[CANCER
RESEARCH
38, 560-565,
March 1978]
Inhibitory Effects of the New Mitotic Inhibitor 5-Chloropyrimidin-2-one
and of Vincristine on Human Cells in Vitro'
Einar Wibe, Reidar Oftebro, Terje Christensen,
Departments
Hospital,
of Tissue Culture
Montebello,
Soren G. Laland, Erik O. Pettersen,
[E. W., P. O., T. C., E. O. P.¡and Biophysics
and Department
ol Biochemistry,
University
[T. L.j, Norsk Hydro's
of Oslo, Blindem,
ABSTRACT
By means of time-lapse microcinematography,
metaphase arrest was studied in synchronized NHIK 3025 cells
in contact with 5-chloropyrimidin-2-one
(NY 3000) or vincristine. A dose-dependent
prolongation of mitosis was
found for both substances, and the fraction of cells able
to escape mitotic arrest declined gradually as the concen
tration of NY 3000 or vincristine was increased. Significant
prolongation of metaphase was observed after treatment
with 0.5 IHM NY 3000 or vincristine (0.5 ng/ml; 0.54 nu),
while total block in metaphase was achieved with 8 DIM
NY 3000 or vincristine (16 ng/ml; 17.3 nu).
The inactivating effect was measured as loss of colonyforming ability after continuous exposure for 12 to 14
days. NY 3000 (0.75 mM) brought total inactivation, al
though the prolongation of metaphase still was small at
this concentration of NY 3000. Vincristine at concentra
tions that cause great prolongation of metaphase still
permitted formation of colonies.
Both drugs demonstrated dose-dependent prolongation
of interphase at concentrations entailing complete meta
phase block. The phase specificity of the interphase
action on synchronized cells was measured by pulsed
incorporation of [ H]thymidine combined with a registra
tion of time of entry into metaphase. NY 3000 resulted in
a prolongation of all stages of interphase. The interphase
effect of vincristine was reflected in a delay in G . This
was confirmed by flow cytometric recording of DNA distri
butions.
These results reveal that the metaphase-arresting
agents NY 3000 and vincristine act also in the interphases
of proliferating cells. However, the mechanism behind
the interphase effect seems to be different for the two
drugs.
INTRODUCTION
1 Supported
by grants
from
The Norwegian
Cancer
Society
Norwegian Research Council for Science and the Humanities.
Received August 15, 1977; accepted November 28, 1977.
560
for Cancer Research,
The Norwegian
Radium
[S. G. L.] Oslo 3, Norway
tensively used in cancer chemotherapy,
also exert effects
on proliferating cells in interphase; this action may account
partly for the inactivating
power of these drugs (4, 7). In
addition, side effects on nondividing body cells have been
observed after treatment with vincristine. Side effects from
vincristine appear among others as dose-dependent
neurotoxic effects (1, 14).
Previously, we studied the metaphase-arresting
ability of
both 5-fluoropyrimidin-2-one
(9) and vincristine (3) on hu
man cells in culture. Metaphase arrest as a function of
drug dose was studied by a technique in which asynchro
nous populations
of cells were kept in contact with the
drug for a certain amount of time, after which the accumu
lated mitoses were differentially
counted. In these early
experiments comparable metaphase arrest was found at a
much lower concentration
of vincristine than of 5-fluoropyrimidin-2-one.
By differential counting of mitotic Chang liver cells, NY
3000,2 chemically
analogous
to 5-fluoropyrimidin-2-one,
has also proved to be a metaphase-arresting
agent. In this
paper, the effects of NY 3000 on NHIK 3025 cells are
reported together with corresponding
data on vincristine.
Data are presented that show the metaphase-arresting
ef
fects as well as the effects on other stages of the cell cycle.
The inactivating effect, measured as loss of reproductive
capacity, was studied separately.
MATERIALS
AND METHODS
Mitotic Inhibitors. The new metaphase inhibitor NY 3000
was synthesized at the Department of Biochemistry,
Blindern, University of Oslo.
