- Email: [email protected]
The response of glycolytic and non-glycolytic mammalian cells to the inhibitory action of fluoropyruvate
Experimental
246
Cell Research 17, 246-255
(1959)
THE RESPONSE OF GLYCOLYTIC AND NON-GLYCOLYTIC MAMMALIAN CELLS TO THE INHIBITORY ACTION OF FLUOROPYRUVATE A. TRAUB
and Y. GINZBURG
Israel Institute for Biological
Research, Ness-Ziona,
Israel
Received September 20, 1958
acid (FCH,COCOOH) has been shown to inhibit respiration in mitochondrial preparations [ 1, 81; it is likely that the inhibition depends on the interaction of essential thiol groups with fluoropyruvate in which the C-F bond has proved to be exceptionally reactive. It was further shown that fluoropyruvate can serve as a substrate of muscle lactic dehydrogenase with concomitant reduction to fluorolactate. Studies in our laboratory revealed that fluoropyruvate inhibits the endogenous respiration of intact mammalian cells, while fluorolactate had no inhibitory effect. It could thus be assumed that fluoropyruvate could serve, under certain conditions, as a specific, inhibitor of the respiration of non-glycolytic cells, while glycolytic cells could overcome the inhibition by a rapid reduction of the inhibitor to fluorolactate. The aim of this investigation has been to determine whether a parallelism exists between the glycolytic activity of cells and their indifference to fluoropyruvate.l FLUOROPYRUVIC
MATERIALS
AND
METHODS
Chemicals.-All chemicals used were reagent grade. Crystalline lactic dehydrogenase was obtained from the Mann Research Laboratories (New York, U.S.A.). Fluoropyruvate and fluorolactate were synthesized according to the methods of Blank, Mager and Bergmann [5], Bergmann and Shahak [4], respectively. Composition of phosphate buffered saline (PBS) in grams per liter distilled water: NaCl, 8, KCl, 0.2, Na,HPO,, 1.15, KH,PO,, 0.2, MgCl, * 6H,O, 0.1, CaCl,, 0.1. Cells and tissues.-Chicken cell suspensions were freshly prepared from 9 days old chick embryos by trypsin treatment according to the method of Dulbecco [6]. Monolayer cultures of chicken fibroblasts were made by growing chicken cell suspensions of 9 days old embryos on Roux bottles for 48 hours at 37X, in Earle’s [7] 1 The following
abbreviations
DPN, diphosphopyridine nucleotide; DPNH, are employed: aminomethane; and PBS, Phosphate buffered saline.
reduced DPN; Tris, tris (hydroxymethyl) Experimental
Cell Research 17
Effect of fluoropyruvafe
on mammalian cells
247
saline containing 0.5 per cent lactalbumin hydrolyzate, 0.1 per cent yeast extract (Difco) and 15 per cent horse serum. The monolayer obtained in this way was washed twice with Earle’s saline ]7], and the cells were removed and dispersed with 0.1 per cent trypsin in Earle’s saline. Rat sarcoma cell suspension.-A cell line-PF-first established by Parker (Toronto) from a rat sarcoma, was grown as a monolayer on Roux bottles in medium 199 [9] with 6 per cent calf serum. Suspensions were prepared by treating the monolayer with a solution of 0.1 per cent sodium Versenate in Earle’s saline from which both the CaCl, and the MgCl, * 6H,O had been omitted. Ehrlich ascites tumor.-The tumor was maintained by weekly intraperitoneal inoculations of 0.2 ml of whole ascitic fluid into the Swiss strain of white mice. The mice were 2-4 months of age. Intact chorioallantoic membranes.-The membranes were removed from 9 days old chick embryos, washed three times with Earle’s saline, dried lightly on filter paper and weighed. The various cell supensions used in the experiments were washed. three times with phosphate buffered saline (PBS). Assay procedures.-The respiration of the tissues was determined manometrically, using the conventional Warburg technique [12]. The cells or membranes were suspended in PBS in the main compartment of a Warburg vessel. Fluoropyruvate or other inhibitors were placed in the side arm and tipped in after ten minutes of temperature equilibration at 37°C. Total volume: 3 ml; gas phase: air. For the assays of fluoropyruvate and lactic acid, the contents of the Warburg vessels were acidified with 0.5 ml of 6 N HCl. Experiments for the measurement of aerobic glycolysis were conducted in the Warburg apparatus. Lactic acid was determined by the method of Barker and Summerson [3], fluoropyruvate by the spectrophotometric method based on its interaction with cysteine [2]: in the presence of an excess of cysteine at an alkaline pH all the fluoropyruvate is converted into a derivative of mercaptopyruvic acid which exhibits a characteristic spectrum in the ultraviolet zone with a peak at the wavelength of 300 mp. The method allows an accurate determination of fluoropyruvate in the range of 5 x 10-s x 10-a M. The reaction mixture of the assay contained: the analyzed sample (or a known amount of fluoropyruvate for the calibration curve), 0.3 ml of 2.5 M KHCO,, 1.5 ml of 0.5 M Tris buffer, pH 8.2, 0.3 ml of 0.1 it4 cysteine. The volume was brought to 3 ml with distilled water. The mixtures were incubated at 58°C for 15 minutes, and the extinction at 300 rnp was measured in silica cells of 1 cm light path, The control cell contained all the components with the exception of fluoropyruvate. The light absorption was measured with a Beckmann spectrophotometer, model DU.
