Download chinese hamster cells can be reversibly blocked before mitosis with

J. Cell Sci. 59, 257-268 (19S3)
Printed in Great Britain © Company of Biologists Limited 1983
257
CHINESE HAMSTER CELLS CAN BE REVERSIBLY
BLOCKED BEFORE MITOSIS WITH THE PROTEIN
SYNTHESIS INHIBITOR, EMETINE
J. TIMOTHY WESTWOOD AND EMILE B. WAGENAAR
Department of Biology, The University ofLethbridge, Lethbridge, Alberta, Canada
T1K3M4
SUMMARY
The inhibition of protein synthesis in eukaryotic cells will prevent them from entering mitosis.
Emetine inhibits peptide elongation. When it was added to asynchronous populations of Chinese
hamster ovary (CHO) cells, the mitotic index decreased sharply 30 to 40 min later. It was found that
the inhibitory effect of emetine could be reversed when it was removed and the reversibility was
dependent on both the initial concentration of emetine and the pH of the medium. Cell populations
that were blocked by emetine for up to 2 h showed a four- to fivefold increase in mitotic index
approximately 1 h after the emetine was removed. These results indicate that there is a point or
period in Gz phase at which critical 'mitotic proteins' are being synthesized, and if their synthesis
is interrupted cells will fail to enter mitosis.
INTRODUCTION
Inhibition of protein synthesis during mid to late Gi phase of the eukaryotic cell
cycle will prevent cells from entering mitosis (Kishimoto & Lieberman, 1964; Tobey,
Peterson, Anderson & Puck, 1966; Donnelly & Sisken, 1967; Wagenaar & Mazia,
1978). During the 'essential' period specific proteins involved in the initiation of
mitosis are probably being synthesized. These 'mitotic proteins' have not as yet been
identified.
The point(s) in the cell cycle at which the essential protein synthetic period occurs
can be determined using inhibitors of protein synthesis. Emetine is a potent inhibitor
of protein synthesis in eukaryotic cells (Grollman, 1966, 1968). If used at low enough
initial concentration, removal of the emetine will result in a reversal of the inhibitory
effect (Gupta & Siminovitch, 1976, 1977; Brinckerhoff &Lubin, 1977). The addition
of emetine to an asynchronous population of dividing cells will perturb the ability of
cells to enter mitosis. By measuring the mitotic index as a function of time after the
addition of emetine one can determine approximately how long before mitosis the
essential protein synthetic period occurs. Additional information on the time-span
between the essential synthetic period and the onset of mitosis can be obtained by
monitoring the recovery of mitotic index after the emetine has been removed from
cells that had been exposed to it.
In this study, the inhibitory effects that emetine exhibits on protein and RNA
synthesis and the reversibility of the protein synthesis inhibition effect were examined
18
CEL 59
258
J. T. Westwood and E. B. Wagenaar
in Chinese hamster ovary (CHO) cells. The rates of inhibition and reversibility, as
well as the effects of drug concentration and the pH on both inhibition and reversibility, were also examined. Finally, the time point at which the final mitotic protein(s)
is synthesized in CHO cells was also determined.
MATERIALS
AND
METHODS
The Chinese hamster ovary cells used in these studies were a gift from Dr T. E. Stubblefield.
Cells were routinely cultured in sterile plastic flasks (Corning) with modified McCoy's 5a medium
(Gibco) supplemented with 10% foetal calf serum and 10mM-Hepes buffer. Gentamycin (50/ig/
ml) was also added to the media. Cells were usually subcultured daily, and removed by a brief
treatment with 0-02 % trypsin in Mg2"1"- and Ca2+-free Hanks' balanced saline. Cells were incubated
in a 5 % CO2 atmosphere at 37 CC. Under these conditions the average doubling time of the cells
was approximately 12-13 h.
Measurement of protein and RNA synthesis
The rate of protein or RNA synthesis was determined by monitoring either [3H]leucine or
[ H]uridine incorporation into the trichloroacetic acid-precipitable fraction of the cells.
