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Experimental
Cell Research
124 (1979) 151-157
ANALYSIS OF THE INTERPHASE ACCUMULATION INDUCED HYDROXYUREA ON PROLIFERATING PLANT CELLS M. H. NAVARRETE, Instituto
B. PEREZ-VILLAMIL
de Biologia
Celular,
CSIC,
BY
and J. F. L6PEZ-SAEZ Madrid-6,
Spain
SUMMARY Cell distribution in different compartments of the cell cycle (Gl, early, middle and late S, G2 and mitosis) has been studied during continuous treatments with hydroxyurea (HU) in onion root meristems by cytophotometric and autoradiographic methods. A sublethal dosis of HU (0.75 mM) has been chosen to allow a good wave of mitotic synchrony during recovery, with a negligible level of chromosomal aberrations. Proliferating cells begin the S period in the presence of HU and are accumulated in early S, where the maximum value (60%) is reached after 8 h of treatment; at the same time middle and late S are practically empty. In the presence of the drug, residual DNA synthesis allows a slow but continuous progress of cells throughout the S period. Differential sensitivity of S cells to HU can be observed; replication is more affected in early S (85 % inhibition) than in the second half of the period (70% inhibition). On the other hand, Gl cells are not apparently affected by HU, while cells in G2 show a delay in their entrance into mitosis.
Hydroxyurea (HU) is an antimitotic and cytotoxic drug which has found a certain application as an antitumor substance and also as synchronising agent on the cell cycle. It is a specific inhibitor of DNA synthesis, as shown in studies on many organisms, from viruses to animal and plant cells. This effect appears to be due to inhibition of the enzyme ribonucleoside diphosphate reductase [23], resulting in depletion of the intracellular pool of DNA precursors [ 11, 16, 17, 201. The biochemical and cytological effects of this drug are highly dependent on the concentration used, the duration of exposure and the sensitivity of the cell system [4, 121. Thus, high concentrations, about 10 mM, completely inhibit semiconservative DNA replication (for review see [18]) and, acting on proliferative cells for about a cycle time, present a strongly cytotoxic action. On the other hand, at lower
concentrations, about 1 mM, hydroxyurea offers certain advantages for synchronizing proliferating cells, for its action is readily reversible by removal of the drug and cytotoxic effects can be avoided by choosing an appropriate incubation time. Partial synchronization has been obtained in animal cells in culture [ 1, 14, 15, 22, 241 and in plants a similar effect has been described in Vicia fuba [8] and Allium sativum [2] root tips and in vitro cultures of Huplopuppus grucilis cells [8]. HU causes proliferative cells to accumulate in interphase and after drug removal a mitotic wave can be detected. However, the location of cells throughout interphase at the time of maximum accumulation has been a bone of contention, and a small number of authors claimed a blocking effect on cells at the Gl-S transition [2, 3, 221 while others demonstrated Exp
Cell Res 124 (I 979)
152
Navarrete,
PCrez-Villamil
and Lbpez-Sbez
that Gl cells enter S phase where they accumulate [4, 12, 211. This paper supplies further evidence on a close approximation to the exact location within the interphase of cells accumulated by HU treatments in onion root meristems. By superimposing autoradiographic and cytophotometric data we analysed the interphase in the Gl, early S, middle S, late S and G2 phases [lo]. The present results demonstrate that at an optimum concentration and exposure for synchronization, HU incubation gives rise to cell accumulation in early S and the partial inhibition of DNA synthesis allows a slow but continuous progress of cells throughout the S period. The sensitivity of proliferating cells to HU is not the same throughout the whole S period. The strongest inhibition of the DNA synthesis rate occurs at the initiation of the replication period. MATERIALS
AND
METHODS
The material used was the root meristem of Allium cepa L. The onion bulbs were grown in the dark at a constant temperature, which is specific for each exneriment, in cvlindrical class recentacles. in tan water which was renewed eveiy 24 h and aerated continuouslv bv bubbling air at 10-20 ml min-r. The bulbs were placed so &at only their bases remained submerged in the water.
Treatments
with hydroxyurea
A 0.75 mM hydroxyurea solution (Sigma Chemical Co) in tap water was used. The onion bulbs were grown in conditions otherwise identical to those in the control bulbs.
Synchronous
binucleate
cell population
The roots, still attached to the bulbs, were submerged in a 5 mM caffeine solution for 1 h. This drug inhibits cytokinesis in cells going through telophase during this time and produces a binucleate cell population in the meristem which enters interphase and goes through the whole cell cycle synchronously [S].
Labelling
with r3H] thymidine
Twenty min pulses with 50 &i ml-l solution of [3H]thymidine ([3H]TdR, 21 mmol-r; Biochemical Centre, Exp CellRes
124(1979)
Amersham) were used just before fixation. The labelling took place on roots cut from the bulb. When treatment with HU was being employed the cut roots were submerged in a solution of 0.75 mM HU and 50 &i/ml PHlTdR for the last 20 min of treatment. &.rashes of labelled roots were made on subbed slides. They were processed for radioautography with a Kodak D-19 developer and fixed with a Kodak ultrarapid acid fixer.
