The Effect of Serum on the Transport and Phosphorylation of 2-Deoxyglucose by Untransformed and Transformed Mouse 3T3 Cells JOHN A. HASSELL.] C L A R E N C E COLBY 2 A N D ANTON10 [ I . K O M A N O Microbiology Sertion, The University of Con necticict, Storrs, Connecticut 06268
ABSTRACT Serum starvation of growing and nongrowing (density-inhibited) mouse 3T3 cells resulted in decreased phosphorylation of 2deoxy-D-glucose, while the time course of transport of this sugar remained unchanged. Serum starvation of SV40 transformed 3T3 cells (SV101) and spontaneously transformed 3T6 cells did not alter either the time course of transport, or phosphorylation of the sugar. Treatment of SVlOl cells with M dibutyryl adenosine cyclic 3':5' monophosphate and 1 0 - 3 M theophylline did not restore the capacity to regulate 2-deoxy-Dglucose phosphorylation when these cells were serum deprived. We conclude that serum factors are involved in the modulation of phosphorylation of 2-deoxy-D-glucose in 3T3 cells rather than its transport. This regulation is operative both in growing as well as nongrowing 3T3 cells. I n contrast, transformed cells do not respond to this regulation of 2-deoxy-D-glucosr phosphorylation. When mouse 3T3 cells are deprived of serum, a n ensemble of alterations in metabolism occurs. These include changes in the overall rate of protein and RNA synthesis, the rate of protein degradation, and the rate of uptake of nucleic acid precursors (Soeiro and Amos, '66; Hershko et al., '71; Hassell and Engelhardt, '73). Those biochemical reactions influenced by serum factors form the pleiotypic program (Kram and Tomkins, '73). In virus-transformed cells the pleiotypic program is relatively insensitive to serum factors (Hershko et al., '71). When 3T3 cells enter the stationary phase of growth their metabolism is altered in much the same manner as occurs after serum starvation (Todaro et al., '65; Cunningham and Pardee, '69; Weber and Rubin, '70). It has been suggested that a limitation of medium factors prevents the growth of 3T3 cells at high cell density (Holley and Kiernan, '71; Dulbecco and Elkington, '73). Hence serum starvation might elicit the same biochemical response by the cell as entry into the stationary phase of the growth curve. It has been reported that the rate of glucose uptake, as measured by the upJ CELL.PHYSIOL., 86. 3 7 4 6 .
take of the analogue 2-deoxyglucose (2dOG), is stimulated by the re-addition of serum to previously starved 3T3 cells. This reaction has been included in tho pleiotypic program (Kram and Tomkins, '73). However, these authors did not separate transport from the subsequent phosphorylation of 2-dOG, and hence it was not clear which process was stimulated by s e r ~ m . Reports from this laboratory (Romano and Colby, '73; Colby and Romano, '74)h i i v f emphasized that the uptake and phosphorylation of 2-dOG are separate and scquential events, and that the apparent enhancement of 2-dOG uptake by 3T3 cells accompanying viral transformation is not due primarily to an effect on the transport process, but to enhanced phosphorylation. The present study was undertaken, therefore, to measure the time course of entry of 2-deoxyglucose into intracellular free sugar and sugar phosphate pools in 3T3 cells and their transformed derivatives. Received Oct. 3, '74. Accepted Jan. 2, '75. * Present address: Cold Spring Harbor Laboratory. P.O. Box 100, Cold Spring Harbor, New York 11724. 2 Present address: Department of Genetics, University of California, Davis, California 95616.
37
38
J. A. HASSELL, C. COLBY A N D A. H. ROMANO
under conditions of serum starvation and serum sufficiency.
and Pardee, '69). Cell cultures were generally used two to four days after seeding. Measurements of the uptake of '"-2MATERIALS A N D METHODS deoxy-D-glucose and its conversion to the Cells were grown in Dulbecco's modified phosphorylated form were made on cells Eagle's medium (DME) (Vogt and Dul- growing on the surface of 2.25 cm2 glass becco, '63) supplemented with calf serum coverslips by a modification of methods to 10% (V/V) (growth medium) in a 5% developed by Foster and Pardee ('69). CovC 0 2 atmosphere. Balb/c 3T3 cells were erslips with adherent cells were transferred maintained by transfer of lo4 cells to 100 with forceps from growth medium through mm Falcon plastic dishes every four days. four successive washings of glucose-free These cells were not allowed to attain con- Hanks solution at 37"C, and'were incufluence before subculturing. SV4[, trans- bated a t 37°C in I4C-2dOG solutions (in formed 3T3 cells (SV101 cells) and 3T6 glucose-free Hanks solution) at the concells were maintained by transfer of 5 X centrations, specific activities and time 10.5 cells to 100 mm plastic Petri dishes, intervals indicated. Coverslips were reand replating after the cells had become moved from the isotopic solutions, rinsed through four successive washings of iceconfluent. The growth of these cells was monitored cold glucose-free Hanks solution, and exas follows: Cells were seeded onto Falcon tracted with 1 ml boiling water for one Petri dishes (19.6 cm2) and 4 ml of growth minute. Free sugar and sugar phosphates medium added. The growth medium was in the extracts were separated by the chrochanged every 48 hours. Cell number was matographic method of Winkler ('60) as determined by rinsing the cells with T.E. follows: 0.5 ml samples of extract were (0.05% trypsin, 0.005 M EDTA in Ca++ applied to columns (4 X 0.6 cm) of BioMg++free phosphate-buffered salt solution), Rad AG1-X2 anion exchange resin (100 to and counting a sample with a hemocy- 200 mesh) which had been converted to tometer. the formate form. Free 2-dOG was eluted To serum deprive cells, the medium was with five 0.5 ml portions of water; the removed, and the cell layer rinsed twice phosphorylated sugar (2-dOG-P) was subwith 5 ml portions of prewamed (37°C) sequently eluted with seven 0.5 ml portions DME; 4 ml of DME was added to some of of 0.5 M ammonium formate in 0.2 M the cultures (serum-deprived cells) while formic acid. Eluted materials were colothers received 4 ml of growth medium lected directly into scintillation vials con(controls). taining 10 ml Bray's fluid (Bray, '60) for Cells growing attached to glass cover- counting. Protein determinations were performed slips (2.25 cm2) were used for uptake measurements (Foster and Pardee, '69). A in quadruplicate on parallel cover slipsuspension of cells in 10 ml of growth me- grown cultures by the method of Lowry et dium was added to coverslips lining the al., ('51). Chemicals were obtained from the folbottoms of 100 mm diameter plastic Petri dishes. A day later these coverslips were lowing sources: DME medium and calf placed in several 100 mm dishes (6 cover- serum, GIBCO; NfiOz-dibutyryl adenosine slips per dish) containing 10 ml of growth cyclic 3':5' monophosphate and theophylmedium, and the growth medium was line, Sigma; uniformly labelled 2-deoxy-Dchanged daily thereafter. The cells were ( I T ) glucose, International Chemical and deprived of serum by removing the growth Nuclear Corp. ; non-radioactive 2-deoxy-Dmedium and rinsing the coverslips twice glucose, SchwarzIMann. with 10 ml portions of prewarmed (37°C) RESULTS DME; 10 ml of DME was added to some Growth response of 3T3 cells to of the cultures (serum-deprived cells) while serum deprivation others (controls) received 10 ml of growth medium. The number of cells per cover3T3 cells growing in DME medium supslip was determined in triplicate on the plement with calf serum (10% ) had a genday preceding, and immediately before eration time of eleven hours and reached their use in uptake experiments (Foster a final saturation density of 7 x 1 0 4 cells/
TRANSFORMATION, SERUM, AND SUGAR PHOSPHORYLATION
cm2, when the medium was changed every 48 hours. Transfer of exponentially growing 3T3 cells to DME medium without serum immediately stopped cell growth, while prolonged serum starvation resulted in cell death (fig. 1). After 24 hours of serum deprivation, cells were still viable as judged by their capacity to resume cell division at the normal rate when stimulated with serum-containing medium (fig. 1). Transport and phosphorylation of 2-deoxy-D-glucose by 3 T 3 cells In previous experiments Romano and Colby ('73) provided evidence that the rate 100 90
80 70
60 50 40
30 20
E
u
1
m
0 x
10 9
2
7
8
S 65 4
0
2
4
6
8
Days Fig. 1 Growth response of 3T3 cells to serum starvation. 3T3 cells were plated at 2.7 X lo3 cells/cmZ on 19.6 cmz Falcon tissue-culture dishes on day 0. On day 2 the medium was changed on all the cultures; some received DME medium plus calf serum (10%) O---O, while others received DME medium only (I---Q. On day 3 the medium was changed on all the cultures and some of those which had been previously serum deprived received DME medium plus calf serum (10%) A-- -A. The medium was changed every 2 days on all the dishes thereafter. Cell number was determined on duplicate dishes as described i n MATERIALS A N D METHODS.
