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J. Cell Sci. 76, 97-104 (1985)
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THE EFFECT OF PARTIAL PROTEIN SYNTHESIS
INHIBITION ON CELL PROLIFERATION IN HIGHER
PLANTS
ANTONIO CUADRADO*, MATILDE H. NAVARRETE AND JOSE
L. CANOVAS
Institute de Biologia Celular, Velazquez, 144, 28006-Madrid, Spain
SUMMARY
Meristematic cells from Allium cepa L. roots can attain a steady state of growth in the presence
of anisomycin at concentrations that effectively reduce the rate of protein synthesis. Under these
conditions the lengths of cell cycle periods increase but not in the same proportion as the generation
time (t). Mitosis is hardly affected and S period is slightly lengthened. Gi increases leas in proportion
to t, while G\ is extended much higher in proportion to /. Natural synchronous populations have
been used to study cell cycle parameters during transition from the physiological steady state to the
new one created by the presence of the drug. Gz was the same during transition as during steady-state
growth. G\ was much shorter during transition. Average cell mass at division was reduced, and a
negative correlation was observed between the length of G% and the size of the cell at termination
of DNA synthesis. We propose that in higher plants, Gi length is regulated by cell mass at completion of DNA synthesis (Gi being shorter in big cells than in small cells), though there is no cell
size requirement for mitosis.
INTRODUCTION
Cell proliferation involves cell growth, genome duplication, and cell division.
Mitchison (1971) has proposed that the cell cycle is composed of two independent but
interacting cycles: the DNA-division cycle, which is concerned with replication and
segregation of DNA, and the growth cycle, which is related to the doubling in cell size.
Relatively little is known about the interaction of both cycles at the level of G\. As
first proposed for bacteria, cells must attain a critical size to initiate DNA replication
(Donachie, 1968; Pritchard, 1968; Helmstetter, Cooper, Pierucci & Revelas, 1968).
This hypothesis explains the negative correlation between cell size at birth and the
length of G\ that has been observed in most materials (for a review, see Baserga, 1984).
Most, if not all, of the G\ period seems to be part of the growth cycle (Cooper, 1979,
1982; Liskay, Leonard & Prescott, 1979). In bacterial cells, the so-called B period
(analogous to G\) may or not be observed depending on cell size at birth. There are
also normal and mutant eukaryotic cell types lacking a measurable G\ period
(Prescott, 1976). This period can be reduced appreciably in yeast (Singer & Johnson,
1981), higher plants (Navarrete, Cuadrado & Cinovas, 1983) and animal cells
(Stancel, Prescott & Liskay, 1981; Rao, Satya-Prakash & Wang, 1984) by growth in
•Author for correspondence.
Key words: cell proliferation, plant cells, anisomycin.
98
A. Cuadrado, M. H. Navarrete andjf. L. Cdnovas
the presence of drugs that reduce the rate of DNA synthesis without altering the
growth rate. It is assumed that, under such conditions, both growth and division
cycles are equalized by lengthening S\ then G\ is partially eliminated as it would be
expected if most of it belonged to the growth cycle.
There is little information about possible interactions between the DNA division
cycle and the growth cycle at other points in the cell cycle. In bacterial (Meacock &
Pritchard, 1975; Puyet & Cdnovas, 1984) and higher plant cells (Navarrete et al.
1983), part of the time between completion of DNA replication and cell division
might belong to the growth cycle, because it is clearly reduced by lengthening the
replication time without altering the growth rate. A reduction in Gz under similar
circumstances can be observed in animal cells, though the authors prefer to consider
it as non-significant (Stancel et al. 1981; Rao et al. 1984). A cell size requirement for
division has been proposed for Amoeba (Hartman, 1928; Prescott, 1956) and yeast
(Nurse, 1975; Fantes & Nurse, 1977).
Our previous suggestion of possible interactions between growth and the DNAdivision cycle at the level of Gz (Navarrete et al. 1983) is tested in this work by using
cells in which protein synthesis has been partially inhibited. The suitability of this
approach has been shown by Liskay et al. (1980) in hamster cells, where slowing
growth by partial protein synthesis inhibition induced a G\ period in Gi-less
mutants. Whether there is a requirement for a critical size for division is especially
considered.
