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Inhibition of mammalian cell division by glyoxals
Q 1968 by Academic Press Inc. Experimental
65
Cell Research 50, 65-72 (1968)
INHIBITION
OF MAMMALIAN BY GLYOXALS
CELL DIVISION
CH. T. GREGG Los Alamos Scientijic Laboratory, University of California, Los Alamos, N. Mex. 87544, USA
Received June 1, 1967
A. SZENT-GYORGYI and his associates have discussed the role [5, 141 of hypothetical growth-promoting and retarding substances in normal growth regulation, wound healing, and neoplastic processes. bfore recently, Egyud and Szent-Gyorgyi [6, 7] and Szent-Gyorgyi, Egyud, and McLaughlin [17] have reported on the effects of methylglyoxal on the growth of a variety of organisms. At 1 mM concentration, this compound stops cell division in a variety of cells; this inhibition is reversed by equimolar quantities of cysteine or ethylenediamine [6, 71. These authors suggested that this reversible inhibition of cell division might induce cells into synchronous growth and division [71. This report concerns the effects of methyl- and propylglyoxal on mammalian cells growing exponentially in suspension culture.2 A primary objective was to determine if the glyoxals induced these cells into synchronous growth. A second goal was to carry out more detailed time-course studies of the action of the glyoxals than was possible with the rapidly growing Escherichia coli [S, 71. The results of these experiments show that glyoxals cannot be used to induce synchronous growth in the two mammalian cell lines used. These compounds appear to act as general inhibitors of protein synthesis, and the effect of this inhibition is to stop cells from continuing through their life cycle, regardless of the location of the cell within the cycle at the time the inhibitor is added. METHODS
AND
MATERIALS
Mouse lymphoma cells (L-5178Y) were used in most of the experiments described here. The cells were the generous gift of Dr Lionel Manson of The Wistar Institute. These cells grow as an ascites tumor
in suitable
mice (strain
DBA-2J)
or in vitro.
1 This work was performed under the auspices of the U.S. Atomic Energy Commission. 0 2 The glyoxals have the structure R-C-CHO, where R is methyl or propyl for the correspondingly named compounds. 5 - 681806
Experimental
Cell Research 50
Ch. T. Gregg Either in viuo or in vifro, the cells have a doubling time of 11-13 h [4] and a modal chromosome number of 41 [IO]. The cells were grown in suspension culture in a New Brunswick 5-liter fermenter with an automatic pH control unit. The medium was modified Fischer’s L-l [4] except that streptomycin was omitted and the concentration of penicillin was reduced one-half. In some experiments, cells derived from the Chinese hamster ovary were used. These cells were grown in ordinary spinner flasks in Ham’s F-10 medium with cysteine omitted [8]. The properties of this cell line are described elsewhere [IZ]. Both cultures were negative when checked at intervals for contamination by mycoplasma (PPLO) by the method of Chanock, Hayflick, and Barile [3]. The lymphoma cells were checked daily under the phase contrast microscope for contamination by yeast or bacteria. Contaminated cultures were occasionally found and were discarded. Cell viability was determined by the trypan blue exclusion test. Cells were counted with a modified Coulter counter, as previously described [IZ]. The experiments described here were done with methyl- and propylglyoxal synthesized and purified by Dr Lazlo Egyud [5]. These compounds were the generous gift of Dr Egyud and Dr Albert Szent-Gyorgyi. Experiments were carried out by diluting the stock cultures into 200 ml of fresh medium in 500-ml spinner flasks. The cell suspensions were kept at 36.5% and stirred continuously during the experiment; aliquots of approximately 3 ml were removed periodically for cell counting. After establishing that the cells were growing normally, aliquots of aqueous solutions of the glyoxals were added to the suspension culture. Because of the relatively short duration of the experiments and the presence of penicillin in the medium, no attempt was made to sterilize the glyoxal solutions; no contamination was every observed. When cysteine was used to reverse inhibition by the glyoxals, it was weighed out, dissolved in equimolar Tris-HCl buffer at pH 7.0, and used immediately. In the tracer experiments 200 ,uc of aH-thymidine or 3H-uridine or 20 ,MZof laCleucine were added to the appropriate vessels at the same time as the glyoxal compound. Duplicate 3-ml aliquots were removed from each flask at intervals, chilled, and rapidly centrifuged in the cold. The cell pellet was resuspended and washed in 0.25 M sucrose. The acid-insoluble fractions were collected on filter paper disks, essentially as described by Byfield and Scherbaum [2], and the disks were counted in a Tri-Carb scintillation counter in Bray’s scintillation mixture [I]. RESULTS Fig. 1 shows a typical experiment carried out with the mouse lymphoma cell. At point A, propylglyoxal was added to final concentrations of 0.03 mM and 0.1 mM (flasks No. 1 and No. 2, respectively). Propylglyoxal at 0.1 mM stopped cell division almost immediately, while 0.03 mM had no effect. Data for a control flask containing no propylglyoxal and for a flask containing 0.01 mM glyoxal are omitted, since they were the same as those of flask No. 1. The relatively narrow concentration range between complete inhibition and no detectable effect, as seen in the data of Fig. 1, is typical for glyoxals Experimental
Cell Research 50
Glyoxal
inhibition
67
in these mammalian cells. In some experiments concentrations below 0.1 mM gave a transient inhibition of cell growth (1 or 2 h), after which the cells spontaneously resumed their normal growth rate. Concentrations higher than 0.2 m&i’ led to complete and irreversible inhibition of cell division and to rapid death of the cells. Szent-Gyorgyi and his colleagues found that higher
5.7 56 55 5.4 53 5.2 5.1 1
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% DEAD CELLS CONTROL 0 CONTROL .-0 2 10 2 I 16
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Fig. 1.