RESTJLTS Aerobic glycolysis.-The rate of production of lactic acid from glucose under aerobic conditions by the various tissues was measured. The results are presented in Fig. 1. The chorioallantoic membranes and the fresh chick embryo cells were characterised by a low aerobic glycolytic activity, while Experimental
Cell Research 17
248
A. Traub and Y. Ginzburg
large amounts of lactic acid were produced by the Ehrlich ascites tumor, the PF cell line and the chick tibroblasts, grown for 48 hours in tissue culture. Effect of fluoropyruvate on respiration.-The amounts of each tissue, used in the following experiments, were chosen so as to get approximately equal oxygen uptakes, and their sensitivity to fluoropyruvate was determined. A concentration of lop3 M fluoropyruvate caused a 75 per cent inhibition in the endogenous respiration of the chick embryo cells, the 48 hours old chick fibroblasts and the Ehrlich ascites tumor. 2 X 1O-3 M fluoropyruvate elicited a similar inhibition in the PF cell line, while 3 x lop3 M was required for the inhibition of the respiration of chorioallantoic membranes to the same
160 150 140 130 o 120 I.&I10 = 100 : 2 90 p c g
80
7
60
2!
50
70
40
Fig. 4.
Fig. 2.
Fig. I.-Aerobic production of lactic acid by various tissues. The experiment was performed in the Warburg apparatus. The tissues were suspended in phosphate buffered saline (PBS) in a total volume of 3 ml and glucose (15 moles/ml) was tipped in after ten minutes of thermoequilibration. The cups were removed at various time intervals, acidified with 0.5 ml. 6 N HCl and analysed membrane; A~ A, chick embryo cells; O-O, for lactic acid. x __ x , chorioallantoic Ehrlich ascites cells; l ~ l . PF cell line; and A~ A , 48 hours old chick fibroblasts. Fig. 2.-Effect of graded amounts of fluoropyruvate on the respiration of Ehrlich ascites tumor. Each cup contained 20 x 106 cells suspended in PBS. Fluoropyruvate was tipped in after ten minutes of thermoequilibration. O---O, control; n -0 , 6 x 1O-4 A4 fluoropyruvate; A~ * , and x ~ x , 2 x 1O-s M fluoro3 x 10-d M fluoropyruvate; * __ A , 1O-3 M fluoropyruvate; pyruvate. Experimental
Cell Research 17
Effect of fluoropyruvate
249
on mammalian cells
extent. These results are summarized in Table I. The effect of graded amounts of fluoropyruvate on the endogenous respiration of Ehrlich ascites cells is presented in Fig. 2. Fluorolactate, even in a ten times higher concentration, did not inhibit the respiration. TABLE
I. Sensitivity Conditions
of the endogenous respiration to fluoropyruvate.
of the experiments
Tissue Chick embryo cells 48 hours old chick fibroblasts Ehrlich ascites tumor PF cell line Chorioallantoic membrane
see under “Materials Number of cells/ Warburg vessel 120 30 25 10
x 106 x 106 x 106 X 106 -
of various tissues and Methods”.
w dry weight/ Warburg vessel
Molar concentration of fluoropyruvate causing 75 % inhibition
12 15 17.5 7 20
10-S 10-S 10-a 12 x 10-S 3 x 10-S
The reversal of the inhibitory action of jluoropyruvate under conditions favouring the production of lactic acid The inhibition by fluoropyruvate was not relieved on addition of Krebs cycle intermediates or various amino acids. But when glucose was added to the respiring cells, in a concentration of 1OV M, the inhibition in the cells with a high aerobic glycolysis, Ehrlich ascites and the PF cell line, was completely abolished; it was partially relieved in the chick tibroblasts grown in tissue culture. The chorioallantoic membranes and the freshly prepared chick embryo cells, which are characterised by a low glycolytic activity, were inhibited even in the presence of glucose. Results are presented in Fig. 3 (a-e). A Crabtree effect (depression of the endogenous respiration in the presence of glycose) was observed in the three tissues which manifest aerobic glycolysis. Among the various sugars tested, only those which serve as substrates of hexokinase (glucose, fructose, mannose), counteracted the inhibition by fluoropyruvate. The non-fermentable sugars (sucrose, ribose, galactose) had no effect. The close correlation between conditions favouring lactic acid production and protective action of glucose, and the fact that only the inhibitory effect of the fluoro analogue of pyruvic acid was relieved by glucose, and not similar Experimental
Cell Research 17
A. Traub and Y. Ginzburg
93
60 r"hnurei
3F
0
0
120
IO
60
90
120
60 MlnLrei
0
Fig. 3 b.
Fig. 3 a. 1
r
90
Fig. 3 c. I
I 170. 160. 150. 140130u 120. 5a 110.
; loos : r
90. so-
r" 70a ; z so-
60.
0
30
60 MlnUW
90
Fig. 3 d.
120
0
15
IO
60 Ml”“M
9D
1211
Fig. 3 e.
Fig. 3. (a-e).-Effect of glucose on the extent of respiratory inhibition produced by fluoropyruvate in various tissues. A ~ A , endogenous; A ~ A , endogenous plus fluoropyruvate; O--O, glucose; and l __ l , glucose plus fluoropyruvate. Each cup contained PBS and lo-* .?f glucose. Cells and fluoropyruvate were added as indicated. (a) Chorioallantoic membranes. Each cup contained 20 mg dry weight of tissue. A concentration of 3 x 1O-3 JI fluoropyruvate was added where indicated. (b) Chick embryo cells. Each cup contained 120 x lo6 cells (12 mg dry weight). A concentration of 1O-3 M fluoropyruvate was added where indicated. (c) 45 hours old chick fibroblasfs. Each cup contained 35 x 1O-6 cells (15 mg dry weight). A concentration of 1O-3 291 fluoropyruvate was added wh’erc indicated. (d) Ehrlich ascites tumor. Each cup contained 25 x lo6 cells (17.5 mg dry weight). A concentration of 1O-3 M fluoropyruvate was added where indicated. (e) PF cell line. Each cup contained 10 x lo6 cells (7 mg dry weight). A concentration of 2 x 1O-3 M fluoropyruvate was added where indicated. Eqxrimental
Cell Research 17
1;
Effect of fluoropyruvate
251
on mammalian cells
inhibitions by other SH-reagents (iodoacetamide, p-chloromercuribenzoate), suggested that fluoropyruvate is eliminated by reduction to fluorolactic acid. This contention is also supported by the following observations. Disappearance of jluoropyruvate conditions favouring
from the reaction mixture lactic acid production
under
The kinetics of the disappearance of fluoropyruv?te from an incubation mixture containing Ehrlich ascites cells, glucose and inhibitor lvere investigated. Results, which show that the disappearance of fluoropyruvate depends on the presence of glucose, are presented in Fig. 4. In the absence of glucose there is an initial sharp drop in the concentration of fluoropyruvate which then remains constant for a long time. In the presence of glucose, the initial sharp drop is followed by a gradual decrease until most of the fluoropyruvate disappears from the reaction mixture. The initial rapid drop, which is independent of the presence of glucose may be due to adsorption of fluoropyruvate by the cells, as it takes place even at 0°C. Fluoropyruvate disappeared from reaction mixtures containing glucose and chick fibroblasts TABLE
II.