Staining
techniques
In order to obtain optimum homogeneity in the proliferating population, the second mm of the root was selected before fixation. For cytological analysis these meristematic segments were fixed in a 3 : 1 ethanol acetic acid mixture. Some meristems were stained with acetic orcein ad modum Tjio 8r Levan [19]. Other meristems were Feulgen-stained after 1 h of hydrolysis with 5 N HCl at 20°C.
Microdensitometry Feulgen-stained nuclei were scored for ontical density with-an MS5 scanning microdensitometer (Vickers Ltd). The readings were taken with a x 100 objective, under masks leaving a visual field of 10-30 pm in diameter, under light of 550 nm wavelength, with a slit width of 30 nm (i.e. wavelength from 520-580 nm) and by using a reading spot 0.4 pm in diameter.
RESULTS
AND
DISCUSSION
In the present experiments, the highest mitotic synchronization was obtained during recovery from treatments with 0.75 mM HU for 14 h at 25”C, 14 h being the average cycle time for onion meristem cells at this temperature in untreated roots. After HU incubation, under these conditions, a mitotic wave (fig. 1) is obtained; the meristem cell population shows a low frequency of chromosomal aberrations or abnormal mitotic pictures, less than 1% of ana-telophases evidencing chromosomal bridges or fragments. A relatively low number of induced aberrations is seen in comparison with other DNA synthesis inhibitors used as synchronizing agents [7]. Fig. 2 (left) shows the fall of the mitotic index (MI) in a continuous 14 h treatment with 0.75 mM HU and (right) the mitotic wave during recovery.
Interphase
sensitivity
to hydroxyurea
153
Fig. 1. Mitotic synchronization induced by a 14 h treatment with 0.75 mM HU (hydroxyurea) at 25°C.
The roots were fixed after 10 h recovery and orcein stained.
The MI starts to fall from the beginning of the treatment, reaching a very low value (ca 1) at the 8th hour; afterwards it progressively increases even in the presence of the drug. We also found that by continuous exposure to HU the MI reached a practically normal value at the 24th hour of the treatment (data not shown in fig. 2). In the recovery the mitotic wave can be seen to reach a maximum MI at the 10th hour, with about 50% of meristem cells in mitosis.
ered it interesting to find out about the cell localization induced by HU in interphase at different times; this can help us locate the HU blockade action. In this study a methodology combining autoradiography and cytophotometry was applied [lo]. Fig. 3 shows the relative frequencies of mitosis and both labelled and unlabelled interphases after a r3H]TdR pulse estimated on radioautography of control or HU-treated Feulgen-stained meristems. In the same preparations cytophotometry was used to determine the frequency of Gl and G2 nuclei [9]. The cell frequency in the Gl compartment decreases progressively with time while in the S compartment it increases so that, in the presence of HU, the cells do not
Cell distribution in cycle compartments The mitotic synchronization observed during the recovery is a result of the action of HU throughout interphase, so we consid-
Exp Cell Res 124 (1979)
154
0
Navarrete,
4
8
PCrez-Villamil
12
0
4
and Lbpez-Sbez
8
12
Fig. 2. Abscissa: time (hours); (left) HU treatment; (right) recovery; ordinate: MI; pointed bar, normal MI value. Mitotic synchronization induced by 0.75 mM HU.
accumulate on the Gl/S fringe but begin DNA synthesis, appearing as a result in the S compartment. In order to avoid problems in the [3H]thymidine incorporation due to DNA synthesis inhibition induced by HU treatments, we chose a high level of radioactivity for the pulses (50 pCi/ml). There is a correlation between radioautographic labelling intensity, [3H]TdR pulse and DNA synthesis rate [4]. Lightly labelled cells in our conditions represent cells synthesizing DNA at low rates during much of what would otherwise be called the Gl phase of the cell cycle. Mak’s approach [9] allowed the frequency of cells in Gl, S and G2 to be estimated in a single sample by combining radioautography and cytophotometry on Feulgenstained meristems. After a [3H]thymidine pulse the relative frequencies of mitoses and both labelled and unlabelled interphases were estimated. In the same preparations cytophotometry allowed estimation of the proportion of unlabelled interphase cells with 2C and 4C DNA content, Exp Cell
Res 124 (1979)
G,
S
G*
M
Fig. 3. Abscissa: interphase compartments (Gl, S and G2) and mitosis (M); ordinate: cell frequency as % of total proliferating cell population. C,-Control meristem; h2, h4, h8 and h14, meristems treated with 0.75 mM HU for 2, 4, 8 and 14 h. Radioautograms of Feulgen-stained meristems were scored after a [3H]TdR pulse. Microdensitometry of unlabelled interphases provided the frequencies of cells in Gl and G2. The entrance and accumulation of cells in the S period in the presence of HU is clearly seen.