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i. LEUCINE INCORPORATION I
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Fig. 2.
Fig. l.-Inhibition of division of L-5178Y cells by propylglyoxal and reversal of inhibition by cysteine. To cells growing exponentially in L-l medium (about lo5 cells/ml), propylglyoxal was added at point A to a final concentration of 0.03 mM (flask No. 1) or 0.1 mM (flask No. 2). Aliquots for electronic cell counting were removed periodically. At point B, 0.125 mM cysteine (final concentration) was added. At points B and C, the percentage of dead cells in each flask was determined by counting the number of cells (per 100 cells counted) that were unable to exclude the dye trypan blue. Data for a control vessel to which propylglyoxal was not added and for a vessel containing 0.01 mM propylglyoxal were omitted, since the cell counts in these two vessels were the same as for flask No. 1. The incubation temperature was 36.5%. The radius of the circles at each point is 1 standard deviation (0.5 per cent). Abscissa: Time (h); ordinate: log of cell number. Fig. 2.-Relative incorporation rates of labeled precursors into DNA, RNA, and protein by L-5178Y cells in the presence of 0.2 mM methylglyoxal. The conditions in this experiment were identical to those of Fig. 1. Cells were growing exponentially in 4 identical flasks. To 2 flasks, 0.2 mM methylglyoxal (final concentration) was added at zero time. At the same time, to one of these, 200 ,UCof SH-thymidine and 20 ,UCof %-leucine were added; to the other were added 200 PC of $H-uridine. The 2 control flasks contained the same isotopic compositions but no methylglyoxal. Duplicate 3-ml aliquots were removed from the flasks at intervals, and the cells were harvested, washed with 0.25 M sucrose, and extracted with cold 10 per cent trichloroacetic acid. The acid-insoluble material was collected on filter paper disks, washed once with cold 10 per cent trichloroacetic acid and twice with ether, then air-dried. The disks were counted in Bray’s scintillation mixture in a Packard Tri-Carb scintillation counter. Abscissa: Time (h); ordinate: y0 control. Experimental
Cell Research 50
Ch. T. Gregg homologs of methylglyoxal (such as the propyl compound) gave a wider margin between the minimum concentration required to reversibly inhibit bacterial. cell division and that which was cytotoxic [16]. However, in the mammalian cell system, methyl- and propylglyoxal behave identically. At point B in Fig. 1, a slight molar excess of cysteine (final concentration 0.125 mM) was added to all reaction vessels. This level of cysteine had no effect on the control cell suspension, but it reversed inhibition of cell division by propylglyoxal and led to a resumption, after several hours, of a growth rate nearly equal to that of the control (a doubling time of 12.7 h versus 12.0 h in the vessels in which growth was not inhibited). In some experiments, cell division was resumed at a nearly normal rate almost immediately after addition of cysteine. However, in many experiments, the pattern shown in Fig. 1 was observed, in which addition of cysteine caused an immediate but transitory return to the normal growth rate, followed by several more hours of slow growth before the normal rate was again resumed. The data of Egyud and Szent-Gyorgyi [6] show a similar lag after addition of cysteine before normal growth is resumed. However, for the bacteria the period of sub-optimal growth is 6 bacterial doubling times but only a third of a doubling time for the mammalian cells. At points B and C of Fig. 1, the percentage of dead cells in each flask was determined by the trypan blue exclusion method. At point B (the time of cysteine addition), there were no detectable dead cells in either the control flask or in flask No. 1, which contained 0.03 mM propylglyoxal. In flask No. 2, 10 per cent of the cells were dead. In all experiments, the amount of the glyoxals required to suppress cell division completely was mildly toxic, killing from 4-10 per cent of the cells. At point C, the proportion of dead cells in flask No. 2 had dropped to 6 per cent, indicating that the death rate of the cells dropped following addition of cysteine. The difference of 4 per cent in viability of the cells in the two vessels at point C is sufficient to account for the difference in doubling times of the two cultures. Identical results were obtained in experiments on the Chinese hamster cell. The results presented in Fig. 1 show an immediate effect of propylglyoxal. Out of 14 experiments, 5 showed no lag between addition of the glyoxal and appearance of its effect on cell division. The average lag in the remaining 9 experiments was 15 min with a range from 4-42 min. In an experiment in which cell counts were taken at 12-min intervals, the lag time was 10 & 3 min. For the cells used in these experiments, the period of mitosis (M) was approximately 45 min, while the G2 period preceding mitosis [9] was approximately 2.6 h [4, 8, 131. The fact that cells stop dividing within 10-l 5 min Experimental
Cell Research 50
Glyoxal
69
inhibition