The effect of glucose on the disappearance incubated with various tissues.
of fluoropyruvate
The tissues were incubated in PBS with or without glucose. The reaction was started by the addition of 1O-3 M fluoropyruvate. Samples were withdrawn at the indicated time intervals and analysed for fluoropyruvate. With incubation time (minutes)
Chorioallantoic membrane 5 mg dry weight/ml Chick embryo cells 15 x lo6 cells/ml 48 hours chick fibroblasts 15 x lo6 cells/ml Ehrlich ascites tumor PF cell line 3 x lo6 cells/ml a Results
are expressed
0
30
0.82a
0.75
0.85
0.72
glucose
Without
A fluoropyruvate
incubation time (minutes)
glucose
0
30
A fluoropyruvate
0.07
0.86
0.76
0.10
0.13
0.84
0.77
0.07
0.60
0.15
0.45
0.62
0.52
0.76
0.16
0.60
0.80
0.70
0.10 0.10
0.85
0.1
0.75
0.90
0.84
0.06
in micromoles
fluoropyruvate
per ml. Experimental
Cell Research 17
252
A. Traub and Y. Ginzburg
or the PF cell line, but not from those containing fresh chick embryo cells or chorioallantoic membranes. Comparative data on the disappearance of fluoropyruvate in the presence of various tissues are presented in Table II. A concentration of 5 X lop3 M iodoacetamide which inhibits glycolysis in Ehrlich ascites cells, also prevented the disappearance of fluoropyruvate. 190 180 i70 160
140 u 130 d;120 '110 z 2100 0" 90 s 80 ;' 2 70 2
60 SO 40 30 20 10 0
tvfinurer Fig. 4.
15
30
45 Mmurer
60
75
90
Fig. 5.
Fig. 4.-Effect of glucose on the disappearance of fluoropyruvate in the presence of Ehrlich ascites cells. The cells were suspended in PBS (8 x 10” cells per ml) with or without 1O-2 M glucose at 37°C. The reaction was started by adding fluoropyruvate in a concentration of 10-z M. Samples for the analysis of fluoropyruvate were withdrawn at the indicated time intervals. A __ a, without glucose; O---O, with glucose. Fig. B.-Reversal of the protective effect Ehrlich ascites cells; 1O-3 A4 glucose and Fluoropyruvate (1O-s M) was tipped in endogenous; A __ A , endogenous plus plus fluoropyruvate; x -x , pyruvate; and n __ n , pyruvate plus FP.
of glucose by pyruvate. Each cup contained 25 x 106 low2 M lithium pyruvate were added where indicated. after 10 minutes of thermoequilibration. A-A, fluoropyruvate; O--O, glucose; l __ l , glucose 0 __ 0 , glucose plus pyruvate plus fluoropyruvate;
Effect of pyruuate.-When lithium pyruvate was added in a concentration of lop2 M to a mixture containing Ehrlich ascites cells, glucose and fluoropyruvate, the inhibitory action of the latter was restored in spite of the presence of glucose (Fig. 5). This effect, which is specific to pyruvate, is ascribed to a competition between fluoropyruvate and pyruvate for the lactic dehydrogenase of the cells. Fig. 6 represents an experiment in which the rates of reduction of fluoropyruvate and pyruvate by DPNH and muscle lactic Experimenfaf
Cell Research 17
253
Effect of fluoropyruvate on mammalian cells dehydrogenase the enzyme rapidly
were compared. in the presence
than
oxidation
It can be seen that oxidation of pyruvate
with fluoropyruvate.
of DPNH was measured
lich ascites
tumor instead
pyruvate, the reduction respiration is inhibited.
of lactic
proceeds
Similar
results
in the presence dehydrogenase.
of fluoropyruvate
almost were
of DPNH
ten times
obtained
Thus,
proceeds
very
more
when
of a sonic extract
by the
of Ehr-
in the presence slowly,
and
of the
l.OO-
-c 5.
ua
0.75-
Y ?! P 0.504 p0
/i::.:.I:
E : 0.25 is
0
5
1 10
15 Mlll”tS
20
Fig. 7. Fig. 6.-Oxidation of DPNH by muscle lactic dehydrogenase in the presence of pyruvate and fluoropyruvate. The reactions were performed in Beckmann silica cells of 1 cm light path. The reaction mixture contained the following components in a 3 ml volume: 10 p moles KH,PO,K,HPO, buffer, pH 7.4, 1 mg crystalline lactic dehydrogenase (Sigma), I p mole of pyruvate or fluoropyruvate and 0,5,u mole of DPNH. The change in the optical density at 340 mp was measured at 15 second intervals, in the Beckmann spectrophotometer. Fig. 7.-Effect of fluoropyruvate on glycolysis of Ehrlich ascites tumor. The cells (8 x lo6 per ml) were suspended in PBS containing 10-Z M glucose. The reaction was started by adding fluoropyruvate in a concentration of 1O-3 M. Samples for analysis of lactic acid were withdrawn at 5 minutes’ intervals.