MATERIALS
AND
METHODS
The root meristem olAllium cepa L. was used. Most of the techniques used have been described
(Navarrete et al. 1983). In order to obtain optimum homogeneity in the proliferating population,
the second half of the meristem (which corresponds to the second mm from the apex of the root)
was selected for cytological and chemical analysis (Gonzilez-Ferndndez, Lopez-Saez, Moreno &
Gim6nez-Martfn, 1968). The protein synthesis inhibitors used were anisomycin and cycloheximide
(Sigma). Growth temperature was 25 °C. Steady-state populations were obtained by growing onion
bulbs in the drug solutions from the beginning of root development. Shift experiments were carried
out with naturally synchronous populations. Bulbs with roots at least 3 cm in length were submerged
in a 5 mM caffeine solution for 1 h. This drug inhibits cytokinesis in cells passing through telophase
and produces a binucleate cell population, which enters interphase and passes through the whole cell
cycle synchronously (Gim6nez-Martm, De la Torre & L6pez-Sfiez, 1977). Roots were washed with
water and then submerged in the drug solution. The generation time of steady-state populations was
measured from the frequency of labelled mitosis by the method of Quastler & Sherman (1959). The
division time of shift-down cells was measured as the time from the end of caffeine treatment to the
point at which the maximum amount of mitosis was detected in the binucleate cell population
(Lo^pez-Siez, Gim6nez-Martfn & Gonzalez-Fernandez, 1966). The relative rate of protein synthesis
was determined by comparing [3H]leucine incorporation into cold trichloroacetic acid-insoluble
material in treated and control roots. Onion bulbs were cut into two pieces: one was grown in water
(control) and the other in the drugsolution. Samples of 30 roots were taken of f the bulb&and incubated,
for 1 h, in the same medium supplemented with 20^Ci ml" 1 of [3H]leucine (46 Ci mmol" 1 ; RadiochemicaJ Centre, Amersham, Bucks, U.K.). Roots were washed three times with water at 4 C C. The
second millimetre from the apex of the root was selected and homogenized in 5 % trichloroacetic
acid at 4°C. After centrifugation, the pellet was washed at 4 C C, first with 80% ethanol, then with
ethanol-ethilic ether (3:1) and finally with 5 % trichloroacetic acid. The pellet was then suspended
in 1 M-NaOH, incubated at 60CC for 1 h, and centrifuged again. In the supernatant, radioactivity
was counted with a liquid scintillation counter and total protein was determined by the method of
Partial protein synthesis inhibition and cell cycle
99
Lowry, Rosebrough, Farr & Randall (1951). Countsmin x jig ' total protein was calculated in
control and test extracts. The relative rate of protein synthesis is expressed as a fraction of the
control.
RESULTS
Selection of adequate conditions for cell cycle analysis
Anysomioin inhibits protein synthesis by acting on 80 S ribosomes (Battaner &
Vizquez, 1972). Bulbs were grown in the presence of anisomycin at concentrations
up to 1'4/igml"1, to determine whether drug concentrations that significantly
increased meristematic cell generation time (t) were appropriate for cell cycle
analysis. We considered that the maximum drug concentration to be used should
not cause inability to attain a steady state of growth or cell disturbances such as
chromosome aberrations. Concentrations below 0-4/igml"1 have no detectable
effects on t, while at concentrations above 1-0 jig ml"1 root response was
not homogeneous and eventually resulted in cell death (empty cell walls were
observed). Cells growing in the presence of 0-4-1-O^gml"1 anisomycin do
not show chromosome aberrations and they appear to maintain the ability to
grow at a steady state. At least, the rate of protein synthesis, the frequency of
cells in S and the mitotic index remain constant at different times of population
growth, as shown in Table 1 for the highest anisomycin concentration to be
used (1-0/igml" 1 ).
Attempts to use cycloheximide as the protein synthesis inhibitor (Battaner &
Vazquez, 1972) did not succeed because cells did not attain a steady state of growth
in the presence of this drug.
Effect of anisomycin on steady-state and shift-down cell populations
As shown in Table 2, there is a reduction of 14-84% in the rate of protein
synthesis in steady-state populations grown in the presence of anisomycin concentrations ranging from 0-4 to 1-0/igml"1. The length of t increases gradually,
while 5 and mitosis (M) are slightly affected. Both G\ and Gt periods are also
Table 1. Constancy of certain cell parameters at different root lengths
Root length (mm)
Parameters
Control
Anisomycin
(1-0/igmr 1 )
20-30
V
U
MI
1-00
45-1
11-5
0-15
11-5
3-2
40-50
V
U
MI
1-00
45-2
12-0
0-16
10-1
3-5
Parameters are: V, relative rate of protein synthesis; MI, frequency of mitotic cells; LI, frequency
of replicating cells.