after addition of 0.1 mM propylglyoxal indicates either that the point of action of this compound is late in the mitotic period (M) or, alternatively, that propylglyoxal blocks a metabolic process essential for cells in all or nearly all portions of the cell cycle, so that the progress of cells around the life cycle stops almost immediately when the inhibitor is added. These two possibilities are easily distinguished. If the action of the glyoxals were specific for cells in late M, then during the course of a 3-h inhibition all cells which were in the earlier portion of M, and most of the cells in G2 at the time the inhibitor was added, would accumulate at the point in M at which the glyoxals act. When inhibition is relieved by addition of cysteine, approximately 19 per cent of the cell population would complete M and divide within a few minutes, giving rise to an abrupt jump in the cell count (i.e., the apparent growth rate would be much higher than that in the control flasks). It is clear from Fig. 1 that such a jump does not occur. On addition of a slight molar excess of cysteine, the inhibited cells, at best, simply resume their normal growth rate. An alternative way to measure production of synchronous division is to determine the mitotic index. In the case discussed above, this index would be expected to rise from about 5 per cent, as observed in the control flask, to about 19 per cent following release of glyoxal inhibition. When mitotic indices were measured in an experiment like that of Fig. 1 (except that inhibition was reversed after 3 h rather than after 4 h), no significant change in mitotic index was observed either following reversal of propylglyoxal inhibition with cysteine or following the initial addition of the glyoxal compound. Thus, those cells which were in mitosis lo-15 min after addition of glyoxal remained in mitosis, but no new cells entered mitosis until the inhibition was released by cysteine. These results convincingly demonstrate that the glyoxals do not inhibit growth of these mammalian cells by acting specifically at some point late in the mitotic period of the cell cycle. However, the data are consistent with the idea that the glyoxal derivatives inhibit a metabolic process essential for cells in all or nearly all regions of the cell cycle. Egyud and Szent-Gyorgyi investigated the incorporation of label into DNA, RNA, and protein in the presence and absence of methylglyoxal [7]. E. coli were exposed to labeled precursors (with or without methylglyoxal) for 2 generation times (60 min). At the end of this time thymine incorporation into DNA was reduced to 50 per cent by methylglyoxal, uracil incorporation into RNA was 75 per cent, and leucine incorporation into protein was only 6 per cent of that found in the absence of methylglyoxal. These authors conExperimenial
Cell Research 50
70
Ch. T. Gregg
eluded that methylglyoxal inhibited cell division in E. coli primarily by its action on protein synthesis. A similar study was undertaken with mammalian cells. In this system it is possible to look at changes in the rates of incorporation of label into the various macromolecules during small fractions of a generation time-something very difficult to do with E. coli, which grows some 24 times faster. The results of such an experiment are shown in Fig. 2. Here 0.2 mM methylglyoxal was added to flasks at zero time, simultaneously with the appropriately labeled compounds, and the rates of incorporation into the acidinsoluble fraction were compared with those of controls to which methylglyoxal was not added. No cysteine was added to reverse the inhibition. After 1 hr in the presence of 0.2 mM methylglyoxal, the rate of incorporation of labeled thymidine had dropped to 40 per cent and the rate of incorporation of leucine to only 5 per cent of the control rate, while the rate of incorporation of uridine into RNA was unaffected. Subsequently the rate of incorporation of label into RNA dropped rapidly. Five hr after addition of methylglyoxal the rate of incorporation of label into DNA was reduced to 25 per cent compared to that of the control and that of RNA to 15 per cent, while the rate of incorporation of leucine into protein remained at 5 per cent of the control rate. DISCUSSION
The only published data available on the effect of glyoxals on cultured mammalian cells are those of Dr Ruth Johnsson, working in Szent-Gyorgyi’s laboratory, who found that methylglyoxal killed KB cells in 24 h at concentrations of 0.1-1.0 mM [15]. All the information on the kinetics of the reaction of cells with the glyoxals refers to work done on E. coli [6, 71. The results presented here show that in two lines of mammalian cells growing in suspension culture methyl- or propylglyoxal at low concentrations quickly and completely stops cell division and that this inhibition is reversed or prevented by approximately equimolar cysteine. This is in complete agreement with the work of Egyud and Szent-Gyorgyi [6, 71 on E. coli. Although the mammalian cells might appear to be much more sensitive to inhibition by glyoxals, this is not the case. Taking a recent value for the protein content of an E. coli cell [14], one finds that, under the conditions used by Egyud and Szent-Gyorgyi [6, 71, reversible inhibition of cell division requires approximately 22 ,umoles of glyoxal per mg of cellular protein. A similar calculation for a typical experiment with mammalian cells yields a Experimental
