Proc. Nat. Acad. Sci. USA Vol. 70, No. 5, pp. 1364-1367, May 1973

Sugar Effects on Murine Sarcoma Virus Transformation (mouse fibroblasts/D-/2-deoxy-D-) MASAKAZU HATANAKA Flow Laboratories, Inc., Rockville, Maryland 20852 Communicated by Herman M. Kalckar, February 12, 1973

ABSTRACT Transformation of NIH Swiss mouse fibro- sion of entry, whereas, cultures grown with D- in blasts by a murine sarcoma virus was influenced by the the medium show full "derepression" of hexose entry. On the type of in the culture medium. Exposure to sarcoma D-mannose and 2-deoxy-D-glucose caused a reduction other hand, cultures fully transformed by murine in the number of transformed colonies in vitro and tumor virus showed very high hexose entry rates even if previously formation in vivo. This is a new type of interference in the grown in medium with D-glucose. transformation process, manifested also by the absence Finally, I shall discuss a new type of interference in en- of the pronounced enhancement of hexose entry into transport by certain ; this is an interference in transformed cells, characteristic of a sarcoma virus in- hanced fection. The uptake of D-glucose, D-mannose, and D- the development of the transformation process. , was repressed by normal mouse cells grown in the presence of D-mannose and D-glucose in the medium, MATERIALS AND METHODS while uptake of the same was derepressed in cells The Harvey strain of mouse sarcoma virus was used grown in media containing D-xylose, L-xylose, or L-arab- cul- inose. D-Galactose had an intermediate effect on hexose throughout (1). The virus was propagated on secondary uptake and permitted derepression. In every case, enhance- tures of NIH Swiss mouse-embryo fibroblasts. Exponentially ment of the primary transport by sarcoma virus trans- growing cell cultures in plastic petri dishes (50 mm in diam- formation was consistently beyond that observed at any eter; Falcon Plastics) were infected with murine sarcoma time in the derepressed control cells, demonstrating the (Harvey). The cells were grown in Eagle's occurrence of another mechanism of enhancement of virus (i\ISV) hexose uptake after sarcoma virus infection. minimal essential medium, with 0.1% glucose, containing 10% fetal-bovine serum for the first 2 days after infection; Enhancement of the uptake of D-glucose, 2-deoxy-D-glucose, then MEM l)lus 2% fetal-bovine serum was used, and the D-mannose, D-galactose, and D-glucosamine was found in cells medium was changed every 2-3 days. Cell cultures were infected with mammalian as well as avian sarcoma viruses washed five times before assay with 10 ml of Hanks' balanced (1-6). Since the phenomenon was detectable at the beginning salt solution without glucose that had been warmed to the of viral transformation irn vitro (5) when glucose concentra- assay temjverature. The cultures were then incubated with tions in untransformed and transformed cells were still high 2.0 ml of warmed solutions of labeled (1 AM, see below) and differed only slightly, we considered the enhancement due in Hank's solution without glucose at 370 for 4-10 min. to the transformation process itself. We emphasize this point After incubation, the cultures were washed five times with because in some recent studies, Martineau et al. (7) described 10 ml of water. The remaining solution was removed by suc- a striking enhancement of hexose entry into chick fibroblasts tion, and the cells were scraped into 1.0 ml of water, transferred by starvation for the specific hexose during growth and to a small tube, and homogenized by vibration with a Vortex demonstrated a strong repression of D-glucose entry into the mixer for 5 sec. Aliquots of 0.2 ml were mixed with 5 ml of cell by high concentrations of glucose in the growth medium, Bray's solution and counted in a Beckman LS-250 liquid but not by D-galactose, whereas, both sugars caused a mod- scintillation counter. Protein was assayed by the method of erate but distinct repression of galactose entry. Lowry et al. (9). In this context, it is also of interest that Kaickar et al. (8) D-[1-14C]Glucose (220 Ci/mol) and D-['4C]mannose (240 concluded that the enhancement of entry of galactose that Ci/mol) were obtained from Schwarz; 2-deoxy-D- [-14C ]- they observed in hamster fibroblasts transformed by polyoma glucose (8.2 Ci/mol), D-[1-14C]galactose (52.5 Ci/mol), and virus, was largely independent of the "repression-derepres- D-[2-'4C]xylose (1000 Ci/mol) from New England Nuclear sion" phenomenon. This interpretation was based on the fact Corp. All unlabeled chemicals were obtained from Sigma. that transformed cultures grown in a medium with galactose that consumed only a minor fraction of the galactose of the RESULTS growth medium still showed full enhancement of galactose Table 1 summarizes the results of several experiments for entry (8). comparison between focus formation in mouse-embryo fibro- In independent studies I also became aware of the problem blasts infected with \ISV (Harvey) cultured in Eagle's of hexose-induced "repression-derepression" (7) of sugar medium with or without D-glucose. D-Xylose and D-galactose entry in normal mouse-embryo fibroblasts. I found that cul- supported the growth of mouse fibroblasts to the same extent tures grown with D-glucose in the medium show strong repres- as D-glucose. Cells grown in medium with L- and D-mannose showed a relatively slow growth rate, while L- Abbreviation: MSV, murine sarcoma virus. xylose and a-methyl-D-glucose did not support cell growth. 1364 Proc. Nat. Acad. Sci. USA 70 (1973) Sugar Effects on MSV Transformation 1365

