Transformed BHK Cells Exhibiting Normal or Subnormal Sugar Uptake F. L. MOOLTEN, D. N. MOOLTEN AND N. J. CAPPARELL Department of Mkrobiology, Boston University School ofkfedkine, Boston, Massachusetts 021 18
ABSTRACT The uptake of deoxyglucose was compared in BHK cells and in DMN4B cells, a conditionally transformed line of BHK cells which exhibits transformed behavior a t 38.5' but not at 32'. At 32', DMN4B cells took up deoxyglucose more slowly than BHK cells, reflecting a higher Km for uptake of this sugar. When both cell lines were grown at 38.5', the Km for DMN4B cells was reduced to a level only slightly greater than for BHK cells, and deoxyglucose uptake became similar in the two cell lines. Growth in glucose-free medium for 22 hours stimulated deoxyglucose uptake in both BHK and DMN4B cells; under these conditions, uptake was equal in the two cells lines, both at 32' and 38.5'. Glycolysis, as measured by lactic acid production, was slower in DMN4B than BHK cells, but in contrast to deoxyglucose uptake, this difference was observed a t 38.5' rather than 32'. The observation that the subnormal deoxyglucose uptake of DMN4B cells in the untransformed state (32') can be normalized by growth a t 38.5', a temperature permissive for transformation, suggests that membrane changes facilitating sugar uptake, which have been found in other transformed cells, are associated with transformation in DMN4B cells as well. However, the failure of uptake to exceed normal in these cells indicates that their transformed behavior is not attributable to excessive sugar uptake per se. A hallmark of malignant transformation is an increase in the rate of cellular uptake of a variety of nutrients and other small molecules. In particular, rates of sugar uptake, as measured by the rate of cellular accumulation of the glucose analogue, 2-deoxy-D-glucose, have been found higher in transformed cells than in their normal counterparts (reviewed by Hatanaka, '74), and it has been suggested that, in fact, increased ability of these cells to take up sugar may be responsible for their uncontrolled growth (Oshiro and DiPaolo, '74; Hatanaka, '74). We now report a transformed line of baby hamster kidney (BHK) cells which displays rates of sugar uptake lower than or equal to those in untransformed BHK cells. Membrane changes associated with increased sugar uptake in other transformed cell lines may be present in these cells as well, but are offset by other changes which reduce the efficiency of sugar transport. The results suggest that changes in sugar uptake may reflect changes in membrane function that are crucial for transformation, but that sugar uptake itself is not the crucial phenomenon. J. CELL. PHYSIOL.. 93: 147-152.
MATERIALS AND METHODS
Cells and media Low-passage BHK cells and the transformed BHK line DMN4B (both obtained from Doctor G. DiMayorca, University of Illinois), BALB/3T3 mouse endothelial cells (clone A31), and A31 cells transformed by Kirsten sarcoma virus (K3T3 cells) were all grown in monolayer culture in Dulbecco's medium containing penicillin (100 unitdml), streptomycin (100 fig/ml), 10% fetal calf serum (FCS), and 25 mM glucose, in a humidified, 5% COz atmosphere. The A31 and K3T3 cells were routinely maintained at 37'. DMN4B cells, a line derived by mutagenesis of BHK cells with dimethylnitrosamine, are conditionally transformed. They exhibit a loss of density-dependent inhibition, a transformed morphology, and the ability to produce colonies in soft agar at 38.5' but not at 32'; they are also tumorigenic in hamsters (DiMayorca et al., '73). Low-passage BHK cells lack these properties (DiMayorca e t al., '73). Because these attriReceived Mar. 25, '77. Accepted Apr. 26, '77.
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rected by subtracting "zero-time'' measurements (3H-deoxyglucosewas added and removed, and the first cold PBS wash was applied within 6-8 seconds). Deoxyglucose uptake measured a t 5 mM concentration was found to be linear with time for at least five to ten minutes with both cell types and both temperatures; therefore, studies performed with other (lower) concentrations utilized 5minute labeling intervals. The "zero-time'' corrections were similar for BHK and DMN4B cells (varying < 15% between the 2 cell types), and always represented < 23% of the total uptake occurring in five minutes. I n some cases, experiments were done with cells "pre-starved" by incubating them for 22 hours in glucose-free Eagle's Minimal Essential Medium (MEM) containing 3% dialyzed Deoxyglucose uptake FCS. To minimize extraneous sources of variaMonolayer cultures in 25 cm2Falcon plastic tion, all comparisons between DMN4B and flasks were grown to confluence-a condition BHK cells (or between A31 and K3T3 cells) that has been reported capable of enhancing involved flasks of both cell types, in duplicate differences in deoxyglucose uptake between to quadruplicate, handled a t the same times transformed and untransformed cells (Bose as part of the same experiment. and Zlotnick, '73). The cultures were used Lactic acid production within one day of reaching the confluent state to prevent cell densities in DMN4B cultures Culture flasks were aspirated and washed from exceeding those in BHK cultures. Under once with HSS. The medium was then rethese conditions, the amount of cellular mate- placed with 5 ml of fresh culture medium, o r rial (measured as protein) was always similar with 5 ml of MEM containing 4%dialyzed FCS in DMN4B and BHK cultures, and differences and glucose a t concentrations of 25, 8, or 4 between the cell lines in deoxyglucose uptake/ mM. After the cells were incubated for three mg protein were always reflected by differ- hours a t the temperature a t which they had ences in uptake/culture flask. For uptake been grown, the medium was removed, and studies, the flasks were aspirated, and washed the cells were dissolved in NaOH for deterthree times with glucose-free Hanks Salt mination of protein concentration. To deterSolution (HSS) pre-warmed to 32" or 38.5" mine the lactic acid concentration of the according to the temperature a t which the aspirated medium, 2 mi of the latter was cells were grown. One milliliter of HSS was added to 4 ml of 8%perchloric acid. The resultthen added, and the flasks were re-incubated ing precipitates were removed by centrifugaat the