39
of phosphorylation of 2-dOG was greater in transformed cells compared to untransformed cells, and suggested that the physiological state of the cell might influence this reaction. Since serum starvation alters the growth rate of the cell (fig. 1) we studied the effect of this parameter on the uptake and phosphorylation of 2-dOG. 3T3 cells at a density of 1-2 x 104 cellslcm' were deprived of serum for 20 hours and the time course of entry of 2-dOG into a free and phosphorylated pool was then measured. The cell number of the control cultures more than doubled, while no increase in cell number occurred in the serum-deprived cultures after 20 hours (data not shown). The time course of entry of 2-dOG into a free intracellular pool was unaffected by serum starvation while the apparent rate of phosphorylation of 2-dOG was reduced to 30% of the control rate (fig. 2A). This result indicates that phosphorylation of the sugar rather than transport is controlled by the availability of serum in the medium. It has been assumed that those reactions which make up the pleiotypic program fluctuate coordinately with changes in the cell growth rate (Hershko et al., '71). We therefore investigated the possibility that nongrowing (density-inhibited) 3T3 cells might phosphorylate 2-dOG at a reduced rate when compared to growing cells. We also inquired whether serum starvation of nongrowing cells would further depress this reaction. Nongrowing 3T3 cells at a density of 7-8 x lo4 cells/cm2 were serum starved for 20 hours and the time course of entry of 2-dOG into a free and phosphorylated pool measured. The results show that nongrowing 3T3 cells transported and phosphorylated 2-dOG at essentially the same rate and to the same extent as growing cells (compare figs. 2A and B). Moreover, serum starvation of nongrowing 3T3 cells for 20 hours elicited a decrease in the rate of phosphorylation of 2-dOG to a level 43% that of the control rate, while the time course of transport remained unchanged (fig. 2B). This decrease in phosphorylation of 2-dOG is of the same order of magnitude as that observed when growing 3T3 cells were serum deprived (compare figs. 2A and B). Twenty hours after the medium change to serum-free or serumcontaining growth medium neither culture
40
J. A. HASSELL. C. COLBY AND A. H. ROMANO 50
zw
A
40
I-
0
rr
a 30 (3
2 \
B 20 J 0
2 0
: 10 z C
10
20
30 0 T I M E IN MINUTES
10
20
30
Fig. 2 Effect of serum deprivation on phosphorylation and transport of 2-deoxy-D-(14C) glucose in (A) growing 3T3 and (B) non-growing 3T3 cells. Cells were seeded at 2.5 x 1 0 3 cellslcmz for growing cells, and at 7 X lo4 cellslcmz for non-growing cells onto glass coverslips distributed over the bottoms of 100 mm Falcon plastic dishes in 10 ml of DME medium plus calf serum (10%).After two days the medium was changed to DME medium only (serumdeprived cells) or DME medium plus calf serum (10% ) (controls). Twenty hours later the coverslip. with adherent cells were washed four times with glucose-free Hanks solution at 37OC and incubated in 2 mM uniforn-ly labelled 2-deoxy-D-(1C) glucose (0.5 &wmole) at 37'C. At appropriate intervals duplicate coverslips were removed and processed as described in MATERIALS AND METHODS to separate and determine free 2-deoxy-D-(14C)glucose and 2-deoxyD-(14C) glucose phosphate. Protein was determined on quadruplicate coverslips as described in MATERIALS AND METHODS. A -A, 2-dOG-P controls; A-A 2-dOG-P serum-deprived cells; O---O free dOG controls; 0 - - 4 free 2-dOG, serum deprived cells.
(serum-deprived nor control) experienced a change in cell number (data not shown). These observations indicate that the response to the absence or presence of serum is the same in growing and nongrowing (density-inhibited) 3T3 cells and suggest that a block in neither of these reactions is of itself primarily responsible for curtailing cell division at high cell density. Growth response of transformed cells to serum deprivation SV40 virus transformed cells do not respond biochemically to serum starvation in a manner analogous to 3T3 cells; instead, the rates of RNA, DNA, protein synthesis, uridine uptake, and protein degradation continue essentially unimpaired after 20 hours of serum starvation (Hershko et al., '71). In these experiments the rate of cell division following serum deprivation was not reported, and hence the re-
latedness of the growth rate of the cell to the reactions of the pleiotypic program was not examined. We therefore measured the growth of SVlOl cells to serum starvation over a 24 hour period. The results depicted in figure 3A show that SVlOl cells grown in DME medium supplemented with calf serum ( 1 0 % ) had a generation time of 11 hours when the medium was changed every 48 hours. Serum starvation of these cells resulted in a continuation of cell division but at a reduced rate. In the continued absence of oeru.m, SVlOl cells ceased dividing and cell death occurred after two days. 3T6 cells also exhibited a generation time of 12 hours under these conditions; in the absence of serum these cells also divided at a reduced rate, but at a much lower rate than SVlOl cells (compare figs. 3A and B). Beside this difference, 3T6 cells grew to a stable final saturation density
TRANSFORMATION, SERUM, AND ;UGAR PHOSPHORYLATION
41
3
Days Fig. 3 Growth response of (A) S V l O l cells and (B) 3T6 cells to serum starvation. S V l O l and 3T6 cells were plated at 1 X 1 0 4 cellslcmz on 19.6 cm* Falcon plastic tissue-culture dishes on day 0. On day 2 the medium was changed to DME medium plus calf serum ( 1 0 % ) -0, or to DME medium alone 0-0. Cell number was determined in duplicate as described i n MATERIALS A N D METHODS,
of 2 x 105 cells/cm2, while SVlOl cells continued to divide until the cell layer disrupted. Transport a n d phosphorylation of 2 -d eoxy- D-g lucos e in transformed cells Serum starvation of transformed cells does not elicit the pleiotypic response (Hershko et al., '71), and does not block cell division (figs. 3 A and B). Since we had found that phosphorylation rather than transport of 2-dOG appeared to be regulated by serum factors in 3T3 cells (figs. 2A and B), we investigated the effect of serum starvation of SVlOl and 3T6 cells on their capacity to take up and phosphorylate the sugar. Figure 4A shows that serum starvation of SVlOl cells for 20 hours did not reduce either the uptake of phosphorylation of 2-dOG. Identical results were obtained when 3T6 cells were serum deprived for 20 hours (fig. 4B). Hence transformed cells are insensitive to serum starvation in that they continue to phosphorylate 2-dOG at the same rate and to the same extent as non-serum-deprived cells (com-
pare (figs. 2 and 4). These results also illustrate that not only virally transformed (SVlOl), but also spontaneously transformed (3T6) cells exhibit serum-independent phosphorylation of 2-dOG). Effect of dibutyryl cyclic A M P o n g r o w t h , transport, a n d phosphorylation of 2-deoxy-D-glucose Cyclic AMP has been implicated as a regulator of cell growth (Heidenek and Ryan, '71; Otten et al., '71; Kram et al., '73). Recent publications reveal that dibutyryl adenosine 3': 5'-cyclic monophosphate (dbcAMP) and theophylline will restore growth control to transformed cells (Johnson et al., '71; Sheppard, '71; Johnson and Pastan, '71). We therefore inquired whether these agents would, when added to SVlOl cells, restore the capacity to cease dividing when serum deprived (a property of 3T3 cells, see fig. 1). Figure 5 shows that 10-4 dbcAMP and 10-3 M theophylline did not reduce the growth rate of SVlOl cells in DME medium supplemented with calf serum (10%). In the absence of serum, SVlOl cells grew at a diminished
42
J. A. HASSELL, C. COLBY AND A. H. ROMANO
P
I
,
I
Time (mtn 1
Fig. 4 Effect of serum starvation on phosphorylation and transport of 2-deoxy-D-(14C) glucose in (A) SVlOl cells and (B) 3T6 cells. Cells were plated at 1-3 x 104 cellslcmz on coverslips distributed over the bottom of 100 mm Falcon plastic dishes and then processed exactly 2-dOG-P controls; A-A, 2-dOG-P serumas described in the legend of figure 1. A-A, deprived cells; 0-- -0, free 2-dOG, controls; @-- -@ free 2-dOG, serum-deprived cells.
