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Thymine starvation by inhibition of uptake and synthesis of thymine-compounds in Tetrahymena
6
1968 by Academic
Experimental
Press Inc.
37
Cell Research 50, 37-46 (1968)
THYMINE UPTAKE
STARVATION
BY INHIBITION
OF
AND SYNTHESIS OF THYMINE-COMPOUNDS IN TETRAHYMENA E. ZEUTHEN Biological
Institute of the Caslsberg Foundation, Copenhagen N, Denmark
Received May 29, 1967
populations the heat-shock induced first synchronous division is closely followed by a synchronous entry of most of the cells into a DNA-synthesis period (S), which itself is a step in the preparation for the second synchronous division. On the other hand, prior to the first synchronous division there is complete asynchrony with respect to the entry of the cells into S [l]. Apparently, in this system synchronous S is a consequence rather than a cause of synchronous cell division. We now want to establish the reverse relationship by directing the synchrony-induction towards the rather than towards cell division. Ultisynthesis of DNA in Tetrahymena, mately, our aim is to apply synchronization procedures for cell division and for DNA-synthesis to the same culture. This limits us to the use of cells synchronization of Tetragrown in complex media, because temperature hymena has been successful only in such media. DNA-synthesis can be synchronized by controlling the supply of thymine is grown in complex compounds for this process [3]. When Tetrahymena media it has access to thymine compounds by synthesis [2] and from the medium. To establish effective thymine starvation both sources must be controlled. No single inhibitor can be expected both to block the synthesis of thymine compounds and to interfere with the use of thymine compounds from the medium. This paper describes how two inhibitors with these separate functions have been sought and found. The inhibitors are methotrexate (RI, a folic acid analogue) and uridine (U). 1~ Tetrahymena
MATERIALS
AND
METHODS
Cultures of Tetrahymena pyriformis, amicronucleate strain GL, were synchronized with seven heat-shocks [6], each of 20 min at 34”C, separated by intervals of 40 min
at 28°C [4, 51. Time zero (EH) is the time when the controlling the last time from control
at 34°C to control
at 28°C. The division
clock switches for index was followed
Experimental
Cell Research 50
E. Zeuthen
38
visually in a number of 1 ml samples removed from the culture flask 5 min after EH for incubation in salt cellars at 28°C. Controls of results with visual estimates were based on cell counts on samples removed at intervals from agitated 150 ml cultures. Agents to be tested for effects on division were added to the I ml samples or to the culture flasks at EH +5 min. The medium [4] was 2 per cent proteosepeptone (Difco), fortified with 0.1 per cent liver extract, fraction L (Wilson Laboratories). Salts were added as described by Kidder and Dewey [2] for their synthetic medium A, omitting the phosphates. The pyrimidines, nucleosides and nucleotides used were from commercial sources. Samples of methotrexate (M, amethopterin; 4-amino-10N-methylpteroylglutamic acid) were donated by Lederle Laboratories, Copenhagen Division. Samples of 5-methyldeoxycytosine (MdC), and of thymineriboside (I-p-n-ribofuranosylthymine, RT) were generously supplied by Dr J. Fox, Sloan Kettering Institute for Cancer Research, Rye, New York. RESULTS
Inhibition
of cell division by methotrexate
plus uridine and release by thymidine
Fig. 1 shows the effect of M + U and of M + U + thymidine(T) on the second synchronous division. The control (I) shows the changing division index in a population of Tetrahymena cells, synchronized for divisions 1, 2 and 3 with seven heat-shocks. The addition of M + U leaves division 1 unaffected, but greatly delays the entry of most cells into division 2. Only a few cells divide at normal time (II). Apparently, M + U produces a nutritional deficiency which relates to the realization, in most cells, of division 2. This deficiency is fully corrected if a 3rd component, thymidine, is added to the medium (III). Apparently, M + U causes shortage of T, and of DNA necessary for division 2. From Fig. 1 is suggested that at the time of division 2 the population is heterogenous. It contains a small group of early and a larger group of later participants. With delay of division 2 as the measure, the small group is insensitive, but the larger group is sensitive to M + U added just after the heat-shocks, We can also suggest from this experiment that in the absence of U, cells treated with M do depend on thymine compounds in the complex medium. Fig. 2 shows controls with M, with U and with T. M alone effects slight delays of divisions 2 and 3. U may by itself slightly delay these divisions. In this experiment such delays are not seen. T alone is non-inhibitory, even in 5 mM concentration. Controls are also shown in which M is combined with T, and U with T. Slight delay by M of division 2 is not corrected by T, and this may be unspecific or due to block by M of methylations other than the one from deoxyuridylate to thymidylate. Comparison of the curves M, T and M + T at the time of division 3 suggests that at this late time the preExperimental
Cell Research 50
Thymine
starvation
in Tetrahymena
39
sence of T may sensitize the cells to the presence of M or vice versa. Other experiments have not, however, shown this effect clearly. The combination U + T has the same effect as U alone. Fig. 3 shows further controls of the experiments described in Fig. 1. Three separate experiments are shown (A, B, C) in which the number of cells per
Fig. l.-Visual estimates of the percent dividing cells in 1 ml samples of a synchronized culture. I, Control; II, addition at EH + 5 min of methotrexate (0.05 m&f) + uridine (5 mM); III, additions as in II, but thymidine (0.5 mM) is also added. Fig. 2.-A series of parallel controls. At EH+5 min are added: 0.05 mM methotrexate (M), 5 mM uridine (U), 5 mM thymidine (T), 0.05 mlM methotrexate, plus 0.5 mlM thymidine (M + T), and 5 mM uridine plus 0.5 mM thymidine (U + T). In the control (Ct) there are no additions. Fig. 3.-Cell multiplication in synchronized control cultures (Ct), and in cultures incubated (from EHt5 min) with 0.05 mil4 methotrexate (M), 5 mM uridine (U), 0.5 mM thymidine (T) and with combinations of these agents.