same temperature for five minutes to tion and the supernatants were analyzed encorrect for any cooling that might have oc- zymatically for lactic acid by measuring the curred during the washing. An additional 1ml quantity of reduced nicotinamide adenine of HSS was then added, containing 1pCi of 2- dinucleotide (NADH) that could be generated deo~y-D-glucose-~H (New England Nuclear, from the oxidized form (NAD) in the presence Boston, Massachusetts) and sufficient non- of lactic dehydrogenase, using a commercially radioactive deoxyglucose to achieve the de- available reagent kit (Sigma Chemical Co., St. sired concentration. The contents were mixed, Louis, Missouri). The values obtained were and the flasks were then re-incubated for corrected by subtracting the values obtained various intervals. They were then aspirated, with unused medium. and washed three times with ice-cold phosRESULTS phate buffered saline (PBS). The cells were At 32" deoxyglucose uptake in DMN4B cells dissolved by addition of 1.7 ml of 0.5 N NaOH, and aliquots were removed for measurements was significantly lower than in BHK cells; of radioactivity and of protein (Lowry et al., Lineweaver-Burk plots of these data are '51). Values of deoxyglucose uptake were cor- shown in figure 1. The reduction in uptake
butes can change with repeated passage, cells from the passage levels used in this study were routinely plated in soft agar in parallel with the experiments on sugar uptake. These tests confirmed the ability of DMN4B cells to form colonies a t 38.5", but not a t 32", and the inability of the BHK cells used in these experiments to form colonies a t either temperature. K3T3 cells produced colonies a t both temperatures. DMN4B and BHK cells were routinely maintained a t 32"; for experiments a t 38.5" they were grown for a t least 3.5 generations a t this higher temperature to permit full expression of the transformed phenotype (DiMayorca et al., '73). All cell lines have been found free of mycoplasma on repeated testing by the method of Schneider et al. ('74).
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Fig. 1 Lineweaver-Burk plots of initial rates of 2-deoxyglucose (2-DOG)uptake by BHK cells (open circles) and DMN4B cells (closed circles) at concentrations between 0.4 and 5 mM. Each point represents the mean of duplicate determinations. Linear regression analysis was used to plot each line, and revealed that the Km value derived from the line for DMN4B cells at 32" differed significantly from the Km for BHK cells at 32" (p < 0.001) and from the Km for DMN4B cells at 38.5' (p < 0.01). TABLE 1
The effectofgrowth conditions on 2-deoxyglucoseuptake by untransformed and transformed hamster and mouse cells Growth conditions
2-deoxyglucose uptake
Temperature
Growth medium
BHK cells
32"
Dulbecco's Glucose-free MEM Dulbecco's Glucose-free MEM
12.3ic1.2(5) 51.6*4.5(4) 16.721.1(6) 85.722.6(4)
38.5'
DMN4B cells
3.5ic0.4(6) 52.2ic6.6(4) 20.7+-1.8(6) 86.0&3.8(4)
A31 cells
K3T3 cells
8.0ic0.113)
17.3t0.613)
7.4t0.2(3)
12.8t0.5(3)
' Cells were exposed to denxyglucose for five minutes a t a concentration of 1 mM. Uptake is expressed as nmolesimg protein/5 min 2 standard error; the number of samples is stated in parentheses. The glucose concentration of the Dulbecco's medium was 25 mM. 'The value for uptake is significantly less than in the corresponding untransformed cells (BHK cells); p < 0.01 by student's t test. 3The value for uptake is significantly greater than in the corresponding untransformed cells (A31 cells); p < 0.01.
was attributable to a higher Km for DMN4B than for BHK cells (10.2 vs 2.2 mM), suggesting a lower affinity of the transport mechanism for the sugar. In contrast, the maximum theoretical rate of uptake, Vmax, was slightly higher in the DMN4B cells (10.2 vs 7.5 nmoledmg protein/min). A t the permissive temperature for transformation in DMN4B cells, 38.5", the differences between the two cell types was much less, primarily reflecting a n increase in Km for BHK cells to 3.2 mM, and a reduction in Km for DMN4B cells to 5.0 mM. Vmax remained slightly higher in DMN4B than in BHK cells (22.2 vs 18.2 nmoleslmg protein/min) . Deoxyglucose uptake has been reported to increase substantially in cells depleted of glucose for many hours (Martineau et al., '72). I t seemed possible, therefore, that failure of DMN4B cells to exceed BHK cells in deoxyglucose uptake when both were grown in
Dulbecco's medium (which contains 25 mM glucose), may have reflected merely a lack of stimulation rather than a lack of capacity. DMN4B and BHK cells were therefore compared after incubation in glucose-free MEM for 22 hours. The results, shown in table 1, indicated that deoxyglucose uptake was in fact greatly enhanced under these conditions. This effect occurred in both types of cells, however, and although sufficient to abolish the disparity between DMN4B and BHK cells a t 32", failed a t either temperature to enhance uptake rates in DMN4B cells to levels exceeding those in BHK cells. In contrast, table 1 also shows that even unstarved cultures of Kirsten sarcoma virus-transformed (K3T3) cells exhibited rates of deoxyglucose uptake about twice as great as those of their normal counterparts. An additional comparison of the ability of DMN4B and BHK cells to utilize sugar was
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F. L. MOOLTEN, D. N. MOOLTEN AND N. J. CAPPARELL TABLE 2
Lactic acid production by DMN4B and BHK cells
Temperature
Incubation medium '
38.5'
Dulbecco's MEM
32"
Dulbecco's MEM
Lactic acid produced lvmoleslmg proteinlhour)
Glucose concentration (mM)
BHK cells
25 25 8 4 25 25 8 4
1.58%0.08(31 1.302 0.0212) 1.0910.07(2) 0.8420.02(2) 0.972 0.06(4) 0.662 0.04(2) 0.5520.03(2) 0.4410.04121
DMN4B cells
0.6920.02(41 0.7120.03(2) 0.63%0.06(2) 0.602 0.02(2) 0.81%0.04(4) 0.70%0.2212) 0.732 O.Ol(2) 0.482 0.08(21
' The Dulbecco's medium contained 10%FCS and the MEM 4%dialyzed FCS. Cells were incubated in these media for three hours at the temperature at which they had been grown. *Results are expressed as mean rt standard error; the number of samples is stated in parentheses. This value differs significantly from the corresponding value for BHK cells (p < 0.01). Combined analysis using values obtained at each glucose concentration show that DMN4B and BHK cells in MEM a t 38.5" differ significantly (p < 0.011.