rate and the presence of M dbcAMP and 1 0 - 3 M theophylline reduced this rate even further. Paul ('73) reported that dbcAMP and theophylline are toxic to SV40 transformed 3T3 cells, and that serum exerted a protective effect against these agents. This toxicity of the drugs might have accounted for the reduced rate of S V l O l cell division observed in the absence of serum. To test this we compared the plating efficiencies of S V l O l cells treated with the drugs in the absence and presence of serum with untreated controls. The results reported in table 1 show that S V l O l cells exposed to M dbcAMP and lo-:' M theophylline for 24 hours were viable as measured by plating efficiency. It has recently been reported that 3T3 cells have a lower capacity for 2-dOG transport than polyoma transformed 3T3 cells. Treatment of polyoma transformed cells with dbcAMP and theophylline conferred the capacity to transport the sugar at the same rate as 3T3 cells (Grimes and Schroe-
TABLE 1
meet of
dbcAMP and theophylline on the viability of SVlO1 cells 2 serum Conditions
Control Control dbcAMP theophylline Serum deprived Serum deprived dbcAMP theophylline
+
+
+
+
Plating efficiency attached cells/ cells plated
0.93
0.90 0.91 0.88
Cells were plated at 1 X 101 cellslcmz into 19.6 Falcon plastic tissueculture dishes and then processed as described in the legend to figure 5. The plating efficiency of these cells was determined by seeding 2 X 105 cells per 19.6 cmz Falcon plastic tissueculture dish in DME medium plus calf serum ( 1 0 % ) at 37°C for twelve hours, and then counting the number of attached cells. Cell number was determined on triplicate dishes as described in MATERIALS AND METHODS.
der, '73). We have previously reported that the rate of transport of 2-dOG was the same in S V l O l and 3T3 cells, but that the rate of phosphorylation of the sugar was enhanced in S V l O l cells (Romano and Colby, '73). It therefore seemed likely that
TRANSFORMATION, SERUM, AND SUGAR PHOSPHORYLATION
43
the phosphorylation reaction might be modified in S V l O l cells by the cyclic AMP analogue. Hence we measured uptake and phorphorylation of 2-dOG by growing S V l O l cells treated with M dbcAMP and 1 0 - 3 M theophylline in comparison to untreated controls. These concentrations of the drugs did not block cell division (fig. 4). Also, table 2 shows that these concentrations of dbcAMP and theophylline had no effect on the amount of 2-dOG entering the free sugar pool or the phosphorylated sugar pool in ten minutes, either in control cells or cells that had been deprived of serum for 20 hours. Thus, these drugs did not restore to the transformed cells the capacity to regulate the phosphorylation of the sugar. I
2
3
DISCUSSION
Doys Fig. 5 Effect of dbcAMP and theophylhne on the growth rate of SV101, cells in the presence and absence of calf serum. Cells were seeded at 1.2 X 104 cells/cm* per 19.6 cmz Falcon plastic tissue-culture dish on day 0. One day later the medium was changed to DME medium plus calf serum ( 1 0 % ) (controls) on half of the cultures while the rest received DME medium plus calf serum ( l o % ) , 10-4 M dbcAMP and 10-3 M theophylline (treated controls). On day 2 both the treated and untreated controls were deprived of serum as indicated. Cell number was determined in duplicate as described in MATERIALS AND METHODS. 0 ,control; , control plus 1 0 - 4 M dbcAMP and 10-3 M theophylline; A , serum deprived; A , serum deprived plus 10-4 M dbcAMP and 1 0 - 3 M theophylline. TABLE 2
meet of dbcAMP and theophylline
on the uptake and phosphorylation of 2-dOG by SVI 01 cells in the presence and absence of serum Nanomoles of Nanomoles of 2-dOG per 2-dOG-Pper milligram milligram of protein of protein
Conditions
Control dbcAMP Control theophylline Serum deprived Serum deprived dbcAMP theophylline
+ +
+
8.0
24
7.4 7.7
25 21
7.1
20
+
Cells were plated at 1 X 1W cells/cm* on coverslips distributed over the bottom of 100 mm Falcon plastic dishes. Cells were then processed exactly as described in the legend to figure 5. Uptake and phosphorylation of Z-deoxy-D-(14C) glucose were determined after ten minutes as described i n the legend of figure 2. Total protein was measured as described in MATERIALS AND METHODS.
