Schedule Dependence of Vincristine Lethality in Sarcoma 180

Cells following Partial Synchronization with Hydroxyurea

Hamza Mujagic, Bruce M. Conger, Charles A. Smith, et al.
Cancer Res 1983;43:3598-3603.

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[CANCER RESEARCH 43. 3598-3603.

August 1983]

Schedule Dependence of Vincristine Lethality in Sarcoma 180 Cells

following Partial Synchronization with Hydroxyurea
Hamza Mujagic,1 Bruce M. Conger, Charles A. Smith, Sandra J. Occhi pinti, William H. Schuette, and
Stanley E. Shackney
Section of Cell Kinetics, Clinical Pharmacology Branch, Division of Cancer Treatment, National Cancer Institute [H. M., B. M. C., C. A. S., S. J. O., S. E. S.], and Applied
Clinical Engineering Section, BiomdicalEngineering and Instrumentation Branch, Division of Research Services [W. H. S.J, NIH, Bethesda, Maryland 20205

ABSTRACT
The effects of exposure to 0.1, 0.5, or 2 pM vincristine for 4
hr were studied in Sarcoma 180 cells at various times after
synchronization with 5 mw hydroxyurea for 1 hr. Maximum
sensitivity to the lethal effects of vincristine was observed at 10
to 14 hr after hydroxyurea exposure at the higher vincristine
concentrations, compared to a period of a maximum sensitivity
to a second dose of hydroxyurea at 8 to 12 hr. Serial flow
cytometry studies indicated that the apparent decrease in sen
sitivity to vincristine at 14 to 18 hr was due to the division of
cells in the leading segment of the synchronized wave and their
entry into the relatively resistant Gphase prior to vincristine
exposure. Synchronized cells that had not divided at the time of
vincristine exposure were blocked transiently in G2. Serial
metaphase index studies suggested that the G2 cells closest to
the end of the cell cycle at the time of vincristine exposure were
likely to exhibit the greatest degree of mitotic disorganization
when they overcame the G2 block and entered metaphase. The
present studies suggest that sensitivity to vincristine increases
progressively as cells approach mitosis. The molecular mecha
nisms underlying this phenomenon are considered in relation to
the increase in cell tubulin content during the course of cell cycle
progression.

MATERIALS AND METHODS

All studies were carried out in Sarcoma 180 (Foley strain CCRF11;
supplied by American Tissue Type Culture, Rockville, Md.) grown in vitro
in Earle's Medium 199 (Flow Laboratories, Rockville, Md.), which was
supplemented with 10% fetal bovine serum, 2 mw t-glutamine,

100 units

of penicillin per ml, and 100 HQ of streptomycin per ml. Cultures were
grown in monolayer in 250-ml plastic tissue culture flasks (growth surface
area, 75 sq cm) (Costar, Cambridge, Mass.) containing 10 ml of medium.
Cells were plated at an initial concentration of 1 x 105 cells/ml. Medium

INTRODUCTION
The Vinca alkaloids, VCR2 and vinblastine, are cytotoxic tubulin-binding agents that are effective in the treatment of leukemias, lymphomas, and a variety of solid tumors in animals and
in humans.
Early studies of the Vinca alkaloids suggested that their lethal
effects were cell-cycle phase-specific (8, 11, 14, 24). These
agents were shown to disrupt the mitotic spindle (8,11,14), and
their lethality was attributed largely to their effects on cells in
mitosis (3, 8, 24). However, in other studies, the Vinca alkaloids
were found to exert their lethal effects on interphase cells (5, 9,
10,12,13, 25). When synchronized cells were exposed to VCR,
a nadir in clonogenic cell survival was observed during S phase
(9, 13) or during late S and G2 (25). In autoradiographic studies
of cells doubly labeled with [3H]dThd and [14C]dThd, it was
shown that cells exposed to VCR during S and G2 were later
arrested in mitosis and subsequently underwent necrosis (5,10).
In our own studies on the effects of VCR in asynchronously
1To whom requests lor reprints should be addressed, at Building 10, Room
12C216, National Cancer Institute, Bethesda, Md. 20205.
2 The abbreviations used are: VCR, vincristine; dThd, thymidine; HU, hydroxy
urea; HBSS. Hanks' balanced salt solution.
Received October 22, 1982; accepted May 5, 1983.