Cell Research 50
Glyokal
71
inhibition
value of 12 ,umoles of glyoxal per mg of cellular protein for reversible inhibition of cell division. The fact that the life cycle of mammalian cells used in these experiments is some 24 times longer than that of E. coli makes it feasible to study the effect of the glyoxals on cell division and on metabolic processes at intervals which are small compared to the doubling time of the cells, and to show that cell division stops lo-15 min after addition of a glyoxal. This observation, when combined with analysis of the cell cycle, with the cell counting rate following release of glyoxal inhibition, and with the measurement of mitotic index before, during, and after release of glyoxal inhibition, indicates that glyoxal inhibition in these mammalian cells does not lead to partially synchronous cell growth and division. The question as to whether inhibition of cell division is a direct consequence of inhibition of DNA, RNA, or protein synthesis is difficult to answer from the data presented by Egyud and Szent-Gyorgyi [7]. In their experiments, measurement of the rates of incorporation of precursors into these three macromolecular fractions was made two bacterial generation times after glyoxal was added to the cell suspension. Under these conditions, it is not possible to decide which process is affected rapidly enough to cause cell division to stop. The data obtained with mammalian cells make the situation somewhat clearer. From the results shown in Fig. 2, inhibition of RNA synthesis can be ruled out as a cause of the stopping of cell division; cell division had stopped completely well before incorporation of label into RNA was affected. Interference with DNA synthesis can also easily be ruled out. In the mammalian cell, DNA synthesis takes place only during the S portion of the cell cycle. Thus, inhibition of DNA synthesis in mammalian cells cannot be responsible for the fact that cell division stops lo-15 min after addition of a glyoxal solution. If interference with DNA synthesis were the basis of inhibition of cell division, there would be no effect of glyoxal on the growth rate until at least 2.5 h after addition of the inhibitor. This would be the time required for the cells already in G2 at the time the glyoxal was added to proceed through M and cytokinesis. A similar argument cannot be made in the case of bacteria, since DNL4 synthesis takes place throughout the cell cycle [ 111. It is clear that, of the three processes studied in the mammalian cell, only protein synthesis is affected early enough to be considered as a possible cause of inhibition of cell division. In addition to their role as a model system for studying the in vivo control of cell division, the glyoxals may prove to be useful tools in studying the Experimental
Cell Research 50
Ch. T. Gregg
72
mechanism of protein synthesis-a process which these compounds reversibly inhibit at some point which is apparently common to 95 per cent of protein synthesis in the growing cell. SUMMARY
Methyl- and propylglyoxal completely inhibited cell division in cultured mammalian cells. This inhibition was reversed by approximately equimolar cysteine, in confirmation of the reports of Szent-Gyorgyi and his colleagues. Cell division stopped within lo-15 min after addition of the glyoxal compound. This fact, taken together with analysis of the cell cycle, with the kinetics of growth after release of glyoxal inhibition, and with the measurement of mitotic index before, during, and aEter release of glyoxal inhibition, indicated that glyoxal treatment did not lead to partially synchronous growth and division in these cells. Tracer experiments on the effect of glyoxals on incorporation of labeled precursors into DNA, RNA, or protein showed that of these processes only protein turnover was affected rapidly enough to be considered a possible cause for the inhibition of cell division. The author wishes to acknowledge the valuable assistance of Patricia LaBauve, Evelyn Campbell, Phyllis Sanders, John Hanners, and especially Susan Carpenter, in various phases of this work. REFERENCES 1. 2. 3. 4. 5. 6.
BRAY, G. A., Anal. Biochem. 1, 279 (1960). BYFIELD, J. E. and SCHERBAUM, 0. H., Anal. Biochem. 17, 434 (1966). CHANOCK, R., HAYFLICK, L. and BARILE, M., Proc. US NatZ Acad. Sci. 48, 41 (1962). DEFENDI, V. and MANSON, L. A., Nature 198, 359 (1963). EGYUD, L. G., Proc. US Nafl Acad. Sci. 54, 200 (1965). EGYUD, L. G. and SZENT-GYORGYI, A., ibid. 55, 388 (1966).
7. __ 8. 9. 10. 11. 12. 13. 14. 15.
16. 17.
ibid. 56, 203 (1966).
HAM, R. G., &pfI’CeZl kes. 29, 515 (1963). HOWARD, A. and PELC, S. R.. Heredifu, Sum~l. 6, 261 (1953). LABA&, P. M. and ~REGG,‘C. T., Unpubi&hed.results. LARK, K. G., in I. L. CAMERON and G. M. PADILLA (eds), Cell Synchrony, p. 54. Academic Press, 1966. PETERSEN, D. F. and ANDERSON, E. C., Nature 203, 642 (1964). PUCK, T. T., SANDERS, P. and PETERSEN, D., Biophys. J. 4, 441 (1964). SCHAIBERGER, G. E., SALLMAN, B. and GIEGAL, J. L., J. Gerontol. 20, 23 (1965). SZENT-GYORGYI, A., Science 149, 34 (1965). __ personal communication. GENT-GYORGYI, A., EGYUD, L. G. and MCLAUGHLIN, J., Science 155, 539 (1967).