We have investigated the uptake and phosphorylation of 2-dOG in untransformed 3T3 cells, spontaneously transformed 3T6 cells, and S V 4 0 virus transformed 3T3 cells, and examined the relatedness of these reactions to the cell growth rate. All of these cells appear to transport 2-dOG at essentially the same rate independently of their growth rate. Furthermore the availability of serum factors does not appear to affect the transport of 2-dOG in these cells. The rate of phosphorylation is altered in transformed cells. 3T6 and S V l O l cells phosphorylated the sugar at a higher rate than did 3T3 cells when these cells were growing exponentially (compare figs. 2 and 4). These observations confirm our previous findings, using both 2-dOG and 3-0-methyl-D-glucose as glucose analogs, that phosphorylation but not transport of hexoses is enhanced in virus-transformed 3T3 cells (Romano and Colby, '73; Colby and Romano, '74). Phosphorylation of 2-dOG by untransformed cells is regulated by the availability of serum factors. This is indicated, since both growing as well as nongrowing (density-inhibited) 3T3 cells had similar capacities to phosphorylate the sugar while serum starvation of these cells elicited a decrease in the rate of this reaction. Hence, the maintenance of a maximal rate of phosphorylation of 2-dOG in 3T3 cells correlates with the availability of serum factors and not primarily with the cell growth
44
J. A. HASSELL, C. COLBY AND A. H. ROMANO
rate. Moreover these findings suggest that different components of serum independently affect cell division and hexose phosphorylation. Similar conclusions have been reached by Thrash and Cunningham ('74). These investigators showed that the initiation of 3T3 cell divisions by cortisol was not preceded by an increase in hexose uptake, while a n increase in hexose uptake was observed when these cells were stimulated to divide by serum addition. Transformed cells (3T6 and S V l O l ) do not regulate the rate of phosphorylation of 2-dOG when serum deprived. These cells exhibit serum-independent phosphorylation of 2-dOG. Treatment of S V l O l cells with dbcAMP and theophylline partially restores growth control to those cells, since these drugs reduce the rate of cell division in the absence of serum. However, the rate of phosphorylation of 2-dOG is unaffected by the drugs in the presence or absence of serum. Therefore control of phosphorylation of 2-dOG appears not to be restored to S V l O l cells treated with dbcAMP and theophylline. Recent findings have focused attention on the relationship between the rate of cell growth and the uptake of 2-dOG. These studies have shown that the rate of uptake of 2-dOG fluctuates concomitantly with changes in the rate of cell division (Sefton and Rubin, '71; Schultz and Culp, '73; Bose and Zlotnick, '73; Oshiro and DiPaolo, '74; Bradley and Culp, '74; Kletzien and Perdue, '74). However, these investigators did not separate the transport process from the subsequent phosphorylation of 2-dOG, and it was not clear which process was affected by changes in the cell growth rate. Additionally many of these studies were conducted with cells growing a t low growth medium to cell ratios. Under these conditions it is likely that serum factors as well a s other components of the medium may be limiting for cell division and sugar uptake. Our studies were performed with cells growing attached to glass coverslips at high growth medium to cell ratios. These differences may account for the disparate results obtained by u s and others. Our experiments indicate that the rate of transport of 2-dOG is the same in untransformed and transformed 3T3 cells and is not subject to regulation by cell density or serum factors. Phosphorylation
of 2-dOG is regulated in 3T3 cells by the availability of serum factors, while transformed cells escape this control mechanism. The fact that transformed cells continue to phosphorylate 2-dOG at the maximal rate in the absence of serum may be due to the production of serum-like factors by these cells, or could be due to cell surface alterations which maintain constant activation of the phosphorylation reaction. This work further indicates that the enhanced rate of glucose metabolism exhibited by transformed cells, or by untransformed cells in response to serum addition, is primarily due to enhanced glycolysis rather than to enhanced transport. It is of interest in this connection that Fodge and Rubin ('73) have shown that serum addition activates phosphofructokinase, a key regulation enzyme in glycolysis, in chick embryo fibroblasts. The resultant lowered intra-cellular levels of glucose-6-phosphate, a known inhibitor of hexokinase of animal cells, may account for the results described here. ACKNOWLEDGMENTS
We thank M. A. Brustalon, M. McLeod and E. Profita for excellent technical assistance. Thanks are also expressed to D. Engelhardt for providing facilities and support for part of this work. This investigation was supported by grants from the Damon Runyon Fund for Cancer Research, Inc. (DRG-1212 and DRG-1191), the National Cancer Institute (NCI-14274), and the National Science Foundation (GB40654). John A. Hassell is a n N.I.H. predoctoral fellow. (G.M.-00317). Clarence Colby is the recipient of a Research Career Development Award from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED Bose, S. K . , and B. J . Zlotnick 1973 Growth and density-dependent inhibition ofdeoxyglucose transport in Balb 3T3 cells and its absence i n cells transformed by murine sarcoma virus. Proc. Nat. Acad. Sci. (U.S.A.), 70: 2374-2378. Bradley, W. E. C., and L. A . Culp 1974 Stimulation of 2-deoxyglucose uptake in growth-inhibited Balb/C 3T3 and revertant SV40-transformed 3T3 cells. Exp. Cell Res., 84: 3 3 5 3 5 0 . Bray, G. A. 1960 A simple efficient liquid scintillation for counting aqueous solutions i n a liquid scintillation counter. Anal. Biochem., I : 279-285. Colby, C., and A. H. Romano 1975 Phosphoryla-
TRANSFORMATION, SERUM, AND SUGAR PHOSPHORYLATION tion but not transport of sugars is enhanced in virus-transformed mouse 3T3 cells. J. Cell. Physiol., 85: 15-24. Cunningham, D. D., and A. B. Pardee 1969 Transport changes rapidly initiated by serum addition to “contact inhibited” 3T3 cells. Proc. Nat. Acad. Sci. (U.S.A.), 64: 1049-1056. Dulbecco, R., and J. Elkington 1973 Conditions limiting multiplication of fibroblastic and epithelial cells in dense culture. Nature, 246: 197199. Fodge, D. W., and H. Rubin Activation of phosphofructokinase by stimulants of cell multiplication. Nature New Biol., 246: 181-183. Foster, D. O., and A. B. Pardee 1969 Transport of amino acids by confluent and nonconfluent 3T3 and polyoma virus-transformed 3T3 cells growing on glass coverslips. J. Biol. Chem., 244: 267.52681. Grimes, W. J., and J. L. Schroeder 1973 Dibutyryl cyclic adenosine 3’5’ monophosphate, sugar transport, and regulatory control of cell division in normal and transformed cells. J. Cell. Biol., 5 6 : 487-491. Hassell, J. A,, and D. L. Engelhardt 1973 Translational inhibition in extracts from serum-deprived animal cells. Biochim. Biophys. Acta, 325: 545-553. Heidinek, M. L., and W. L. Ryan 1970 Cyclic nucleotides on cell growth in nitro Cancer Res., 30: 37C378. Hershko, A,, P. Mamont, R. Shields and G. M. Tomkins 1971 Pleiotypic response. Nature New Biology, 232: 206-211. Holley, R. W., and J. Kiernan 1971 Studies of serum factors required by 3T3 and SV3T3 cells. In: Growth Control of Cell Culture. G. E. W. Wolstenholme and J. Knight, eds. Churchill Livingstone, Edinburgh a n d London, pp. 3-15. Johnson, G. S., R. M. Friedman and I . Pastan 1971 Restoration of several morphological characteristics of normal fibroblasts in sarcoma cells treated with adenosine-3’5’-cyclic monophosphate and its derivatives. Proc. Nat. Acad. Sci. ( U . S. A,), 68: 4 2 5 4 2 9 . Kletzien, R. F., and J. F. Perdue 1974 Sugar transport in chick embryo fibroblasts. 1. A functional change in the plasma membrane associated with the rate of cell growth. J. Biol. Chem., 249: 3366-3374. Kram, R., P. Mamont and G. M. Tomkins 1973 Pleiotypic control by adenosine 3’:5‘-cyclic mono-
45
phosphate: A model for growth control in animal cells. Proc. Nat. Acad. Sci. (U.S.A.), 70: 1432-1436. Kram, R., and G. M. Tomkins 1973 Pleiotypic control by cyclic AMP: Interaction with cyclic GMP and possible role of microtubules. Proc. Nat. Acad. Sci. (U.S.A.), 70: 1659-1663. Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Oshiro, Y., and J. A. DiPaolo 1974 Changes in the uptake of 2-deoxy-D-glucose i n Balb/3T3 cells chemically transformed in culture. J. Cell. Physiol., 8 3 : 193-202. Otten, J., G. S . Johnson and I. Pasten 1971 Cyclic AMP levels in fibroblast: Relationship to growth rate and contact inhibition of growth. Biochem. Biophys. Res. Comm., 4 4 : 1192-1198. Paul. D. 1972 Effects of cyclic AMP on SV3T3 cells in culture. Nature New Biol., 240: 179-181. Romano, A. H., and C. Colby 1973 SV40 virus transformation of mouse 3T3 cells does not specifically enhance sugar transport. Science, 179: 1238-1240. Schultz, A. R., and L. A. Culp 1973 Contactinhibited revertant cell lines isolated from SV40transformed cells. V. Contact inhibition of sugar transport. Exp. Cell Res., 81 : 95-103. Sefton, B. M., and H. Rubin 1971 Stimulation of glucose transport in cultures of density-inhibited chick embryo cells. Proc. Nat. Acad. Sci. (U.S.A.), 68: 3 1 5 4 3 1 5 7 , Sheppard, J. R. 1971 Restoration of contactinhibited growth to transformed cells by dibutyryl adenosine 3’:5’-cyclic monophosphate. Proc. Nat. Acad. Sci. (U.S.A.), 68: 1316-1320. Soeiro, R., and H. Amos 1966 Arrested protein synthesis in polysomes of cultured chick embryo cells. Science, 154: 662-665. Todaro, G. J., G. K. Lazar a n d H. Green 1965 The initiation of cell division i n a contact-inhibited mammalian cell line. J. Cell. and Comp. Phys., 6 6 : 315-333. Vogt, M., and R. Dulbecco 1963 Steps i n the neoplastic transformation of hamster embryo cells by polyoma virus. Proc. Nat. Acad. Sci. (U.S.A.), 4 9 : 171-179. Weber, M. J., and H. Rubin 1970 Uridine transport and RNA synthesis in growing and in density-inhibited animal cells. J. Cell. Physiol., 77: 157-168.