Approximately 24 h before each experiment, identical monolayer cultures of CHO cells were
grown in the bottoms of glass scintillation vials (Kimble) by seeding each vial with 2 ml of a cell
suspension. Prior to each experiment, the medium was poured from the vials and the vials were
rinsed once with Hanks' balanced saline solution. Then, 2 ml of leucine-free McCoy's Sa medium
supplemented with 10% dialysed foetal calf serum (FCS) was added to each vial. At the specific
sample times 50 jA of balanced saline solution containing 40/iCi/ml [3H]leucine (New England
Nuclear, sp. act. 5-0 Ci/mmol) was added to the respective vials, resulting in a final concentration
of 1 lid/ ml. After 15 min, the medium was poured from each vial and 5 ml of cold 5 % trichloroacetic acid was added for 5 min at 4 C C. For the determination of RNA synthesis, cultures were continuously labelled in medium containing 2-OflCi/ml [3H]uridine (New England Nuclear, sp. act.
25-0 Ci/mmol) and fixed with cold trichloroacetic acid as just described. After three 5-min washes
with cold acid, cultures were rinsed twice with absolute ethanol (5—10 ml each rinse) followed by two
rinses with toluene. Scintiverse (Fisher Scientific) (10 ml) was then added and the vials were
allowed to stand overnight in the dark before being counted on a Beckman model LS200C liquid
scintillation counter. All samples were done in either triplicate or quadruplicate.
3
Inhibition and reversibility ofprotein synthesis
Effect of drug concentration. Stock solutions of emetine-HCl (1 X 10~ 3 M) (Sigma Chemical, St
Louis, Mo.) were made in Hanks' balanced saline solution. Leucine-free medium containing the
appropriate concentration of emetine was added to the respective vials at * = 0 min. At t = 30 min,
fH]leucine was added to the vials for a 15-min pulse label. In the case of [3H]uridine incorporation,
cells were continuously labelled. Reversibility was checked by exposing the cells to emetine for
30 min, removing the medium, rinsing the vials once with 2 ml of Hanks' balanced saline, and then
adding 2 ml of inhibitor and leucine-free medium to each of the vials. [3H] leucine was added 30 min
later for a 15-min pulse label.
Rates of inhibition and reversibility. Emetine (5 X 10~7) was added to the cultures at Omin,
followed by the addition of [3H]leucine (for a 10-min pulse) at 0, 2, 5 and 10 min. For reversibility,
the drug-containing medium was removed, rinsed once with Hanks' balanced saline, and replaced
with inhibitor and leucine-free medium at time zero. [3H]leucine was added for 10-min pulses at 5,
10, 20 and 30 min.
Effect ofpH. Leucine-free 5a medium containing 20mM-Hepes buffer (with dialysed FCS) was
adjusted to 6'0, 6-5, 7-0 and 7-5 using HC1 or NaOH. Approximately 40 min before the start of the
experiment the medium was poured off the cells and the pH-adjusted medium added. Approximately 30 min before the start of the experiment, emetine stock solution was added to the appropriate
vials to a final concentration of 5 X 10" 7 M.
Entry into mitosis can be reversibly blocked
259
Effect of emetine on cells entering mitosis
Asynchronous populations of CHO cells were grown to about 70-90% confluency in plastic T25 flasks. Emetine in conditioned medium (taken from flasks in which cells had been growing) was
added to the respective flasks at Omin at a final concentration of 5 X 10~7 or 1 X 10~*M. Samples
were taken at 10-min intervals after the addition of emetine. In a second experiment, cells were
exposed to emetine (5 X 10~7 M) for either 60 or 120 min after which time the emetine medium was
poured off, the flasks rinsed once and conditioned medium added. Samples were taken at 15-min
intervals after the removal of emetine. Approximately 5 min before each sample time in both
experiments, cells were scraped from the flasks using a rubber policeman and the cell suspension
was poured into a 15 ml conical centrifuge tube. The cells were pelleted in a clinical centrifuge, and
at the sample time were fixed with 3 : 1 , methanol/glacial acetic acid mixture. Air-dried slides were
prepared and stained with 2% aceto-orcein (Gibco). The mitotic indexes were determined by
separately counting prophase, metaphase, anaphase and telophase, and interphase cells and dividing
the total number of cells in the first four groups (mitotic cells) by the total number of cells in all
groups. A minimum of 1000 cells were counted to determine the mitotic index of each sample. Initial
experiments involved blind controls; that is, the experimenter did not know the identity of the
sample being counted.