i.e. in Gl and G2. Fig. 3 shows the cell cycle distribution in control roots and during HU treatment at 2 h, 4 h, 8 h and 14 h. Parallel Feulgen preparations of meristems not processed for autoradiography were used to estimate the DNA content in a sample of randomly chosen interphase nuclei. These nuclei were grouped in three intervals (I, II, III), each with increased DNA content. Interval I is the first part of the interphase, Gl and early S; interval II, the mid-S phase and interval III the final S and G2 phases. Fig. 4 shows the frequency of the interphase nuclei in each interval in control bulbs and meristems after 2 h, 4 h, 8 h, and 14 h HU treatment. Interval I increases with the incubation time and reaches its maximum value at the 8th hour, coinciding with the minimum frequency of cells in mitosis. In interval II the values remain similar after different treatment times.
Interphase G,
sensitivity
6
s
i t
I
i5
to hydroxyurea
II
i !
5-O
155
Gz t M
I t t
m
75
M
100
Fig. 5. Cell cycle compartments in control me&em. The central bar is the integration of both sets of data contained in the two external lines: the upper line from fig. 3, the lower from fig. 4, corresponding to the control meristems; the compartments are ordered in sequence and their relative frequencies are accumulated. Three artificial compartments are obtained within the S period (early, middle and late S). Numbered line, cumulative frequency.
Fig. 4. Abscissa: interphase intervals according to their DNA content (I, II and III) and mitosis (M); ordinate: cell frequency as % of total proliferating cell population. C, h2, h4, h8, and h14 as in fig. 3. The DNA content of Feulgen-stained interphase nuclei was recorded. The maximum and minimum readings for these DNA contents were conventionally called 1 and 2. The frequencies of cells having values of I, 1.0-1.33; II, 1.33-1.66; and III, 1.66-2.0 were estimated.
The variation in interval III is similar to that of the mitotic frequency. As the order of compartments is known in the cell cycle they can be presented in a sequence with cumulative frequencies. By superimposing control data from figs 3 and 4 (ordered in sequence) we obtain a graphic representation of the cell cycle showing six different compartments, three corresponding to the S period (fig. 5, middle bar). The upper line corresponds to the control in fig. 3; the lower line to that in fig. 4. The modification of the six compartments during continuous treatment with HU is presented in fig. 6. Cell progression throughout interphase The major event of HU incubation is doubtedly the continuous accumulation proliferative cells in the S period (fig. The DNA synthesis inhibition induced
unof 6). by
the treatment probably slows down the cell progression throughout this period. However, the cells progress from the Gl to the S period, and accumulate in early S period (Sl) where the meristem population grows from about 10 % in control conditions up to 60 % after 8 h treatment. On the other hand, a continuous depletion is seen in the rest of the S period, the S2+S3 compartment, from the beginning to the 8th hour. This observation suggests that the DNA synthesis rate may be more strongly inhibited in Sl than in the other parts of the S period, making the input into S2+S3 lower than the output. Later, from the 8th hour on, the S2 compartment begins to grow, for a significant fraction of Sl cells reach this level of DNA content in spite of the partial inhibition of replication. The MI which clearly falls with treatment of the cells suggests that this cycle period is not essentially affected. In a similar way, the Gl cells are apparently not biased by hydroxyurea in their cycle progression. On the other hand, the rough constancy (or steadiness) of the G2 frequency throughout the treatment can only be explained by admitting an important slowing down of the cell progression. As a matter of fact, the output of the S period is severely depressed Exp Cell
Res 124 (1979)
156
Navarrete,
PPrez-Villamil
and Lbpez-SBez
35.
3.04h
I”
Bh I”
//J :.
H.U.
H.U
25
l&h I” H.U.
4, 0
25
50
75
100
Fig. 6. Modification of the cell cycle compartments in continuous treatment with 0.75 mM HU at 25°C. The numbered line indicates cumulative frequency. Each bar was obtained as described in fig. 5. After 14 h 70% of the meristematic cells are located within the S period. The progressive accumulation in Sl can be observed, reaching its maximum value after 8 h.
by HU, while the mitotic entrance is not affected. Thus, if the G2 input decreases and the cell frequency is roughly constant, an important depression of the cycle rate must take place in this period. Recently, Schneidermann et al. [ 131 demonstrated, with CHO cells, that HU does not prevent G2 cells from reaching mitosis but does retard their progression rate, so that cells in G2 at the time of HU treatment are clearly delayed in entering mitosis. Differential sensitivity of S cells
When roots are incubated in 5 mM caffeine for 1 h, cells traversing cytokinesis during treatment become binucleate. This population is easily distinguishable from mononucleate cells and develops a cycle with parameters similar to normal mononucleate cells [6]. In order to estimate the DNA synthesis inhibition induced by HU at different points in the S period we made two parallel exExp Cell Res 124 (1979)
8
15
22
Fig. 7. Abscissa: time (hours) after treatment with 5 mM caffeine at lPC, 8, 15 and 22 correspond to the initial, middle and final S period in a control binucleate cell population at this temperature; or&rare: DNA content/nucleus of binucleate cell expressed in C, telophase nucleus being 2C. 0, Control meristem; A, 0, treatment with 0.75 mM HU from A, the gth to 15th hour; 0, the 15th to 22nd hour; !3, residual DNA synthesis. In the first experiment (A) the DNA synthesis inhibition reaches 85 %, while in the second experiment (0) it is only 70 % of the control rate. HU acts more intensively at the beginning of the replication process than during the second half of the S phase.