ABSTRACT Serum starvation of growing and nongrowing (density-inhibited) mouse 3T3 cells resulted in decreased phosphorylation of 2deoxy-D-glucose, while the time course of transport of this sugar remained unchanged. Serum starvation of SV40 transformed 3T3 cells (SV101) and spontaneously transformed 3T6 cells did not alter either the time course of transport, or phosphorylation of the sugar. Treatment of SVlOl cells with M dibutyryl adenosine cyclic 3':5' monophosphate and 1 0 - 3 M theophylline did not restore the capacity to regulate 2-deoxy-Dglucose phosphorylation when these cells were serum deprived. We conclude that serum factors are involved in the modulation of phosphorylation of 2-deoxy-D-glucose in 3T3 cells rather than its transport. This regulation is operative both in growing as well as nongrowing 3T3 cells. I n contrast, transformed cells do not respond to this regulation of 2-deoxy-D-glucosr phosphorylation. When mouse 3T3 cells are deprived of serum, a n ensemble of alterations in metabolism occurs. These include changes in the overall rate of protein and RNA synthesis, the rate of protein degradation, and the rate of uptake of nucleic acid precursors (Soeiro and Amos, '66; Hershko et al., '71; Hassell and Engelhardt, '73). Those biochemical reactions influenced by serum factors form the pleiotypic program (Kram and Tomkins, '73). In virus-transformed cells the pleiotypic program is relatively insensitive to serum factors (Hershko et al., '71). When 3T3 cells enter the stationary phase of growth their metabolism is altered in much the same manner as occurs after serum starvation (Todaro et al., '65; Cunningham and Pardee, '69; Weber and Rubin, '70). It has been suggested that a limitation of medium factors prevents the growth of 3T3 cells at high cell density (Holley and Kiernan, '71; Dulbecco and Elkington, '73). Hence serum starvation might elicit the same biochemical response by the cell as entry into the stationary phase of the growth curve. It has been reported that the rate of glucose uptake, as measured by the upJ CELL.PHYSIOL., 86. 3 7 4 6 .
take of the analogue 2-deoxyglucose (2dOG), is stimulated by the re-addition of serum to previously starved 3T3 cells. This reaction has been included in tho pleiotypic program (Kram and Tomkins, '73). However, these authors did not separate transport from the subsequent phosphorylation of 2-dOG, and hence it was not clear which process was stimulated by s e r ~ m . Reports from this laboratory (Romano and Colby, '73; Colby and Romano, '74)h i i v f emphasized that the uptake and phosphorylation of 2-dOG are separate and scquential events, and that the apparent enhancement of 2-dOG uptake by 3T3 cells accompanying viral transformation is not due primarily to an effect on the transport process, but to enhanced phosphorylation. The present study was undertaken, therefore, to measure the time course of entry of 2-deoxyglucose into intracellular free sugar and sugar phosphate pools in 3T3 cells and their transformed derivatives. Received Oct. 3, '74. Accepted Jan. 2, '75. * Present address: Cold Spring Harbor Laboratory. P.O. Box 100, Cold Spring Harbor, New York 11724. 2 Present address: Department of Genetics, University of California, Davis, California 95616.
37
38
J. A. HASSELL, C. COLBY A N D A. H. ROMANO
under conditions of serum starvation and serum sufficiency.
and Pardee, '69). Cell cultures were generally used two to four days after seeding. Measurements of the uptake of '"-2MATERIALS A N D METHODS deoxy-D-glucose and its conversion to the Cells were grown in Dulbecco's modified phosphorylated form were made on cells Eagle's medium (DME) (Vogt and Dul- growing on the surface of 2.25 cm2 glass becco, '63) supplemented with calf serum coverslips by a modification of methods to 10% (V/V) (growth medium) in a 5% developed by Foster and Pardee ('69). CovC 0 2 atmosphere. Balb/c 3T3 cells were erslips with adherent cells were transferred maintained by transfer of lo4 cells to 100 with forceps from growth medium through mm Falcon plastic dishes every four days. four successive washings of glucose-free These cells were not allowed to attain con- Hanks solution at 37"C, and'were incufluence before subculturing. SV4[, trans- bated a t 37°C in I4C-2dOG solutions (in formed 3T3 cells (SV101 cells) and 3T6 glucose-free Hanks solution) at the concells were maintained by transfer of 5 X centrations, specific activities and time 10.5 cells to 100 mm plastic Petri dishes, intervals indicated. Coverslips were reand replating after the cells had become moved from the isotopic solutions, rinsed through four successive washings of iceconfluent. The growth of these cells was monitored cold glucose-free Hanks solution, and exas follows: Cells were seeded onto Falcon tracted with 1 ml boiling water for one Petri dishes (19.6 cm2) and 4 ml of growth minute. Free sugar and sugar phosphates medium added. The growth medium was in the extracts were separated by the chrochanged every 48 hours. Cell number was matographic method of Winkler ('60) as determined by rinsing the cells with T.E. follows: 0.5 ml samples of extract were (0.05% trypsin, 0.005 M EDTA in Ca++ applied to columns (4 X 0.6 cm) of BioMg++free phosphate-buffered salt solution), Rad AG1-X2 anion exchange resin (100 to and counting a sample with a hemocy- 200 mesh) which had been converted to tometer. the formate form. Free 2-dOG was eluted To serum deprive cells, the medium was with five 0.5 ml portions of water; the removed, and the cell layer rinsed twice phosphorylated sugar (2-dOG-P) was subwith 5 ml portions of prewamed (37°C) sequently eluted with seven 0.5 ml portions DME; 4 ml of DME was added to some of of 0.5 M ammonium formate in 0.2 M the cultures (serum-deprived cells) while formic acid. Eluted materials were colothers received 4 ml of growth medium lected directly into scintillation vials con(controls). taining 10 ml Bray's fluid (Bray, '60) for Cells growing attached to glass cover- counting. Protein determinations were performed slips (2.25 cm2) were used for uptake measurements (Foster and Pardee, '69). A in quadruplicate on