performed by comparing the rates a t which they produced lactic acid from glucose present in the medium. Table 2 shows that BHK cells grown at 38.5' and exposed at that temperature to a variety of glucose concentrations produced more lactic acid than DMN4B cells subjected to the same conditions. Cells grown and incubated at 32" produced less lactic acid than a t 38.5', and no unequivocal pattern of differences between DMN4B and BHK cells was discernible. DISCUSSION
Radioactive 2-deoxyglucose is extensively used for studies of sugar uptake by cells because it shares a common transport mechanism with glucose, but unlike glucose, is not catabolized after phosphorylation to products which can be quickly lost from the cell (Kipnis and Cori, '59; Smith and Gorski, '68). Although evidence has been reported suggesting that cells may differ in rates of deoxyglucose uptake primarily because of differences in their rates of phosphorylating this sugar (Romano and Colby, '73; Hassell et al., '751, it is likely that most differences involving initial rates of deoxyglucose uptake reflect differences in transport per se (Hatanaka, '74; Kletzien and Perdue, '74). Almost universally, comparison between transformed cells and their normal counterparts has revealed higher rates of deoxyglucose uptake in the former. The present study confirms these findings in the case of the sarcoma virus-transformed K3T3 cells. Such evidence has led to the suggestion that acceler-
ated sugar uptake may account for the loss of the growth restraint characteristic of malignant cells (Oshiro and DiPaolo, '74; Hatanaka, '741, and possibly for their abnormally high rates of glycolysis (Gregg, '72). A recent exception to this evidence is the report of Patterson e t al. ('76), who found that WI-38 human fibroblasts transformed by SV40 virus took up deoxyglucose a t the same rate as untransformed WI-38 cells. The SV40-transformed WI-38 cells are a permanent cell line exhibiting in vitro manifestations of transformation (i.e., loss of density-dependent inhibition) ; however, since they are human cells, tests to confirm in vivo tumorigenicity in the species of origin are not feasible. The most significant finding to emanate from the current study is that a transformed (and tumorigenic) line of BHK cells (DMN4B) takes up deoxyglucose no more rapidly than untransformed BHK cells, and under some circumstances, less rapidly. This is paralleled by evidence that DMN4B cells exhibit rates of glycolysis which are no more rapid than in BHK cells, and at times less rapid, as measured by lactic acid production. No attempt was made to distinguish aerobic from anaerobic glycolysis, since, although the former is a conspicuous feature of most malignant cells, both are characteristically elevated in such cells (Gregg, '72). An unexplained discrepancy between the two results is that differences in deoxyglucose uptake were most conspicuous a t 32", whereas differences in lactic acid production were evident primarily a t 38.5". Presumably, the balance between glyco-
SUGAR UPTAKE IN TRANSFORMED BHK CELLS
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lytic and non-glycolytic metabolic pathways is affected differently by t e m p e r a t u r e in DMN4B and BHK cells. Our results suggest that untransformed cells (at least untransformed BHK cells) need not acquire an enhanced capacity to take up sugar (or metabolize i t to lactic acid) in order to undergo conversion to a transformed state in vitro or become tumorigenic in vivo. Presumably, their growth is regulated by other factors. It is conceivable, of course, that DMN4B cells are exceptional in this regard and have become transformed by a mechanism totally different from the mechanisms responsible for transformation in other cells. Our kinetic data provide suggestive evidence against this possibility, however. Like other transformed cells, DMN4B cells appear to have undergone membrane changes affecting sugar transport. In previous reports, increased transport has been correlated in some cases with reductions in Km (Hatanaka et al., '69; Hatanaka et al., '70; Hatanaka and Gilden, '70; Hatanaka and Hanafusa, '70; May et al., '731, whereas in other cases, increases in Vmax have been found, without changes in Km (Isselbacher, '72; Weber, '73; Plagemann, '73; Kuroki and Yamakawa, '74; Kletzien and Perdue, '74; Siddiqi and Iype, '75). In the case of DMN4B cells, small increases in Vmax were observed at both 32" and 38.5". The larger changes, however, were in Km. At 32", Km was s u b s t a n t i a l l y higher t h a n i n u n transformed BHK cells. In contrast, a t 38.5", Km in DMN4B cells was reduced significantly, to a level only slightly higher than in BHK cells. Thus, DMN4B cells, in changing from the untransformed (32") to the transformed s t a t e (38.5'1, exhibit reductions in Km analogous to those reported for a variety of other transformed cells. Because however, the Km which characterizes their untransformed state is so high, the reduction observed in the transformed state is sufficient only to normalize their deoxyglucose uptake, rather than raise it to supernormal levels. DMN4B cells thus appear to exhibit both membrane alterations similar to those of other transformed cells, and other alterations which may uniquely impair their ability to take up sugar. If the common membrane alterations are responsible for cellular transformation, the present results indicate that this effect is probably mediated by something other than an abnormally high sugar uptake. Whether