Experimenfal
Cell Research 50
65
Cell Research 50, 65-72 (1968)
INHIBITION
OF MAMMALIAN BY GLYOXALS
CELL DIVISION
CH. T. GREGG Los Alamos Scientijic Laboratory, University of California, Los Alamos, N. Mex. 87544, USA
Received June 1, 1967
A. SZENT-GYORGYI and his associates have discussed the role [5, 141 of hypothetical growth-promoting and retarding substances in normal growth regulation, wound healing, and neoplastic processes. bfore recently, Egyud and Szent-Gyorgyi [6, 7] and Szent-Gyorgyi, Egyud, and McLaughlin [17] have reported on the effects of methylglyoxal on the growth of a variety of organisms. At 1 mM concentration, this compound stops cell division in a variety of cells; this inhibition is reversed by equimolar quantities of cysteine or ethylenediamine [6, 71. These authors suggested that this reversible inhibition of cell division might induce cells into synchronous growth and division [71. This report concerns the effects of methyl- and propylglyoxal on mammalian cells growing exponentially in suspension culture.2 A primary objective was to determine if the glyoxals induced these cells into synchronous growth. A second goal was to carry out more detailed time-course studies of the action of the glyoxals than was possible with the rapidly growing Escherichia coli [S, 71. The results of these experiments show that glyoxals cannot be used to induce synchronous growth in the two mammalian cell lines used. These compounds appear to act as general inhibitors of protein synthesis, and the effect of this inhibition is to stop cells from continuing through their life cycle, regardless of the location of the cell within the cycle at the time the inhibitor is added. METHODS
AND
MATERIALS
Mouse lymphoma cells (L-5178Y) were used in most of the experiments described here. The cells were the generous gift of Dr Lionel Manson of The Wistar Institute. These cells grow as an ascites tumor
in suitable
mice (strain
DBA-2J)
or in vitro.
1 This work was performed under the auspices of the U.S. Atomic Energy Commission. 0 2 The glyoxals have the structure R-C-CHO, where R is methyl or propyl for the correspondingly named compounds. 5 - 681806
Experimental
Cell Research 50
Ch. T. Gregg Either in viuo or in vifro, the cells have a doubling time of 11-13 h [4] and a modal chromosome number of 41 [IO]. The cells were grown in suspension culture in a New Brunswick 5-liter fermenter with an automatic pH control unit. The medium was modified Fischer’s L-l [4] except that streptomycin was omitted and the concentration of penicillin was reduced one-half. In some experiments, cells derived from the Chinese hamster ovary were used. These cells were grown in ordinary spinner flasks in Ham’s F-10 medium with cysteine omitted [8]. The properties of this cell line are described elsewhere [IZ]. Both cultures were negative when checked at intervals for contamination by mycoplasma (PPLO) by the method of Chanock, Hayflick, and Barile [3]. The lymphoma cells were checked daily under the phase contrast microscope for contamination by yeast or bacteria. Contaminated cultures were occasionally found and were discarded. Cell viability was determined by the trypan blue exclusion test. Cells were counted with a modified Coulter counter, as previously described [IZ]. The experiments described here were done with methyl- and propylglyoxal synthesized and purified by Dr Lazlo Egyud [5]. These compounds were the generous gift of Dr Egyud and Dr Albert Szent-Gyorgyi. Experiments were carried out by diluting the stock cultures into 200 ml of fresh medium in 500-ml spinner flasks. The cell suspensions were kept at 36.5% and stirred continuously during the experiment; aliquots of approximately 3 ml were removed periodically for cell counting. After establishing that the cells were growing normally, aliquots of aqueous solutions of the glyoxals were added to the suspension culture. Because of the relatively short duration of the experiments and the presence of penicillin in the medium, no attempt was made to sterilize the glyoxal solutions; no contamination was every observed. When cysteine was used to reverse inhibition by the glyoxals, it was weighed out, dissolved in equimolar Tris-HCl buffer at pH 7.0, and used immediately. In the tracer experiments 200 ,uc of aH-thymidine or 3H-uridine or 20 ,MZof laCleucine were added to the appropriate vessels at the same time as the glyoxal compound. Duplicate 3-ml aliquots were removed from each flask at intervals, chilled, and rapidly centrifuged in the cold. The cell pellet was resuspended and washed in 0.25 M sucrose. The acid-insoluble fractions were collected on filter paper disks, essentially as described by Byfield and Scherbaum [2], and the disks were counted in a Tri-Carb scintillation counter in Bray’s scintillation mixture [I]. RESULTS Fig. 1 shows a typical experiment carried out with the mouse lymphoma cell. At point A, propylglyoxal was added to final concentrations of 0.03 mM and 0.1 mM (flasks No. 1 and No. 2, respectively). Propylglyoxal at 0.1 mM stopped cell division almost immediately, while 0.03 mM had no effect. Data for a control flask containing no propylglyoxal and for a flask containing 0.01 mM glyoxal are omitted, since they were the same as those of flask No. 1. The relatively narrow concentration range between complete inhibition and no detectable effect, as seen in the data of Fig. 1, is typical for glyoxals Experimental
Cell Research 50
Glyoxal
inhibition
67
in these mammalian cells. In some experiments concentrations below 0.1 mM gave a transient inhibition of cell growth (1 or 2 h), after which the cells spontaneously resumed their normal growth rate. Concentrations higher than 0.2 m&i’ led to complete and irreversible inhibition of cell division and to rapid death of the cells. Szent-Gyorgyi and his colleagues found that higher
5.7 56 55 5.4 53 5.2 5.1 1
/!