parallel cover slipsuspension of cells in 10 ml of growth me- grown cultures by the method of Lowry et dium was added to coverslips lining the al., ('51). Chemicals were obtained from the folbottoms of 100 mm diameter plastic Petri dishes. A day later these coverslips were lowing sources: DME medium and calf placed in several 100 mm dishes (6 cover- serum, GIBCO; NfiOz-dibutyryl adenosine slips per dish) containing 10 ml of growth cyclic 3':5' monophosphate and theophylmedium, and the growth medium was line, Sigma; uniformly labelled 2-deoxy-Dchanged daily thereafter. The cells were ( I T ) glucose, International Chemical and deprived of serum by removing the growth Nuclear Corp. ; non-radioactive 2-deoxy-Dmedium and rinsing the coverslips twice glucose, SchwarzIMann. with 10 ml portions of prewarmed (37°C) RESULTS DME; 10 ml of DME was added to some Growth response of 3T3 cells to of the cultures (serum-deprived cells) while serum deprivation others (controls) received 10 ml of growth medium. The number of cells per cover3T3 cells growing in DME medium supslip was determined in triplicate on the plement with calf serum (10% ) had a genday preceding, and immediately before eration time of eleven hours and reached their use in uptake experiments (Foster a final saturation density of 7 x 1 0 4 cells/
TRANSFORMATION, SERUM, AND SUGAR PHOSPHORYLATION
cm2, when the medium was changed every 48 hours. Transfer of exponentially growing 3T3 cells to DME medium without serum immediately stopped cell growth, while prolonged serum starvation resulted in cell death (fig. 1). After 24 hours of serum deprivation, cells were still viable as judged by their capacity to resume cell division at the normal rate when stimulated with serum-containing medium (fig. 1). Transport and phosphorylation of 2-deoxy-D-glucose by 3 T 3 cells In previous experiments Romano and Colby ('73) provided evidence that the rate 100 90
80 70
60 50 40
30 20
E
u
1
m
0 x
10 9
2
7
8
S 65 4
0
2
4
6
8
Days Fig. 1 Growth response of 3T3 cells to serum starvation. 3T3 cells were plated at 2.7 X lo3 cells/cmZ on 19.6 cmz Falcon tissue-culture dishes on day 0. On day 2 the medium was changed on all the cultures; some received DME medium plus calf serum (10%) O---O, while others received DME medium only (I---Q. On day 3 the medium was changed on all the cultures and some of those which had been previously serum deprived received DME medium plus calf serum (10%) A-- -A. The medium was changed every 2 days on all the dishes thereafter. Cell number was determined on duplicate dishes as described i n MATERIALS A N D METHODS.
39
of phosphorylation of 2-dOG was greater in transformed cells compared to untransformed cells, and suggested that the physiological state of the cell might influence this reaction. Since serum starvation alters the growth rate of the cell (fig. 1) we studied the effect of this parameter on the uptake and phosphorylation of 2-dOG. 3T3 cells at a density of 1-2 x 104 cellslcm' were deprived of serum for 20 hours and the time course of entry of 2-dOG into a free and phosphorylated pool was then measured. The cell number of the control cultures more than doubled, while no increase in cell number occurred in the serum-deprived cultures after 20 hours (data not shown). The time course of entry of 2-dOG into a free intracellular pool was unaffected by serum starvation while the apparent rate of phosphorylation of 2-dOG was reduced to 30% of the control rate (fig. 2A). This result indicates that phosphorylation of the sugar rather than transport is controlled by the availability of serum in the medium. It has been assumed that those reactions which make up the pleiotypic program fluctuate coordinately with changes in the cell growth rate (Hershko et al., '71). We therefore investigated the possibility that nongrowing (density-inhibited) 3T3 cells might phosphorylate 2-dOG at a reduced rate when compared to growing cells. We also inquired whether serum starvation of nongrowing cells would further depress this reaction. Nongrowing 3T3 cells at a density of 7-8 x lo4 cells/cm2 were serum starved for 20 hours and the time course of entry of 2-dOG into a free and phosphorylated pool measured. The results show that nongrowing 3T3 cells transported and phosphorylated 2-dOG at essentially the same rate and to the same extent as growing cells (compare figs. 2A and B). Moreover, serum starvation of nongrowing 3T3 cells for 20 hours elicited a decrease in the rate of phosphorylation of 2-dOG to a level 43% that of the control rate, while the time course of transport remained unchanged (fig. 2B). This decrease in phosphorylation of 2-dOG is of the same order of magnitude as that observed when growing 3T3 cells were serum deprived (compare figs. 2A and B). Twenty hours after the medium change to serum-free or serumcontaining growth medium neither culture
40
J. A. HASSELL. C. COLBY AND A. H. ROMANO 50
zw
A
40
I-
0
rr
a 30 (3
2 \
B 20 J 0
2 0
: 10 z C
10
20
30 0 T I M E IN MINUTES
10
20
30
Fig. 2 Effect of serum deprivation on phosphorylation and transport of 2-deoxy-D-(14C) glucose in (A) growing 3T3 and (B) non-growing 3T3 cells. Cells were seeded at 2.5 x 1 0 3 cellslcmz for growing cells, and at 7 X lo4 cellslcmz for non-growing cells onto glass coverslips distributed over the bottoms of 100 mm Falcon plastic dishes in 10 ml of DME medium plus calf serum (10%).After two days the medium was changed to DME medium only (serumdeprived cells) or DME medium plus calf serum (10% ) (controls). Twenty hours later the coverslip. with adherent cells were washed four times with glucose-free Hanks solution at 37OC and incubated in 2 mM uniforn-ly labelled 2-deoxy-D-(1C) glucose (0.5 &wmole) at 37'C. At appropriate intervals duplicate coverslips were removed and processed as described in MATERIALS AND METHODS to separate and determine free 2-deoxy-D-(14C)glucose and 2-deoxyD-(14C) glucose phosphate. Protein was determined on quadruplicate coverslips as described in MATERIALS AND METHODS. A -A, 2-dOG-P controls; A-A 2-dOG-P serum-deprived cells; O---O free dOG controls; 0 - - 4 free 2-dOG, serum deprived cells.
