely to be the primary event in causing transformation-specific changes in sugar metabolism. We have also examined uptake into the internal pools of both the phosphorylated and non-phosphorylated forms of 2-deoxyglucose in normal cells and in cells transformed by the wild-type virus. These data indicate a corresponding increase in the rate of accumulation of the free sugar in transformed cells and point to transport as the rate limiting step in the accumulation of 2-deoxyglucose in both normal and transformed chicken embryo cells. Malignant transformation of cultured fibroblasts results in an increased rate of uptake of hexoses, whether the transforming agent is an RNA tumor virus (Hatanaka and Hanafusa, '70) a DNA tumor Virus (Eckhart and Weber, '74; Colby and Romano, '75) or a chemical carcinogen (Oshiro and DiPaolo, '74). The increased rate of hexose uptake thus serves as an excellent marker of the transformed state for this cell type. I t is widely thought that increased transport across the cell membrane accounts for this change in uptake rate based largely on the observation that uptake of 3-0methylglucose, a glucose analog which is transported, but not further metabolized, shows increased uptake in transformed cells (Weber, '73). However, there have been several suggestions that transport is not rate-limiting for hexose uptake, and that an increased r a t e of post-transport phosphorylation accounts for the increased rate of accumulation J. CELL. PHYSIOL. (1978)94: 315-320.

of hexoses in transformed cells. In particular, Colby and Romano ('75) and Romano ('76) reported that uptake of 2-deoxyglucose into the free sugar pool was not increased in 3T3 cells transformed by SV40. Romano, in addition, raises the possibility that 3-0-methylglucose may not be an appropriate hexose analog to use in transport studies ('76). Another criticism of the conclusion that increased hexose uptake is due to increased transport is implicit in the work of Singh et al. ('74) and Fodge and Rubin ('73). Since measurements of hexose transport in transformed cells have been performed with fully transformed cells, it remains a possibility that alterations in post-transport metabolism (such as phosphofructokinase or hexokinase activity) are primary events in transformation-specific alterations in sugar metabolism, and Received Aug. 15. '77. Accepted Oct. 18, "77.

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3

6

9

12

3

6

9

12

Hours Fig. 1 Uptake rates of 1 mM 31Hl-2-deoxyglucose(A) and 1 mM 3[H1-3-O-methylglucose(B) in cells infected with a temperature-conditional mutant of Rous sarcoma virus. Cells grown at 42°C (A-A 1, shifted from 42°C to 36OC (0-0) or shifted from 3 6 T to 42°C (0-0). All data is expressed as a percentage of the 36°C (transformed) control.

t h a t the increased rate of hexose transport is a later, secondary consequence of the altered metabolism. In this communication we address both of these questions. We demonstrate that, for chicken embryo fibroblasts, either normal or transformed by Rous sarcoma virus, transport across the cell membrane is rate limiting for uptake of 2-deoxyglucose, and that similar results are obtained whether one measures uptake of 3-0-methylglucoseor uptake of 2-deoxyglucose into the unphosphorylated pool. In addition, we demonstrate, using a mutant of Rous sarcoma virus which is temperature-conditional for transformation, that alterations in the rate of 3-0-methylglucose transport across the cell membrane occur rapidly in response to the switching on or switching off of viral oncogenic information and thus are unlikely to be secondary consequences of alterations in hexose metabolism.

took advantage of the temperature conditional mutant NY-ts68 (Kawai and Hanafusa, '71). Cells infected with this mutant are phenotypically normal when held at the restrictive temperature of 42"C, but rapidly and reversibly become transformed when shifted to 36OC. Kinetics of changes in 2-deoxyglucose and 3-0-methylglucose uptake following a temperature shift a r e shown in figure 1. Uptake of both 2-deoxyglucose and of 3-0methylglucose increased rapidly following a shift from 42°C to 36OC, and reached t h e level characteristic of fully transformed cells within approximately nine hours. When cells which were held at 36°C were shifted to 42OC they began rapidly to decrease their rates of uptake of both hexose analogs and within six hours displayed transport rates characteristic of normal, uninfected cells.