The direct measurement of the production by the presence of a large excess of lactic
of fluorolactate was hampered acid in the reaction mixture,
though,
determined
oxidation
when
alone,
fluorolactate
with yeast lactic
can
dehydrogenase
be
(Sh.
Dickstein,
by
an
enzymatic
to be published).
The sensitivity of the glycolytic system to jluoropyruvate The ability of cells with a high aerobic glycolysis to reduce fluoropyruvate to fluorolactate and thus protect their respiration, implies that the glycolytic system is less sensitive to the inhibitor than the respiratory one. This was Experimental
Cell Research 17
25
254 proven caused Ehrlich almost 75 per
A. Traub and Y. Ginzburg to be the case. From Fig. 7 it can be seen that lop3 M fluoropyruvate only a 20 per cent inhibition in the production of lactic acid by ascites tumor. At this concentration, the endogenous respiration is completely inhibited; 3 X 10P3 M fluoropyruvate was required for cent inhibition of the glycolytic activity.
DISCUSSION
The fact that fluoropyruvate, in the presence of glucose, acts as a specific inhibitor of the respiration of non-glycolysing cells is of interest for students of the metabolism of the neoplastic tissue and of mammalian cells growing in tissue culture. The ability- of this compound to act as a selective inhibitor is due to the presence of two chemically reactive groups in the molecule. The alkylating property which leads to the interaction with SH-groups, resides in the C-F bond, and the reactivity of this bond depends on the presence of the u-carbonyl group. Thus the reduction of fluoropyruvate to fluorolactate eliminates the alkylating activity and with it the ability to act as an inhibitor of respiration. This is in contrast to other common SHreagents like iodoacetate or p-chloro mercuribenzoate which are not changed by the metabolic activity of the cell. One of the intriguing problems in the biochemistry of cancer and also that of mammalian cells grown in tissue culture is the probable relationship between the high glycolytic activities of these cells and their rapid growth and proliferation [ll]. One may approach such a problem by a study of cells with different glycolytic activities and observe if there is a quantitative correlation between the extent of glycolysis and their rate of growth. Fluoropyruvate may thus serve as a tool for the selection of cells with different glycolytic activities. As shown in this study, the resistance of cells to the action of fluoropyruvate depends on their ability to reduce it to fluorolactate; it may thus be used for a selection of highly glycolysing cell clones from a population composed of a mixture of cells with a wide spectrum of glycolytic activity. Experiments along these lines are being carried out in our laboratory. If a tumor is characterized by a pronounced aerobic glycolysis as compared with most normal tissues (with the exception of brain and retina), we have in fluoropyruvate an inhibitor which acts preferentially on the respiration of normal cells. The capacity of a tissue to respire in the presence of glucose and fluoropyruvate may constitute an additional measure of its neoplastic state. Experimental
Cell Research 17
Effect of fluoropyruvate
on mammalian cells
255
SUMMARY A study on the effect of fluoropyruvate on the respiration of various mammalian tissues is reported. 1. The endogenous respiration of chorioallantoic membranes, freshly prepared chick embryo cells, chick fibroblasts grown in tissue culture, Ehrlich tumor and a rat sarcoma cell line (PF line) are inhibited by small amounts of fluoropyruvate. 2. In the presence of glucose, fructose or mannose the inhibition is abolished in cells which manifest a high aerobic glycolysis (chick fibroblasts, Ehrlich ascites tumor and PF cell line). Cells of low glycol,ytic activity (chorioallantoic membrane and chick embryo cells) are equally inhibited in the presence and in the absence of these sugars. 3. The protective action of the sugars against inhibition by fluoropyruvate is tentatively explained by a rapid reduction of the inhibitor to fluorolactate by the glycolytic system of the cells. 4. The use of fluoropyruvate for the selection of cell lines with high glycolytic activity is suggested. The authors are indebted to Dr. Y. Avi-Dor for helpful advice during the course of this work. The capable technical assistance of Miss Esther Burstein and Miss Sarah Per1 is gratefully acknowledged. This investigation was supported by a grant from the United States Public Health Service. REFERENCES Y. and MAGER, J., Biochem. J. 63, 613 (1956). 1. AVI-DOR, J. Biol. Chem. 222, 249 (1956). 2. ~ S. B. and SUMMERSON, W. H., J. Biot. Chem. 138, 535 (1956). 3. BARKER, 4. BERGMANN, E. D. and SHAHAK, I., Unpublished results. 5. BLANK, I., MAGER, J. and BERGMANN, E. D., J. Chem. Sot. 2190 (1955). 6. DULBECC~, R., Proc. Natl. Acad. Sci: 38, 747 (1952). 7. EARLE, W. R., J. Natl. Cancer Inst. 4, 165 (1943). 8. GAL, E. M., PETERS, R. A. and WAKE&N, F\. W.; Bioehem. J. 64,161 (1956). 9. MORGAN, J. F., MORTON, H. J. and PARKER, R. C., Proc. Sot. Exptl. Biol. Med. 73, 1 (1950). 10. PETERS, R. A. and HALL, R. J., Biochim. et Biophys. Acta 26, 433 (1957). 11. POTTER, VAN R., Cancer Research 16, 658 (1956). 12. UMBREIT, W. W., BURRIS, R. H. and STAUFFER, J. F., Manometric techniques for the study of tissue Metabolism. Burgess Publishing Co., Minneapolis, Minn., U.S.A. 1947.