3598

growing Sarcoma 180 cells (16), VCR produced a transient block

of interphase cells in G2. We also found that not all cells that
were lethally damaged by VCR accumulated in mitosis, and that
many of the cells that did accumulate in mitosis did not become
necrotic but went on to become polyploid cells.
In order to explore these observations in greater detail, we
have studied the effects of VCR on cell survival, cell cycle
progression, DMA synthesis, and metaphase accumulation in
Sarcoma 180 cells that were synchronized by prior exposure to
hydroxyurea for 1 hr. The results of these studies are described
in this paper. We have found that the lethal effects of VCR
became more pronounced as cells progressed through the cell
cycle and approached mitosis. The mechanism underlying this
observation may be related to the reduplication of cell tubulin
content late in the cell cycle.

was changed on Days 2 and 4, and cells were subcultured on Day 5.

Cell Survival Studies. Two-day-old log-phase cell cultures were in
cubated at 37with 5 rriM HU for 1 hr. At the end of the drug exposure
period, the medium containing drug was removed, and the cells were
rinsed 3 times with 5 ml of HBSS. Cells were then resuspended in
Medium 199. A drug-free control flask was included and was treated
identically in each experiment. At various intervals after HU pretreatment,
VCR was added at final concentrations of 0.1, 0.5, or 2 UM for 4 hr. For
reference purposes, time intervals between drugs are reported from the
onset of exposure to each drug.
At the end of the VCR exposure period, the medium was removed,
and the cells were washed 4 times with HBSS. The cells were then
harvested by incubation (37) with 0.25% trypsin for 8 min. Controls
treated with HU alone were also obtained at each time point. Total cell
counts in each flask were determined using a Coulter Counter (Coulter
Electronics, Hialeah, Fla.). Cell suspensions were diluted with Medium
199, and known numbers of cells were cloned in soft agar. Nine-day-old
colonies were fixed, stained with Giemsa stain, and counted visually.
Clonogenic cell yield for each flask, defined as the number of colonies
enumerated divided by the number of cells plated, was averaged for 5
replicate flasks at each drug concentration. The viable cell count per
flask was calculated as the total cell number per flask multiplied by the
clonogenic cell yield at a given time point. The surviving viable cell
fraction following VCR exposure was calculated as the number of viable
HU-treated cells per flask divided by the number of viable HU-treated
control cells per flask, harvested at the same time, Thus, the reported

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RESEARCH

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Vincristine Schedule Dependence