151
this conclusion also applies to the uptake of other substrates remains to be determined experimentally. ACKNOWLEDGMENT
This work was supported in part by NIH Grants CA12404 and CA15848. LITERATURE CITED Bose, S. K., and B. J. Zlotnick 1973 Growth and densitydependent inhibition of deoxyglucose transport in Balb 3T3 cells and its absence in cells transformed by murine sarcoma virus. Proc. Nat. Acad. Sci., 70: 2374-2378. DiMayorca, G., M. Greenblatt, T. Trauthen, A. Soller and R. Giordano 1973 Malignant transformation of BHKPl Clone 13 cells in vitro by nitrosamines-a conditional state. Proc. Nat. Acad. Sci., 70: 46-49. Gregg, C. T. 1972 Some aspects of the energy metabolism of mammalian cells. In: Growth, Nutrition, and Metaholism of Cells in Culture. G . H. Rothblat and V. J. Cristofalo, eds. Academic Press, New York and London, pp. 83-136. Hassell, J. A., C. Colby and A. H. Romano 1975 The effect of serum on the transport and phosphorylation of 2-deoxyglucose by untransformed and transformed mouse 3T3 cells. J. Cell. Physiol., 86: 37-45. Hatanaka, M. 1974 Transport of sugars in tumor cell membranes. Biochim. Biophys. Acta, 355: 77-104. Hatanaka, M., and R. V. Gilden 1970 Virus-specified changes in the sugar-transport kinetics of r a t embryo cells infected with murine sarcoma virus. J. Nat. Cancer Inst., 45: 87-89. 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. Hatanaka, M., R. J. Huebner and R. V. Gilden 1969 Alterations in the characteristics of sugar uptake by mouse cells transformed by murine sarcoma viruses. J. Nat. Cancer Inst., 43: 1091-1096. Hatanaka, M., G. J. Todaro and R. V. Gilden 1970 Altered glucose transport kinetics in murine sarcoma virustransformed BALB/3T3 clones. Internat. J. Cancer, 5: 224-228. Isselbacher, K. J. 1972 Increased uptake of amino acids and 2-deoxy-D-glucoseby virus-transformed cells in culture. Proc. Nat. Acad. Sci., 69: 585-589. Kipnis, D. M., and C. F. Cori 1959 Studies of tissue permeability. V. The penetration and phosphorylation of 2deoxyglucose in the r a t diaphragm. J. Biol. Chem., 234: 171-177. Kletzien, R. F., and J. J. Perdue 1974 Sugar transport in chick embryo fibroblasts. 11. Alterations in transport following transformation by a temperature-sensitive mutant of the Rous sarcoma virus. J. Biol. Chem., 249: 3375-3382. Kuroki, T., and S. Yamakawa 1974 Kinetics of uptake of 2deoxy-D-glucoseand 2-aminoisobutyric acid in chemically transformed cells. Internat. J. Cancer, 13: 240-245. Lowry, O., N. Rosebrough, A. Farr and R. Randall 1951 Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Martineau, R., M. Kohlbacher, S. N. Shaw and H. Amos 1972 Enhancement of hexose entry into chick fibroblasts by starvation: differential effect on galactose and glucose. Proc. Nat. Acad. Sci., 69: 3407-3411.
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May, J. T., K. D. Somers and S. Kit 1973 Temperaturedependent alterations in 2-deoxyglucose uptake in rat cells transformed by a cold-sensitive murine sarcoma virus mutant. Internat. J. Cancer, 11: 377-384. Oshiro, Y., and J. A. DiPaolo 1974 Changes in the uptake of 2-deoxy-D-glucosein Balb/3T3 cells chemically transformed in culture. J. Cell. Physiol., 83: 193-202. Patterson, M. K., P. J. Birckbichler, E. Conway and G. R. Orr 1976 Amino acid and hexose transport of normal and Simian Virus 40-transformed human cells. Cancer Res., 36: 394-397. Plagemann, P. G. W. 1973 Deoxyglucose transport by uninfected, murine sarcoma virus-transformed, and murine leukemia virus-infected mouse cells. J. Cell. Physiol., 82: 421-433. Romano, A. H., and C. Colby 1973 SV40 virus transforma-
tion of mouse 3T3 cells does not specifically enhance sugar transport. Science, 179: 1238-1240. Schneider, E. L., E. J. Stanhridge and C. J. Epstein 1974 Incorporation of 3H-uridine and 3H-uracil into RNA. A simple technique for the detection of mycoplasma contamination of cultured cells. Exptl. Cell Res., 84:311-318. Siddiqi, M., and P. T. Iype 1975 Studies on the uptake of 2deoxy-D-glucose in normal and malignant rat epithelial liver cells in culture. Internat. J. Cancer, 15: 773-780. Smith, D. E., and J. Gorski 1968 Estrogen control of uterine glucose metabolism. An analysis based on the transport and phosphorylation of 2-deoxyglucose.J. Biol. Chem., 243: 4169-4174. Weber, M. J. 1973 Hexose transport in normal and Rous sarcoma virus-transformed cells. J. Biol. Chem., 248: 2978-2983.