% DEAD CELLS CONTROL 0 CONTROL .-0 2 10 2 I 16
I 20
Fig. 1.
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Fig. 2.
Fig. l.-Inhibition of division of L-5178Y cells by propylglyoxal and reversal of inhibition by cysteine. To cells growing exponentially in L-l medium (about lo5 cells/ml), propylglyoxal was added at point A to a final concentration of 0.03 mM (flask No. 1) or 0.1 mM (flask No. 2). Aliquots for electronic cell counting were removed periodically. At point B, 0.125 mM cysteine (final concentration) was added. At points B and C, the percentage of dead cells in each flask was determined by counting the number of cells (per 100 cells counted) that were unable to exclude the dye trypan blue. Data for a control vessel to which propylglyoxal was not added and for a vessel containing 0.01 mM propylglyoxal were omitted, since the cell counts in these two vessels were the same as for flask No. 1. The incubation temperature was 36.5%. The radius of the circles at each point is 1 standard deviation (0.5 per cent). Abscissa: Time (h); ordinate: log of cell number. Fig. 2.-Relative incorporation rates of labeled precursors into DNA, RNA, and protein by L-5178Y cells in the presence of 0.2 mM methylglyoxal. The conditions in this experiment were identical to those of Fig. 1. Cells were growing exponentially in 4 identical flasks. To 2 flasks, 0.2 mM methylglyoxal (final concentration) was added at zero time. At the same time, to one of these, 200 ,UCof SH-thymidine and 20 ,UCof %-leucine were added; to the other were added 200 PC of $H-uridine. The 2 control flasks contained the same isotopic compositions but no methylglyoxal. Duplicate 3-ml aliquots were removed from the flasks at intervals, and the cells were harvested, washed with 0.25 M sucrose, and extracted with cold 10 per cent trichloroacetic acid. The acid-insoluble material was collected on filter paper disks, washed once with cold 10 per cent trichloroacetic acid and twice with ether, then air-dried. The disks were counted in Bray’s scintillation mixture in a Packard Tri-Carb scintillation counter. Abscissa: Time (h); ordinate: y0 control. Experimental
Cell Research 50
Ch. T. Gregg homologs of methylglyoxal (such as the propyl compound) gave a wider margin between the minimum concentration required to reversibly inhibit bacterial. cell division and that which was cytotoxic [16]. However, in the mammalian cell system, methyl- and propylglyoxal behave identically. At point B in Fig. 1, a slight molar excess of cysteine (final concentration 0.125 mM) was added to all reaction vessels. This level of cysteine had no effect on the control cell suspension, but it reversed inhibition of cell division by propylglyoxal and led to a resumption, after several hours, of a growth rate nearly equal to that of the control (a doubling time of 12.7 h versus 12.0 h in the vessels in which growth was not inhibited). In some experiments, cell division was resumed at a nearly normal rate almost immediately after addition of cysteine. However, in many experiments, the pattern shown in Fig. 1 was observed, in which addition of cysteine caused an immediate but transitory return to the normal growth rate, followed by several more hours of slow growth before the normal rate was again resumed. The data of Egyud and Szent-Gyorgyi [6] show a similar lag after addition of cysteine before normal growth is resumed. However, for the bacteria the period of sub-optimal growth is 6 bacterial doubling times but only a third of a doubling time for the mammalian cells. At points B and C of Fig. 1, the percentage of dead cells in each flask was determined by the trypan blue exclusion method. At point B (the time of cysteine addition), there were no detectable dead cells in either the control flask or in flask No. 1, which contained 0.03 mM propylglyoxal. In flask No. 2, 10 per cent of the cells were dead. In all experiments, the amount of the glyoxals required to suppress cell division completely was mildly toxic, killing from 4-10 per cent of the cells. At point C, the proportion of dead cells in flask No. 2 had dropped to 6 per cent, indicating that the death rate of the cells dropped following addition of cysteine. The difference of 4 per cent in viability of the cells in the two vessels at point C is sufficient to account for the difference in doubling times of the two cultures. Identical results were obtained in experiments on the Chinese hamster cell. The results presented in Fig. 1 show an immediate effect of propylglyoxal. Out of 14 experiments, 5 showed no lag between addition of the glyoxal and appearance of its effect on cell division. The average lag in the remaining 9 experiments was 15 min with a range from 4-42 min. In an experiment in which cell counts were taken at 12-min intervals, the lag time was 10 & 3 min. For the cells used in these experiments, the period of mitosis (M) was approximately 45 min, while the G2 period preceding mitosis [9] was approximately 2.6 h [4, 8, 131. The fact that cells stop dividing within 10-l 5 min Experimental
Cell Research 50
Glyoxal
69
inhibition