RESULTS
The effect of emetine concentration on inhibition of [3H]leucine incorporation into
CHO cells and the reversibility of this inhibition is shown in Fig. 1. Increasing the
concentration of emetine results in a decreasing amount of [ H]leucine incorporation
into cells. Approximately 90 % inhibition of [ H]leucine incorporation occurs in cells
treated with 5 X 10~7 M-emetine, while 2 X 10~7 M-emetine results in 75 % inhibition.
The ability of the cells to resume protein synthesis once the emetine is removed is also
affected by the initial concentration of emetine used. Inhibition is virtually 100%
reversible at 2 X 10~ 7 M, 95 % reversible at 5 X 10~ 7 M, but less than 60% reversible
at 1 X 10~6M-emetine (see Fig. 1). These results indicate that the concentration range
at which emetine provides adequate protein synthetic inhibition (>90 %) and retains
reversibility properties (i.e. >90% reversible) is quite narrow and lies between 5 X
10~ 7 M and
1 X 10~ 6 M.
The rate at which emetine inhibits protein synthesis and the rate of recovery from
protein synthesis inhibition once the emetine is removed is illustrated in Fig. 2. At a
concentration of 5 X 10~7 M-emetine, 75% inhibition of [3H]leucine incorporation
occurs by 7 min and 90 % inhibition occurs at 15 min after the drug is added. When
the emetine is removed, incorporation of [3H]leucine reaches approximately 75 % of
control levels by 15 min and greater than 90% by 25 min. Taking into account the
degree of inhibition and recovery from inhibition that occurs at 5 X 10~7 M-emetine
it appears that maximum inhibition is achieved within 15 min after drug addition and
maximum recovery from inhibition is attained 15-25 min after drug removal.
Altering the pH of the medium affected both the inhibitory and reversibility
properties of emetine. Fig. 3 shows that as the pH of the medium is lowered the degree
of protein synthesis inhibition in the presence of 5 X 10~7 M-emetine decreases. At
pH7-5, 5 X 10~7 M-emetine induces over 90% inhibition of protein synthesis, while
at pH6 - 0 there is only 40% inhibition at the same concentration of emetine. In
respect to the reversibility of emetine inhibition, decreasing the pH increases the
260
jf. T. Westwood and E. B. Wagenaar
100
•
90
c
% Inhibh
o
70
100
% Recovery
X :
40
10"7
5x10" 7
Emetine concentration (M)
10~6
Fig. 1. Effect of emetine concentration on protein synthesis. For the % inhibition results
various concentrations of emetine were added to a number of identical cultures (see
Materials and Methods). Twenty minutes after drug addition [3H] leucine (1 flCi/ml final
concentration) was added for a 15-min pulse label. Cultures were processed as described
in Materials and Methods and the amount of incorporation into the trichloroacetic acidprecipitable fraction of the cells was determined. For the % recover)' results cultures were
exposed to the various concentrations of emetine for 30 min after which the emetine was
removed and replaced with normal medium. Thirty minutes after emetine removal
[3H]leucine was added to the cells for a 15-min pulse label. For this experiment and for
the experiments represented in Figs 2, 4 and 5 and Tables 1, 2 and 3 protein synthesis
inhibition experiments were carried out in medium at pH 7-4 and recovery experiments
in medium at pH 6-5. % Inhibition = 100 — (c.p.m. of sample/c.p.m. of controls) X 100;
% recovery = (c.p.m. sample/c.p.m. of controls X 100). Control cultures (0% inO)
hibition, 100% recovery) had incorporated approximately 20000 c.p.m. (O
Cultures in the presence of emetine; ( •
D) cultures 30 min after emetine removal.
amount of reversibility. When emetine (5 X 10~ 7 M) is removed from cells at pH7-5
there is virtually no recovery from the inhibitory effect, while at pH 6"0 approximately
100% recovery occurs. Another interesting finding is the decrease in [3H]leucine
incorporation that occurs in control populations when the pH of the medium is
lowered below pH 7-0. [3H]leucine incorporation at pH 6-0 is approximately 65 % of
the incorporation at pH 7-0 (results not shown). On the basis of these observations the
experiments represented in Figs 1, 2, 4 and 5 and Table 1 were carried out at pH 7-4
when cells were being inhibited by emetine, and at approximately pH6-5 when
reversal of inhibition was desired.