surviving cell fractions after VCR exposure reflected the lethal effects
produced by VCR alone. Each experiment was performed at least 3
times, and the reported results represented the log means of replicate
studies.
For comparison, flasks of cells exposed initially to 5 rriM HU for 1 hr
were exposed to a second dose of HU at the same concentration for 1
hr at the same time intervals at which VCR exposures were initiated.
Metaphase Index Studies. Serial metaphase indices were obtained
at intervals during and after HU and HU plus VCR exposure. Aliquots of
cells were cytocentrifuged on glass slides and stained with acetic orcein.
Only those cells containing condensed, discrete chromosomes in the
absence of a nuclear membrane were included in the numerator of the
metaphase index. Metaphases were further classified on the basis of the
degree of disruption of normal metaphase chromosome patterns. Meta
phases in which the chromosomes were organized in a single ring (or
band-like structure, when viewed on edge) were considered to have
intact kinetochore microtubules (2) and were classified as organized for
our purposes. Metaphases in which chromosomes were spread ran
domly throughout the cell, and/or in which there were multiple-ring
structures, each involving 2 to 3 chromosomes, were classified as
disorganized metaphases. Two thousand cells were counted at each
time point. Each experiment was performed 3 times, and reported results
represent the means of replicate studies.
Flow Cytometry Studies. Serial DNA histograms were obtained be
fore, during, and after exposure to HU and HU plus VCR. Cells were
washed with HBSS, harvested by trypsinization, fixed in cold 70%
ethanol, and stained with mithramycin (100 ^g/ml) in 15 mM MgCl.. and
0.15 M NaCI at a final cell concentration of 1 x 10s cells/ml. Nuclear
fluorescence was measured with a Los Alamos cell sorter. Data were
recorded, stored, and analyzed on a DEC 11/40 computer system using
software developed at the Los Alamos Scientific Laboratory, Los Alamos,
N. M. At least 20,000 cells were measured in each sample. All DNA
histograms were normalized with respect to total cells analyzed per
sample for display purposes.
Prior to the measurement of each drug-treated sample, a sample of
untreated control cells was run, and amplifier gain settings were adjusted
so that the G, peak of the control cells appeared in the same reference
channel (Channel 60 of 256). Immediately after the measurement of each
drug-treated sample, the position of the Gpeak of control cells was
rechecked to ensure that it had not drifted during the course of the
measurement.
DNA Histogram Analysis. Because of the presence of large numbers
of nuclear fragments and polyploid cells at various times after VCR
exposure (see Chart 4, and Ref. 15), conventional methods of DNA
histogram analysis could not be used. In order to quantitate population
shifts among the cell cycle phases, and in order to quantitate the
development of nuclear fragmentation and polyploidy during and at
various times after drug exposure, the DNA histogram was analyzed by
a modification of the method of Alabaster and Cassidy (1). The DNA
histogram was divided into bounded regions as follows: pre-G,. Channels
<50; G,Channels 50 to 70; S, Channels 71 to 99; G2 M, Channels 100
to 140; and post-G2-M, Channels >140. The fraction of cells in each
region was calculated for each histogram. Because the gain settings
were adjusted to an external reference standard within one channel of
60 for each sample, valid comparisons could be made among sequential
histograms, and corresponding data from multiple experiments could be
grouped for analysis. The reported results represent the means of 3
experiments. Although by this method the cell fraction in each region
was contaminated to some extent by cells from adjacent regions, this
proved to be of little practical consequence. Major shifts of cells from
one region of the histogram to another that were apparent on visual
inspection were faithfully reflected in the processed data.

RESULTS
Cell Survival Studies. Chart 1 shows the effects on cell
survival of VCR exposure for 4 hr, commencing at various time

10

12

14

16

18

TIME, HR
Chart 1. Surviving clonogenic cell fraction following exposure to VCR for 4 hr,
initiated at various intervals after exposure to 5 RIM HU for 1 hr. Data are normalized
with respect to time-matched controls treated initially with HU alone and reflect the
effects of VCR administration only.
,0.1 uVCR; *,
0.5 MMVCR;
.2 MVCR. Effects on cell survival of a second dose of 5 mM HU for 1 hr
at various intervals after the first are shown for comparison (
,shaded
region). For discussion, see text. Data represent the means of 3 experiments. Bars,
S.E.

intervals after an exposure for 1 hr to 5 mw HU. The effects of

exposure to a second dose of HU are shown for comparison.
Since the data are normalized with respect to time-matched
controls treated with HU alone (see "Materials and Methods"),
they reflect the effects of administration of the second drug only
on cell survival.
When cells were exposed to 0.1 MMVCR for 4 hr, starting at
2 to 10 hr after exposure to HU, the surviving clonogenic cell
fraction ranged from 0.70 to 0.75 (Chart 1). When cells were
exposed to 0.1 ^M VCR at 12 to 14 hr after exposure to HU, the
surviving cell fraction fell to 0.5. Exposure to 0.1 /UMVCR at 16
and 18 hr produced surviving cell fractions of 0.6 and 0.75,
respectively.
When cells were exposed to 0.5 ^M VCR at various intervals
after exposure to HU, the schedule-dependent effects on cell
survival were more pronounced. The nadir in the surviving cell
fraction (0.25 to 0.31) was observed at 12 to 14 hr (Chart 1).
Cell killing was also greater at 6 to 10 hr with 0.5 //M VCR than
with 0.1 UM VCR. The effects of 2 >IMVCR were similar to those
observed at 0.5 mw VCR. The nadir in the surviving cell fraction
(0.22 to 0.27) also occurred at 12 to 14 hr. The decrease in
surviving cell fraction at 10 hr was greater with exposure to 2
M
VCR than with 0.5 MVCR.
By comparison, the nadir in the surviving cell fraction following
exposure to a second dose of HU was observed between 6 and
12 hr and ranged from 0.45 to 0.55 (see Chart 1, shaded region).
While there was extensive overlap in the intervals of maximum
sensitivity to a second dose of HU and to VCR, respectively, the
interval of maximum sensitivity to VCR extended 2 to 4 hr later
than that for a second dose of HU, and the nadir was lower at
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H Mu/agicet al
VCR concentrations of 0.5 (iu or greater.
In earlier radioautographic studies of the effects of exposure
of 5 m** HU for 1 hr, peak rates of DMA synthesis during the
pos ttreat men t period were observed at 6 to 10 hr. Concorri i-