Vincristine sulfate (Oncovin) (referred to as vincristine in
this report) was purchased from Eli Lilly & Co., Indianapolis,
Ind. Stock solutions were made by dissolving vincristine in
Hanks' solution. They were stored at 4°for not more than 1
week before use. In this report "ng/ml"
is chosen as
concentration
The probability of successful cancer chemotherapy
can
be enhanced by investigation of the action of the selected
drugs at the cellular level. A detailed knowledge of the
mode of action of such drugs on the individual cell could
facilitate construction
of rational schemes for combination
therapy with different drugs and for the combination
of
drugs and radiation.
Metaphase-arresting
agents like vincristine, which is ex-
Institute
and Tore Lindmo
unit for vincristine
(1 ng/ml
= 1.08 nM).
Cell Line NHIK 3025 and Synchronization Method. The
cell line NHIK 3025 originates from a human cervix carci
noma in situ and has been grown in vitro for 10 years.
Stock cultures were grown in 75 sq cm Falcon plastic
flasks and kept in almost continuous logarithmic growth by
trypsinization
3 times per week. Medium E2a (13), contain
ing 40% synthetic mixture, 30% Hanks' solution, 20% hu
man serum, and 10% horse serum, was used.
The cells were kept at a constant temperature
of 37°.
and The
2 The abbreviation used is: NY 3000, S-chloropyrimidin^-one.
CANCER
RESEARCH
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Effect
Under these conditions, the average durations of G,, S, G2,
and mitosis for untreated NHIK 3025 cells are 6.5, 8, 2.5,
and 1 hr, respectively (11).
Suspensions
of synchronized
cells were obtained by
selection of mitotic cells from populations trypsinized the
day before. The selection was performed by shaking culture
flasks on a reciprocal shaker (11).
Time-Lapse
Experiments. The newly selected mitotic
cells were allowed to attach to circular Corning coverslips
on the bottoms of 2 Retri dishes and were incubated in a
CO2 incubator. After the attachment period of 2 hr, the
medium in one of the dishes was replaced by fresh medium
containing a mitotic inhibitor, while the other dish received
fresh control medium.
Six hr after mitotic selection, 1 coverslip from each dish
was transferred to Emdeco model 101-700 tissue culture
chambers previously filled with medium from the dishes.
Both chambers were mounted on the stages of phasecontrast microscopes
(Carl Zeiss, Oberkochen,
W. Ger
many) fitted with a Zeiss micro cine camera, and located
inside a 37°heat box during the time of observation.
Each chamber contained about 30,000 cells. By use of
x10 objectives,
microscopic
fields containing
about 25
cells from each chamber could be selected on the 2 micro
scopes. The microscope-time-lapse
unit made it possible
to obtain simultaneous
exposures from the 2 microscopic
fields on the respective halves of each frame.
Filming was carried out with 35-mm Kodak Eastman
Plus-X Negative film. The cells were continuously
illumi
nated. To avoid unwanted heating of the cells, we used
heat protection filters on both microscopes and kept the
light intensity at a very low level (exposure time, 30 sec).
Exposures were made at regular intervals about 15 min
apart.
In most experiments filming was stopped when the sec
ond generation of control cells was about to enter mitosis,
e.g., 30 hr after mitotic selection. In some experiments
filming continued up to 50 hr after selection in order to
check whether any of the cells arrested in metaphase were
able to escape mitosis after the point of time when filming
normally ended.
In experiments in which the filming was prolonged up to
50 hr, no difference appeared between the duration of the
first and second generations of control cells. This finding
indicates that growth conditions in the Emdeco chambers
are stable within the period of observation.
The films were analyzed by means of an Acmade 35-mm
film analyzer. The life history of each cell present in the
initial frame and the birth and fate of daughter cells were
followed. Each cell was recorded as having entered meta
phase when it had assumed a circular outline and as having
completed division when daughter cells, delimited by an
unbroken contour, had appeared. Time spent in mitosis for
each cell was measured as the interval between entry into
metaphase and completion
of division. This definition of
the mitotic event is not according to cytological standards.
However, it was chosen for practical purposes, because all
divisions entailed cytokinesis,
whereas the state of the
nucleus was difficult to ascertain in these films. The average
duration of the mitotic event defined in this way was always
between 30 and 40 min for control cells.