periments using these synchronous cell populations. In the first the bulbs were treated with HU from the 8th to the 15th hour of the cell cycle, which corresponds to the first half of the S period in a control population. In the 2nd experiment the treatment occurred from the 15th to the 22nd hour, which corresponds to the second half of the S period. Roots were fixed at 8, 15 and 22 h in control bulbs, in the first experiment with HU at 8 and 15 h, and in the 2nd experiment at 15 and 22 h; i.e. at the beginning and the end of the incubations. By cytophotometry of Feulgen-stained binucleate cells from each sample we estimated the DNA content (fig. 7). According to the results the DNA synthesis rate appears to be uniform throughout the S period
Interphase
in controls. However, the inhibition of DNA replication induced by HU in the first half of the S period (85 %) is clearly stronger than that induced in the second half (70%). The residual DNA synthesis is represented by dark triangles. It seems logical to assume that the action of HU on the enzyme ribonucleoside diphosphate reductase must be the same at any point in the cell cycle, but the resulting depletion of intracellular pools of deoxyribonucleotides may depend on a given moment in the S period. We propose that a growing pool may be characteristic of early S and more sensitive to inhibition of DNA precursor formation.
sensitivity
to hydroxyurea
157
REFERENCES
A cycle time incubation results in a clear interphase accumulation in early S period. Thus, HU does not prevent plant cells in Gl from entering the S phase. Semiconservative DNA replication is fairly strongly inhibited but not completely blocked, and early S replication is more affected (85 % inhibition) than that of the second half of the period (70 % inhibition). Lastly, not only does HU prevent the progression of S phase cells into G2-it also retards the rate of G2 cell entry into mitosis.
1. Adams, R L P & Lindsay, J G, J biol them 242 (1967) 1314. 2. Brulfett, A & Deysson, G, Compt rend acad sci Paris ser D, 273 (1971) 146. 3. Camevali, F & Mariotti, D, Chromosoma 63 (1977) 33. 4. Ford, S S & Shackney, S E, Cancer res 37 (1977) 2628. 5. Gimtnez-Martin, G, Gonzalez-Fem&ndez, A & L6pezS&ez, J F, J cell bio126 (1%5) 305. 6. Gimenez-Martin, G, De la Torre, C & LbpezSbz, J F, Mechanisms and control of cell division (ed T L Rost & E M Gifford, jr). Dowden, Hutchinson & Ross, Stroudsburg, Pa (1977). Karon, M&Benedict, W F, Science 178 (1972) 62. ii: Kihlman, B A, Eriksson, T & Odmark, G, Hereditas 55 (1966) 386. 9. Mak, S, Exp cell res 39 (1%5) 286. 10. Navarrete, M H, De la Torre, C & Schwartzman, J B, Cell biol int rep 2 (1978) 607. 11. Plagemann, P GW & Erbe, J, J cell physiol 83 (1974) 321. 12. Ramseier. H P. Burkhalter. M & Gautschi. I J R.I Exp cell res 105 (1977) 445. 13. Schneiderman, M H, Kimler, B F, Leeper, D B & Dewey, W C, Exp cell res 115 (1978) 465. 14. Sinclair. W K. Science 150 (1965) 1729. 15. - Cancer res 27 (1%7) 297: 16. Skoog, L & Nordenskjold, B, Eur j biochem 19 (1971) 81. 17. Skoog, L & Bjursell, G, J biol them 249 (1974) 6434. 18. Timson, J, Mutation res 32 (1975) 115. 19. Tjio, J H & Levan, A, Ann estac exp Aula Dei 2 (1950) 21. 20. Walters, R A, Tobey, R A & Ratliff, R L, Biochim biophys acta 319 (1973) 336. 21. Walters, R A, Tobey, R A & Hildebrand, C E, Biochem biophys res commun 69 (1976) 212. 22. Wheeler, G P, Bowden, B J, Adamson, D J & Vail, M H, Cancer res 32 (1972) 2661. Young, C W & Hodas, S, Science 146 (1964) 1172. 2 Yu, C K & Sinclair, W K, J cell physiol 72 (1968) 39.
The authors are indebted to Miss. M. L. Martinez and Miss 0. Partearroyo for technical assistance. This work was partially supported by the Comisi6n Asesora para Investigacibn Cientifica y Ttcnica, Spain.