Experimental
Cell Research 17
246
Cell Research 17, 246-255
(1959)
THE RESPONSE OF GLYCOLYTIC AND NON-GLYCOLYTIC MAMMALIAN CELLS TO THE INHIBITORY ACTION OF FLUOROPYRUVATE A. TRAUB
and Y. GINZBURG
Israel Institute for Biological
Research, Ness-Ziona,
Israel
Received September 20, 1958
acid (FCH,COCOOH) has been shown to inhibit respiration in mitochondrial preparations [ 1, 81; it is likely that the inhibition depends on the interaction of essential thiol groups with fluoropyruvate in which the C-F bond has proved to be exceptionally reactive. It was further shown that fluoropyruvate can serve as a substrate of muscle lactic dehydrogenase with concomitant reduction to fluorolactate. Studies in our laboratory revealed that fluoropyruvate inhibits the endogenous respiration of intact mammalian cells, while fluorolactate had no inhibitory effect. It could thus be assumed that fluoropyruvate could serve, under certain conditions, as a specific, inhibitor of the respiration of non-glycolytic cells, while glycolytic cells could overcome the inhibition by a rapid reduction of the inhibitor to fluorolactate. The aim of this investigation has been to determine whether a parallelism exists between the glycolytic activity of cells and their indifference to fluoropyruvate.l FLUOROPYRUVIC
MATERIALS
AND
METHODS
Chemicals.-All chemicals used were reagent grade. Crystalline lactic dehydrogenase was obtained from the Mann Research Laboratories (New York, U.S.A.). Fluoropyruvate and fluorolactate were synthesized according to the methods of Blank, Mager and Bergmann [5], Bergmann and Shahak [4], respectively. Composition of phosphate buffered saline (PBS) in grams per liter distilled water: NaCl, 8, KCl, 0.2, Na,HPO,, 1.15, KH,PO,, 0.2, MgCl, * 6H,O, 0.1, CaCl,, 0.1. Cells and tissues.-Chicken cell suspensions were freshly prepared from 9 days old chick embryos by trypsin treatment according to the method of Dulbecco [6]. Monolayer cultures of chicken fibroblasts were made by growing chicken cell suspensions of 9 days old embryos on Roux bottles for 48 hours at 37X, in Earle’s [7] 1 The following
abbreviations
DPN, diphosphopyridine nucleotide; DPNH, are employed: aminomethane; and PBS, Phosphate buffered saline.
reduced DPN; Tris, tris (hydroxymethyl) Experimental
Cell Research 17
Effect of fluoropyruvafe
on mammalian cells
247
saline containing 0.5 per cent lactalbumin hydrolyzate, 0.1 per cent yeast extract (Difco) and 15 per cent horse serum. The monolayer obtained in this way was washed twice with Earle’s saline ]7], and the cells were removed and dispersed with 0.1 per cent trypsin in Earle’s saline. Rat sarcoma cell suspension.-A cell line-PF-first established by Parker (Toronto) from a rat sarcoma, was grown as a monolayer on Roux bottles in medium 199 [9] with 6 per cent calf serum. Suspensions were prepared by treating the monolayer with a solution of 0.1 per cent sodium Versenate in Earle’s saline from which both the CaCl, and the MgCl, * 6H,O had been omitted. Ehrlich ascites tumor.-The tumor was maintained by weekly intraperitoneal inoculations of 0.2 ml of whole ascitic fluid into the Swiss strain of white mice. The mice were 2-4 months of age. Intact chorioallantoic membranes.-The membranes were removed from 9 days old chick embryos, washed three times with Earle’s saline, dried lightly on filter paper and weighed. The various cell supensions used in the experiments were washed. three times with phosphate buffered saline (PBS). Assay procedures.-The respiration of the tissues was determined manometrically, using the conventional Warburg technique [12]. The cells or membranes were suspended in PBS in the main compartment of a Warburg vessel. Fluoropyruvate or other inhibitors were placed in the side arm and tipped in after ten minutes of temperature equilibration at 37°C. Total volume: 3 ml; gas phase: air. For the assays of fluoropyruvate and lactic acid, the contents of the Warburg vessels were acidified with 0.5 ml of 6 N HCl. Experiments for the measurement of aerobic glycolysis were conducted in the Warburg apparatus. Lactic acid was determined by the method of Barker and Summerson [3], fluoropyruvate by the spectrophotometric method based on its interaction with cysteine [2]: in the presence of an excess of cysteine at an alkaline pH all the fluoropyruvate is converted into a derivative of mercaptopyruvic acid which exhibits a characteristic spectrum in the ultraviolet zone with a peak at the wavelength of 300 mp. The method allows an accurate determination of fluoropyruvate in the range of 5 x 10-s x 10-a M. The reaction mixture of the assay contained: the analyzed sample (or a known amount of fluoropyruvate for the calibration curve), 0.3 ml of 2.5 M KHCO,, 1.5 ml of 0.5 M Tris buffer, pH 8.2, 0.3 ml of 0.1 it4 cysteine. The volume was brought to 3 ml with distilled water. The mixtures were incubated at 58°C for 15 minutes, and the extinction at 300 rnp was measured in silica cells of 1 cm light path, The control cell contained all the components with the exception of fluoropyruvate. The light absorption was measured with a Beckmann spectrophotometer, model DU.