(serum-deprived nor control) experienced a change in cell number (data not shown). These observations indicate that the response to the absence or presence of serum is the same in growing and nongrowing (density-inhibited) 3T3 cells and suggest that a block in neither of these reactions is of itself primarily responsible for curtailing cell division at high cell density. Growth response of transformed cells to serum deprivation SV40 virus transformed cells do not respond biochemically to serum starvation in a manner analogous to 3T3 cells; instead, the rates of RNA, DNA, protein synthesis, uridine uptake, and protein degradation continue essentially unimpaired after 20 hours of serum starvation (Hershko et al., '71). In these experiments the rate of cell division following serum deprivation was not reported, and hence the re-
latedness of the growth rate of the cell to the reactions of the pleiotypic program was not examined. We therefore measured the growth of SVlOl cells to serum starvation over a 24 hour period. The results depicted in figure 3A show that SVlOl cells grown in DME medium supplemented with calf serum ( 1 0 % ) had a generation time of 11 hours when the medium was changed every 48 hours. Serum starvation of these cells resulted in a continuation of cell division but at a reduced rate. In the continued absence of oeru.m, SVlOl cells ceased dividing and cell death occurred after two days. 3T6 cells also exhibited a generation time of 12 hours under these conditions; in the absence of serum these cells also divided at a reduced rate, but at a much lower rate than SVlOl cells (compare figs. 3A and B). Beside this difference, 3T6 cells grew to a stable final saturation density
TRANSFORMATION, SERUM, AND ;UGAR PHOSPHORYLATION
41
3
Days Fig. 3 Growth response of (A) S V l O l cells and (B) 3T6 cells to serum starvation. S V l O l and 3T6 cells were plated at 1 X 1 0 4 cellslcmz on 19.6 cm* Falcon plastic tissue-culture dishes on day 0. On day 2 the medium was changed to DME medium plus calf serum ( 1 0 % ) -0, or to DME medium alone 0-0. Cell number was determined in duplicate as described i n MATERIALS A N D METHODS,
of 2 x 105 cells/cm2, while SVlOl cells continued to divide until the cell layer disrupted. Transport a n d phosphorylation of 2 -d eoxy- D-g lucos e in transformed cells Serum starvation of transformed cells does not elicit the pleiotypic response (Hershko et al., '71), and does not block cell division (figs. 3 A and B). Since we had found that phosphorylation rather than transport of 2-dOG appeared to be regulated by serum factors in 3T3 cells (figs. 2A and B), we investigated the effect of serum starvation of SVlOl and 3T6 cells on their capacity to take up and phosphorylate the sugar. Figure 4A shows that serum starvation of SVlOl cells for 20 hours did not reduce either the uptake of phosphorylation of 2-dOG. Identical results were obtained when 3T6 cells were serum deprived for 20 hours (fig. 4B). Hence transformed cells are insensitive to serum starvation in that they continue to phosphorylate 2-dOG at the same rate and to the same extent as non-serum-deprived cells (com-
pare (figs. 2 and 4). These results also illustrate that not only virally transformed (SVlOl), but also spontaneously transformed (3T6) cells exhibit serum-independent phosphorylation of 2-dOG). Effect of dibutyryl cyclic A M P o n g r o w t h , transport, a n d phosphorylation of 2-deoxy-D-glucose Cyclic AMP has been implicated as a regulator of cell growth (Heidenek and Ryan, '71; Otten et al., '71; Kram et al., '73). Recent publications reveal that dibutyryl adenosine 3': 5'-cyclic monophosphate (dbcAMP) and theophylline will restore growth control to transformed cells (Johnson et al., '71; Sheppard, '71; Johnson and Pastan, '71). We therefore inquired whether these agents would, when added to SVlOl cells, restore the capacity to cease dividing when serum deprived (a property of 3T3 cells, see fig. 1). Figure 5 shows that 10-4 dbcAMP and 10-3 M theophylline did not reduce the growth rate of SVlOl cells in DME medium supplemented with calf serum (10%). In the absence of serum, SVlOl cells grew at a diminished
42
J. A. HASSELL, C. COLBY AND A. H. ROMANO
P
I
,
I
Time (mtn 1
Fig. 4 Effect of serum starvation on phosphorylation and transport of 2-deoxy-D-(14C) glucose in (A) SVlOl cells and (B) 3T6 cells. Cells were plated at 1-3 x 104 cellslcmz on coverslips distributed over the bottom of 100 mm Falcon plastic dishes and then processed exactly 2-dOG-P controls; A-A, 2-dOG-P serumas described in the legend of figure 1. A-A, deprived cells; 0-- -0, free 2-dOG, controls; @-- -@ free 2-dOG, serum-deprived cells.
rate and the presence of M dbcAMP and 1 0 - 3 M theophylline reduced this rate even further. Paul ('73) reported that dbcAMP and theophylline are toxic to SV40 transformed 3T3 cells, and that serum exerted a protective effect against these agents. This toxicity of the drugs might have accounted for the reduced rate of S V l O l cell division observed in the absence of serum. To test this we compared the plating efficiencies of S V l O l cells treated with the drugs in the absence and presence of serum with untreated controls. The results reported in table 1 show that S V l O l cells exposed to M dbcAMP and lo-:' M theophylline for 24 hours were viable as measured by plating efficiency. It has recently been reported that 3T3 cells have a lower capacity for 2-dOG transport than polyoma transformed 3T3 cells. Treatment of polyoma transformed cells with dbcAMP and theophylline conferred the capacity to transport the sugar at the same rate as 3T3 cells (Grimes and Schroe-
TABLE 1
meet of
dbcAMP and theophylline on the viability of SVlO1 cells 2 serum Conditions
Control Control dbcAMP theophylline Serum deprived Serum deprived dbcAMP theophylline
+
+
+
+
Plating efficiency attached cells/ cells plated
0.93
0.90 0.91 0.88
Cells were plated at 1 X 101 cellslcmz into 19.6 Falcon plastic tissueculture dishes and then processed as described in the legend to figure 5. The plating efficiency of these cells was determined by seeding 2 X 105 cells per 19.6 cmz Falcon plastic tissueculture dish in DME medium plus calf serum ( 1 0 % ) at 37°C for twelve hours, and then counting the number of attached cells. Cell number was determined on triplicate dishes as described in MATERIALS AND METHODS.
der, '73). We have previously reported that the rate of transport of 2-dOG was the same in S V l O l and 3T3 cells, but that the rate of phosphorylation of the sugar was enhanced in S V l O l cells (Romano and Colby, '73). It therefore seemed likely that
TRANSFORMATION, SERUM, AND SUGAR PHOSPHORYLATION
43
the phosphorylation reaction might be modified in S V l O l cells by the cyclic AMP analogue. Hence we measured uptake and phorphorylation of 2-dOG by growing S V l O l cells treated with M dbcAMP and 1 0 - 3 M theophylline in comparison to untreated controls. These concentrations of the drugs did not block cell division (fig. 4). Also, table 2 shows that these concentrations of dbcAMP and theophylline had no effect on the amount of 2-dOG entering the free sugar pool or the phosphorylated sugar pool in ten minutes, either in control cells or cells that had been deprived of serum for 20 hours. Thus, these drugs did not restore to the transformed cells the capacity to regulate the phosphorylation of the sugar. I
2
3
DISCUSSION
Doys Fig. 5 Effect of dbcAMP and theophylhne on the growth rate of SV101, cells in the presence and absence of calf serum. Cells were seeded at 1.2 X 104 cells/cm* per 19.6 cmz Falcon plastic tissue-culture dish on day 0. One day later the medium was changed to DME medium plus calf serum ( 1 0 % ) (controls) on half of the cultures while the rest received DME medium plus calf serum ( l o % ) , 10-4 M dbcAMP and 10-3 M theophylline (treated controls). On day 2 both the treated and untreated controls were deprived of serum as indicated. Cell number was determined in duplicate as described in MATERIALS AND METHODS. 0 ,control; , control plus 1 0 - 4 M dbcAMP and 10-3 M theophylline; A , serum deprived; A , serum deprived plus 10-4 M dbcAMP and 1 0 - 3 M theophylline. TABLE 2
meet of dbcAMP and theophylline
on the uptake and phosphorylation of 2-dOG by SVI 01 cells in the presence and absence of serum Nanomoles of Nanomoles of 2-dOG per 2-dOG-Pper milligram milligram of protein of protein
Conditions
Control dbcAMP Control theophylline Serum deprived Serum deprived dbcAMP theophylline
+ +
+
8.0
24
7.4 7.7
25 21
7.1
20
+
Cells were plated at 1 X 1W cells/cm* on coverslips distributed over the bottom of 100 mm Falcon plastic dishes. Cells were then processed exactly as described in the legend to figure 5. Uptake and phosphorylation of Z-deoxy-D-(14C) glucose were determined after ten minutes as described i n the legend of figure 2. Total protein was measured as described in MATERIALS AND METHODS.