ABSTRACT The uptake of deoxyglucose was compared in BHK cells and in DMN4B cells, a conditionally transformed line of BHK cells which exhibits transformed behavior a t 38.5' but not at 32'. At 32', DMN4B cells took up deoxyglucose more slowly than BHK cells, reflecting a higher Km for uptake of this sugar. When both cell lines were grown at 38.5', the Km for DMN4B cells was reduced to a level only slightly greater than for BHK cells, and deoxyglucose uptake became similar in the two cell lines. Growth in glucose-free medium for 22 hours stimulated deoxyglucose uptake in both BHK and DMN4B cells; under these conditions, uptake was equal in the two cells lines, both at 32' and 38.5'. Glycolysis, as measured by lactic acid production, was slower in DMN4B than BHK cells, but in contrast to deoxyglucose uptake, this difference was observed a t 38.5' rather than 32'. The observation that the subnormal deoxyglucose uptake of DMN4B cells in the untransformed state (32') can be normalized by growth a t 38.5', a temperature permissive for transformation, suggests that membrane changes facilitating sugar uptake, which have been found in other transformed cells, are associated with transformation in DMN4B cells as well. However, the failure of uptake to exceed normal in these cells indicates that their transformed behavior is not attributable to excessive sugar uptake per se. A hallmark of malignant transformation is an increase in the rate of cellular uptake of a variety of nutrients and other small molecules. In particular, rates of sugar uptake, as measured by the rate of cellular accumulation of the glucose analogue, 2-deoxy-D-glucose, have been found higher in transformed cells than in their normal counterparts (reviewed by Hatanaka, '74), and it has been suggested that, in fact, increased ability of these cells to take up sugar may be responsible for their uncontrolled growth (Oshiro and DiPaolo, '74; Hatanaka, '74). We now report a transformed line of baby hamster kidney (BHK) cells which displays rates of sugar uptake lower than or equal to those in untransformed BHK cells. Membrane changes associated with increased sugar uptake in other transformed cell lines may be present in these cells as well, but are offset by other changes which reduce the efficiency of sugar transport. The results suggest that changes in sugar uptake may reflect changes in membrane function that are crucial for transformation, but that sugar uptake itself is not the crucial phenomenon. J. CELL. PHYSIOL.. 93: 147-152.
MATERIALS AND METHODS
Cells and media Low-passage BHK cells and the transformed BHK line DMN4B (both obtained from Doctor G. DiMayorca, University of Illinois), BALB/3T3 mouse endothelial cells (clone A31), and A31 cells transformed by Kirsten sarcoma virus (K3T3 cells) were all grown in monolayer culture in Dulbecco's medium containing penicillin (100 unitdml), streptomycin (100 fig/ml), 10% fetal calf serum (FCS), and 25 mM glucose, in a humidified, 5% COz atmosphere. The A31 and K3T3 cells were routinely maintained at 37'. DMN4B cells, a line derived by mutagenesis of BHK cells with dimethylnitrosamine, are conditionally transformed. They exhibit a loss of density-dependent inhibition, a transformed morphology, and the ability to produce colonies in soft agar at 38.5' but not at 32'; they are also tumorigenic in hamsters (DiMayorca et al., '73). Low-passage BHK cells lack these properties (DiMayorca e t al., '73). Because these attriReceived Mar. 25, '77. Accepted Apr. 26, '77.
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F. L. MOOLTEN, D. N. MOOLTEN AND N. J. CAPPARELL
rected by subtracting "zero-time'' measurements (3H-deoxyglucosewas added and removed, and the first cold PBS wash was applied within 6-8 seconds). Deoxyglucose uptake measured a t 5 mM concentration was found to be linear with time for at least five to ten minutes with both cell types and both temperatures; therefore, studies performed with other (lower) concentrations utilized 5minute labeling intervals. The "zero-time'' corrections were similar for BHK and DMN4B cells (varying < 15% between the 2 cell types), and always represented < 23% of the total uptake occurring in five minutes. I n some cases, experiments were done with cells "pre-starved" by incubating them for 22 hours in glucose-free Eagle's Minimal Essential Medium (MEM) containing 3% dialyzed Deoxyglucose uptake FCS. To minimize extraneous sources of variaMonolayer cultures in 25 cm2Falcon plastic tion, all comparisons between DMN4B and flasks were grown to confluence-a condition BHK cells (or between A31 and K3T3 cells) that has been reported capable of enhancing involved flasks of both cell types, in duplicate differences in deoxyglucose uptake between to quadruplicate, handled a t the same times transformed and untransformed cells (Bose as part of the same experiment. and Zlotnick, '73). The cultures were used Lactic acid production within one day of reaching the confluent state to prevent cell densities in DMN4B cultures Culture flasks were aspirated and washed from exceeding those in BHK cultures. Under once with HSS. The medium was then rethese conditions, the amount of cellular mate- placed with 5 ml of fresh culture medium, o r rial (measured as protein) was always similar with 5 ml of MEM containing 4%dialyzed FCS in DMN4B and BHK cultures, and differences and glucose a t concentrations of 25, 8, or 4 between the cell lines in deoxyglucose uptake/ mM. After the cells were incubated for three mg protein were always reflected by differ- hours a t the temperature a t which they had ences in uptake/culture flask. For uptake been grown, the medium was removed, and studies, the flasks were aspirated, and washed the cells were dissolved in NaOH for deterthree times with glucose-free Hanks Salt mination of protein concentration. To deterSolution (HSS) pre-warmed to 32" or 38.5" mine the lactic acid concentration of the according to the temperature a t which the aspirated medium, 2 mi of the latter was cells were grown. One milliliter of HSS was added to 4 ml of 8%perchloric acid. The resultthen added, and the flasks were re-incubated ing precipitates were removed by centrifugaat the same temperature for five minutes to tion and the supernatants