RESTJLTS Aerobic glycolysis.-The rate of production of lactic acid from glucose under aerobic conditions by the various tissues was measured. The results are presented in Fig. 1. The chorioallantoic membranes and the fresh chick embryo cells were characterised by a low aerobic glycolytic activity, while Experimental
Cell Research 17
248
A. Traub and Y. Ginzburg
large amounts of lactic acid were produced by the Ehrlich ascites tumor, the PF cell line and the chick tibroblasts, grown for 48 hours in tissue culture. Effect of fluoropyruvate on respiration.-The amounts of each tissue, used in the following experiments, were chosen so as to get approximately equal oxygen uptakes, and their sensitivity to fluoropyruvate was determined. A concentration of lop3 M fluoropyruvate caused a 75 per cent inhibition in the endogenous respiration of the chick embryo cells, the 48 hours old chick fibroblasts and the Ehrlich ascites tumor. 2 X 1O-3 M fluoropyruvate elicited a similar inhibition in the PF cell line, while 3 x lop3 M was required for the inhibition of the respiration of chorioallantoic membranes to the same
160 150 140 130 o 120 I.&I10 = 100 : 2 90 p c g
80
7
60
2!
50
70
40
Fig. 4.
Fig. 2.
Fig. I.-Aerobic production of lactic acid by various tissues. The experiment was performed in the Warburg apparatus. The tissues were suspended in phosphate buffered saline (PBS) in a total volume of 3 ml and glucose (15 moles/ml) was tipped in after ten minutes of thermoequilibration. The cups were removed at various time intervals, acidified with 0.5 ml. 6 N HCl and analysed membrane; A~ A, chick embryo cells; O-O, for lactic acid. x __ x , chorioallantoic Ehrlich ascites cells; l ~ l . PF cell line; and A~ A , 48 hours old chick fibroblasts. Fig. 2.-Effect of graded amounts of fluoropyruvate on the respiration of Ehrlich ascites tumor. Each cup contained 20 x 106 cells suspended in PBS. Fluoropyruvate was tipped in after ten minutes of thermoequilibration. O---O, control; n -0 , 6 x 1O-4 A4 fluoropyruvate; A~ * , and x ~ x , 2 x 1O-s M fluoro3 x 10-d M fluoropyruvate; * __ A , 1O-3 M fluoropyruvate; pyruvate. Experimental
Cell Research 17
Effect of fluoropyruvate
249
on mammalian cells
extent. These results are summarized in Table I. The effect of graded amounts of fluoropyruvate on the endogenous respiration of Ehrlich ascites cells is presented in Fig. 2. Fluorolactate, even in a ten times higher concentration, did not inhibit the respiration. TABLE
I. Sensitivity Conditions
of the endogenous respiration to fluoropyruvate.
of the experiments
Tissue Chick embryo cells 48 hours old chick fibroblasts Ehrlich ascites tumor PF cell line Chorioallantoic membrane
see under “Materials Number of cells/ Warburg vessel 120 30 25 10
x 106 x 106 x 106 X 106 -
of various tissues and Methods”.
w dry weight/ Warburg vessel
Molar concentration of fluoropyruvate causing 75 % inhibition
12 15 17.5 7 20
10-S 10-S 10-a 12 x 10-S 3 x 10-S
The reversal of the inhibitory action of jluoropyruvate under conditions favouring the production of lactic acid The inhibition by fluoropyruvate was not relieved on addition of Krebs cycle intermediates or various amino acids. But when glucose was added to the respiring cells, in a concentration of 1OV M, the inhibition in the cells with a high aerobic glycolysis, Ehrlich ascites and the PF cell line, was completely abolished; it was partially relieved in the chick tibroblasts grown in tissue culture. The chorioallantoic membranes and the freshly prepared chick embryo cells, which are characterised by a low glycolytic activity, were inhibited even in the presence of glucose. Results are presented in Fig. 3 (a-e). A Crabtree effect (depression of the endogenous respiration in the presence of glycose) was observed in the three tissues which manifest aerobic glycolysis. Among the various sugars tested, only those which serve as substrates of hexokinase (glucose, fructose, mannose), counteracted the inhibition by fluoropyruvate. The non-fermentable sugars (sucrose, ribose, galactose) had no effect. The close correlation between conditions favouring lactic acid production and protective action of glucose, and the fact that only the inhibitory effect of the fluoro analogue of pyruvic acid was relieved by glucose, and not similar Experimental
Cell Research 17
A. Traub and Y. Ginzburg
93
60 r"hnurei
3F
0
0
120
IO
60
90
120
60 MlnLrei
0
Fig. 3 b.
Fig. 3 a. 1
r
90
Fig. 3 c. I
I 170. 160. 150. 140130u 120. 5a 110.
; loos : r
90. so-
r" 70a ; z so-
60.
0
30
60 MlnUW
90
Fig. 3 d.
120
0
15
IO
60 Ml”“M
9D
1211
Fig. 3 e.
Fig. 3. (a-e).-Effect of glucose on the extent of respiratory inhibition produced by fluoropyruvate in various tissues. A ~ A , endogenous; A ~ A , endogenous plus fluoropyruvate; O--O, glucose; and l __ l , glucose plus fluoropyruvate. Each cup contained PBS and lo-* .?f glucose. Cells and fluoropyruvate were added as indicated. (a) Chorioallantoic membranes. Each cup contained 20 mg dry weight of tissue. A concentration of 3 x 1O-3 JI fluoropyruvate was added where indicated. (b) Chick embryo cells. Each cup contained 120 x lo6 cells (12 mg dry weight). A concentration of 1O-3 M fluoropyruvate was added where indicated. (c) 45 hours old chick fibroblasfs. Each cup contained 35 x 1O-6 cells (15 mg dry weight). A concentration of 1O-3 291 fluoropyruvate was added wh’erc indicated. (d) Ehrlich ascites tumor. Each cup contained 25 x lo6 cells (17.5 mg dry weight). A concentration of 1O-3 M fluoropyruvate was added where indicated. (e) PF cell line. Each cup contained 10 x lo6 cells (7 mg dry weight). A concentration of 2 x 1O-3 M fluoropyruvate was added where indicated. Eqxrimental
Cell Research 17
1;
Effect of fluoropyruvate
251
on mammalian cells
inhibitions by other SH-reagents (iodoacetamide, p-chloromercuribenzoate), suggested that fluoropyruvate is eliminated by reduction to fluorolactic acid. This contention is also supported by the following observations. Disappearance of jluoropyruvate conditions favouring
from the reaction mixture lactic acid production
under
The kinetics of the disappearance of fluoropyruv?te from an incubation mixture containing Ehrlich ascites cells, glucose and inhibitor lvere investigated. Results, which show that the disappearance of fluoropyruvate depends on the presence of glucose, are presented in Fig. 4. In the absence of glucose there is an initial sharp drop in the concentration of fluoropyruvate which then remains constant for a long time. In the presence of glucose, the initial sharp drop is followed by a gradual decrease until most of the fluoropyruvate disappears from the reaction mixture. The initial rapid drop, which is independent of the presence of glucose may be due to adsorption of fluoropyruvate by the cells, as it takes place even at 0°C. Fluoropyruvate disappeared from reaction mixtures containing glucose and chick fibroblasts TABLE
II.