Transport and phosphorylation of 2-deoxyglucose MATERIALS AND METHODS We have examined the distribution of the Cell culture and transport measurements free and phosphorylated forms of 2-deoxygluwere as described (Weber, '73). Separation of cose to determine whether transport or phosfree and phosphorylated 2-deoxyglucose was phorylation is increased in Rous-transformed cells. For these experiments, cells transformed as described by Colby and Romano ('75). by the wild-type Schmidt-Ruppin strain of RESULTS RSV were used. Figure 2 shows the results of Kinetics of changes in hexose transport rate a n experiment comparing the distribution of To determine whether transformation-spe- radioactivity into the free sugar and phosphocific changes in transport were secondary to rylated sugar pools in normal density inhibalterations in post-transport metabolism, we ited cells, in normal growing cells and in cells

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Minutes Fig. 2 Uptake of 2 mM 31HI-2-deoxyglucoseinto the intracellular free sugar pool (0-0 1 and into 2-deoxyglucose-6-P04(0- -01. Thearrow (- f indicates the level ofequilibriumwith themediaconcentration.

transformed by RSV. Density inhibited cells show very low rates of uptake or phosphorylation of 2-deoxyglucose. In these cells, the level of free 2-deoxyglucose inside the cell did not reach the level in the medium after as much as 15 minutes of incubation. In both the norm a l growing and t h e transformed cells, however, there was a small but reproducible transient overshoot in the free 2-deoxyglucose level inside the cell compared to the outside concentration. This overshoot may be a consequence of counter transport. The overshoot appeared more rapidly and was dissipated more rapidly in transformed cells than in normal cells. Note t h a t for all three cell types the initial rate of uptake of 2-deoxyglucose into the free sugar pool showed the same qualitative differences seen with 3-0-methylglucose and for the accumulation of 2-deoxyglucose-6phosphate. Inall cell types, the level of free 2-deoxyglucose was less than t h e outside concentration of sugar once the overshoot had been dissipated. This is shown in table 1,where internal concentrations of 2-deoxyglucose and 2-deoxyglucose-6-P04are shown. DISCUSSION

The data presented in this report indicate that the increase in hexose transport observed

TABLE 1

Levels of the free and phosphorylated forms of 2-DG in density inhibited, normal growing,and transformed chicken embryo fibroblasts Intracellular concentration (mM) '

Free Z-deoxyglucose Z-deoxyglucose&PO,

Cell type Densityinhibited

Growing

0.28

0.39

1.07

2.38

Transformed

0.60 15.7

Cells incubated with 2 m M 2-deoxyglucose for ten minutes at 37°C were rinsed with cold buffer and extracted with boiling water. Data were calculated using an intracellular space value of 4.3 p f g cell protein (Weher, '73). 1

in RSV infected chicken embryo cells is a n early event in the sequence of events leading to transformation of these cells. When cells were infected with NY-ts68, a temperature sensitive variant of RSV, and shifted from restrictive to permissive conditions a n increase in 3-0-methylglucose transport coincided in time with the previously reported (Martin et al., '71; Kawaii and Hanafusa, "71)increase in deoxyglucose uptake. Since 3-0-methylglucose is transported but unlike 2-deoxyglucose is not phosphorylated by intracellular kinases (Weber, '73) these data indicate that there is a n early increase in the actual transport of the

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free sugar across the membrane. Combined with the findings of Bissell (’76) demonstrating that inhibition of glucose transport in transformed cells could restore perturbations in sugar metabolism to normal, and the findings of Fodge and Rubin (‘73) that increased activity of phosphofructokinase occurs concomitantly with increased sugar uptake, these data provide persuasive evidence that changes in glucose transport across the cell membrane are the cause of subsequent changes in sugar metabolism and underlie the heightened “aerobic glycolysis” thought to be associated with tumor cells since the early reports of Warburg (‘30). The above interpretation rests on experiments in which 3-0-methylglucose has been used to measure the activity of the glucose transport system. Romano (‘76) has presented evidence indicating that in mouse 3T3 cells transformed by either RNA or DNA viruses, increased hexose uptake is a consequence of enhanced phosphorylation of 2-deoxyglucose, and entry of 2-deoxyglucose into the free, unphosphorylated pool is not affected by transformation. However, the size of the free sugar pool a t any time is a reflection not only of the rate of transport of the free sugar into the cell but also of the removal of sugar from this pool due to its phosphorylation by intracellular kinases. Therefore when the entry of isotopic labeled hexose into the free sugar pool is shown to be equivalent in normal and transformed cells, the data can be interpreted in one of two ways. Either the rate of transport of the free sugar into the cell must be increased to balance the increased rate of its removal due to phosphorylation or the transport step is not rate limiting for uptake in these cells. The fact that in 3T3 cells (either normal or transformed) the free sugar pool inside the cell rapidly equilibrates with the external concentration suggests (but does not prove) that transport may not be the rate limiting step in glucose metabolism in these cells. When we examined the distribution of radioactivity into the free and phosphorylated pools in chicken cells, several differences from the 3T3 system were noted. The equilibrium concentration of free 2-deoxyglucose inside the cell was well below the external concentration, indicating t h a t phosphorylation reactions were occurring more rapidly than transport, and thus that transport was the rate-limiting step for accumulation of this hexose inside these cells. Moreover, the initial rates of entry