were analyzed encorrect for any cooling that might have oc- zymatically for lactic acid by measuring the curred during the washing. An additional 1ml quantity of reduced nicotinamide adenine of HSS was then added, containing 1pCi of 2- dinucleotide (NADH) that could be generated deo~y-D-glucose-~H (New England Nuclear, from the oxidized form (NAD) in the presence Boston, Massachusetts) and sufficient non- of lactic dehydrogenase, using a commercially radioactive deoxyglucose to achieve the de- available reagent kit (Sigma Chemical Co., St. sired concentration. The contents were mixed, Louis, Missouri). The values obtained were and the flasks were then re-incubated for corrected by subtracting the values obtained various intervals. They were then aspirated, with unused medium. and washed three times with ice-cold phosRESULTS phate buffered saline (PBS). The cells were At 32" deoxyglucose uptake in DMN4B cells dissolved by addition of 1.7 ml of 0.5 N NaOH, and aliquots were removed for measurements was significantly lower than in BHK cells; of radioactivity and of protein (Lowry et al., Lineweaver-Burk plots of these data are '51). Values of deoxyglucose uptake were cor- shown in figure 1. The reduction in uptake
butes can change with repeated passage, cells from the passage levels used in this study were routinely plated in soft agar in parallel with the experiments on sugar uptake. These tests confirmed the ability of DMN4B cells to form colonies a t 38.5", but not a t 32", and the inability of the BHK cells used in these experiments to form colonies a t either temperature. K3T3 cells produced colonies a t both temperatures. DMN4B and BHK cells were routinely maintained a t 32"; for experiments a t 38.5" they were grown for a t least 3.5 generations a t this higher temperature to permit full expression of the transformed phenotype (DiMayorca et al., '73). All cell lines have been found free of mycoplasma on repeated testing by the method of Schneider et al. ('74).
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2.5
mM-'
Fig. 1 Lineweaver-Burk plots of initial rates of 2-deoxyglucose (2-DOG)uptake by BHK cells (open circles) and DMN4B cells (closed circles) at concentrations between 0.4 and 5 mM. Each point represents the mean of duplicate determinations. Linear regression analysis was used to plot each line, and revealed that the Km value derived from the line for DMN4B cells at 32" differed significantly from the Km for BHK cells at 32" (p < 0.001) and from the Km for DMN4B cells at 38.5' (p < 0.01). TABLE 1
The effectofgrowth conditions on 2-deoxyglucoseuptake by untransformed and transformed hamster and mouse cells Growth conditions
2-deoxyglucose uptake
Temperature
Growth medium
BHK cells
32"
Dulbecco's Glucose-free MEM Dulbecco's Glucose-free MEM
12.3ic1.2(5) 51.6*4.5(4) 16.721.1(6) 85.722.6(4)
38.5'
DMN4B cells
3.5ic0.4(6) 52.2ic6.6(4) 20.7+-1.8(6) 86.0&3.8(4)
A31 cells
K3T3 cells
8.0ic0.113)
17.3t0.613)
7.4t0.2(3)
12.8t0.5(3)
' Cells were exposed to denxyglucose for five minutes a t a concentration of 1 mM. Uptake is expressed as nmolesimg protein/5 min 2 standard error; the number of samples is stated in parentheses. The glucose concentration of the Dulbecco's medium was 25 mM. 'The value for uptake is significantly less than in the corresponding untransformed cells (BHK cells); p < 0.01 by student's t test. 3The value for uptake is significantly greater than in the corresponding untransformed cells (A31 cells); p < 0.01.
was attributable to a higher Km for DMN4B than for BHK cells (10.2 vs 2.2 mM), suggesting a lower affinity of the transport mechanism for the sugar. In contrast, the maximum theoretical rate of uptake, Vmax, was slightly higher in the DMN4B cells (10.2 vs 7.5 nmoledmg protein/min). A t the permissive temperature for transformation in DMN4B cells, 38.5", the differences between the two cell types was much less, primarily reflecting a n increase in Km for BHK cells to 3.2 mM, and a reduction in Km for DMN4B cells to 5.0 mM. Vmax remained slightly higher in DMN4B than in BHK cells (22.2 vs 18.2 nmoleslmg protein/min) . Deoxyglucose uptake has been reported to increase substantially in cells depleted of glucose for many hours (Martineau et al., '72). I t seemed possible, therefore, that failure of DMN4B cells to exceed BHK cells in deoxyglucose uptake when both were grown in
Dulbecco's medium (which contains 25 mM glucose), may have reflected merely a lack of stimulation rather than a lack of capacity. DMN4B and BHK cells were therefore compared after incubation in glucose-free MEM for 22 hours. The results, shown in table 1, indicated that deoxyglucose uptake was in fact greatly enhanced under these conditions. This effect occurred in both types of cells, however, and although sufficient to abolish the disparity between DMN4B and BHK cells a t 32", failed a t either temperature to enhance uptake rates in DMN4B cells to levels exceeding those in BHK cells. In contrast, table 1 also shows that even unstarved cultures of Kirsten sarcoma virus-transformed (K3T3) cells exhibited rates of deoxyglucose uptake about twice as great as those of their normal counterparts. An additional comparison of the ability of DMN4B and BHK cells to utilize sugar was
150
F. L. MOOLTEN, D. N. MOOLTEN AND N. J. CAPPARELL TABLE 2
Lactic acid production by DMN4B and BHK cells
Temperature
Incubation medium '
38.5'
Dulbecco's MEM
32"
Dulbecco's MEM
Lactic acid produced lvmoleslmg proteinlhour)
Glucose concentration (mM)
BHK cells
25 25 8 4 25 25 8 4
1.58%0.08(31 1.302 0.0212) 1.0910.07(2) 0.8420.02(2) 0.972 0.06(4) 0.662 0.04(2) 0.5520.03(2) 0.4410.04121
DMN4B cells
0.6920.02(41 0.7120.03(2) 0.63%0.06(2) 0.602 0.02(2) 0.81%0.04(4) 0.70%0.2212) 0.732 O.Ol(2) 0.482 0.08(21
' The Dulbecco's medium contained 10%FCS and the MEM 4%dialyzed FCS. Cells were incubated in these media for three hours at the temperature at which they had been grown. *Results are expressed as mean rt standard error; the number of samples is stated in parentheses. This value differs significantly from the corresponding value for BHK cells (p < 0.01). Combined analysis using values obtained at each glucose concentration show that DMN4B and BHK cells in MEM a t 38.5" differ significantly (p < 0.011.