100
A. Cuadrado, M. H. Navarrete andjf. L. Cdnovas
Table 2. Cell cycle parameters during steady-state growth in the presence of different
concentrations of anisomydn
Anisomycin
(/igml- 1 )
V
t
G,
S
G2
M
0-0
0-4
0-6
0-8
1-0
1-00
0-86
0-67
0-33
0-16
14-0
16-8
24-2
31-1
55-6
3-1
5-3
11-2
16-7
40-2
6-1
6-8
6-8
7-6
8-0
2-6
2-6
3-7
4-1
4-9
2-2
2-1
2-5
2-7
2-5
Parameters values are expressed in hours except V, which is the relative rate of protein synthesis.
increased though to different extents. Apparently, the increase in t is mainly produced by an increase in Gi. Gz contributes to such lengthening to a less significant
degree.
Naturally synchronous populations were used to analyse the cell cycle during
transition from the physiological steady state to the new one created by the presence
of the drug. A high concentration of anisomycin (0-8 ^g ml"1) was used. Changes in
cell cycle parameters are shown in Table 3. These changes should be compared with
those observed in steady-state populations grown with the same drug concentration
(Table 2). The effect on 5 and Gi is practically the same in both populations. However, the increase in the length of G\ is quite different: it is 6-8 h in shift-down cells
and 13-6 h in steady-state cells. Consequently, the increase in the cycle time was also
lower in shift-down cells.
Average cell mass at different stages of the cell cycle can be calculated using the
general formula:
Mx=My^/\
(1)
in which My is mass at a certain point in the cell cycle, (usually at the beginning;
Mb), Mx is mass at a subsequent event, x is the time between these two events, and
ris the mass doubling time. In the shift-down experiments, Mb is the same as in the
control. Obviously, t and X are the same in steady-state cells. However, in shiftdown cells x equals the t value of steady-state populations grown with the same drug
Table 3. Values of cell cycle parameters (hours) following protein synthesis shift-down
Experimental
conditions
Control
Anisomycin
(0-8/Jgmr 1 )
G\
S
Gi
t
2-9
6-1
2-8
14
9-7
8-0
4-1
24
Gz was calculated from measured G^+M values by considering that M remains constant as shown
by the analysis of steady-state populations.
Partial protein synthesis inhibition and cell cycle
101
Table 4. Relative values of average cell mass at certain points of the cell cycle
following protein synthesis shift-down
Experimental conditions
Mb
M\
Mt
Mi
Control
Anisomycin
(0-8^gmr')
1-00
1-17
1-58
2-00
1-00
1-24
1-47
1-71
Mass jit^ Mb, birth; M\, beginning of S; Mt; end of S; Mi, division. The value 1 was given to
control Mb-
concentrations, because the reduction in the rate of protein synthesis caused by
the drug is immediate (experiments not shown). Therefore, as both Mb and T are
known, this equation can be used, without any kind of assumption, to calculate the
relative values of the average mass at initiation of DNA replication (Mi), at the
end of S (Mt), and at cell division (Md). As shown in Table 4, following a reduction in the rate of protein synthesis, M, appears to increase, while Mt and Md are
reduced.
DISCUSSION
The generation time and the cell cycle phases have been measured in A. cepa cells
growing with a reduced rate of protein synthesis. The lengths of these periods
increase but not in proportion to the increase in t. Mitosis is hardly affected, S and
Gz increase less in proportion to t, and there is a lengthening of G\ that is much
higher in proportion to the increase in t. Small changes in S could be due to drug
toxicity. It has been reported, using the same and also other biological materials,
that cells in the S period are not affected by inhibition of protein synthesis (GarcfaHerdugo, Gonz£lez-Fern£ndez & L<5pez-S6ez, 1976; Garcfa-Herdugo, 1977; Liskay
et al. 1980).
The variation in the patterns of G\ and Gz, in comparison to t may be interpreted
by assuming that the cell cycle is composed of two independent cycles (Mitchison,
1971), the DNA-division cycle and the growth cycle, which interact in such a way that
both G\ (Cooper, 1979) and Gi (Navarrete et al. 1983) are cell-size-controlled by
means of a negative correlation.
Theoretical values of G\ in steady-state populations grown in the presence of
anisomycin can be calculated using equation (1), by assuming that both Mb and M\ are
not affected by the drug. However, these values do not fit at all with experimental
values (Table 5). On the other hand, the shift-down experiment described in Table
4 shows that mass parameters do change with growth in the presence of anisomycin.