MARCH
of NY 3000 and Vincristine
in Vitro
Studies of Colony-forming Ability. Cell survival after the
presence of a mitotic inhibitor in the medium for about 2
weeks was measured by counting the number of macro
scopic colonies on the dishes. An exponentially
growing
population of cells was trypsinized to obtain a suspension
of single cells. The cells were then plated and allowed to
attach to the bottom of the plastic Petri dishes (Falcon
Plastics, Oxnard, Calif.). After 4 hr the medium was re
placed by fresh medium containing different concentrations
of NY 3000 or vincristine.
The medium in the dishes was again replaced by fresh
medium, containing the same amount of mitotic inhibitor,
6 days after plating. After the required length of incubation
(12 to 14 days), the colonies were fixed and stained as
described in a previous report (12). Colonies containing
less than 40 cells were not counted.
Studies of Interphase Effect. The effect of the 2 mitotic
inhibitors on the progress of the cells through the cell
cycle was investigated by measuring the amount of incor
porated [3H]thymidine at set times after mitotic selection.
Suspensions of newly selected mitotic cells were trans
ferred to 25-sq cm plastic Nunclon flasks for attachment of
the cells to the bottom of the flasks. At a fixed time the
medium in the flasks was replaced by fresh medium con
taining the mitotic inhibitor. The amount of incorporated
[3H]thymidine was measured by a pulse-labeling technique
(15-min pulse; 5 /xCi/flask; 5 Ci/mmole).
This technique
was described previously (11).
Parallel to the mapping of DNA synthesis, observation
was performed in an inverted microscope with phase-con
trast equipment (Zeiss) of cells attached to the bottom of
25-sq cm plastic Nunclon flasks. About 100 viable cells
from each experimental group were observed, and the time
of entry into metaphase for each cell was recorded. After
incorporation
of [3H]thymidine and observation in a micro
scope, the onset and duration of each of the phases of the
cell cycle for treated and untreated populations were mea
sured for the first generation after selection.
Flow Cytometric Measurement
of DNA Distributions.
DNA histograms showing the distribution
of cells in the
various phases of the cell cycle were obtained from popu
lations harvested at 2-hr intervals after mitotic selection.
Cells were stained with the DNA-specific fluorescent stain
mithramycin
(100 /ng/ml) (Charles Pfizer & Co., Inc., New
York, N. Y.) without previous fixation, as described by
Crissman and Tobey (2). The staining reaction was allowed
at least 3 hr at room temperature
to reach equilibrium
before the sample was analyzed. The DNA histograms were
measured on a laboratory-built
flow cytometer (5, 11), with
the use of the 457 nm line of a 4-watt argon laser for
excitation of mithramycin fluorescence.
RESULTS
The data presented in Chart 1 demonstrate how treatment
with NY 3000 or vincristine
influences the duration of
mitosis. In the dose range of these curves, a high fraction
of the treated cells completed division (>50%; see Chart
2). For higher doses [in the experiment with vincristine (8
ng/ml) and in 1 of the 2 experiments with 4 mw NY 3000],
1978
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561
E. Wibe et al.
ig
1
Q
05
1.0
1.5
CONCENTRATION OF NY 3000
2.0
2.5
(mM)
20
S
15
•z.
g
et 10
Ãœs
=>
0123
CONCENTRATION 0F VINCRISTINE
4
(ng/ml)
Chart 1. Average duration of mitosis for the fraction of treated NHIK 3025
cells able to escape mitotic arrest at different concentrations of NY 3000 or
vincristine. Each plotted value represents a separate experiment and shows
the ratio between the durations of mitosis for treated and untreated cultures.
only a few cells escaped mitotic arrest, and because of the
poor statistics the duration of mitosis at these concentra
tions is not indicated in Chart 1.
Cells that had spent several hr in metaphase often went
through irregular divisions, resulting in more than 2 daugh
ter cells. Almost without exception such divisions were
followed by a fusion of the daughter cells, in most cases
into 1 cell (Fig. 1). Nevertheless, cells going through such
divisions were scored as dividing cells because they had
managed to escape mitotic arrest. Other cells arrested in
mitosis disintegrated within time of filming, and these cells
were recorded as irreversibly arrested in mitosis.
For demonstration of the typical events analyzed in such
experiments, parts of the 2 microscopic fields in one of the
experiments performed with 2 mw NY 3000 are shown in
Fig. 1. Fig. 1, A to D, demonstrates control cells at different
stages in the cell cycle, while Fig. 1, £to H, shows cells
treated with 2 mM NY 3000. Pictures mounted opposite to
each other are taken simultaneously.