Received March 27, 1979 Accepted May 18, 1979
CONCLUSIONS
Exp Cell Res 124 (1979)
Experimental
Cell Research
124 (1979) 151-157
ANALYSIS OF THE INTERPHASE ACCUMULATION INDUCED HYDROXYUREA ON PROLIFERATING PLANT CELLS M. H. NAVARRETE, Instituto
B. PEREZ-VILLAMIL
de Biologia
Celular,
CSIC,
BY
and J. F. L6PEZ-SAEZ Madrid-6,
Spain
SUMMARY Cell distribution in different compartments of the cell cycle (Gl, early, middle and late S, G2 and mitosis) has been studied during continuous treatments with hydroxyurea (HU) in onion root meristems by cytophotometric and autoradiographic methods. A sublethal dosis of HU (0.75 mM) has been chosen to allow a good wave of mitotic synchrony during recovery, with a negligible level of chromosomal aberrations. Proliferating cells begin the S period in the presence of HU and are accumulated in early S, where the maximum value (60%) is reached after 8 h of treatment; at the same time middle and late S are practically empty. In the presence of the drug, residual DNA synthesis allows a slow but continuous progress of cells throughout the S period. Differential sensitivity of S cells to HU can be observed; replication is more affected in early S (85 % inhibition) than in the second half of the period (70% inhibition). On the other hand, Gl cells are not apparently affected by HU, while cells in G2 show a delay in their entrance into mitosis.
Hydroxyurea (HU) is an antimitotic and cytotoxic drug which has found a certain application as an antitumor substance and also as synchronising agent on the cell cycle. It is a specific inhibitor of DNA synthesis, as shown in studies on many organisms, from viruses to animal and plant cells. This effect appears to be due to inhibition of the enzyme ribonucleoside diphosphate reductase [23], resulting in depletion of the intracellular pool of DNA precursors [ 11, 16, 17, 201. The biochemical and cytological effects of this drug are highly dependent on the concentration used, the duration of exposure and the sensitivity of the cell system [4, 121. Thus, high concentrations, about 10 mM, completely inhibit semiconservative DNA replication (for review see [18]) and, acting on proliferative cells for about a cycle time, present a strongly cytotoxic action. On the other hand, at lower
concentrations, about 1 mM, hydroxyurea offers certain advantages for synchronizing proliferating cells, for its action is readily reversible by removal of the drug and cytotoxic effects can be avoided by choosing an appropriate incubation time. Partial synchronization has been obtained in animal cells in culture [ 1, 14, 15, 22, 241 and in plants a similar effect has been described in Vicia fuba [8] and Allium sativum [2] root tips and in vitro cultures of Huplopuppus grucilis cells [8]. HU causes proliferative cells to accumulate in interphase and after drug removal a mitotic wave can be detected. However, the location of cells throughout interphase at the time of maximum accumulation has been a bone of contention, and a small number of authors claimed a blocking effect on cells at the Gl-S transition [2, 3, 221 while others demonstrated Exp
Cell Res 124 (I 979)
152
Navarrete,
PCrez-Villamil
and Lbpez-Sbez
that Gl cells enter S phase where they accumulate [4, 12, 211. This paper supplies further evidence on a close approximation to the exact location within the interphase of cells accumulated by HU treatments in onion root meristems. By superimposing autoradiographic and cytophotometric data we analysed the interphase in the Gl, early S, middle S, late S and G2 phases [lo]. The present results demonstrate that at an optimum concentration and exposure for synchronization, HU incubation gives rise to cell accumulation in early S and the partial inhibition of DNA synthesis allows a slow but continuous progress of cells throughout the S period. The sensitivity of proliferating cells to HU is not the same throughout the whole S period. The strongest inhibition of the DNA synthesis rate occurs at the initiation of the replication period. MATERIALS
AND
METHODS
The material used was the root meristem of Allium cepa L. The onion bulbs were grown in the dark at a constant temperature, which is specific for each exneriment, in cvlindrical class recentacles. in tan water which was renewed eveiy 24 h and aerated continuouslv bv bubbling air at 10-20 ml min-r. The bulbs were placed so &at only their bases remained submerged in the water.
Treatments
with hydroxyurea
A 0.75 mM hydroxyurea solution (Sigma Chemical Co) in tap water was used. The onion bulbs were grown in conditions otherwise identical to those in the control bulbs.
Synchronous
binucleate
cell population
The roots, still attached to the bulbs, were submerged in a 5 mM caffeine solution for 1 h. This drug inhibits cytokinesis in cells going through telophase during this time and produces a binucleate cell population in the meristem which enters interphase and goes through the whole cell cycle synchronously [S].
Labelling
with r3H] thymidine
Twenty min pulses with 50 &i ml-l solution of [3H]thymidine ([3H]TdR, 21 mmol-r; Biochemical Centre, Exp CellRes
124(1979)
Amersham) were used just before fixation. The labelling took place on roots cut from the bulb. When treatment with HU was being employed the cut roots were submerged in a solution of 0.75 mM HU and 50 &i/ml PHlTdR for the last 20 min of treatment. &.rashes of labelled roots were made on subbed slides. They were processed for radioautography with a Kodak D-19 developer and fixed with a Kodak ultrarapid acid fixer.