TABLE 1. Cell growth and focus formation in the presence In the presence of 0.1% D-glucose, cell growth was similar to of various sugars that obtained with or without 0.1% of other sugars, except for 6-deoxy-D-galactose and 2-deoxy-D-glucose, which were Cell growth Focus formation highly toxic. The extent of focus formation by MSV (Harvey) With- With- was decreased in most cases in media with sugars other than out With out With D-glucose (D-galactose, L-arabinose, D-mannose, L-xylose, Additions to medium D-Glu n-Glu D-Glu n-Glu a-methyl-D-glucose, L-, and f-D-thioglucose), possibly because of interference with D-glucose uptake. In contrast, D-Glucose + + ++++ ++++ L-mannose, L-glucose, and D-Xylose + + ++++ ++++ D-, D-mannoheptulose had D-Galactose + + + + + + no effect on focus formation. In experiments where the total iArabinose Slow + + + + sugar concentration in the medium was maintained at 0.1% D-Mannose Slow + - + and equal proportions of the different sugars were added, no iXylose Static + - + + change in the pattern shown in Table 1 was observed (data a-Methyl-D- not shown). D-Mannose suppressed focus formation, while glucose Static + - + + addition of L-mannose to Eagle's medium did not affect focus i-Mannose - + - ++++ formation (Table 2). In addition to focus reduction, the up- isGlucose - + - ++++ take of D-mannose, D-glucose, and D-galactose was repressed D-Inulin - + - ++++ in uninfected cells as well as in cells infected with MSV D-Mannoheptulose - + - + + + + (Harvey) cultured in Eagle's medium with D-mannose (Table i-Fucose - + - + 2). flD-Thioglucose - + - + MSV (Harvey) progeny formation was also prevented in 6-Deoxy-D- cells grown in medium with D-mannose. The medium from galactose - Toxic - cell cultures grown in D-mannose for 7 days after infection by 2-Deoxy-i-glucose - Toxic - MSV (Harvey) was filtered through a 1.2-Am Millipore filter and used to infect fresh mouse-embryo fibroblasts cultured in Secondary cultures of fibroblasts from NIH Swiss mouse media with either D-glucose or D-mannose. Up to 8 days after embryos (4 X 105 cells per plate) were cultured with 0.1% of infection, no transformed foci were observed in either culture, additional sugar in Eagle's MEM with or without 0.1% D-glu- while the control culture grown in D-glucose, then transferred cose and 2% fetal-bovine serum. Cell growth (measured by count- to medium with D-glucose, showed extensive focus formation ing cells per plate) was compared to that in Eagle's MEM with (>400 focus-forming units). Again, MSV (Harvey) grown in 0.1%o-glucose. "+" Means roughly similar growth rate as ob- medium with D-glucose did not induce focus formation when served in Eagle's MEM; "slow" means less than half the growth plated on cells grown in medium with D-mannose (data not rate of the cells in Eagle's MEM; "static" means cells are in- shown). tact but did not increase in number; "-" means most cells did Mouse cells cultured in medium with D-xylose not grow and eventually died; "toxic" means cell death with derepressed vacuolation. The cells cultured in the media indicated were the uptake of D-glucose, D-mannose, and D-galactose, although inoculated with 0.1 ml [400 focus-forming units of MSV (Har- the rate of hexose uptake did not reach that obtained in cells vey)]. 8 Days after inoculation, the foci were counted. "+ + + +" infected with MSV (Harvey), indicating that an additional Indicates 100% of the cells were transformed; "+ + " indicates mechanism(s) other than derepression, is necessary in case of about 200 foci per plate; "+ " indicates about 100 foci per plate; hexose entry induced by MSV (Harvey). D-Galactose also in- "-" indicates no foci at all. duced derepression of hexose uptake by mouse cells (Table 3).