ml culture is followed for 6 h past EH. When no agents are added to the medium there are approximate doublings of cell numbers at the times of synchronous divisions 1 and 2 (curves Ct). There is no or little measurable change when M, or U, or T are added at EH + 5 min. When M and U are added together (M + U) division 1 is unaffected, but later multiplication Experimental
Cell Research 50
40
E. Zeuthen
is much slowed. This inhibition is almost fully corrected with respect to division 2, and less completely with regard to division 3, when T is given together with M and U. Selection of concentrations
of methotrexate,
uridine,
and thymidine
The concentrations of M, U, and T used in the experiments of Figs 1, 2, and 3 are chosen on the basis of the information contained in Table 1. The results pertain to the fraction of cells which in division 2 demonstrates sensitivity to M + U. M is found to be maximally effective in concentrations above 0.005 mM. Then, with M fixed at 0.05 mM (underlined in the table), and T at 0.5 mM, it is found that U must be above 1 mM if we shall obtain the effect of M + U shown in Fig. 1. For U below 1 mlM the effect of M + U is absent, or weak and not fully reproducible. Above 20 mill, U may sometimes by itself be inhibitory of the same fraction of cells, which in the 2nd TABLE
1. Experiments to find proper concentrations of M + U to inhibit division, and of T to release inhibition e M
i T
U
%I + u
Mt T
U+T
0.5 0.05
5 5
0.5 0.5
+ +
-
-
-
Gz5 0.0005 0.05 0.05 0.05 0.05
5 5 0.05 0.5 1 5
0.5 0.5 0.5 0.5 0.5 0.5
+ -
-
(+) + +
-
-
-
0.5 0.5 0.5 0.0005 0.005 0.05 0.5
+ + + + + + +
-
-
-
-
(11 -
ii--
+
-
-
-
0.05 0.05 0.05 0.05 0.05 0.05 0.05
lo 20 30 5 5 5 5
0.05
5
c, Concentration in mM. i, Inhibition as indicated by delay of division 2. U, Uridine. T, Thymidine. M, Methotrexate. - , No inhibition. + , Inhibition. Additions of M, U, and T at EH + 5 min. Experimenfal
M+U+T
Cell Research 50
+
cell
Thymine
starvation
41
in Tetrahymena
division are registered as sensitive to M + U (5 mM). Inhibition by 20 and 30 mM U is corrected by 0.5 mM T, the same way as is the effect of 5 mM U applied together with M. For combination with 0.05 mM methotrexate, U is therefore used in the 5 mM concentration which is underlined in the table. Finally, with M fixed at 0.05 mM and U at 5 mM it is found that T overcomes TABLE
2.
Tests of various uracil-compounds for capacity to delay division when combined with methotrexate (M) Uracil U Uracil Uridine UMP
compound
(U)
M: 005 mM+
Cont. of U MM)
Delay of division 2 (min)
5 5 5
0 120 0
UDP and UTP have not been tested because they hydrolyze solutions.