region in the presence of drug through the 14-hr time point (Chart
2D, shaded region), ana the fraction of cells in the Gz-M region
remained elevated or continued to rise for at least 2 hr after drug
removal. Peak fractions of VCR-treated cells in the Gz-M region

tantly, a wave of recruited eels was shown by flow cytometry to

move through early S at 6 hr, mid-S at 8 to 10 hr, and late Searly G2 at 12 hr (6). Preliminary studies of changes in the
metaphase index following exposure to 5 IM HU for 1 hr indicated
a decrease in the metaphase index during the first 10 to 12 hr,
and a rise at 14 to 18 hr. That is, exposure to HU for 1 hr was
followed by the appearance of a cohort of rapidly cycling, syn
chronized ceOsthat moved through mid-S at 8 to 10 hr, traversed
late S and 62 at 12 to 14 hr, and underwent mitosis between 14
and 18 hr. Thus, one interpretation of the cell survival studies
shown in Chart 1 might be that susceptibility to VCR peaked in
late S and earty Gz and then decreased late in G2. Alternatively,
it may be that cells in late Gz were no less susceptible to VCR
effects than were eels in late S and early G.. and that the
apparent decrease in drug effectiveness at 14 to 18 hr was due
to the vanguard of the partially synchronized wave of cells
dividing and entering a relatively resistant G, phase at this time.
In order to distinguish between these 2 possibilities, we chose
the 10-and 14-hr time points after HU exposure for more detailed

ranged from 0.8 to 0.9. However, concomitant metaphase index

studies showed that the fraction of cells in metaphase at 10 to
16 hr was less than 0.05 at all drug concentrations (see below
and Chart 4 and associated text). Thus, the cells that accumu
lated in the Gz-M region of the ONA histogram between 12 and
18 hr were almost all premi tot ic cells in Gz. A similar VCRinduced G, block was observed in asynchronously growing
sarcoma 180 cells as well (16).
The Gz block induced by VCR was transient, and its duration
was dependent on VCR concentration. Following exposure to
0.1 MVCR, the fraction of cells in 62 fed to control values by
22 hr (Chart 2D). Following exposure to 0.5 and 2 /IM VCR, a
significant decrease in the G2 fraction was observed at 22 hr,
but return to control values did not occur until 26 hr.
The progressive and sustained increase in the G, cell fraction
of 0.2 to 0.3 above the HU-treated controls between 12 and 18
hr at VCR concentrations of 0.5 and 2 <
M (Chart 2D) would
suggest that VCR induced a 62 block, not only in all cells that
were in G2 at the time of drug exposure, but also in S-phase
ceils that were within 6 to 8 hr of mitosis at the time of initial
drug exposure. This is supported by the observations that VCRtreated cells continue to empty out of the S region at up to 18
hr (Chart 2C) but do not proceed from the Gz-M region into the
Gregion until after 18 hr (Chart 2B. 0.5 and 2 ^M VCR). This
would suggest that the wave of cells recruited by HU that was
passing through G. at the time of VCR exposure had a trailing
segment that extended well back into S and included approxi
mately 20 to 30% of the population, i.e., that, initially, HU induced
only partial synchronization of these cells.
Cells arrested in Gz by VCR underwent one of several subse
quent fates. A small fraction of the cells was arrested transiently
in metaphase at 18 and 22 hr (see below); presumably, these
metaphases were included in the descending limb of the wave
of cells in the G2-M region of the histogram (Chart 2D). Some
cells resumed cycling and reappeared in the G, region of the
histogram. A larger proportion of cells exposed to 0.1 MM VCR
recovered than after exposure to 0.5 or 2 UM VCR, and recovery
occurred more rapidly at the lowest drug concentration (Chart
2).Some G2-arrested cells subsequently underwent fragmen
tation and appeared in the pre-Gi region of the histogram (Chart
2A), white others went on to become polyploid cells that ap
peared in the post-Gz-M region of the histogram (Chart 2).
Chart 3 shows the effects on the fractions of cells in different
regions of the DNA histogram of a 4-hr exposure to VCR initiated
at 14 hr after HU exposure. The patterns are somewhat more
complex, due to the fact that cells in the leading segment of the
HU-recruited wave had divided and reappeared in G, between
12 and 14 hr, just prior to VCR exposure (Chart 3B), while cells
in the broader trailing segment of the HU-recruited wave had not
divided yet when VCR was introduced. While VCR promptly
blocked the entry of new cells into d, it did not prevent the 20%
of the total population that had already reentered Gfrom pro
gressing out of the Gregion between 14 and 22 hr (Chart 3,
shaded region and beyond). This subgroup of recruited cells that
had escaped the effects of VCR could be seen passing through
and leaving the S region between 16 and 24 hr (Chart 3C).
Presumably, it was this subgroup of recruited, partially synchro-