Entry into mitosis can be reversibly blocked
0
10
20
Time after emetine
addition (min)
261
Time after emetine
removal (min)
Fig. 2. Rates of inhibition by emetine and recovery from inhibition. To determine the rate
of inhibition, 5 X 10~7 M-emetine was first added to identical cultures at Omin. At 0, 2, 5
and 10 min [3H]leucine (1 ^Ci/ml final concentration) was added to cultures for a 10-min
pulse label. Cultures were processed as described in Materials and Methods and results
calculated as described in the legend to Fig. 1. The result obtained for a particular 10-min
pulse period was plotted 5 min (midway) after the [3H]leucine was added (e.g. results
plotted at 5 min for samples in which [3Hlleucine was added at 0 min). For the % recovery
results cultures were exposed to 5 x 10 M-emetine for 30 min, after which the emetine
was removed and replaced with normal medium. [3H]leucine was added to the cultures at
various times after emetine removal and the results plotted as described above. Cultures
showed 11 % recovery in the presence of emetine (0 min) because only 89 % inhibition was
obtained by 5 X 10"' M-emetine. Control cultures (0% inhibition, 100% recovery) had
incorporated approximately 10000 c.p.m. (O
O) Cultures in the presence of
emetine; ( •
D) cultures after emetine had been removed.
The effect of emetine concentration and length of emetine exposure on [3H]uridine
incorporation is shown in Table I. At lower concentrations (5 X 10~7 and 2X
3
10~ 6 M) [ H]uridine incorporation was 85 % of control levels at lOmin after addition
and 70 % of controls at 150 min after the addition of emetine. Increasing the emetine
concentration to 10~ 5 M decreased [3H]uridine incorporation only slightly.
When emetine (5 X 10~7 and 2 x 10~ 6 M) is added to CHO cells in log-phase
growth, a slight depression in mitotic index occurs about 30 min after the addition of
the drug (see Fig. 4). An examination of the cells in each of the various stages of
mitosis reveals that the number of metaphases in the population is reduced by half
(when compared to control populations) between 20 and 30 min after the addition of
emetine (see Table 2). By 40 min, the overall mitotic index is reduced to approximately one-fourth of the control level and significant reductions are apparent in all the
stages of mitosis. Beyond 40 min, the mitotic index remains depressed and by 2 h after
the addition of emetine, the mitotic index is approximately 10% of that of control
values.
J . T. Westwood and E. B. Wagenaar
6.0
6.5
7.0
7.5
pH of media
Fig. 3
20
40
60
80
100
Time after the addition of emetine (min)
Fig. 4
Entry into mitosis can be reversibly blocked
263
Table 1. Effect of emetine on [3H]uridine incorporation
°ki Incorporation of [3H]uridine*
Time after
emetine addition
(min)
SX 10"7M
2 x 10" 6 M
1 X 10"5 M
10
30
90
120
84- 1+9-3
79- 8113-0
77- 9 1 2 - 3
69- 0 1 3 - 2
82-618-2
N/Af
80-813-2
69-214-4
79-6 + 4-2
68-613-8
N/Af
61-2 + 5-2
Emetine concentrations:
•Emetine and 2/iCi/ml [3H]uridine were added to the cultures at Omin. The cultures were
continuously labelled with samples taken at the indicated times and processed as described in
Materials and Methods. 100% incorporation is the amount of [3H]uridine incorporated by control
(non-emetine treated) cultures.
f N / A , no samples taken at these time points.