The effect of glucose on the disappearance incubated with various tissues.
of fluoropyruvate
The tissues were incubated in PBS with or without glucose. The reaction was started by the addition of 1O-3 M fluoropyruvate. Samples were withdrawn at the indicated time intervals and analysed for fluoropyruvate. With incubation time (minutes)
Chorioallantoic membrane 5 mg dry weight/ml Chick embryo cells 15 x lo6 cells/ml 48 hours chick fibroblasts 15 x lo6 cells/ml Ehrlich ascites tumor PF cell line 3 x lo6 cells/ml a Results
are expressed
0
30
0.82a
0.75
0.85
0.72
glucose
Without
A fluoropyruvate
incubation time (minutes)
glucose
0
30
A fluoropyruvate
0.07
0.86
0.76
0.10
0.13
0.84
0.77
0.07
0.60
0.15
0.45
0.62
0.52
0.76
0.16
0.60
0.80
0.70
0.10 0.10
0.85
0.1
0.75
0.90
0.84
0.06
in micromoles
fluoropyruvate
per ml. Experimental
Cell Research 17
252
A. Traub and Y. Ginzburg
or the PF cell line, but not from those containing fresh chick embryo cells or chorioallantoic membranes. Comparative data on the disappearance of fluoropyruvate in the presence of various tissues are presented in Table II. A concentration of 5 X lop3 M iodoacetamide which inhibits glycolysis in Ehrlich ascites cells, also prevented the disappearance of fluoropyruvate. 190 180 i70 160
140 u 130 d;120 '110 z 2100 0" 90 s 80 ;' 2 70 2
60 SO 40 30 20 10 0
tvfinurer Fig. 4.
15
30
45 Mmurer
60
75
90
Fig. 5.
Fig. 4.-Effect of glucose on the disappearance of fluoropyruvate in the presence of Ehrlich ascites cells. The cells were suspended in PBS (8 x 10” cells per ml) with or without 1O-2 M glucose at 37°C. The reaction was started by adding fluoropyruvate in a concentration of 10-z M. Samples for the analysis of fluoropyruvate were withdrawn at the indicated time intervals. A __ a, without glucose; O---O, with glucose. Fig. B.-Reversal of the protective effect Ehrlich ascites cells; 1O-3 A4 glucose and Fluoropyruvate (1O-s M) was tipped in endogenous; A __ A , endogenous plus plus fluoropyruvate; x -x , pyruvate; and n __ n , pyruvate plus FP.
of glucose by pyruvate. Each cup contained 25 x 106 low2 M lithium pyruvate were added where indicated. after 10 minutes of thermoequilibration. A-A, fluoropyruvate; O--O, glucose; l __ l , glucose 0 __ 0 , glucose plus pyruvate plus fluoropyruvate;
Effect of pyruuate.-When lithium pyruvate was added in a concentration of lop2 M to a mixture containing Ehrlich ascites cells, glucose and fluoropyruvate, the inhibitory action of the latter was restored in spite of the presence of glucose (Fig. 5). This effect, which is specific to pyruvate, is ascribed to a competition between fluoropyruvate and pyruvate for the lactic dehydrogenase of the cells. Fig. 6 represents an experiment in which the rates of reduction of fluoropyruvate and pyruvate by DPNH and muscle lactic Experimenfaf
Cell Research 17
253
Effect of fluoropyruvate on mammalian cells dehydrogenase the enzyme rapidly
were compared. in the presence
than
oxidation
It can be seen that oxidation of pyruvate
with fluoropyruvate.
of DPNH was measured
lich ascites
tumor instead
pyruvate, the reduction respiration is inhibited.
of lactic
proceeds
Similar
results
in the presence dehydrogenase.
of fluoropyruvate
almost were
of DPNH
ten times
obtained
Thus,
proceeds
very
more
when
of a sonic extract
by the
of Ehr-
in the presence slowly,
and
of the
l.OO-
-c 5.
ua
0.75-
Y ?! P 0.504 p0
/i::.:.I:
E : 0.25 is
0
5
1 10
15 Mlll”tS
20
Fig. 7. Fig. 6.-Oxidation of DPNH by muscle lactic dehydrogenase in the presence of pyruvate and fluoropyruvate. The reactions were performed in Beckmann silica cells of 1 cm light path. The reaction mixture contained the following components in a 3 ml volume: 10 p moles KH,PO,K,HPO, buffer, pH 7.4, 1 mg crystalline lactic dehydrogenase (Sigma), I p mole of pyruvate or fluoropyruvate and 0,5,u mole of DPNH. The change in the optical density at 340 mp was measured at 15 second intervals, in the Beckmann spectrophotometer. Fig. 7.-Effect of fluoropyruvate on glycolysis of Ehrlich ascites tumor. The cells (8 x lo6 per ml) were suspended in PBS containing 10-Z M glucose. The reaction was started by adding fluoropyruvate in a concentration of 1O-3 M. Samples for analysis of lactic acid were withdrawn at 5 minutes’ intervals.