TABLE 2. Repression of sugar uptake in medium with D-mannose

Experiment 1 Experiment 2 Additions to 1-Glucose D-Glucose Cells Eagle's MEM Foci D-Mannose uptake D-Galactose Foci uptake None 0* 9.46t 19.2 4.98 0 20.1 Control D-Mannose 0 4.90 11.2 2.15 0 10.4 L-Mannose 0 20.0 0 20.5 None 60, 65 31.8 59.3 14.4 (400) 186.0 Infected with MSV D-Mannose 5, 4 8.18 18.2 9.14 10, 8 46.9 (Harvey) L-.-Iannose 60, 63 - 63.5 (400) 195.0

Cells cultured in Eagle's MIEM with 0.1% D- or -mannose were infected with MSV (Harvey) (62 focus-forming units for Exp. 1, and 400 focus-forming units for Exp. 2). The foci were counted at day 6 after infection, and sugar uptake was assayed as follows: 2 ml of 1 ,gM labeled sugar was added to the plate after the removal of culture medium and washing three times with Hank's balanced salt solution without glucose. After a 10-min incubation at 37°, cells were washed five times each with 10 ml of H20, and the residual solution was removed by suction. 1 ml of H20 was added, and the cells were scraped with a rubber policeman and transferred to a glass tube. The sus- pension was mixed for 10 sec with a Vortex mixer, and 0.1 ml was counted for radioactivity in Bray's solution and 0.1 ml was assayed for protein content by Lowry et al.'s method (9). "* Indicates foci per plate; "+ " indicates pmol of sugar uptake per mg of protein per min at 370; parentheses indicate expected focus-forming units, actually too many to count. 1366 Cell Biology: Hatanaka Proc. Nat. Acad. Sci. USA 70 (1973) TABLE 3. Derepression of sugar uptake by r-xylose, ixylose, Larabinose, andD-galactose

Sugar uptake pmol/mg of protein per min 1-Glucose D-Mannose D-Galactose Cells cultured in Control MSV (Harvey) Control MSV (Harvey) Control MSV (Harvey) )-Glucose 19.2 69.7 9.68 31.8 5.32 13.2 n-Mannose 8.97 18.6 6.58 12.6 4.45 9.14 D-Galactose 67.4 186.0 57.8 131.0 12.4 28.6 D-XylOSe 138.0 225.0 95.7 129.0 22.3 37.5 LXylose 135.0 175.0 91.6 198.0 19.9 29.3 L-Arabinose 115.0 188.0 65.4 113.0 20.5 38.4

Secondary mouse cells cultured in Eagle's medium without glucose, but with added 0.1% each of -glucose, D-mannose, D-galactose. D-xylose, L-xylose, or iarabinose and with 10% dialyzed fetal-bovine serum were infected with 100 focus-forming units of MSV (Harvey). 5 Days after infection the sugar uptake was assayed as described in Table 2.