2
U
too quickly
in neutral
aqueous
the effect of M + U only in concentrations well above 0.05 mM. T in a concentration of 0.5 mM (underlined in Table 1) has therefore been chosen for the experiments of Figs 1, 2 and 3. Thymine (0.5 mM) tested in place of thymidine has no releasing effect. It has incomplete releasing effect in 5 mM concentration. Basis on which
uridine
is selected for combination
with methotrexate
In Table 2 are shown results of experiments in which uracil and two of its derivatives have been tested in 5 mM concentration for their capacity to delay division 2 when added together with 0.05 mM methotrexate at EH + 5 min. As is seen in the table only the nucleoside possesses this capacity. In the next experiment (Table 3) a series of ribonucleosides and deoxyribonucleosides were tested in 5 or 0.5 mM concentration for their capacity to delay division 2 when combined with M in 0.05 mM concentration. Among the agents tested uridine is the most potent, and deoxyuridine comes second. The two methylated compounds, thymidine (T) and 5-methyldeoxycytidine (MdC) have no effect. Is thymidine methotrexate
a specific releaser of the inhibition plus uridine?
of division
2 produced
by
Some nucleosides (Table 4) have been tested in 0.5 mM concentration for capacity to overcome the effect of M + U on division 2. Only thymidine Experimenfal
Cell Research 50
42
E. Zeuthen
(T) and Smethyldeoxycytidine (MdC) have this capacity. The two are chemically closely related: MdC needs only deamination at position 6 to be transformed into T. Thymineriboside (RT) will not replace T as a releaser of the inhibition produced by M + U (due to shortage of this compound we have made only one test). These results support the view expressed above TABLE
3. Tests of various nucleosides for capacity to delay division combined with methotrexate (M, in 0.05 mM concentration) Nucleoside (N) N U dU C dC T MdC A dA G dG U T MdC RT
Cont. of N (mW 5 5 5 5 5 5 5 5 5 5 0.5 0.5 0.5 0.5
2 when
M+N Delay of division 2a (min) 135 125 20 30 0 0 50 30 50 30 20 0 0 0
a Relative to M; M itself delayed 5 min relative to control without additions.
that the combination M + U creates a nutritional deficiency for thymine compounds essential for preparation of division 2 in most cells. Effects of methotrexate, of uridine, and of the two together, on the incorporation of exogenous 3H-thymidine into DNA In the experiments to be reported M (0.05 mM), U (5 mM), or M + U (0.05 mM + 5 mM) were added at EH + 5 min. Tritiated thymidine was supplied in high specific activity and in tracer amounts at EH + 75 min. Samples were taken for autoradiography at 95 and at 115 min. The grains above between 11 and 20 labelled macronuclei were counted in each sample. The autoradiographic procedures are described in ref. [l]. The choice of the time for the addition of the labelled compound was based on the recent Experimental
Cell Research 50
Thymine
starvation
43
in Tetrahymena
4. Tests of various nucleosides for capacity to release inhibition of division 2, produced by methotrexate (AI, 0.05 mM) + uridine (U, 5 mM)
TABLE
M+U+N Delay of division 2 (min)
Nucleoside (N) Cont. of N @Ml
N 0 dU c dC T MdC RT A dA G dG
125+10 125-125-125-0 0 125-125-125 -125-125--
0
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Tests with N in 5 mM concentration gave identical results (no test with RT), or delayed further (dU, G). TABLE
5. Labelling Incorporaiion
of macronuclei intervals,
min past EH
75-115
%b
c/mnC
nd
%
cjmn
n
95-115 (by difference) cjmn
45 54 46
28+5e 4314 25+3
I2 20 I3
60 59 49
11218 1353-7 3053
14 II 15
84$9 92+8 514
75-95
Control Mf M+U’
with 3H-thynCdinea
a Added final concentration: 2 x 10-s mM. Methyl-tritiated thymidine from Radiochemical Centre, Amersham, England; 0.1 mc/0.25 ml, 1000 mc/mM. Upon receipt diluted IO x , stored under sterile conditions at 4°C for 25 days. In the experiment IO ~1 of dilute label was added to 200 ~1 cell suspension. b %: % macronuclei labelled, 200 cells counted. ’ c/mn: Grains counted per average labelled macronucleus. ’ n: Number of macronuclei over which grains were counted. Counts are given after subtraction of cytoplasmic background. Xd2 ___. 1/-n (n-l) ’ M: 0.05 mM methotrexate, added at EH + 5 min. B U: 5 mM uridine, added at EH + 5 min.
e+
Experimental
Cell Research 50
44
E. Zeuthen
finding [l ] that over a period of only 30 min, beginning at EH + 75-80 min, 60 per cent or so of the populations’ macronuclei enter DNA-synthesis. Previous [l] and parallel [8] tests with DNase made in our laboratory have shown that 3H-thymidine gets incorporated mostly into macronuclear DNA. In Table 5 the control shows the labelling pattern between 75 and 115 min past EH of those macronuclei which engage in DNA-synthesis early in this time interval. This labelling is fastest in the 95-115 min interval. Methotrexate (M) increases, and methotrexate + uridine (M + U) causes inhibition of this labelling. However, relative to the control without additions (except tracer) the inhibition by M + U is limited to the time after EH + 95 min, and it is strong. Relative to the effect of M alone it is even stronger, and weak inhibition is apparent also in the interval between 75 and 95 min past EH.