The pictures taken 12 hr and 53 min after mitotic selection
demonstrate untreated (Fig. 1/4) and treated (Fig. 1£)cells
in late S. In Fig. 18, 1 anaphase, 1 telophase, and 4 earlyG, cells can be seen. The 4 treated cells were all arrested
in metaphase at that time (Fig. 1F). Fig. 1C shows 8 normal
daughter cells, the result of normal divisions of the 4
control cells in Fig. 1/4. In Fig. 1G the result of an abnormal
division of a treated cell can be seen. Three daughter cells
arose (arrow), and in Fig. 1W the fusion of the daughter
cells is completed.
Chart 2 shows the fractions of first-generation cells that
are able to escape mitotic arrest in the presence of different
562
16hr48min
22 hr 32 mm
D
25hr
H
9min
Fig. 1. Untreated NHIK 3025 cells (A to D) and cells treated with 2 mM NY
3000 (E to H). Time after mitotic selection is indicated below photographs.
For details, see text, x 390.
concentrations of NY 3000 or vincristine (b curves). For
comparison, surviving fractions as measured by the ability
to form colonies after continuous exposure to the mitotic
CANCER
RESEARCH
VOL.
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38
Effect
01
0.1
!
2
CONCENTRATION
3
OF NY3000(mM)
g 0.1
0.1
u
53
öS
im
SS
-s
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01
0.01
234567
CONCENTRATION OF VINCRISTINE
of NY 3000 and Vincristine
in Vitro
By the same methods as those for 8 mM NY 3000, the
interphase action of vincristine (64 ng/ml) was examined
(Chart 46). The [3H]thymidine incorporation curves indicate
only a minor or no effect on cells in G, or S. The presence
of vincristine in G, seemed to have virtually no influence on
the rate of progress through interphase (the 2-hr curve
coincides with the 6-hr curve). However, from the curves
showing the onset of metaphase, a 3-hr prolongation of
interphase can be estimated. This prolongation then must
arise in G^ or at the G^-mitosis transition.
For ensurance that the DNA synthesis curves in Chart 4
were not only a result of pool effects, the distribution of
the DNA content of individual cells at various times after
mitotic selection was measured by flow cytometry. In Chart
5A DNA histograms are shown for cells treated with 8 mw
NY 3000, compared with histograms for untreated cells.
The delay in interphase seemed to increase proportionally
to the time passed with NY 3000 present in the medium.
Chart 5ß shows DNA histograms that support the
[3H]thymidine incorporation curves in Chart 4. The DNA
histograms demonstrate that the presence of vincristine
(64 ng/ml) has very little influence on the progression of
(
Chart 2. •.
fraction of NHIK 3025 cells escaping mitotic arrest at different
concentrations of NY 3000 or vincristine divided by the fraction of dividing
control cells. Each plotted value represents a separate experiment. O,
surviving fractions, measured as ability to form macroscopic colonies after
10 to 12 days of incubation in the presence of different concentrations of
NY 3000 or vincristine.
inhibitors for 12 to 14 days are shown in the same chart (a
curves).
The duration of interphase for cells kept in the presence
of different concentrations of NY 3000 or vincristine as
measured in the time-lapse experiments is shown in Chart
3. The number of cells that did not enter mitosis during the
time of observation was always close to or equal to zero,
except for the 2 experiments performed with 8 mw NY 3000
and 1 of the experiments performed with vincristine (16
ng/ml), for which the rate of progress through the cell
cycle was extremely slowed down for a considerable frac
tion of the cell population (see legend to Chart 3).
For a detailed picture of the specificity of the interphase
action of NY 3000 and vincristine, pulsed incorporation of
[3H]thymidine was performed. Chart 4 shows the rate of
DNA synthesis at set times after mitotic selection for un
treated cells and for cells treated with 8 HIM NY 3000. For
parallel populations the time of onset of metaphase was
measured by observation in a microscope. Cells treated
with 8 rriM NY 3000 from 2 hr after mitotic selection were
more delayed than were cells treated from the onset of S (6
hr after mitotic selection). This demonstrates an effect on
cells in G,. Moreover, the total prolongation of interphase
seemed to increase proportionally to the duration of the
presence of NY 3000, regardless of cell cycle phase. This
was confirmed by additional experiments (data not shown)
in which NY 3000 was added and removed at different
stages in the cell cycle; the prolongation of interphase was
not phase specific.