study by means of flow cytometry.

Row Cytometry Studies. Chart 2 shows the effects on the
fractions of cete in Afferent regions of the DMA histogram of a
4-hr exposure to VCR that was initiated at 10 hr after HU
exposure. The effects of VCR concentrations of 0.1, 0.5, and 2
ftu are shown in comparison with the effects of initial exposure
to HU alone. The presence of VCR did not prevent the continued
progression of the wave of synchronized cells through the S
region and into the Gz-M region of the DNA histogram at any of
the VCR concentrations studied (Chart 2C, shaded region).
At ail 3 VCR concentrations, cells accumulated in the G2-M

02

10 14 18 22 26 30

TIME, HR
Chart 2. Changes in the tractions of cete as a function of time in diffrent
regions of the DNA histogram in cete exposed to 5 nu* HU for 1 hr and to various
concentrations of VCR at 10 hr. A. PRE-G, (eel fragments) region. 8. G, region. C.
S region. O. G^M region. E. POST GJA region ipredormnanUy pofyploid cete). .
HU controls; T, 0.1 *u VCR; A. 0.5 IM VCR; ,2 jriwVCR. Shaded region, time
of exposure to VCR. Al data points represent the means of 3 experiments. Bars,
S.E. For discussion, see text

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VOL. 43

Vincristine Schedule Dependence

observed at 4 to 8 hr after the removal of VCR.
When Chart 4 is considered together with Charts 2D and 3D,
it becomes apparent that premitotic cells were blocked tran
siently in G2 during VCR exposure and for 2 to 4 hr after drug
removal. Thus, the delayed appearance of the peaks in the
metaphase index until 4 to 8 hr after drug removal, regardless
of the time of VCR exposure (Chart 4), is directly attributable to
prior VCR-induced G2 block.

~0 2

10 14 18 22 26 30 34 38

02

10 14 18 22 28 30 34 38

TIME. HR

Chart 3. Changes in the fractions of cells as a function of time in cells exposed

to 5 mu HU for 1 hr, and to 0.1, 0.5, and 2 IM
VCR at 14 hr. Legend as in Chart
2. Shaded region, time of exposure to VCR. All data represent the means of 3
experiments. Bars, S.E. For discussion, see text.