When emetine (5 X 10 7 M ) was added to cultures for either 60 or 120 min the
mitotic index was significantly lower than control values, as one would expect (see
Fig. 5). When the emetine was removed from these cultures, the cells rinsed with
saline and then replaced with emetine-free medium, the mitotic index remained low
until approximately 60 min after emetine removal. In the cultures that had received
a 60-min treatment with emetine, the mitotic index increased approximately
threefold between 45 and 60 min after removal of emetine. Cultures that received
a 120-min treatment with emetine showed a fivefold increase in mitotic index
between 60 and 75 min after emetine removal (see Table 3). In both cases, the sharp
increase in mitotic index was due primarily to large increases in the number of
prophase and metaphase cells (see Table 3). For example, in the cultures that
received a 60-min treatment with emetine, the number of prophase configurations
increased from 1 to 19 between 45 and 60 min after removal of emetine. For both
sets of cultures, the mitotic index increased to control levels only upon removal of
emetine and maintained this level for approximately 1 h, after which the mitotic
Fig. 3. Effect of pH on emetine inhibition and recovery. Hepes-buffered medium was
adjusted to a variety of pH values and added to cultures. [3H]leucine incorporation at
these pH values were determined both in the presence of 5 X 10~7M-emetine and 30 min
after its removal. Cultures were processed as described in Materials and Methods and
results calculated as described in the legend to Fig. 1. (O
O) Cultures in the
presence of emetine; (D
• ) cultures 30min after emetine removal.
Fig. 4. The relationship between inhibition of protein synthesis and mitotic index.
Emetine (1 X 10"6 or 5 X 10~ 7 M) was added to asynchronous cultures and the mitotic
index scored at various times after emetine addition (see Materials and Methods). The
number of cells in each of the various stages of mitosis for all of the samples is shown
in Table 2. (O
O) Control cultures (no emetine added); ( •
• ) cultures in
the presence of 2 X 10~6M-emetine; (A
A) cultures in the presence of 5 X 10~ 7 M
-emetine.
264
J. T. Westwood and E. B. Wagenaar
Table 2. Effect ofprotein synthesis inhibition on mitotic index*
Number of cells
Time of
sampling Mitoticf
(min)
index
Prophase Metaphase Anaphase and Interphase
telophase
Control
10
30
60
90
4-90
5-05
4-82
5-54
2
2
6
6
36
30
35
35
16
22
11
20
1010
1016
1027
1041
5 X 10~7M-emetine
30
45
60
75
90
4-15
2-32
1-61
1-40
1-45
2
5
1
1
1
14
13
7
8
9
28
6
9
6
6
1022
1009
1042
1056
1087
2 x 10~6M-emetine
10
20
30
40
50
60
75
90
105
4-60
4-97
3-80
1-39
1-45
1-35
2-04
1-32
1-14
1
1
3
0
3
1
3
1
0
32
37
16
6
10
8
13
7
8
18
15
22
9
2
5
6
6
4
1059
1013
1037
1064
1022
1023
1058
1049
1042
• T w o different emetine concentrations (5 X 10 7 and 2 X 10 6 M ) were added to asynchronous
cultures at 0 min and samples were taken at the indicated times. Cultures were fixed and scored for
mitotic index as described in Materials and Methods.
t Mitotic index _= no. of prophase+metaphase+anaphase and telophase cells (X 100)
Total no. of cells
30
60
90
120
Time after the removal of emetine (min)
Fig. 5
150
Entry into mitosis can be reversibly blocked
265
Table 3. Changes in mitotic index after the removal of emetine*
Number of cells
A
1 1111C
Control
Emetine-treated
(120 min)
Emetine (continual)
r
Prophase Metaphase Anaphase and Interphase
telophase
sampling
(min)
Mitotic
index
-It
3-58
3-65
3-92
3-59
4-00
6
7
6
9
7
22
22
22
20
25
10
12
12
11
12
1024
1083
980
1073
1061
-It
1-32
0-88
2-54
313
2-88
2-50
2-51
1
1
19
9
6
4
3
5
4
5
17
16
13
15
8
4
4
7
8
9
9
1045
1010
1071
1021
1010
1016
1047
-It
45
60
75
90
120
150
0-59
0-77
0-76
3-57
3-29
2-66
2-31
0
3
5
17
5
2
2
3
3
2
17
22
19
18
3
2
1
4
10
6
4
1009
1009
1034
1025
1087
987
1052
90
150
0-79
0-30
0
0
6
1
1
2
1008
1012
60
90
120
150
Emetine-treated
(60min)
Ul
45
60
75
90
120
150
• Asynchronous cultures were treated with 5 X 10 7 M-emetine for either 60 or 120 min. At 0 min
the emetine medium was removed and replaced with ordinary medium. In the emetine continuous
samples emetine was added at —120 min and was not removed at 0 min. Samples were taken at the
indicated times and the mitotic scored as described in the legend to Table 2.