DISCUSSION
This report supplies evidence that the folic acid analogue methotrexate (0.05 mM) much slows or fully blocks the synthesis of thymine compounds in Tetrahymena. In the presence of this inhibitor the cells become fully dependent on exogenous thymine compounds for use in the synthesis of DNA. Because in the presence of methotrexate the cells are capable of several undisturbed divisions (Figs 2 and 3), and of uninhibited DNA-synthesis (Table 5), we can conclude that the proteose-peptone liver extract used contains thymine compounds for a number of divisions. The availability of these compounds for DNA-synthesis seems to be strongly interfered with of by nucleosides, and especially by 5 mA4 uridine (Table 3). Inhibition cell division by the combined addition of methotrexate and of uridine can be released only with 5-methylpyrimidinedeoxyribosides (thymidine and 5-methyldeoxycytidine, see Table 4). This may suggest that the thymine compound which from the food vacuole enters the cytoplasm is mostly thymidine. This compound may be present as such in the growth medium or it may be liberated from chemical complexes by digestive enzymes in the food vacuole. In combination with methotrexate, uridine inhibits cell division more strongly than does any other normal nucleoside tested (Table 3). Deoxyuridine is also highly active, and the four normal purine nucleosides have some, though weaker effect. If in all cases we refer the measured delays of division to effects on the synthesis of DNA in preparation of a cell division it appears that these effects are shown by several purine- and pyrimidine nucleosides. Experimenfaf
Cell Research 50
Thymine
starvation
in Tetrahymena
45
When 3H-thymidine is added to the medium it can be seen that this compound enters DNA at rates which are 4 X (control) or 3 X (methotrexate present) increased after the lapse of the first 20 min, of S (Table 5). In methotrexate-treated cells thymine compounds for the synthesis of DNA must be available either from intracellular stores, or they must be absorbed from the medium. In these cells the further addition of uridine almost blocks the incorporation of 3H-thymidine into DNA. However, this is so only after S has been in progress for 20 min. It is proposed that the transport of thymidine from the medium to the macronucleus is by mechanisms which change fundamentally 20 min or so after DNA-synthesis has been initiated. Further, that only the transport mechanisms then activated are sensitive to the presence in the medium of nucleosides in high concentration. Thymidine kinase has been mentioned as one enzyme possibly involved in this transport [7]. When from the end of the last heat-shock populations with heat-shock induced synchronous division are incubated with methotrexate plus uridine there is inhibition of the second division in most cells, but the first synchronous division is completely unaffected. It can be suggested that thymine compounds and DNA for the first, and in a few cells also for the second division are accumulated during the period with heat-shocks. In those cells which carry these compounds for only one division more accumulation can be demonstrated to take place after EH and before the second synchronous division. Previous [l] and present results strongly suggest that this accumulation includes DNA-synthesis which is initiated at the time of the first synchronous division. SUMMARY In Tetrahymena populations, which are heat-synchronized in a proteosepeptone, liver medium, addition after the heat-shocks of a folic acid analogue, 0.05 mM methotrexate (M), plus 5 mM uridine (U), inhibit the second synchronous division in most cells. This inhibition can be specifically overcome with thymidine. M slightly stimulates DNA-synthesis as measured by autoradiography with 3H-thymidine. M + U inhibits DNA-synthesis strongly, but .only after initial stages of S have been passed. Uridine may interfere with mechanisms which transport thymidine into the cell or into the macronucleus. The skillful and patient technical assistance of Miss Helga Langelo is acknowlwedged. For critical reading of the manuscript, I thank Magister Ole Westergaard and Dr Ronald Pearlman. Experimental
Cell Research 50
E. Zeuthen REFERENCES 1. HJELM, K. K. and ZEUTHEN, E., Compt. Rend. Trav. Lab. Carlsberg 36, 127 (1967). 2. KIDDER, G. W. and DEWEY, V. C., in A. LWOFF (ed.), Biochemistry and Physiology of Protozoa, vol. 1, p. 323. Academic Press, New York, 1951. 3. NEWTON, ALISON A., in E. ZEUTHEN (ed.), Synchrony in Cell Division and Growth, pp. 441, 601 and 605. Interscience, New York, 1964. 4. PLESNER, P., RASMUSSEN, L. and ZEUTHEN, E., in E. ZEUTHEN (ed.), Synchrony in Cell Division and Growth, p. 543. Interscience, New York, 1964. 5. RASMUSSEN, L. and ZEUTHEN, E., Compt. Rend. Trav. Lab. Carlsberg 32, 333 (1962). 6. SCHERBAUM, 0. and ZEUTHEN, E., Ezptl Cell Res. 6, 221 (1954). 7. STONE, G. E. and PRESCOTT, D. M., J. Cell Biol. 21, 275 (1964). 8. WAND, M., ZEUTHEN, E. and EVANS, E. A., Science 157, 436 (1967).