Staining
techniques
In order to obtain optimum homogeneity in the proliferating population, the second mm of the root was selected before fixation. For cytological analysis these meristematic segments were fixed in a 3 : 1 ethanol acetic acid mixture. Some meristems were stained with acetic orcein ad modum Tjio 8r Levan [19]. Other meristems were Feulgen-stained after 1 h of hydrolysis with 5 N HCl at 20°C.
Microdensitometry Feulgen-stained nuclei were scored for ontical density with-an MS5 scanning microdensitometer (Vickers Ltd). The readings were taken with a x 100 objective, under masks leaving a visual field of 10-30 pm in diameter, under light of 550 nm wavelength, with a slit width of 30 nm (i.e. wavelength from 520-580 nm) and by using a reading spot 0.4 pm in diameter.
RESULTS
AND
DISCUSSION
In the present experiments, the highest mitotic synchronization was obtained during recovery from treatments with 0.75 mM HU for 14 h at 25”C, 14 h being the average cycle time for onion meristem cells at this temperature in untreated roots. After HU incubation, under these conditions, a mitotic wave (fig. 1) is obtained; the meristem cell population shows a low frequency of chromosomal aberrations or abnormal mitotic pictures, less than 1% of ana-telophases evidencing chromosomal bridges or fragments. A relatively low number of induced aberrations is seen in comparison with other DNA synthesis inhibitors used as synchronizing agents [7]. Fig. 2 (left) shows the fall of the mitotic index (MI) in a continuous 14 h treatment with 0.75 mM HU and (right) the mitotic wave during recovery.
Interphase
sensitivity
to hydroxyurea
153
Fig. 1. Mitotic synchronization induced by a 14 h treatment with 0.75 mM HU (hydroxyurea) at 25°C.
The roots were fixed after 10 h recovery and orcein stained.
The MI starts to fall from the beginning of the treatment, reaching a very low value (ca 1) at the 8th hour; afterwards it progressively increases even in the presence of the drug. We also found that by continuous exposure to HU the MI reached a practically normal value at the 24th hour of the treatment (data not shown in fig. 2). In the recovery the mitotic wave can be seen to reach a maximum MI at the 10th hour, with about 50% of meristem cells in mitosis.
ered it interesting to find out about the cell localization induced by HU in interphase at different times; this can help us locate the HU blockade action. In this study a methodology combining autoradiography and cytophotometry was applied [lo]. Fig. 3 shows the relative frequencies of mitosis and both labelled and unlabelled interphases after a r3H]TdR pulse estimated on radioautography of control or HU-treated Feulgen-stained meristems. In the same preparations cytophotometry was used to determine the frequency of Gl and G2 nuclei [9]. The cell frequency in the Gl compartment decreases progressively with time while in the S compartment it increases so that, in the presence of HU, the cells do not
Cell distribution in cycle compartments The mitotic synchronization observed during the recovery is a result of the action of HU throughout interphase, so we consid-
Exp Cell Res 124 (1979)
154
0
Navarrete,
4
8
PCrez-Villamil
12
0
4
and Lbpez-Sbez
8
12
Fig. 2. Abscissa: time (hours); (left) HU treatment; (right) recovery; ordinate: MI; pointed bar, normal MI value. Mitotic synchronization induced by 0.75 mM HU.
accumulate on the Gl/S fringe but begin DNA synthesis, appearing as a result in the S compartment. In order to avoid problems in the [3H]thymidine incorporation due to DNA synthesis inhibition induced by HU treatments, we chose a high level of radioactivity for the pulses (50 pCi/ml). There is a correlation between radioautographic labelling intensity, [3H]TdR pulse and DNA synthesis rate [4]. Lightly labelled cells in our conditions represent cells synthesizing DNA at low rates during much of what would otherwise be called the Gl phase of the cell cycle. Mak’s approach [9] allowed the frequency of cells in Gl, S and G2 to be estimated in a single sample by combining radioautography and cytophotometry on Feulgenstained meristems. After a [3H]thymidine pulse the relative frequencies of mitoses and both labelled and unlabelled interphases were estimated. In the same preparations cytophotometry allowed estimation of the proportion of unlabelled interphase cells with 2C and 4C DNA content, Exp Cell
Res 124 (1979)
G,
S
G*
M
Fig. 3. Abscissa: interphase compartments (Gl, S and G2) and mitosis (M); ordinate: cell frequency as % of total proliferating cell population. C,-Control meristem; h2, h4, h8 and h14, meristems treated with 0.75 mM HU for 2, 4, 8 and 14 h. Radioautograms of Feulgen-stained meristems were scored after a [3H]TdR pulse. Microdensitometry of unlabelled interphases provided the frequencies of cells in Gl and G2. The entrance and accumulation of cells in the S period in the presence of HU is clearly seen.