f — 1 min samples taken just before the emetine was removed.
index decreased slightly. Cells that were in the continual presence of emetine showed
no fluctuation in mitotic index and maintained a mitotic index approximately onesixth of that of controls.
Cytological observations of emetine-arrested cells did not reveal any abnormalities
in the mitotic configurations that were present.
Fig. 5. Changes in mitotic index after the removal of emetine. Emetine (5 x 10 7 M) was
added to cells for either a 60 or 120-min treatment. At Omin the emetine media was
removed and replaced with normal medium and the mitotic index scored at various times
after removal. The number of cells in each of the stages of mitosis is shown in Table 3.
(O
O) Control cultures (no emetine added); (D
• ) cultures that received a
60-min emetine treatment; (A
A) cultures that received a 120-min emetine treatment; ( O
O ) cultures in the continual presence of emetine.
266
J. T. Westwood and E. B. Wagenaar
DISCUSSION
Early studies on the use of emetine as an inhibitor of protein synthesis in mammalian cells had indicated that emetine was a very effective inhibitor but irreversible
even at low concentrations ( 1 0 ~ 7 - 1 0 ~ 6 M ) (Grollman, 1966, 1968). More recent
studies have shown that the inhibitory effect of emetine could be reversed at low
concentrations ( 2 X 1 0 ~ 7 M ) (Gupta & Siminovitch, 1976, 1977; Brinckerhoff &
Lubin, 1977). The results of this study have demonstrated that emetine is a potent
and fast-acting reversible inhibitor of protein synthesis in mammalian cells. However,
the conditions under which emetine can be used as a reversible inhibitor are quite
stringent. First, the concentration range at which emetine exhibits sufficient of both
inhibitory and reversibility properties lies between 5 X 10~ 7 M and 1 X 10~ 6 M for
CHO cells. Second, the pH of the medium has a dramatic effect on both inhibition
and the reversibility capacities of the drug. Inhibition of protein synthesis by emetine
is significantly reduced when the pH of the medium is lowered below 7-0. Also, the
reversibility characteristics of emetine are affected by pH, reversibility decreasing as
the pH is raised above 6'5. At pH7 - 5 or greater, emetine appears to act as an irreversible inhibitor of protein synthesis. The reason why pH has such a profound effect on
both the inhibition and reversibility properties of emetine may be related to the drug's
mechanism of action, more specifically, its ability to bind to ribosomes. It has
previously been demonstrated that emetine's inhibitory effect is presumably a result
of its binding to the 40 S ribosomal subunit in eukaryotic cells (Gupta & Siminovitch,
1977; Boersma, McGill, Mollenkamp & Roufa, 1979). Therefore, as the pH of the
medium is changed the conformation of the ribosome or the microenvironment of the
emetine binding site might also be changed in such a way that emetine binding is
significantly reduced at lower pH values. The fact that relatively small changes in the
pH of the medium have such a profound effect on the reversibility of the inhibitory
action of emetine may explain why an earlier investigator (Grollman, 1966, 1968)
described the action of this drug as being irreversible.
Because emetine exhibits good reversibility of its inhibitory effect, it may prove to
be a useful drug for studying events related to protein synthesis in a number of other
eukaryotic systems.