Experimental
Cell Research 50
1968 by Academic
Experimental
Press Inc.
37
Cell Research 50, 37-46 (1968)
THYMINE UPTAKE
STARVATION
BY INHIBITION
OF
AND SYNTHESIS OF THYMINE-COMPOUNDS IN TETRAHYMENA E. ZEUTHEN Biological
Institute of the Caslsberg Foundation, Copenhagen N, Denmark
Received May 29, 1967
populations the heat-shock induced first synchronous division is closely followed by a synchronous entry of most of the cells into a DNA-synthesis period (S), which itself is a step in the preparation for the second synchronous division. On the other hand, prior to the first synchronous division there is complete asynchrony with respect to the entry of the cells into S [l]. Apparently, in this system synchronous S is a consequence rather than a cause of synchronous cell division. We now want to establish the reverse relationship by directing the synchrony-induction towards the rather than towards cell division. Ultisynthesis of DNA in Tetrahymena, mately, our aim is to apply synchronization procedures for cell division and for DNA-synthesis to the same culture. This limits us to the use of cells synchronization of Tetragrown in complex media, because temperature hymena has been successful only in such media. DNA-synthesis can be synchronized by controlling the supply of thymine is grown in complex compounds for this process [3]. When Tetrahymena media it has access to thymine compounds by synthesis [2] and from the medium. To establish effective thymine starvation both sources must be controlled. No single inhibitor can be expected both to block the synthesis of thymine compounds and to interfere with the use of thymine compounds from the medium. This paper describes how two inhibitors with these separate functions have been sought and found. The inhibitors are methotrexate (RI, a folic acid analogue) and uridine (U). 1~ Tetrahymena
MATERIALS
AND
METHODS
Cultures of Tetrahymena pyriformis, amicronucleate strain GL, were synchronized with seven heat-shocks [6], each of 20 min at 34”C, separated by intervals of 40 min
at 28°C [4, 51. Time zero (EH) is the time when the controlling the last time from control
at 34°C to control
at 28°C. The division
clock switches for index was followed
Experimental
Cell Research 50
E. Zeuthen
38
visually in a number of 1 ml samples removed from the culture flask 5 min after EH for incubation in salt cellars at 28°C. Controls of results with visual estimates were based on cell counts on samples removed at intervals from agitated 150 ml cultures. Agents to be tested for effects on division were added to the I ml samples or to the culture flasks at EH +5 min. The medium [4] was 2 per cent proteosepeptone (Difco), fortified with 0.1 per cent liver extract, fraction L (Wilson Laboratories). Salts were added as described by Kidder and Dewey [2] for their synthetic medium A, omitting the phosphates. The pyrimidines, nucleosides and nucleotides used were from commercial sources. Samples of methotrexate (M, amethopterin; 4-amino-10N-methylpteroylglutamic acid) were donated by Lederle Laboratories, Copenhagen Division. Samples of 5-methyldeoxycytosine (MdC), and of thymineriboside (I-p-n-ribofuranosylthymine, RT) were generously supplied by Dr J. Fox, Sloan Kettering Institute for Cancer Research, Rye, New York. RESULTS
Inhibition
of cell division by methotrexate
plus uridine and release by thymidine
Fig. 1 shows the effect of M + U and of M + U + thymidine(T) on the second synchronous division. The control (I) shows the changing division index in a population of Tetrahymena cells, synchronized for divisions 1, 2 and 3 with seven heat-shocks. The addition of M + U leaves division 1 unaffected, but greatly delays the entry of most cells into division 2. Only a few cells divide at normal time (II). Apparently, M + U produces a nutritional deficiency which relates to the realization, in most cells, of division 2. This deficiency is fully corrected if a 3rd component, thymidine, is added to the medium (III). Apparently, M + U causes shortage of T, and of DNA necessary for division 2. From Fig. 1 is suggested that at the time of division 2 the population is heterogenous. It contains a small group of early and a larger group of later participants. With delay of division 2 as the measure, the small group is insensitive, but the larger group is sensitive to M + U added just after the heat-shocks, We can also suggest from this experiment that in the absence of U, cells treated with M do depend on thymine compounds in the complex medium. Fig. 2 shows controls with M, with U and with T. M alone effects slight delays of divisions 2 and 3. U may by itself slightly delay these divisions. In this experiment such delays are not seen. T alone is non-inhibitory, even in 5 mM concentration. Controls are also shown in which M is combined with T, and U with T. Slight delay by M of division 2 is not corrected by T, and this may be unspecific or due to block by M of methylations other than the one from deoxyuridylate to thymidylate. Comparison of the curves M, T and M + T at the time of division 3 suggests that at this late time the preExperimental
Cell Research 50
Thymine
starvation
in Tetrahymena
39
sence of T may sensitize the cells to the presence of M or vice versa. Other experiments have not, however, shown this effect clearly. The combination U + T has the same effect as U alone. Fig. 3 shows further controls of the experiments described in Fig. 1. Three separate experiments are shown (A, B, C) in which the number of cells per
Fig. l.-Visual estimates of the percent dividing cells in 1 ml samples of a synchronized culture. I, Control; II, addition at EH + 5 min of methotrexate (0.05 m&f) + uridine (5 mM); III, additions as in II, but thymidine (0.5 mM) is also added. Fig. 2.-A series of parallel controls. At EH+5 min are added: 0.05 mM methotrexate (M), 5 mM uridine (U), 5 mM thymidine (T), 0.05 mlM methotrexate, plus 0.5 mlM thymidine (M + T), and 5 mM uridine plus 0.5 mM thymidine (U + T). In the control (Ct) there are no additions. Fig. 3.-Cell multiplication in synchronized control cultures (Ct), and in cultures incubated (from EHt5 min) with 0.05 mil4 methotrexate (M), 5 mM uridine (U), 0.5 mM thymidine (T) and with combinations of these agents.