MARCH
23456
CONCENTRATION OF NY 3000
(mM)
M
I'3
z
o
1.1
<
S '°
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£ 0.9
I
J_
16
J_
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I
32
CONCENTRATION OF VINCRISTINE
I
56
(ng/ml)
Chart 3. Average duration of ¡nterphasefor NHIK 3025 cells after treat
ment with different concentrations of NY 3000 or vincristine. Each plotted
value represents 1 experiment and shows the ratio between the duration of
interphase for treated and untreated cultures. In the 2 experiments per
formed with 8 rnw NY 3000, only 50 and 80%, respectively, of the cells
reached metaphase during the time of observation (30 hr) In 1 of the
experiments performed with vincristine (16 ng/ml) only 60% of the cells
reached metaphase within 30 hr after mitotic selection. The plotted value
from this experiment was ignored when the curve in B was drawn.
1978
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563
E. Wibe et al.
From Chart 2 it appears that the inactivating effect of NY
3000 is much more potent after long-term exposure than
what would be expected from the time-lapse experiments.
After the presence of 0.75 mM NY 3000 in the medium
during the time of incubation, the survival was too low to
6
S 10 12 K 16 18 20 22 24
TIME AFTER MITOTIC SELECTION (hr)
5hr
5hr
CONTROL
NY 3000
7hr
CONTROL
9 hr
NV 3000
11 hr
CONTROL
15 hr
NY 3000
13hr
CONTROL
19 hr
NY 3000
26
tooi
90
z
E
<->
S
80 S
70 m
6° fÜ
2
50
S
40 r
30 m
20 >
i
o
2
4
io 5
o ifi
6
8
10
TIME AFTER
12
U.
16
18
20
22
2¿ 26
MITOTIC SELECTION (hr)
Chart 4. Lapse of cell cycle for treated and untreated NHIK 3025 cells
measured by incorporation of [3H]thymidine and observation in microscope
of time of entry into metaphase. A. cpm in thousands for control cells (O)
and cells treated with 8 mM NY 3000 from 2 (A) or 6 (D) hr after mitotic
selection. Arrows, time of addition of NY 3000. Percentage of cells having
entered metaphase is also indicated for each separate population (filled
symbols). B, experiment identical with that presented in A, but with vincristine instead of NY 3000. Vincristine (64 ng/ml) was added 2 (A, A) or 6
(D, •)hr after mitotic selection.
Q
"b
ÃœJ
u
ce
cells through G, and S. Fifteen hr after selection, a small
fraction of the control cells had divided (G,), while the
histogram for treated cells demonstrates accumulation of
cells in Grmitosis.
—
U
O
Ü>
rr
DISCUSSION
It has been established that vincristine is a spindle inhib
itor (4, 7). The arrangements of chromosomes during me
taphase arrest of NHIK 3025 cells caused by vincristine
were reported previously (3). The same type of chromo
somal distribution in metaphase was observed after treat
ment with different concentrations of NY 3000.
The results plotted in Chart 1 show that both 0.5 mM NY
3000 and vincristine (0.5 ng/ml; 0.54 nw) involve significant
prolongation of mitosis. One mM NY 3000 causes nearly
the same prolongation of mitosis as does vincristine (1 ng/
ml). The lowest concentrations that entailed total block of
NHIK 3025 cells in metaphase were 8 mw NY 3000 and
vincristine (16 ng/ml; 17.3 nM).
The results from the experiments performed with vincris
tine are in agreement with the previously reported results
of Dahl era/. (3), in which the metaphase index of asynchronously growing populations of NHIK 3025 cells after treat
ment with different concentrations of vincristine was mea
sured. According to those results the metaphase index
after 6 hr treatment had reached the maximum level at a
vincristine concentration of 8 ng/ml. This investigation,
however, reveals that vincristine at this concentration does
not result in a complete block, but rather an average
duration of metaphase longer than 6 hr for cells able to
escape from the arrest.