of 2-deoxyglucose into the cells showed differences between the various cell types similar to those obtained with 3-0-methylglucose. Since we find that rates of uptake of glucose, 2-deoxyglucose and 3-0-methylglucose vary coordinately as cellular physiology changes and that these three sugars compete with each other for entry (unpublished) it seems highly probable that 3-0-methylglucose uptake is a valid measure of the activity of the glucose transport system in these cells. Moreover, since Eckhart and Weber find that polyoma infection of 3T3 cells stimulates uptake of both 3-0-methylglucose and 2-deoxyglucose (Eckhart and Weber, ’74) transport is probably affected in this system as well. Thus, the findings of Colby and Romano (‘75) are most likely attributable to secondary alterations arising in their established cell lines with continued passaging. It is interesting, nonetheless, that the cell lines used by Romano (‘76) have maintained an enhanced rate of sugar uptake, even if the mechanism differs from freshly infected cultures. Why different transformed cell types establish and maintain enhanced hexose uptake rates by differing mechanisms is a question which deserves further study. LITERATURE CITED Bissell, M. J. 1976 Transport a s a rate limiting step in glucose metabolism in virus-transformed cells: Studies with Cytochalasin B. J. Cell. Physiol., 89: 701-710. Colby, C., and H. Romano 1975 Phosphorylation but not transport of sugars is enhanced in virus-transformed mouse 3T3 cells. J. Cell. Physiol., 85: 15-23. Eckhart, W., and M. J. Weber 1974 Uptake of 2-deoxyglucose by BALBT3 cells: Changes after polyoma infection. Virology, 61: 223-228. Fodge, D. W., and H. Rubin 1973Activation of phosphofructokinase by stimulants of cell multiplication. Nature New Biol., 246: 181-183. Hatanaka, M., and H. Hanafusa 1970 Analysis of a functional change in membrane in the process of cell transformation by Rous Sarcoma virus; Alteration in the characteristics of sugar transport. Virology, 41: 647-652. Kawai, S., and H. Hanafusa 1971 The effects of reciprocal changes in temperature on the transformed state of cells infected with a Rous Sarcoma virus mutant. Virology,46: 470-479. Martin, G. S., S. Venuta, M. J. Weber and H. Rubin 1971 Temperature-dependent alterations in sugar transport in cells infected by a temperature-sensitive mutant of Rous sarcoma virus. Proc. Nat. Acad. Sci. (U.S.A.), 68: 2739-2741. Oshiro, Y., an d J. A. DiPaolo 1974 Changes in the uptake of 2-DG in BalbT3 cells chemically transformed in culture. J. Cell. Physiol., 83: 193-201. Romano, A. H. 1976 Is glucose transport enhanced in virus-transformed mammalian cells? A dissenting view. J. Cell. Physiol., 89: 737-744.

HEXOSE TRANSPORT IN RSV TRANSFORMED CELLS Singh, V. N., M. Singh, J. T. August and B. L. Horecker 1974Alterations in glucose metabolism in chick-embryo c e l l s transformed by Rous sarcoma virus: Intracellular levels of glycolytic intermediates. Proc. Nat. Acad. Sci. (U.S.A.),71: 4129-4132.

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Warburg, 0. 1930 The Metabolism of Tumors. Constable, London. Weber, M. J. 1973 Hexose transport in normal and Rous sarcoma virus-transformed cells. J. Biol. Chem., 248: 2978-2983.

Increased membrane transport of 2-deoxyglucose and 3-O-methylglucose is an early event in the transformation of chick embryo fibroblasts by Rous sarcoma virus.

Increased Membrane Transport of 2-Deoxyglucose and 3-0-Methylglucose Is an Early Event in the Transformation of Chick Embryo Fibroblasts by Rous Sarco...
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