ml culture is followed for 6 h past EH. When no agents are added to the medium there are approximate doublings of cell numbers at the times of synchronous divisions 1 and 2 (curves Ct). There is no or little measurable change when M, or U, or T are added at EH + 5 min. When M and U are added together (M + U) division 1 is unaffected, but later multiplication Experimental
Cell Research 50
40
E. Zeuthen
is much slowed. This inhibition is almost fully corrected with respect to division 2, and less completely with regard to division 3, when T is given together with M and U. Selection of concentrations
of methotrexate,
uridine,
and thymidine
The concentrations of M, U, and T used in the experiments of Figs 1, 2, and 3 are chosen on the basis of the information contained in Table 1. The results pertain to the fraction of cells which in division 2 demonstrates sensitivity to M + U. M is found to be maximally effective in concentrations above 0.005 mM. Then, with M fixed at 0.05 mM (underlined in the table), and T at 0.5 mM, it is found that U must be above 1 mM if we shall obtain the effect of M + U shown in Fig. 1. For U below 1 mlM the effect of M + U is absent, or weak and not fully reproducible. Above 20 mill, U may sometimes by itself be inhibitory of the same fraction of cells, which in the 2nd TABLE
1. Experiments to find proper concentrations of M + U to inhibit division, and of T to release inhibition e M
i T
U
%I + u
Mt T
U+T
0.5 0.05
5 5
0.5 0.5
+ +
-
-
-
Gz5 0.0005 0.05 0.05 0.05 0.05
5 5 0.05 0.5 1 5
0.5 0.5 0.5 0.5 0.5 0.5
+ -
-
(+) + +
-
-
-
0.5 0.5 0.5 0.0005 0.005 0.05 0.5
+ + + + + + +
-
-
-
-
(11 -
ii--
+
-
-
-
0.05 0.05 0.05 0.05 0.05 0.05 0.05
lo 20 30 5 5 5 5
0.05
5
c, Concentration in mM. i, Inhibition as indicated by delay of division 2. U, Uridine. T, Thymidine. M, Methotrexate. - , No inhibition. + , Inhibition. Additions of M, U, and T at EH + 5 min. Experimenfal
M+U+T
Cell Research 50
+
cell
Thymine
starvation
41
in Tetrahymena
division are registered as sensitive to M + U (5 mM). Inhibition by 20 and 30 mM U is corrected by 0.5 mM T, the same way as is the effect of 5 mM U applied together with M. For combination with 0.05 mM methotrexate, U is therefore used in the 5 mM concentration which is underlined in the table. Finally, with M fixed at 0.05 mM and U at 5 mM it is found that T overcomes TABLE
2.
Tests of various uracil-compounds for capacity to delay division when combined with methotrexate (M) Uracil U Uracil Uridine UMP
compound
(U)
M: 005 mM+
Cont. of U MM)
Delay of division 2 (min)
5 5 5
0 120 0
UDP and UTP have not been tested because they hydrolyze solutions.