564
50
B
100
no
50
11 hr
CONTROL
llhr
VINCRISTINE
15 hr
CONTROL
15 hr
VINCRISTINE
V
O
50
100
O
50
100
CHANNEL NUMBER (PROPORTIONAL TO CELLULAR DNA-CONTENT)
Chart 5. A, DNA histograms for NHIK 3025 cells treated with 8 mM NY
3000 from 2 hr after mitotic selection compared to histograms for control
cells. Time passed after mitotic selection is indicated in each chart. Histo
grams showing similar DNA distributions are mounted opposite to each
other. B, DNA histograms for cells treated with vincristine (64 ng/ml) from 2
hr after mitotic selection compared to histograms for control cells. The
results presented in Charts 48 and 5ßare from the same experiment. G,
cells are not recorded in the same channels in A and B because of different
instrument settings.
CANCER
RESEARCH
VOL.
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38
Effect of NY 3000 and Vincristine in Vitro
be measured. Consequently, 0.5 HIM was the highest con
centration of NY 3000 that allowed cells to form macro
scopic colonies. At this concentration of NY 3000, the
percentage of cells able to perform the first division after
mitotic selection was as high as for control populations
(Chart 2), and the duration of the metaphase was only
slightly longer than for untreated cells (Chart 1). However,
only about 7% were able to form colonies in the presence
of 0.5 mM NY 3000. This supports the view that the appar
ently normal divisions at this concentration of NY 3000
conceal latent damage in the daughter cells.
In comparison, a vincristine concentration of 2 ng/ml
permitted formation of colonies (Chart 2). This is a rather
surprising result if one takes into consideration the long
duration of metaphase arrest at this concentration of vin
cristine (Chart 1). At 0.5 ng/ml, where the prolongation of
metaphase still was considerable, the colony-forming ability
was nearly 100%. Consequently, some of the cells that
spend about 6 hr in metaphase arrest in the presence of
vincristine are still able to make viable progenies. The
mechanisms of action in metaphase of NY 3000 and vincris
tine may therefore be different.
The curves in Chart 3 demonstrate that treatment with
NY 3000 or vincristine in the concentration range that
entails complete mitotic arrest involves dose-dependent
prolongation of interphase. The prolongation of interphase
caused by the presence of NY 3000 is dramatically in
creased when the concentration exceeds that resulting in
complete mitotic arrest. Treatment with increasing concen
trations of vincristine entails a more moderate prolongation
of interphase.
In a review article from 1972, Marsden (7) concludes that
the mechanisms of vincristine action are unknown. He
points out the conflicting results from different laboratories
concerning the influence of vincristine on DNA and RNA
synthesis. Thésituation seems to be the same today.
Madoc-Jones and Mauro (6) found that synchronized
HeLa cells continuously exposed to vincristine (10 ng/ml)
were 3 hr late in reaching mitosis compared to untreated
cells. The same delay was found whether the drug was
added in G, or in G,. This means that the prolongation of
interphase must arise in G-,,which is in agreement with our
results. Furthermore, Madoc-Jones and Mauro found that
cells exposed to vincristine (100 ng/ml) only in S proceeded
at a normal rate to mitosis, where they were arrested
irreversibly. In these experiments vincristine was removed
by washing before the cells reached G.,, and no delay in G.,
was observed. This demonstrates that the G2 delay is not
induced in S but depends on the presence of a considerable
concentration of vincristine in the cells during G,.
Inasmuch as the G2-prophase transition was not recorded
in our experiments, the curves in Chart 46 do not exclude
the possibility that the delay resulting from contact with
vincristine (64 ng/ml) appears in prophase rather than in
G-,. However, the previously reported results of Dahl ef al.
(3) do not indicate any accumulation of NHIK 3025 cells in
prophase when cells are treated with this concentration of
MARCH
vincristine.
NY 3000 (0.5 mM) and vincristine (0.5 ng/ml; 0.54 nw)
both involve significant prolongation of metaphase. Total
block in metaphase is achieved at a NY 3000 concentration
of 8 mM and a vincristine concentration of 16 ng/ml (17.3
nM). Consequently, the ratio between the molar concentra
tions of NY 3000 and vincristine sufficient for total block in
metaphase is about 500,000.
There is dose-dependent prolongation of interphase at
concentrations of NY 3000 or vincristine that entail total
block in metaphase. The prolongation of interphase follow
ing treatment with NY 3000 is proportional to the duration
of the exposure to this drug and seems to be rather
nonspecific with respect to cell age. The delay in interphase
caused by treatment with vincristine is due to a prolonged
G,.