after addition of 0.1 mM propylglyoxal indicates either that the point of action of this compound is late in the mitotic period (M) or, alternatively, that propylglyoxal blocks a metabolic process essential for cells in all or nearly all portions of the cell cycle, so that the progress of cells around the life cycle stops almost immediately when the inhibitor is added. These two possibilities are easily distinguished. If the action of the glyoxals were specific for cells in late M, then during the course of a 3-h inhibition all cells which were in the earlier portion of M, and most of the cells in G2 at the time the inhibitor was added, would accumulate at the point in M at which the glyoxals act. When inhibition is relieved by addition of cysteine, approximately 19 per cent of the cell population would complete M and divide within a few minutes, giving rise to an abrupt jump in the cell count (i.e., the apparent growth rate would be much higher than that in the control flasks). It is clear from Fig. 1 that such a jump does not occur. On addition of a slight molar excess of cysteine, the inhibited cells, at best, simply resume their normal growth rate. An alternative way to measure production of synchronous division is to determine the mitotic index. In the case discussed above, this index would be expected to rise from about 5 per cent, as observed in the control flask, to about 19 per cent following release of glyoxal inhibition. When mitotic indices were measured in an experiment like that of Fig. 1 (except that inhibition was reversed after 3 h rather than after 4 h), no significant change in mitotic index was observed either following reversal of propylglyoxal inhibition with cysteine or following the initial addition of the glyoxal compound. Thus, those cells which were in mitosis lo-15 min after addition of glyoxal remained in mitosis, but no new cells entered mitosis until the inhibition was released by cysteine. These results convincingly demonstrate that the glyoxals do not inhibit growth of these mammalian cells by acting specifically at some point late in the mitotic period of the cell cycle. However, the data are consistent with the idea that the glyoxal derivatives inhibit a metabolic process essential for cells in all or nearly all regions of the cell cycle. Egyud and Szent-Gyorgyi investigated the incorporation of label into DNA, RNA, and protein in the presence and absence of methylglyoxal [7]. E. coli were exposed to labeled precursors (with or without methylglyoxal) for 2 generation times (60 min). At the end of this time thymine incorporation into DNA was reduced to 50 per cent by methylglyoxal, uracil incorporation into RNA was 75 per cent, and leucine incorporation into protein was only 6 per cent of that found in the absence of methylglyoxal. These authors conExperimenial
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Ch. T. Gregg
eluded that methylglyoxal inhibited cell division in E. coli primarily by its action on protein synthesis. A similar study was undertaken with mammalian cells. In this system it is possible to look at changes in the rates of incorporation of label into the various macromolecules during small fractions of a generation time-something very difficult to do with E. coli, which grows some 24 times faster. The results of such an experiment are shown in Fig. 2. Here 0.2 mM methylglyoxal was added to flasks at zero time, simultaneously with the appropriately labeled compounds, and the rates of incorporation into the acidinsoluble fraction were compared with those of controls to which methylglyoxal was not added. No cysteine was added to reverse the inhibition. After 1 hr in the presence of 0.2 mM methylglyoxal, the rate of incorporation of labeled thymidine had dropped to 40 per cent and the rate of incorporation of leucine to only 5 per cent of the control rate, while the rate of incorporation of uridine into RNA was unaffected. Subsequently the rate of incorporation of label into RNA dropped rapidly. Five hr after addition of methylglyoxal the rate of incorporation of label into DNA was reduced to 25 per cent compared to that of the control and that of RNA to 15 per cent, while the rate of incorporation of leucine into protein remained at 5 per cent of the control rate. DISCUSSION
The only published data available on the effect of glyoxals on cultured mammalian cells are those of Dr Ruth Johnsson, working in Szent-Gyorgyi’s laboratory, who found that methylglyoxal killed KB cells in 24 h at concentrations of 0.1-1.0 mM [15]. All the information on the kinetics of the reaction of cells with the glyoxals refers to work done on E. coli [6, 71. The results presented here show that in two lines of mammalian cells growing in suspension culture methyl- or propylglyoxal at low concentrations quickly and completely stops cell division and that this inhibition is reversed or prevented by approximately equimolar cysteine. This is in complete agreement with the work of Egyud and Szent-Gyorgyi [6, 71 on E. coli. Although the mammalian cells might appear to be much more sensitive to inhibition by glyoxals, this is not the case. Taking a recent value for the protein content of an E. coli cell [14], one finds that, under the conditions used by Egyud and Szent-Gyorgyi [6, 71, reversible inhibition of cell division requires approximately 22 ,umoles of glyoxal per mg of cellular protein. A similar calculation for a typical experiment with mammalian cells yields a Experimental
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Glyokal