i.e. in Gl and G2. Fig. 3 shows the cell cycle distribution in control roots and during HU treatment at 2 h, 4 h, 8 h and 14 h. Parallel Feulgen preparations of meristems not processed for autoradiography were used to estimate the DNA content in a sample of randomly chosen interphase nuclei. These nuclei were grouped in three intervals (I, II, III), each with increased DNA content. Interval I is the first part of the interphase, Gl and early S; interval II, the mid-S phase and interval III the final S and G2 phases. Fig. 4 shows the frequency of the interphase nuclei in each interval in control bulbs and meristems after 2 h, 4 h, 8 h, and 14 h HU treatment. Interval I increases with the incubation time and reaches its maximum value at the 8th hour, coinciding with the minimum frequency of cells in mitosis. In interval II the values remain similar after different treatment times.
Interphase G,
sensitivity
6
s
i t
I
i5
to hydroxyurea
II
i !
5-O
155
Gz t M
I t t
m
75
M
100
Fig. 5. Cell cycle compartments in control me&em. The central bar is the integration of both sets of data contained in the two external lines: the upper line from fig. 3, the lower from fig. 4, corresponding to the control meristems; the compartments are ordered in sequence and their relative frequencies are accumulated. Three artificial compartments are obtained within the S period (early, middle and late S). Numbered line, cumulative frequency.
Fig. 4. Abscissa: interphase intervals according to their DNA content (I, II and III) and mitosis (M); ordinate: cell frequency as % of total proliferating cell population. C, h2, h4, h8, and h14 as in fig. 3. The DNA content of Feulgen-stained interphase nuclei was recorded. The maximum and minimum readings for these DNA contents were conventionally called 1 and 2. The frequencies of cells having values of I, 1.0-1.33; II, 1.33-1.66; and III, 1.66-2.0 were estimated.
The variation in interval III is similar to that of the mitotic frequency. As the order of compartments is known in the cell cycle they can be presented in a sequence with cumulative frequencies. By superimposing control data from figs 3 and 4 (ordered in sequence) we obtain a graphic representation of the cell cycle showing six different compartments, three corresponding to the S period (fig. 5, middle bar). The upper line corresponds to the control in fig. 3; the lower line to that in fig. 4. The modification of the six compartments during continuous treatment with HU is presented in fig. 6. Cell progression throughout interphase The major event of HU incubation is doubtedly the continuous accumulation proliferative cells in the S period (fig. The DNA synthesis inhibition induced
unof 6). by
the treatment probably slows down the cell progression throughout this period. However, the cells progress from the Gl to the S period, and accumulate in early S period (Sl) where the meristem population grows from about 10 % in control conditions up to 60 % after 8 h treatment. On the other hand, a continuous depletion is seen in the rest of the S period, the S2+S3 compartment, from the beginning to the 8th hour. This observation suggests that the DNA synthesis rate may be more strongly inhibited in Sl than in the other parts of the S period, making the input into S2+S3 lower than the output. Later, from the 8th hour on, the S2 compartment begins to grow, for a significant fraction of Sl cells reach this level of DNA content in spite of the partial inhibition of replication. The MI which clearly falls with treatment of the cells suggests that this cycle period is not essentially affected. In a similar way, the Gl cells are apparently not biased by hydroxyurea in their cycle progression. On the other hand, the rough constancy (or steadiness) of the G2 frequency throughout the treatment can only be explained by admitting an important slowing down of the cell progression. As a matter of fact, the output of the S period is severely depressed Exp Cell
Res 124 (1979)
156
Navarrete,
PPrez-Villamil
and Lbpez-SBez
35.
3.04h
I”
Bh I”
//J :.
H.U.
H.U
25
l&h I” H.U.
4, 0
25
50
75
100
Fig. 6. Modification of the cell cycle compartments in continuous treatment with 0.75 mM HU at 25°C. The numbered line indicates cumulative frequency. Each bar was obtained as described in fig. 5. After 14 h 70% of the meristematic cells are located within the S period. The progressive accumulation in Sl can be observed, reaching its maximum value after 8 h.
by HU, while the mitotic entrance is not affected. Thus, if the G2 input decreases and the cell frequency is roughly constant, an important depression of the cycle rate must take place in this period. Recently, Schneidermann et al. [ 131 demonstrated, with CHO cells, that HU does not prevent G2 cells from reaching mitosis but does retard their progression rate, so that cells in G2 at the time of HU treatment are clearly delayed in entering mitosis. Differential sensitivity of S cells
When roots are incubated in 5 mM caffeine for 1 h, cells traversing cytokinesis during treatment become binucleate. This population is easily distinguishable from mononucleate cells and develops a cycle with parameters similar to normal mononucleate cells [6]. In order to estimate the DNA synthesis inhibition induced by HU at different points in the S period we made two parallel exExp Cell Res 124 (1979)
8
15
22
Fig. 7. Abscissa: time (hours) after treatment with 5 mM caffeine at lPC, 8, 15 and 22 correspond to the initial, middle and final S period in a control binucleate cell population at this temperature; or&rare: DNA content/nucleus of binucleate cell expressed in C, telophase nucleus being 2C. 0, Control meristem; A, 0, treatment with 0.75 mM HU from A, the gth to 15th hour; 0, the 15th to 22nd hour; !3, residual DNA synthesis. In the first experiment (A) the DNA synthesis inhibition reaches 85 %, while in the second experiment (0) it is only 70 % of the control rate. HU acts more intensively at the beginning of the replication process than during the second half of the S phase.