An inactivating effect, measured as lack of colony-form
ing ability after continuous exposure for 2 weeks, is seen
even at NY 3000 concentrations at which the prolongation
of metaphase is negligible. Vincristine given in concentra
tions that entail great prolongation of metaphase still per
mits cells to form macroscopic colonies.
REFERENCES
1. Burdman, J. A. A Note on the Selective Toxicity of Vincristine Sulfate
on Chick-Embryo Sensory Ganglia in Tissue Culture. J. Nati. Cancer
lnst.,37: 331-335, 1966.
2. Crissman, H. A., and Tobey, R. A. Cell Cycle Analysis in 20 Minutes.
Science. 784: 1297-1299, 1974.
3. Dahl, W. N., Oftebro, R., Pettersen, E. 0., and Brustad, T. Inhibitory
and Cytotoxic Effects of Oncovin (Vincristine Sulfate) on Cells of Human
Line NHIK 3025. Cancer Res., 36: 3101-3105, 1976.
4. Himes, R. H., Kersey, R. N., Heller-Bettinger, I., and Samson, F. E.
Action of the Vinca Alkaloids Vincristine, Vinblastine, and Desacetyl
Vinblastine Amide on Microtubules in Vitro. Cancer Res.. 36: 37983802,1976.
5. Lindmo, T.. and Steen, H. B. Flow Cytometric Measurement of the
Polarization of Fluorescence from Intracellular Fluorescein in Mammal
ian Cells. Biophys. J., 18: 173-187, 1977.
6. Madoc-Jones, H., and Mauro, F. Interphase Action of Vinblastine and
Vincristine: Differences in Their Lethal Action through the Mitotic Cycle
of Cultured Mammalian Cells. J. Cellular Physiol., 72: 185-196, 1968.
7. Marsden, J. H. Mechanism of Action of the Vinca Alkaloids. In: I.
Brodsky and S. B. Kahn (eds.), Cancer Chemotherapy, Vol. 2, pp. 3340. New York: Gruñe& Stratton. Inc., 1972.
8. Nordbye, K.. and Oftebro, R. Establishment of Four New Cell Strains
from Human Uterine Cervix. I. Exptl. Cell Res., 58: 458, 1969.
9. Oftebro, R., Grimmer, 0., Oyen, T. B., and Laland, S. G. 5-Fluoropyrimidin-2-one. a New Metaphase Arresting Agent. Biochem. Pharmacol., 21: 2451-2456, 1972.
10. Oftebro, R., and Nordbye, K. Establishment of Four New Cell Strains
from Human Uterine Cervix. II. Exptl. Cell Res.. 58: 459-460, 1969.
11. Pettersen, E. O., Bakke, 0., Lindmo, T., and Oftebro, R. Cell Cycle
Characteristics of Synchronized and Asynchronous Populations of Hu
man Cells and Effect of Cooling of Selected Mitotic Cells. Cell Tissue
Kinet., 10: 511-522, 1977.
12. Pettersen, E. 0., Oftebro, R.. and Brustad, T. X-ray Inactivation of
Human Cells in Tissue Culture under Aerobic and Extremely Hypoxie
Conditions in the Presence and Absence of TMPN. Intern. J. Radiation
Biol.. 24: 285-296, 1973.
13. Puck, T. T., Cieciura, S. J., and Fisher, H. W. Clonal Growth in Vitro of
Human Cells with Fibroblastic Morphology. J. Exptl. Med., 706: 145165, 1957.
14. Winkelman, A. C., and Mancali, E. L. Toxic Effects of Cancer Chemo
therapy Agents on the Nervous System. In: I. Brodsky and S. B. Kahn
(eds.), Cancer Chemotherapy, Vol. 2. pp. 231-237. New York: Gruñe&
Stratton, Inc.. 1972.
1978
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1978 American Association for Cancer Research.
565
Inhibitory Effects of the New Mitotic Inhibitor
5-Chloropyrimidin-2-one and of Vincristine on Human Cells in
Vitro
Einar Wibe, Reidar Oftebro, Terje Christensen, et al.
Cancer Res 1978;38:560-565.
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