The direct measurement of the production by the presence of a large excess of lactic
of fluorolactate was hampered acid in the reaction mixture,
though,
determined
oxidation
when
alone,
fluorolactate
with yeast lactic
can
dehydrogenase
be
(Sh.
Dickstein,
by
an
enzymatic
to be published).
The sensitivity of the glycolytic system to jluoropyruvate The ability of cells with a high aerobic glycolysis to reduce fluoropyruvate to fluorolactate and thus protect their respiration, implies that the glycolytic system is less sensitive to the inhibitor than the respiratory one. This was Experimental
Cell Research 17
25
254 proven caused Ehrlich almost 75 per
A. Traub and Y. Ginzburg to be the case. From Fig. 7 it can be seen that lop3 M fluoropyruvate only a 20 per cent inhibition in the production of lactic acid by ascites tumor. At this concentration, the endogenous respiration is completely inhibited; 3 X 10P3 M fluoropyruvate was required for cent inhibition of the glycolytic activity.
DISCUSSION
The fact that fluoropyruvate, in the presence of glucose, acts as a specific inhibitor of the respiration of non-glycolysing cells is of interest for students of the metabolism of the neoplastic tissue and of mammalian cells growing in tissue culture. The ability- of this compound to act as a selective inhibitor is due to the presence of two chemically reactive groups in the molecule. The alkylating property which leads to the interaction with SH-groups, resides in the C-F bond, and the reactivity of this bond depends on the presence of the u-carbonyl group. Thus the reduction of fluoropyruvate to fluorolactate eliminates the alkylating activity and with it the ability to act as an inhibitor of respiration. This is in contrast to other common SHreagents like iodoacetate or p-chloro mercuribenzoate which are not changed by the metabolic activity of the cell. One of the intriguing problems in the biochemistry of cancer and also that of mammalian cells grown in tissue culture is the probable relationship between the high glycolytic activities of these cells and their rapid growth and proliferation [ll]. One may approach such a problem by a study of cells with different glycolytic activities and observe if there is a quantitative correlation between the extent of glycolysis and their rate of growth. Fluoropyruvate may thus serve as a tool for the selection of cells with different glycolytic activities. As shown in this study, the resistance of cells to the action of fluoropyruvate depends on their ability to reduce it to fluorolactate; it may thus be used for a selection of highly glycolysing cell clones from a population composed of a mixture of cells with a wide spectrum of glycolytic activity. Experiments along these lines are being carried out in our laboratory. If a tumor is characterized by a pronounced aerobic glycolysis as compared with most normal tissues (with the exception of brain and retina), we have in fluoropyruvate an inhibitor which acts preferentially on the respiration of normal cells. The capacity of a tissue to respire in the presence of glucose and fluoropyruvate may constitute an additional measure of its neoplastic state. Experimental
Cell Research 17
Effect of fluoropyruvate
on mammalian cells
255
SUMMARY A study on the effect of fluoropyruvate on the respiration of various mammalian tissues is reported. 1. The endogenous respiration of chorioallantoic membranes, freshly prepared chick embryo cells, chick fibroblasts grown in tissue culture, Ehrlich tumor and a rat sarcoma cell line (PF line) are inhibited by small amounts of fluoropyruvate. 2. In the presence of glucose, fructose or mannose the inhibition is abolished in cells which manifest a high aerobic glycolysis (chick fibroblasts, Ehrlich ascites tumor and PF cell line). Cells of low glycol,ytic activity (chorioallantoic membrane and chick embryo cells) are equally inhibited in the presence and in the absence of these sugars. 3. The protective action of the sugars against inhibition by fluoropyruvate is tentatively explained by a rapid reduction of the inhibitor to fluorolactate by the glycolytic system of the cells. 4. The use of fluoropyruvate for the selection of cell lines with high glycolytic activity is suggested. The authors are indebted to Dr. Y. Avi-Dor for helpful advice during the course of this work. The capable technical assistance of Miss Esther Burstein and Miss Sarah Per1 is gratefully acknowledged. This investigation was supported by a grant from the United States Public Health Service. REFERENCES Y. and MAGER, J., Biochem. J. 63, 613 (1956). 1. AVI-DOR, J. Biol. Chem. 222, 249 (1956). 2. ~ S. B. and SUMMERSON, W. H., J. Biot. Chem. 138, 535 (1956). 3. BARKER, 4. BERGMANN, E. D. and SHAHAK, I., Unpublished results. 5. BLANK, I., MAGER, J. and BERGMANN, E. D., J. Chem. Sot. 2190 (1955). 6. DULBECC~, R., Proc. Natl. Acad. Sci: 38, 747 (1952). 7. EARLE, W. R., J. Natl. Cancer Inst. 4, 165 (1943). 8. GAL, E. M., PETERS, R. A. and WAKE&N, F\. W.; Bioehem. J. 64,161 (1956). 9. MORGAN, J. F., MORTON, H. J. and PARKER, R. C., Proc. Sot. Exptl. Biol. Med. 73, 1 (1950). 10. PETERS, R. A. and HALL, R. J., Biochim. et Biophys. Acta 26, 433 (1957). 11. POTTER, VAN R., Cancer Research 16, 658 (1956). 12. UMBREIT, W. W., BURRIS, R. H. and STAUFFER, J. F., Manometric techniques for the study of tissue Metabolism. Burgess Publishing Co., Minneapolis, Minn., U.S.A. 1947.
Experimental
Cell Research 17