2
U
too quickly
in neutral
aqueous
the effect of M + U only in concentrations well above 0.05 mM. T in a concentration of 0.5 mM (underlined in Table 1) has therefore been chosen for the experiments of Figs 1, 2 and 3. Thymine (0.5 mM) tested in place of thymidine has no releasing effect. It has incomplete releasing effect in 5 mM concentration. Basis on which
uridine
is selected for combination
with methotrexate
In Table 2 are shown results of experiments in which uracil and two of its derivatives have been tested in 5 mM concentration for their capacity to delay division 2 when added together with 0.05 mM methotrexate at EH + 5 min. As is seen in the table only the nucleoside possesses this capacity. In the next experiment (Table 3) a series of ribonucleosides and deoxyribonucleosides were tested in 5 or 0.5 mM concentration for their capacity to delay division 2 when combined with M in 0.05 mM concentration. Among the agents tested uridine is the most potent, and deoxyuridine comes second. The two methylated compounds, thymidine (T) and 5-methyldeoxycytidine (MdC) have no effect. Is thymidine methotrexate
a specific releaser of the inhibition plus uridine?
of division
2 produced
by
Some nucleosides (Table 4) have been tested in 0.5 mM concentration for capacity to overcome the effect of M + U on division 2. Only thymidine Experimenfal
Cell Research 50
42
E. Zeuthen
(T) and Smethyldeoxycytidine (MdC) have this capacity. The two are chemically closely related: MdC needs only deamination at position 6 to be transformed into T. Thymineriboside (RT) will not replace T as a releaser of the inhibition produced by M + U (due to shortage of this compound we have made only one test). These results support the view expressed above TABLE
3. Tests of various nucleosides for capacity to delay division combined with methotrexate (M, in 0.05 mM concentration) Nucleoside (N) N U dU C dC T MdC A dA G dG U T MdC RT
Cont. of N (mW 5 5 5 5 5 5 5 5 5 5 0.5 0.5 0.5 0.5
2 when
M+N Delay of division 2a (min) 135 125 20 30 0 0 50 30 50 30 20 0 0 0
a Relative to M; M itself delayed 5 min relative to control without additions.
that the combination M + U creates a nutritional deficiency for thymine compounds essential for preparation of division 2 in most cells. Effects of methotrexate, of uridine, and of the two together, on the incorporation of exogenous 3H-thymidine into DNA In the experiments to be reported M (0.05 mM), U (5 mM), or M + U (0.05 mM + 5 mM) were added at EH + 5 min. Tritiated thymidine was supplied in high specific activity and in tracer amounts at EH + 75 min. Samples were taken for autoradiography at 95 and at 115 min. The grains above between 11 and 20 labelled macronuclei were counted in each sample. The autoradiographic procedures are described in ref. [l]. The choice of the time for the addition of the labelled compound was based on the recent Experimental
Cell Research 50
Thymine
starvation
43
in Tetrahymena
4. Tests of various nucleosides for capacity to release inhibition of division 2, produced by methotrexate (AI, 0.05 mM) + uridine (U, 5 mM)
TABLE
M+U+N Delay of division 2 (min)
Nucleoside (N) Cont. of N @Ml
N 0 dU c dC T MdC RT A dA G dG
125+10 125-125-125-0 0 125-125-125 -125-125--
0
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Tests with N in 5 mM concentration gave identical results (no test with RT), or delayed further (dU, G). TABLE
5. Labelling Incorporaiion
of macronuclei intervals,
min past EH
75-115
%b
c/mnC
nd
%
cjmn
n
95-115 (by difference) cjmn
45 54 46
28+5e 4314 25+3
I2 20 I3
60 59 49
11218 1353-7 3053
14 II 15
84$9 92+8 514
75-95
Control Mf M+U’
with 3H-thynCdinea
a Added final concentration: 2 x 10-s mM. Methyl-tritiated thymidine from Radiochemical Centre, Amersham, England; 0.1 mc/0.25 ml, 1000 mc/mM. Upon receipt diluted IO x , stored under sterile conditions at 4°C for 25 days. In the experiment IO ~1 of dilute label was added to 200 ~1 cell suspension. b %: % macronuclei labelled, 200 cells counted. ’ c/mn: Grains counted per average labelled macronucleus. ’ n: Number of macronuclei over which grains were counted. Counts are given after subtraction of cytoplasmic background. Xd2 ___. 1/-n (n-l) ’ M: 0.05 mM methotrexate, added at EH + 5 min. B U: 5 mM uridine, added at EH + 5 min.
e+
Experimental
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44
E. Zeuthen
finding [l ] that over a period of only 30 min, beginning at EH + 75-80 min, 60 per cent or so of the populations’ macronuclei enter DNA-synthesis. Previous [l] and parallel [8] tests with DNase made in our laboratory have shown that 3H-thymidine gets incorporated mostly into macronuclear DNA. In Table 5 the control shows the labelling pattern between 75 and 115 min past EH of those macronuclei which engage in DNA-synthesis early in this time interval. This labelling is fastest in the 95-115 min interval. Methotrexate (M) increases, and methotrexate + uridine (M + U) causes inhibition of this labelling. However, relative to the control without additions (except tracer) the inhibition by M + U is limited to the time after EH + 95 min, and it is strong. Relative to the effect of M alone it is even stronger, and weak inhibition is apparent also in the interval between 75 and 95 min past EH.