performed by comparing the rates a t which they produced lactic acid from glucose present in the medium. Table 2 shows that BHK cells grown at 38.5' and exposed at that temperature to a variety of glucose concentrations produced more lactic acid than DMN4B cells subjected to the same conditions. Cells grown and incubated at 32" produced less lactic acid than a t 38.5', and no unequivocal pattern of differences between DMN4B and BHK cells was discernible. DISCUSSION
Radioactive 2-deoxyglucose is extensively used for studies of sugar uptake by cells because it shares a common transport mechanism with glucose, but unlike glucose, is not catabolized after phosphorylation to products which can be quickly lost from the cell (Kipnis and Cori, '59; Smith and Gorski, '68). Although evidence has been reported suggesting that cells may differ in rates of deoxyglucose uptake primarily because of differences in their rates of phosphorylating this sugar (Romano and Colby, '73; Hassell et al., '751, it is likely that most differences involving initial rates of deoxyglucose uptake reflect differences in transport per se (Hatanaka, '74; Kletzien and Perdue, '74). Almost universally, comparison between transformed cells and their normal counterparts has revealed higher rates of deoxyglucose uptake in the former. The present study confirms these findings in the case of the sarcoma virus-transformed K3T3 cells. Such evidence has led to the suggestion that acceler-
ated sugar uptake may account for the loss of the growth restraint characteristic of malignant cells (Oshiro and DiPaolo, '74; Hatanaka, '741, and possibly for their abnormally high rates of glycolysis (Gregg, '72). A recent exception to this evidence is the report of Patterson e t al. ('76), who found that WI-38 human fibroblasts transformed by SV40 virus took up deoxyglucose a t the same rate as untransformed WI-38 cells. The SV40-transformed WI-38 cells are a permanent cell line exhibiting in vitro manifestations of transformation (i.e., loss of density-dependent inhibition) ; however, since they are human cells, tests to confirm in vivo tumorigenicity in the species of origin are not feasible. The most significant finding to emanate from the current study is that a transformed (and tumorigenic) line of BHK cells (DMN4B) takes up deoxyglucose no more rapidly than untransformed BHK cells, and under some circumstances, less rapidly. This is paralleled by evidence that DMN4B cells exhibit rates of glycolysis which are no more rapid than in BHK cells, and at times less rapid, as measured by lactic acid production. No attempt was made to distinguish aerobic from anaerobic glycolysis, since, although the former is a conspicuous feature of most malignant cells, both are characteristically elevated in such cells (Gregg, '72). An unexplained discrepancy between the two results is that differences in deoxyglucose uptake were most conspicuous a t 32", whereas differences in lactic acid production were evident primarily a t 38.5". Presumably, the balance between glyco-
SUGAR UPTAKE IN TRANSFORMED BHK CELLS
-
lytic and non-glycolytic metabolic pathways is affected differently by t e m p e r a t u r e in DMN4B and BHK cells. Our results suggest that untransformed cells (at least untransformed BHK cells) need not acquire an enhanced capacity to take up sugar (or metabolize i t to lactic acid) in order to undergo conversion to a transformed state in vitro or become tumorigenic in vivo. Presumably, their growth is regulated by other factors. It is conceivable, of course, that DMN4B cells are exceptional in this regard and have become transformed by a mechanism totally different from the mechanisms responsible for transformation in other cells. Our kinetic data provide suggestive evidence against this possibility, however. Like other transformed cells, DMN4B cells appear to have undergone membrane changes affecting sugar transport. In previous reports, increased transport has been correlated in some cases with reductions in Km (Hatanaka et al., '69; Hatanaka et al., '70; Hatanaka and Gilden, '70; Hatanaka and Hanafusa, '70; May et al., '731, whereas in other cases, increases in Vmax have been found, without changes in Km (Isselbacher, '72; Weber, '73; Plagemann, '73; Kuroki and Yamakawa, '74; Kletzien and Perdue, '74; Siddiqi and Iype, '75). In the case of DMN4B cells, small increases in Vmax were observed at both 32" and 38.5". The larger changes, however, were in Km. At 32", Km was s u b s t a n t i a l l y higher t h a n i n u n transformed BHK cells. In contrast, a t 38.5", Km in DMN4B cells was reduced significantly, to a level only slightly higher than in BHK cells. Thus, DMN4B cells, in changing from the untransformed (32") to the transformed s t a t e (38.5'1, exhibit reductions in Km analogous to those reported for a variety of other transformed cells. Because however, the Km which characterizes their untransformed state is so high, the reduction observed in the transformed state is sufficient only to normalize their deoxyglucose uptake, rather than raise it to supernormal levels. DMN4B cells thus appear to exhibit both membrane alterations similar to those of other transformed cells, and other alterations which may uniquely impair their ability to take up sugar. If the common membrane alterations are responsible for cellular transformation, the present results indicate that this effect is probably mediated by something other than an abnormally high sugar uptake. Whether