nized cells that contributed to the late rise and fall in the G2-M
region between 22 and 30 hr (compare Charts 2D and 3D). If
this is so, then the wave of cells in the G2-M region between 14
and 30 hr (Chart 3D) actually consisted of 2 components, namely,
the static VCR-blocked trailing segment of the initial HU-recruited
wave, upon which was superimposed the moving wave of cells
that had already escaped into d by the time of onset of VCR
exposure and had traversed the cell cycle once more over the
succeeding 10 to 12 hr.
It is of some interest that, in this series of experiments, one
can identify clearly a cohort of recruited cells treated with HU
alone in which synchrony was sustained through a second cell
cycle. An initial wave of HU-treated controls could be seen
entering Gat 14 to 16 hr (Chart 30), traversing S between 16
and 24 hr (Chart 3C), and traversing the G2-M region between
22 and 30 hr (Chart 3D). In retrospect, sustained synchrony may
also have been present among the HU controls in Chart 2, to
D, but the synchronized waves are not as well defined.
In summary, Charts 2 and 3 show that the leading segment of
the synchronized wave began to divide between 10 and 14 hr
after HU exposure. Cells that had not divided by the time of
initiation of VCR exposure were promptly blocked in G2; in
contrast, cells that had divided and entered Gat the time of
introduction of VCR were not prevented from traversing the cell
cycle.
Metaphase Index Studies. During the course of the flow
cytometry studies, aliquots of cells were also taken for parallel
determinations of the metaphase index at each time point. The
results are shown in Chart 4. Whether VCR was added at 10 hr
after HU (Chart 4/4) or at 14 hr after HU (Chart 4),there were
only minimal changes in the metaphase index after 4 hr of drug
exposure, and for 2 hr thereafter. Increases in the metaphase
index above 0.1 were observed only in cells exposed to 0.5 and
2 UMVCR at 10 hr after HU exposure (Chart 4/4); peak metaphase
indices ranging from 0.10 to 0.15 were seen at 4 to 8 hr after
VCR removal. When 0.5 and 2 UM VCR were added at 14 hr
after HU, there were modest rises in the metaphase index above
control values; peak values ranging from 0.05 to 0.075 were

AUGUST

The differences in the ranges of peak values of the metaphase

index in Chart 4, A and B, are attributable to differences in the
proportions of cells in the population that were susceptible to
VCR-induced metaphase arrest in the 2 drug schedules. When
VCR was introduced at 10 hr after HU exposure, the HU-recruited
wave was just entering G2 (Chart 2D); when VCR was introduced
at 14 hr, many of the HU-recruited cells had already divided and
entered d (Chart 3B).
It is clear that, of all the cells that were blocked transiently in
G2 by VCR, only a small proportion were subsequently arrested
in metaphase after 4 hr of exposure to the drug. To explore the
possibility that the cells that were closest to mitosis at the time
of VCR addition were those that were more likely to undergo
metaphase arrest once the G2 block was overcome, metaphases
were subclassified with respect to the degree of disruption of
spatial organization of the chromosomes in the metaphase figure
(see "Materials and Methods"). Organized and disorganized
metaphases were then scored separately. The total metaphase
index and organized metaphase index are shown in Chart 5 for
each VCR concentration and drug schedule. At concentrations
of 0.1 UM VCR. nearly all metaphases were organized, whether
the drug was administered at 10 or 14 hr after HU (Chart 5, A
and B, respectively). At the higher drug concentrations, disor
ganized metaphases first appeared at the end of the 4-hr drug
exposure period and peaked at 4 hr after the termination of drug
exposure, regardless of drug schedule (Chart 5, /42,/43, B2, and
B3). In contrast, at 8 hr after the termination of drug exposure,
the metaphase peaks consisted almost entirely of organized
metaphases. These data are consistent with the premise that
the cells closest to mitosis were the first to overcome VCRinduced G2 block and, subsequently, exhibited the greatest
degree of disruption of metaphase organization. Presumably,
cells that were 2 to 4 hr away from mitosis at the time of VCR
exposure overcame VCR-induced G2 block at 4 to 8 hr after

26X02

61014182226303438
TIME,

HR

Chart 4. Changes in the metaphase index as a function of time in cells exposed

to 5 rriM HU for 1 hr, and to 0.1, 0.5, and 2 ^M VCR at 10 hr (A) or at 14 hr (fl).
Legend as in Chart 2. Shaded region, time of exposure to VCR. All data points
represent the means of 3 experiments. Bars, S.E. For discussion, see text.

3601

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H.Mujagic
etal.
0.15

0.10

6 10 14 18 22 26 30

02

6 10 14 18 22 26 30 34 38

TIME,HR
Charts. Changes in thtotal metaphase index ( )and in the organized
metaphase index (
)as a function of time after exposure to 5 mw HU for
1 hr and 0.1, 0.5, or 2 UM VCR at 10 hr (A,, A,, and A3, respectively) and 0.1, 0.5,
or 2 put VCR at 14 hr (,,B,and B3, respectively). Difference between total and
organized metaphase index at each time point represents the fraction of disorga
nized metaphases. Shaded region, time of exposure to VCR. Each data point
represents the mean of 3 experiments. Bars, S.E. For discussion, see text.

drug removal, and their metaphases exhibited a greater degree

of organization.