We have investigated the uptake and phosphorylation of 2-dOG in untransformed 3T3 cells, spontaneously transformed 3T6 cells, and S V 4 0 virus transformed 3T3 cells, and examined the relatedness of these reactions to the cell growth rate. All of these cells appear to transport 2-dOG at essentially the same rate independently of their growth rate. Furthermore the availability of serum factors does not appear to affect the transport of 2-dOG in these cells. The rate of phosphorylation is altered in transformed cells. 3T6 and S V l O l cells phosphorylated the sugar at a higher rate than did 3T3 cells when these cells were growing exponentially (compare figs. 2 and 4). These observations confirm our previous findings, using both 2-dOG and 3-0-methyl-D-glucose as glucose analogs, that phosphorylation but not transport of hexoses is enhanced in virus-transformed 3T3 cells (Romano and Colby, '73; Colby and Romano, '74). Phosphorylation of 2-dOG by untransformed cells is regulated by the availability of serum factors. This is indicated, since both growing as well as nongrowing (density-inhibited) 3T3 cells had similar capacities to phosphorylate the sugar while serum starvation of these cells elicited a decrease in the rate of this reaction. Hence, the maintenance of a maximal rate of phosphorylation of 2-dOG in 3T3 cells correlates with the availability of serum factors and not primarily with the cell growth
44
J. A. HASSELL, C. COLBY AND A. H. ROMANO
rate. Moreover these findings suggest that different components of serum independently affect cell division and hexose phosphorylation. Similar conclusions have been reached by Thrash and Cunningham ('74). These investigators showed that the initiation of 3T3 cell divisions by cortisol was not preceded by an increase in hexose uptake, while a n increase in hexose uptake was observed when these cells were stimulated to divide by serum addition. Transformed cells (3T6 and S V l O l ) do not regulate the rate of phosphorylation of 2-dOG when serum deprived. These cells exhibit serum-independent phosphorylation of 2-dOG. Treatment of S V l O l cells with dbcAMP and theophylline partially restores growth control to those cells, since these drugs reduce the rate of cell division in the absence of serum. However, the rate of phosphorylation of 2-dOG is unaffected by the drugs in the presence or absence of serum. Therefore control of phosphorylation of 2-dOG appears not to be restored to S V l O l cells treated with dbcAMP and theophylline. Recent findings have focused attention on the relationship between the rate of cell growth and the uptake of 2-dOG. These studies have shown that the rate of uptake of 2-dOG fluctuates concomitantly with changes in the rate of cell division (Sefton and Rubin, '71; Schultz and Culp, '73; Bose and Zlotnick, '73; Oshiro and DiPaolo, '74; Bradley and Culp, '74; Kletzien and Perdue, '74). However, these investigators did not separate the transport process from the subsequent phosphorylation of 2-dOG, and it was not clear which process was affected by changes in the cell growth rate. Additionally many of these studies were conducted with cells growing a t low growth medium to cell ratios. Under these conditions it is likely that serum factors as well a s other components of the medium may be limiting for cell division and sugar uptake. Our studies were performed with cells growing attached to glass coverslips at high growth medium to cell ratios. These differences may account for the disparate results obtained by u s and others. Our experiments indicate that the rate of transport of 2-dOG is the same in untransformed and transformed 3T3 cells and is not subject to regulation by cell density or serum factors. Phosphorylation
of 2-dOG is regulated in 3T3 cells by the availability of serum factors, while transformed cells escape this control mechanism. The fact that transformed cells continue to phosphorylate 2-dOG at the maximal rate in the absence of serum may be due to the production of serum-like factors by these cells, or could be due to cell surface alterations which maintain constant activation of the phosphorylation reaction. This work further indicates that the enhanced rate of glucose metabolism exhibited by transformed cells, or by untransformed cells in response to serum addition, is primarily due to enhanced glycolysis rather than to enhanced transport. It is of interest in this connection that Fodge and Rubin ('73) have shown that serum addition activates phosphofructokinase, a key regulation enzyme in glycolysis, in chick embryo fibroblasts. The resultant lowered intra-cellular levels of glucose-6-phosphate, a known inhibitor of hexokinase of animal cells, may account for the results described here. ACKNOWLEDGMENTS
We thank M. A. Brustalon, M. McLeod and E. Profita for excellent technical assistance. Thanks are also expressed to D. Engelhardt for providing facilities and support for part of this work. This investigation was supported by grants from the Damon Runyon Fund for Cancer Research, Inc. (DRG-1212 and DRG-1191), the National Cancer Institute (NCI-14274), and the National Science Foundation (GB40654). John A. Hassell is a n N.I.H. predoctoral fellow. (G.M.-00317). Clarence Colby is the recipient of a Research Career Development Award from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED Bose, S. K . , and B. J . Zlotnick 1973 Growth and density-dependent inhibition ofdeoxyglucose transport in Balb 3T3 cells and its absence i n cells transformed by murine sarcoma virus. Proc. Nat. Acad. Sci. (U.S.A.), 70: 2374-2378. Bradley, W. E. C., and L. A . Culp 1974 Stimulation of 2-deoxyglucose uptake in growth-inhibited Balb/C 3T3 and revertant SV40-transformed 3T3 cells. Exp. Cell Res., 84: 3 3 5 3 5 0 . Bray, G. A. 1960 A simple efficient liquid scintillation for counting aqueous solutions i n a liquid scintillation counter. Anal. Biochem., I : 279-285. Colby, C., and A. H. Romano 1975 Phosphoryla-
TRANSFORMATION, SERUM, AND SUGAR PHOSPHORYLATION tion but not transport of sugars is enhanced in virus-transformed mouse 3T3 cells. J. Cell. Physiol., 85: 15-24. Cunningham, D. D., and A. B. Pardee 1969 Transport changes rapidly initiated by serum addition to “contact inhibited” 3T3 cells. Proc. Nat. Acad. Sci. (U.S.A.), 64: 1049-1056. Dulbecco, R., and J. Elkington 1973 Conditions limiting multiplication of fibroblastic and epithelial cells in dense culture. Nature, 246: 197199. Fodge, D. W., and H. Rubin Activation of phosphofructokinase by stimulants of cell multiplication. Nature New Biol., 246: 181-183. Foster, D. O., and A. B. Pardee 1969 Transport of amino acids by confluent and nonconfluent 3T3 and polyoma virus-transformed 3T3 cells growing on glass coverslips. J. Biol. Chem., 244: 267.52681. Grimes, W. J., and J. L. Schroeder 1973 Dibutyryl cyclic adenosine 3’5’ monophosphate, sugar transport, and regulatory control of cell division in normal and transformed cells. J. Cell. Biol., 5 6 : 487-491. Hassell, J. A,, and D. L. Engelhardt 1973 Translational inhibition in extracts from serum-deprived animal cells. Biochim. Biophys. Acta, 325: 545-553. Heidinek, M. L., and W. L. Ryan 1970 Cyclic nucleotides on cell growth in nitro Cancer Res., 30: 37C378. Hershko, A,, P. Mamont, R. Shields and G. M. Tomkins 1971 Pleiotypic response. Nature New Biology, 232: 206-211. Holley, R. W., and J. Kiernan 1971 Studies of serum factors required by 3T3 and SV3T3 cells. In: Growth Control of Cell Culture. G. E. W. Wolstenholme and J. Knight, eds. Churchill Livingstone, Edinburgh a n d London, pp. 3-15. Johnson, G. S., R. M. Friedman and I . Pastan 1971 Restoration of several morphological characteristics of normal fibroblasts in sarcoma cells treated with adenosine-3’5’-cyclic monophosphate and its derivatives. Proc. Nat. Acad. Sci. ( U . S. A,), 68: 4 2 5 4 2 9 . Kletzien, R. F., and J. F. Perdue 1974 Sugar transport in chick embryo fibroblasts. 1. A functional change in the plasma membrane associated with the rate of cell growth. J. Biol. Chem., 249: 3366-3374. Kram, R., P. Mamont and G. M. Tomkins 1973 Pleiotypic control by adenosine 3’:5‘-cyclic mono-
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