151
this conclusion also applies to the uptake of other substrates remains to be determined experimentally. ACKNOWLEDGMENT
This work was supported in part by NIH Grants CA12404 and CA15848. LITERATURE CITED Bose, S. K., and B. J. Zlotnick 1973 Growth and densitydependent inhibition of deoxyglucose transport in Balb 3T3 cells and its absence in cells transformed by murine sarcoma virus. Proc. Nat. Acad. Sci., 70: 2374-2378. DiMayorca, G., M. Greenblatt, T. Trauthen, A. Soller and R. Giordano 1973 Malignant transformation of BHKPl Clone 13 cells in vitro by nitrosamines-a conditional state. Proc. Nat. Acad. Sci., 70: 46-49. Gregg, C. T. 1972 Some aspects of the energy metabolism of mammalian cells. In: Growth, Nutrition, and Metaholism of Cells in Culture. G . H. Rothblat and V. J. Cristofalo, eds. Academic Press, New York and London, pp. 83-136. Hassell, J. A., C. Colby and A. H. Romano 1975 The effect of serum on the transport and phosphorylation of 2-deoxyglucose by untransformed and transformed mouse 3T3 cells. J. Cell. Physiol., 86: 37-45. Hatanaka, M. 1974 Transport of sugars in tumor cell membranes. Biochim. Biophys. Acta, 355: 77-104. Hatanaka, M., and R. V. Gilden 1970 Virus-specified changes in the sugar-transport kinetics of r a t embryo cells infected with murine sarcoma virus. J. Nat. Cancer Inst., 45: 87-89. 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. Hatanaka, M., R. J. Huebner and R. V. Gilden 1969 Alterations in the characteristics of sugar uptake by mouse cells transformed by murine sarcoma viruses. J. Nat. Cancer Inst., 43: 1091-1096. Hatanaka, M., G. J. Todaro and R. V. Gilden 1970 Altered glucose transport kinetics in murine sarcoma virustransformed BALB/3T3 clones. Internat. J. Cancer, 5: 224-228. Isselbacher, K. J. 1972 Increased uptake of amino acids and 2-deoxy-D-glucoseby virus-transformed cells in culture. Proc. Nat. Acad. Sci., 69: 585-589. Kipnis, D. M., and C. F. Cori 1959 Studies of tissue permeability. V. The penetration and phosphorylation of 2deoxyglucose in the r a t diaphragm. J. Biol. Chem., 234: 171-177. Kletzien, R. F., and J. J. Perdue 1974 Sugar transport in chick embryo fibroblasts. 11. Alterations in transport following transformation by a temperature-sensitive mutant of the Rous sarcoma virus. J. Biol. Chem., 249: 3375-3382. Kuroki, T., and S. Yamakawa 1974 Kinetics of uptake of 2deoxy-D-glucoseand 2-aminoisobutyric acid in chemically transformed cells. Internat. J. Cancer, 13: 240-245. Lowry, O., N. Rosebrough, A. Farr and R. Randall 1951 Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Martineau, R., M. Kohlbacher, S. N. Shaw and H. Amos 1972 Enhancement of hexose entry into chick fibroblasts by starvation: differential effect on galactose and glucose. Proc. Nat. Acad. Sci., 69: 3407-3411.
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May, J. T., K. D. Somers and S. Kit 1973 Temperaturedependent alterations in 2-deoxyglucose uptake in rat cells transformed by a cold-sensitive murine sarcoma virus mutant. Internat. J. Cancer, 11: 377-384. Oshiro, Y., and J. A. DiPaolo 1974 Changes in the uptake of 2-deoxy-D-glucosein Balb/3T3 cells chemically transformed in culture. J. Cell. Physiol., 83: 193-202. Patterson, M. K., P. J. Birckbichler, E. Conway and G. R. Orr 1976 Amino acid and hexose transport of normal and Simian Virus 40-transformed human cells. Cancer Res., 36: 394-397. Plagemann, P. G. W. 1973 Deoxyglucose transport by uninfected, murine sarcoma virus-transformed, and murine leukemia virus-infected mouse cells. J. Cell. Physiol., 82: 421-433. Romano, A. H., and C. Colby 1973 SV40 virus transforma-
tion of mouse 3T3 cells does not specifically enhance sugar transport. Science, 179: 1238-1240. Schneider, E. L., E. J. Stanhridge and C. J. Epstein 1974 Incorporation of 3H-uridine and 3H-uracil into RNA. A simple technique for the detection of mycoplasma contamination of cultured cells. Exptl. Cell Res., 84:311-318. Siddiqi, M., and P. T. Iype 1975 Studies on the uptake of 2deoxy-D-glucose in normal and malignant rat epithelial liver cells in culture. Internat. J. Cancer, 15: 773-780. Smith, D. E., and J. Gorski 1968 Estrogen control of uterine glucose metabolism. An analysis based on the transport and phosphorylation of 2-deoxyglucose.J. Biol. Chem., 243: 4169-4174. Weber, M. J. 1973 Hexose transport in normal and Rous sarcoma virus-transformed cells. J. Biol. Chem., 248: 2978-2983.