periments using these synchronous cell populations. In the first the bulbs were treated with HU from the 8th to the 15th hour of the cell cycle, which corresponds to the first half of the S period in a control population. In the 2nd experiment the treatment occurred from the 15th to the 22nd hour, which corresponds to the second half of the S period. Roots were fixed at 8, 15 and 22 h in control bulbs, in the first experiment with HU at 8 and 15 h, and in the 2nd experiment at 15 and 22 h; i.e. at the beginning and the end of the incubations. By cytophotometry of Feulgen-stained binucleate cells from each sample we estimated the DNA content (fig. 7). According to the results the DNA synthesis rate appears to be uniform throughout the S period
Interphase
in controls. However, the inhibition of DNA replication induced by HU in the first half of the S period (85 %) is clearly stronger than that induced in the second half (70%). The residual DNA synthesis is represented by dark triangles. It seems logical to assume that the action of HU on the enzyme ribonucleoside diphosphate reductase must be the same at any point in the cell cycle, but the resulting depletion of intracellular pools of deoxyribonucleotides may depend on a given moment in the S period. We propose that a growing pool may be characteristic of early S and more sensitive to inhibition of DNA precursor formation.
sensitivity
to hydroxyurea
157
REFERENCES
A cycle time incubation results in a clear interphase accumulation in early S period. Thus, HU does not prevent plant cells in Gl from entering the S phase. Semiconservative DNA replication is fairly strongly inhibited but not completely blocked, and early S replication is more affected (85 % inhibition) than that of the second half of the period (70 % inhibition). Lastly, not only does HU prevent the progression of S phase cells into G2-it also retards the rate of G2 cell entry into mitosis.
1. Adams, R L P & Lindsay, J G, J biol them 242 (1967) 1314. 2. Brulfett, A & Deysson, G, Compt rend acad sci Paris ser D, 273 (1971) 146. 3. Camevali, F & Mariotti, D, Chromosoma 63 (1977) 33. 4. Ford, S S & Shackney, S E, Cancer res 37 (1977) 2628. 5. Gimtnez-Martin, G, Gonzalez-Fem&ndez, A & L6pezS&ez, J F, J cell bio126 (1%5) 305. 6. Gimenez-Martin, G, De la Torre, C & LbpezSbz, J F, Mechanisms and control of cell division (ed T L Rost & E M Gifford, jr). Dowden, Hutchinson & Ross, Stroudsburg, Pa (1977). Karon, M&Benedict, W F, Science 178 (1972) 62. ii: Kihlman, B A, Eriksson, T & Odmark, G, Hereditas 55 (1966) 386. 9. Mak, S, Exp cell res 39 (1%5) 286. 10. Navarrete, M H, De la Torre, C & Schwartzman, J B, Cell biol int rep 2 (1978) 607. 11. Plagemann, P GW & Erbe, J, J cell physiol 83 (1974) 321. 12. Ramseier. H P. Burkhalter. M & Gautschi. I J R.I Exp cell res 105 (1977) 445. 13. Schneiderman, M H, Kimler, B F, Leeper, D B & Dewey, W C, Exp cell res 115 (1978) 465. 14. Sinclair. W K. Science 150 (1965) 1729. 15. - Cancer res 27 (1%7) 297: 16. Skoog, L & Nordenskjold, B, Eur j biochem 19 (1971) 81. 17. Skoog, L & Bjursell, G, J biol them 249 (1974) 6434. 18. Timson, J, Mutation res 32 (1975) 115. 19. Tjio, J H & Levan, A, Ann estac exp Aula Dei 2 (1950) 21. 20. Walters, R A, Tobey, R A & Ratliff, R L, Biochim biophys acta 319 (1973) 336. 21. Walters, R A, Tobey, R A & Hildebrand, C E, Biochem biophys res commun 69 (1976) 212. 22. Wheeler, G P, Bowden, B J, Adamson, D J & Vail, M H, Cancer res 32 (1972) 2661. Young, C W & Hodas, S, Science 146 (1964) 1172. 2 Yu, C K & Sinclair, W K, J cell physiol 72 (1968) 39.
The authors are indebted to Miss. M. L. Martinez and Miss 0. Partearroyo for technical assistance. This work was partially supported by the Comisi6n Asesora para Investigacibn Cientifica y Ttcnica, Spain.
Received March 27, 1979 Accepted May 18, 1979
CONCLUSIONS
Exp Cell Res 124 (1979)