DISCUSSION
This report supplies evidence that the folic acid analogue methotrexate (0.05 mM) much slows or fully blocks the synthesis of thymine compounds in Tetrahymena. In the presence of this inhibitor the cells become fully dependent on exogenous thymine compounds for use in the synthesis of DNA. Because in the presence of methotrexate the cells are capable of several undisturbed divisions (Figs 2 and 3), and of uninhibited DNA-synthesis (Table 5), we can conclude that the proteose-peptone liver extract used contains thymine compounds for a number of divisions. The availability of these compounds for DNA-synthesis seems to be strongly interfered with of by nucleosides, and especially by 5 mA4 uridine (Table 3). Inhibition cell division by the combined addition of methotrexate and of uridine can be released only with 5-methylpyrimidinedeoxyribosides (thymidine and 5-methyldeoxycytidine, see Table 4). This may suggest that the thymine compound which from the food vacuole enters the cytoplasm is mostly thymidine. This compound may be present as such in the growth medium or it may be liberated from chemical complexes by digestive enzymes in the food vacuole. In combination with methotrexate, uridine inhibits cell division more strongly than does any other normal nucleoside tested (Table 3). Deoxyuridine is also highly active, and the four normal purine nucleosides have some, though weaker effect. If in all cases we refer the measured delays of division to effects on the synthesis of DNA in preparation of a cell division it appears that these effects are shown by several purine- and pyrimidine nucleosides. Experimenfaf
Cell Research 50
Thymine
starvation
in Tetrahymena
45
When 3H-thymidine is added to the medium it can be seen that this compound enters DNA at rates which are 4 X (control) or 3 X (methotrexate present) increased after the lapse of the first 20 min, of S (Table 5). In methotrexate-treated cells thymine compounds for the synthesis of DNA must be available either from intracellular stores, or they must be absorbed from the medium. In these cells the further addition of uridine almost blocks the incorporation of 3H-thymidine into DNA. However, this is so only after S has been in progress for 20 min. It is proposed that the transport of thymidine from the medium to the macronucleus is by mechanisms which change fundamentally 20 min or so after DNA-synthesis has been initiated. Further, that only the transport mechanisms then activated are sensitive to the presence in the medium of nucleosides in high concentration. Thymidine kinase has been mentioned as one enzyme possibly involved in this transport [7]. When from the end of the last heat-shock populations with heat-shock induced synchronous division are incubated with methotrexate plus uridine there is inhibition of the second division in most cells, but the first synchronous division is completely unaffected. It can be suggested that thymine compounds and DNA for the first, and in a few cells also for the second division are accumulated during the period with heat-shocks. In those cells which carry these compounds for only one division more accumulation can be demonstrated to take place after EH and before the second synchronous division. Previous [l] and present results strongly suggest that this accumulation includes DNA-synthesis which is initiated at the time of the first synchronous division. SUMMARY In Tetrahymena populations, which are heat-synchronized in a proteosepeptone, liver medium, addition after the heat-shocks of a folic acid analogue, 0.05 mM methotrexate (M), plus 5 mM uridine (U), inhibit the second synchronous division in most cells. This inhibition can be specifically overcome with thymidine. M slightly stimulates DNA-synthesis as measured by autoradiography with 3H-thymidine. M + U inhibits DNA-synthesis strongly, but .only after initial stages of S have been passed. Uridine may interfere with mechanisms which transport thymidine into the cell or into the macronucleus. The skillful and patient technical assistance of Miss Helga Langelo is acknowlwedged. For critical reading of the manuscript, I thank Magister Ole Westergaard and Dr Ronald Pearlman. Experimental
Cell Research 50
E. Zeuthen REFERENCES 1. HJELM, K. K. and ZEUTHEN, E., Compt. Rend. Trav. Lab. Carlsberg 36, 127 (1967). 2. KIDDER, G. W. and DEWEY, V. C., in A. LWOFF (ed.), Biochemistry and Physiology of Protozoa, vol. 1, p. 323. Academic Press, New York, 1951. 3. NEWTON, ALISON A., in E. ZEUTHEN (ed.), Synchrony in Cell Division and Growth, pp. 441, 601 and 605. Interscience, New York, 1964. 4. PLESNER, P., RASMUSSEN, L. and ZEUTHEN, E., in E. ZEUTHEN (ed.), Synchrony in Cell Division and Growth, p. 543. Interscience, New York, 1964. 5. RASMUSSEN, L. and ZEUTHEN, E., Compt. Rend. Trav. Lab. Carlsberg 32, 333 (1962). 6. SCHERBAUM, 0. and ZEUTHEN, E., Ezptl Cell Res. 6, 221 (1954). 7. STONE, G. E. and PRESCOTT, D. M., J. Cell Biol. 21, 275 (1964). 8. WAND, M., ZEUTHEN, E. and EVANS, E. A., Science 157, 436 (1967).
Experimental
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