Theoretical calculation of G\ in steady-state cells should be made as a function of the
Mi value measured in the transition cycle and of an Mb value equal to half theMd value
in the transition cycle. For cells grown with 0-8/igml"1 anisomycin, a theoretical
G\ value of 16-8 h is then obtained that fits perfectly to the experimental value
102
A. Cuadrado, M. H. Navarrete andj. L. Cdnovas
Table 5. Theoretical analysts of the effect of protein synthesis limitation on Gi, G2 and
average cell mass at the beginning (M,) and at the end (M() of the S period
Predicted
Observed
Assumption 1
Anisomycin
(^gml- 1 )
G,
Gz
G,
0-0
0-4
0-6
0-8
10
3-1
5-3
11-2
16-7
40-2
2-6
2-6
3-7
4-1
4.9
3-1
3-8
5-5
7-0
12-6
2-6
3-5
8-0
11-5
26-1
Assumption 2
M,
Assumption 3
Mt
117
1-24
1-38
1-45
1-65
1-58
1-55
1-42
1-39
1-29
Calculations were parried out using equation (1) on the basis of three different assumptions.
Assumption 1: both, Mi and birth mass (Mb) remain constant. This assumption allows us to calculate
G\ and Gi by using control values of M; and Mb reported in Table 4 as well as experimental values
of t, S and M shown in Table 2 (observed G\ and Gi values are also included for comparison).
Assumption 2: Mb remains constant. M; can_be calculated by assuming M b ^ 1, with experimental
t and G\ valuesJTable 2). Assumption 3: M; remains constant. Predicted Mt values were obtained
by assuming Mi =1-17 (Table 4), with experimental t and S values (Table 2).
(16-7 h). The observed difference in G\ between steady-state cells and shift-down cells
is also explained by considering that Mb is different in both populations. An aspect
that might be significant is that M; does not appear to be a constant parameter that is
only species-dependent, but is growth-dependent as reported for bacterial cells
(Churchward, Estiva & Bremer, 1981).
From previous observations (Navarrete et al. 1983), cell size regulation of Gz may
be explained by any of the following hypotheses: (1) cell division requires the attainment of a certain size, as proposed for yeast (Nurse, 1975; Fantes & Nurse, 1977) and
Amoeba (Hartman, 1928; Prescott, 1956); (2) cell size at termination of DNA replication determines per se the length of Gz by means of a negative correlation. In our
opinion, the results described in this article are not compatible with the first
hypothesis. Theoretical values of Gz in steady-state populations grown in the presence
of anisomycin can be calculated using equation (1), by assuming that both M\ and
MA (and therefore Mb) do not change. Predicted values do not fit at all with experimental values (Table 5). It is also improbable that Mi at least, and therefore Mb,
remains constant, because changes in M\ would be too large (Table 5); the increase
in Mi for cells growing with 0-8 yi% ml"1 anisomycin was about 6 % (Table 4), which
is very different from the predicted 24% increase. On the other hand, an actual
reduction in Mb of about 17% has been observed in shift-down cells (Table 4). In
contrast, the reported results are consistent with the second hypothesis. Experimental
Gz values and calculated Mt values (Table 5) suggest the existence of a negative
correlation between these two parameters. Such correlation is clearly observed when
Gi and Mt values are compared in control cells and in shift-down cells treated with
0-8/igml"1 anisomycin (Tables 3 and 4). Regulation of Gz by Mt also explains that (in
Partial protein synthesis inhibition and cell cycle
103
contrast to G\) Gi in shift-down cells equals Gi in steady-state cells: as the values of
M\ and 5 (and therefore of Mt) corresponding to the new growth conditions appear to
be reached during shift-down, Gz should be the same in both populations.
We propose, therefore, that Gz length is regulated by cell mass at completion of
DNA replication, Gz being shorter in big cells, though there is not a size requirement
for mitosis. The nature of cell size controls of G\ and Gz appears to be different. The
former might be related to the attainment of a critical size needed to initiate DNA
replication. The latter might result from cell mass modulation of the timing of the
processes that take place in GzThis work was supported in part by the Comisi6n Asesora de Investigaci6n Cientffica y T6cnica
(grant no. 31/1982) and by the Acuerdo de Cooperaci6n Cientffica entre el CSIC y la Universidad
de Chile. A.C. is the recipient of a fellowship from the Caja de Ahorros y M.P. de Madrid. J. F.
L6pez-Sfiez is thanked for suggestions and criticism.
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(Received 14 November 1984 -Accepted 7December 1984)