Among the several isomers tested, L-xylose and L-arabinose, these sugars on viral transformation in vivo. Newborn mice like D-xylose, derepressed the uptake of D-glucose, D-mannose, were inoculated subcutaneously with 0.1 ml of 104 focus- and D-galactose (Table 3). D-Galactose had an intermediate forming units of MSV (Harvey), followed by 0.1 ml of 2- derepressing effect, and in contrast, D-mannose showed a deoxy-D-glucose or D-mannose every day until palpable greater repressing effect than D-glucose. Again, the rate of tumors were observed. Most tumor-bearing mice died 3-5 hexose uptake in uninfected cells was lower than that in cells days after tumor onset. As shown in Table 5, 100 /.tg of 2- infected with MSV (Harvey). deoxy-D-glucose or D-mannose delayed the onset and reduced 2-Deoxy-D-glucose is a D-glucose analog that is taken up the incidence of tumors. quite well in cells infected with MSV (Harvey) (2). The in- cubation of cells infected with MSV (Harvey) with 2-deoxy- DISCUSSION D-glucose and D-glucose resulted in a reduction of transformed It is clear from these experiments that the enhancement of foci as shown in Table 4. The uptake of D-mannose and D- sugar transport in transformed cells was observed even in glucose was also reduced when the cells were cultured in cultures grown in derepressing sugars such as D-xylose, L- medium with 2-deoxy-D-glucose and D-glucose. 1 mM 2- xylose, L-arabinose, and D-galactose (Table 6), all of which deoxy-D-glucose was cytotoxic but 0.01 mM of the same sugar allow transformation of cells by MSV (Harvey). The rate analog in the presence of 5.5 mM D-glucose allowed normal of sugar uptake induced by virus infection was not based on cell growth, but still inhibited both transformation and en- hanced uptake of D-glucose and D-mannose. TABLE 5. Reduced viral transformation in vivo by 2-deoxy-D- The reduction of focus-formation by 2-deoxy-D-glucose glucose and D-mannose and D-mannose in vitro led us to investigate the effects of Sugar TABLE 4. Reducedfociformation by cells grown in medium with admin- Tumor incidence 2-deoxy-D-glucose istered No. of (pg) animals 17 day 20 day 37 day Cells 0 15 14/15* 15/15 cult- 26 11/26 25/26 ured in Sugar uptake pmol/mg of 2-Deoxy-n-glucose

2- Foci protein per min 1 6 6/6 6/6 deoxy- DGlucose DMannose 10 10 4/10 9/10 D-glu- Aver- 100 21 3/21 8/21 10/21 cose age MSV MSV 29 11/29 5/29 20/29 (M) no. % Control (Harvey) Control (Harvey) 1000 15 0/6t 0/6 0/6 10-3 0 0 10.2 16.4 5.08 16.1 D-Mannose 10-4 58 35.6 10.2 89.9 8.13 84.1 100 13 3/13 4/13 11/13 10-5 62 38.0 11.5 128.0 10.5 98.4 1000 17 3/17 6/17 10/17 10-6 118 73.4 14.0 281.0 8.57 220.0 10-7 120 73.6 14.0 249.0 8.27 260.0 Newborn NIH Swiss mice were inoculated subcutaneously with 0 163 100.0 13.8 288.0 8.37 279.0 0.1 ml of 104 focus-forming units of MSV (Harvey), followed by 0.1 ml of sugar every day, until palpable tumors were observed. Cells cultured in Eagle's medium with the indicated amount Most tumor-bearing mice died within 3-5 days after the formation of 2-deoxy-D-glucose and 10% fetal-bovine serum were infected of palpable tumors. with MSV (Harvey) (163 focus-forming unit per plate). The foci * Tumor-bearing mice to total number of mice. were counted 5 days after infection, and sugar uptake was as- t Nine mice died 2 days after beginning the inoculation of sayed as described in Table 2. Numbers are average focus counts high doses of 2-deoxy-D-glucose, probably due to the toxic effect. of three plates. $ Killed by tumor. Proc. Nat. Acad. Sci. USA 70 (1978) Sugar Effects on MSV Transformation 1367 TABLE 6. Repression and derepression of sugar uptake The mechanism of D-mannose effect remains to be studied, although the repression of uptake of this sugar might prevent Sugar uptake Cells cultured with viral transformation or alternatively, the catabolite products could further suppress de- Allowing derepression D-Xylose of D-mannose in transforming cells L-Xylose velopment of transformation. L-Arabinose D-Galactose I thank Drs. H. M. Kalckar and R. V. Gilden for helpful sug- gestions in preparing the manuscript. This work was supported Maintaining repression D-MIannose by Contract NIH-NCI-E-71-2097 of the Special Virus Cancer D-Glucose Program of the National Cancer Institute, National Institutes of 2-Deoxy-D-glucose Health, Bethesda, Md. 20014