71
inhibition
value of 12 ,umoles of glyoxal per mg of cellular protein for reversible inhibition of cell division. The fact that the life cycle of mammalian cells used in these experiments is some 24 times longer than that of E. coli makes it feasible to study the effect of the glyoxals on cell division and on metabolic processes at intervals which are small compared to the doubling time of the cells, and to show that cell division stops lo-15 min after addition of a glyoxal. This observation, when combined with analysis of the cell cycle, with the cell counting rate following release of glyoxal inhibition, and with the measurement of mitotic index before, during, and after release of glyoxal inhibition, indicates that glyoxal inhibition in these mammalian cells does not lead to partially synchronous cell growth and division. The question as to whether inhibition of cell division is a direct consequence of inhibition of DNA, RNA, or protein synthesis is difficult to answer from the data presented by Egyud and Szent-Gyorgyi [7]. In their experiments, measurement of the rates of incorporation of precursors into these three macromolecular fractions was made two bacterial generation times after glyoxal was added to the cell suspension. Under these conditions, it is not possible to decide which process is affected rapidly enough to cause cell division to stop. The data obtained with mammalian cells make the situation somewhat clearer. From the results shown in Fig. 2, inhibition of RNA synthesis can be ruled out as a cause of the stopping of cell division; cell division had stopped completely well before incorporation of label into RNA was affected. Interference with DNA synthesis can also easily be ruled out. In the mammalian cell, DNA synthesis takes place only during the S portion of the cell cycle. Thus, inhibition of DNA synthesis in mammalian cells cannot be responsible for the fact that cell division stops lo-15 min after addition of a glyoxal solution. If interference with DNA synthesis were the basis of inhibition of cell division, there would be no effect of glyoxal on the growth rate until at least 2.5 h after addition of the inhibitor. This would be the time required for the cells already in G2 at the time the glyoxal was added to proceed through M and cytokinesis. A similar argument cannot be made in the case of bacteria, since DNL4 synthesis takes place throughout the cell cycle [ 111. It is clear that, of the three processes studied in the mammalian cell, only protein synthesis is affected early enough to be considered as a possible cause of inhibition of cell division. In addition to their role as a model system for studying the in vivo control of cell division, the glyoxals may prove to be useful tools in studying the Experimental
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Ch. T. Gregg
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mechanism of protein synthesis-a process which these compounds reversibly inhibit at some point which is apparently common to 95 per cent of protein synthesis in the growing cell. SUMMARY
Methyl- and propylglyoxal completely inhibited cell division in cultured mammalian cells. This inhibition was reversed by approximately equimolar cysteine, in confirmation of the reports of Szent-Gyorgyi and his colleagues. Cell division stopped within lo-15 min after addition of the glyoxal compound. This fact, taken together with analysis of the cell cycle, with the kinetics of growth after release of glyoxal inhibition, and with the measurement of mitotic index before, during, and aEter release of glyoxal inhibition, indicated that glyoxal treatment did not lead to partially synchronous growth and division in these cells. Tracer experiments on the effect of glyoxals on incorporation of labeled precursors into DNA, RNA, or protein showed that of these processes only protein turnover was affected rapidly enough to be considered a possible cause for the inhibition of cell division. The author wishes to acknowledge the valuable assistance of Patricia LaBauve, Evelyn Campbell, Phyllis Sanders, John Hanners, and especially Susan Carpenter, in various phases of this work. REFERENCES 1. 2. 3. 4. 5. 6.
BRAY, G. A., Anal. Biochem. 1, 279 (1960). BYFIELD, J. E. and SCHERBAUM, 0. H., Anal. Biochem. 17, 434 (1966). CHANOCK, R., HAYFLICK, L. and BARILE, M., Proc. US NatZ Acad. Sci. 48, 41 (1962). DEFENDI, V. and MANSON, L. A., Nature 198, 359 (1963). EGYUD, L. G., Proc. US Nafl Acad. Sci. 54, 200 (1965). EGYUD, L. G. and SZENT-GYORGYI, A., ibid. 55, 388 (1966).
7. __ 8. 9. 10. 11. 12. 13. 14. 15.
16. 17.
ibid. 56, 203 (1966).
HAM, R. G., &pfI’CeZl kes. 29, 515 (1963). HOWARD, A. and PELC, S. R.. Heredifu, Sum~l. 6, 261 (1953). LABA&, P. M. and ~REGG,‘C. T., Unpubi&hed.results. LARK, K. G., in I. L. CAMERON and G. M. PADILLA (eds), Cell Synchrony, p. 54. Academic Press, 1966. PETERSEN, D. F. and ANDERSON, E. C., Nature 203, 642 (1964). PUCK, T. T., SANDERS, P. and PETERSEN, D., Biophys. J. 4, 441 (1964). SCHAIBERGER, G. E., SALLMAN, B. and GIEGAL, J. L., J. Gerontol. 20, 23 (1965). SZENT-GYORGYI, A., Science 149, 34 (1965). __ personal communication. GENT-GYORGYI, A., EGYUD, L. G. and MCLAUGHLIN, J., Science 155, 539 (1967).
Experimenfal
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