The effect emetine exhibits on other macromolecular systems, including DNA and
RNA synthesis, has been well documented by previous investigators. DNA synthesis,
for example, is reduced by about 80 % within 15 min of addition of emetine to HeLa
cells (Grollman, 1966, 1968). The effect of emetine on RNA synthesis varies depending on the concentration of emetine used. At high concentrations (approx. 10~3 M) all
types of RNA synthesis (i.e. 4S, 5S and 18 S) are inhibited, whereas at low concentrations (10~7-lCr6M), emetine reduces the synthesis of 18S RNA only (preribosomal RNA), with practically no effect on 4S and 5 S RNA synthesis (Gilead &
Becker, 1971). The results obtained in this study for'low'emetine concentrations (i.e.
20 % reduction in RNA synthesis after 30 min) are in agreement with those obtained
by Gilead & Becker (1971). Because only low concentrations of emetine were used in
this study, we assume that the effects emetine is exhibiting are probably a result of
Entry into mitosis can be reversibly blocked
267
emetine's primary effect on protein synthesis rather than on RNA synthesis, since
others have shown that messenger and transfer RNA synthesis appear not to be
inhibited at low concentrations of emetine (Gilead & Becker, 1971).
When emetine was added to asynchronously growing populations of CHO cells the
mitotic index began to decrease approximately 30-40 min after the drug was added.
This suggests that protein synthesis is necessary for cells to initiate mitosis and that
there is a point, roughly 30 min or so before metaphase, where one or more 'mitotic
proteins' are being synthesized. This finding is not completely in agreement with the
findings of previous investigators, who reported a similar essential protein synthetic
period occurring approximately 10 min before mitosis in CHO cells (Tobey et al.
1966). It should be noted, however, that those authors used the rate of cell division
rather than mitotic index to determine the effects of the protein synthesis inhibitor,
puromycin.
In the experiment in which emetine was added to growing cells for either 60 or
120 min and then removed, the mitotic index increased sharply approximately 60 min
after the drug was removed. If one includes a 20- to 25-min lag time for protein
synthesis to resume after drug removal, the period at which the mitotic proteins are
synthesized occurs roughly 40 min before mitosis. In addition, there was very little
difference in the time it took for the mitotic index to recover from either a 60 or
120-min emetine pulse. The fact that the mitotic index resumed at almost control
levels 60-75 min after a 2-h inhibition of protein synthesis indicates that the period
during which the mitotic proteins are being synthesized is probably quite short, since
a long synthetic period would most likely result in a longer delay in the recovery of
mitotic index (i.e. equal to the amount of time for which protein synthesis was
inhibited).
It is evident from the results of this study that a mitotic protein (or proteins) is
synthesized during a period approximately 30 min prior to mitosis in CHO cells.
Without the synthesis of these proteins cells will not initiate mitosis. The mitotic
protein synthetic period has been implicated in previous studies using mammalian
cells, but not clearly defined (Kishimoto & Lieberman, 1964; Tobey et al. 1966;
Donelly & Sisken, 1967). Wagenaar & Mazia (1978) have reported a mitotic protein
synthetic period in the sea urchin Strongylocentrotus purpuratus. In their study, the
addition of emetine any time between fertilization and 50 min prior to the first
cleavage division will prevent chromosome condensation in the first mitosis.
The function of these mitotic proteins is still not known. Some authors (Wagenaar
& Mazia, 1978) have suggested that such a protein may be involved in either
chromosome condensation or nuclear membrane breakdown, for example. It has
already been established by Sunkara and co-workers (Sunkara, Wright & Rao, 1979)
that the factor responsible for premature chromosome condensation is a protein and
is present in late Gz phase as well as mitotic HeLa cells.
The authors would like to thank Dr Ron L. Brown and Dr T. E. Stubblefield for their help and
the use of their facilities at M. D. Anderson Hospital in Houston. Thanks also goes to Dr R. B.
Church for his assistance in the preparation of this manuscript. This work was supported by a
National Science and Engineering Council of Canada Scholarship and grant A4926.
268
jf. T. Westivood and E. B. Wagenaar
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(Received 23 July 1982)