Mouse cells cultured in medium containing the above sugars 1. Hatanaka, M., Huebner, R. J. & Gilden, R. V. (1969) "Al- were assayed after 1-6 days of cultivation for transport of D- terations in the characteristics of sugar uptake by mouse cells [4C] glucose, D- [ 14C] mannose, and D- [14C] galactose, as described transformed by murine sarcoma viruses," J. Nat. Cancer Inst. in Methods. 43, 1091-1096. 2. Hatanaka, M., Augl, C. & Gilden, R. V. (1970) "Evidence for a functional change in the plasma membrane of murine sar- the derepression effect alone (Table 3). Derepression by coma virus-infected mouse embryo cells," J. Biol. Chem. 245, lowering the concentrations of repressing sugars such as 714-717. D-glucose was not the factor causing the enhancement of 3. Hatanaka, M., Todaro, G. J. & Gilden, R. V. (1970) "Altered sugar uptake by sarcoma virus infection, due to the fact that glucose transport kinetics in murine sarcoma virus-trans- started at the beginning of transforma- formed BALB/3T3 clones," Intern. J. Cancer 5, 224-228. the enhanced uptake 4. Hatanaka, M. & Gilden, R. V. (1970) "Virus-specified tion, when the extracellular D-glucose concentration did not changes in the sugar transport kinetics of rat-embryo cells change significantly, and was maintained well over the Km infected with murine sarcoma virus," J. Nat. Cancer Inst. concentration of sugar uptake (5). Transport enhancement by 45, 87-89. transformation was also reported by Kalckar et al. (8). 5. Hatanaka, M. & Hanafusa, H. (1970) "Analysis of a func- tional change in membrane in the process of cell transforma- The "repression-derepression" phenomena of sugar trans- tion by Rous sarcoma virus; alteration in the characteristics port observed in chick fibroblasts (7) appear similar to that of sugar transport," Virology 41, 647-652. observed in mouse fibroblasts, as shown here, with some 6. Hatanaka, M., Gilden, R. V. & Kelloff, G. (1971) "Induction quantitative differences. In the case of mouse fibroblasts, of sugar uptake by a hamster pseudotype sarcoma virus," hexose uptake is derepressed most by D-xylose, L-xylose, and Virology 43, 734-736. 7. Martineau, R., Kohlbacher, M., Shaw, S. N. & Amos, H. L-arabinose, followed by D-galactose, which causes derepres- (1972) "Enhancement of hexose entry into chick fibroblasts sion of hexose uptake in chick fibroblasts (7). In the case by starvation: differential effect on galactose and glucose," of repressing sugars (Table 6), 2-deoxy-D-glucose is not catabo- Proc. Nat. Acad. Sci. USA 69, 3407-3411. lized, thus the accumulation of this sugar in cells infected 8. Kalckar, H. M., Ullrey, D., Kijomoto, S. & Hakomori, S. (1973) Proc. Nat. Acad. Sci. USA 70, 839-843. with MSV (Harvey) could be fatal. Another repressing sugar, 9. Lowry, 0. H., Rosebrough, N. J., Farr, L. & Randall, R. J. D-mannose (Table 6), inhibited focus-formation and repressed (1951) "Protein measurement with the Folin phenol rea- sugar uptake in both uninfected and infected cells (Table 3). gent," J. Biol. Chem. 193, 265-275.