DISCUSSION
Effects of VCR in Relation to the Cell Cycle. Published
studies on the cell cycle-dependent lethality of VCR have yielded
seemingly conflicting results. Madoc-Jones and Mauro (13) and
Hill and Whelan (9) have claimed that VCR is S-phase-specific
on the basis of their studies in HU-synchronized and mitotically
selected cells, respectively. That is, VCR sensitivity was thought
to peak during S and decline thereafter as cells approached
mitosis. In contrast, the studies of Wibe (25) in mitotically se
lected cells suggested that sensitivity to the lethal effects of VCR
increased progressively as cells traversed the cell cycle, i.e., that
cells in G2 were more sensitive to VCR than were cells in S.
Studies in asynchronously growing cells double-labeled with
[3H]dThd and [14C]dThd have suggested that cells in both S and
G2, at the time of VCR exposure, were later arrested in meta
phase and subsequently went on to become necrotic (5, 10).
These findings were subsequently confirmed in time-lapse cine
matography studies (12).
Cell survival studies in synchronized cells must be interpreted
with care. Artifacts associated with the synchronization proce
3602

dure itself can lead to erroneous conclusions regarding the phase

specificity of drug lethality. For example, the mitotic shake tech
nique might select for the most rapidly proliferating component
of a population, and this selected cohort might traverse the cell
cycle more rapidly than expected. On the other hand, synchro
nizing agents such as HU are cytotoxic and might introduce
unwanted retardant effects on cell cycle progression. Regardless
of which method is chosen, the degree of synchronization can
be expected to diminish progressively as cells traverse the cell
cycle (22), and this, too, can affect the interpretation of cell
survival studies.
Since the technique of mitotic selection does not provide
sufficient numbers of cells for analysis by flow cytometry, we
used HU synchronization in order to examine directly the behav
ior of synchronized sarcoma 180 cells before, during, and after
exposure to VCR for 4 hr. Although the cell survival patterns
superficially resembled those that might be expected for a cycle
phase-specific drug (Chart 1), a more detailed analysis of the
data showed that the sensitivity of cells to the lethal effects of
VCR increased progressively as they traversed S and G2. It was
apparent from our serial flow cytometry studies that the HUsynchronized cohort of cells actually traversed the late portion
of cell cycle in a broad wave with a relatively long trailing segment
(Charts 2 and 3). The apparent decrease in VCR lethality between
12 and 18 hr after exposure to HU (Chart 1) could be attributed
to the division of an increasingly larger proportion of cells in the
HU-recruited cohort and their entry into a relatively resistant G
phase during this period (Charts 2B and 38). There were striking
differences in the subsequent behavior of cells that had just
divided prior to VCR exposure and cells that were approaching
mitosis but had not yet divided at the time of VCR addition. VCR
had no effect on the movement out of d of cells that had already
divided (Chart 3)and had no effect on their subsequent move
ment through S (Chart 2C). However, the entry of new cells into
d was halted rapidly (Charts 2B and 3B), and cells began to
accumulate in the G2-M region of the histogram (Charts 2D and
3D) soon after the introduction of VCR.
In the present study, there were several other indications that
cells approaching mitosis were especially sensitive to the effects
of VCR. The transient blockade of cells in G2 (Charts 2D and 3D)
is itself a manifestation of this phenomenon. Our metaphase
index data are also consistent with the premise that the G2 cells
closest to the end of the cell cycle at the time of initial exposure
to VCR were the cells that were likely to exhibit the greatest
degree of mitotic disorganization when they overcame the G2
block and entered metaphase (Chart 5).
Mechanisms of VCR Lethality. The finding that sensitivity to
VCR lethality increases progressively as cells traverse the late
portion of the cell cycle may have important implications with
regard to understanding the relation between known molecular
mechanisms of VCR action and drug lethality. VCR is known to
bind specifically to tubulin (17-19), a major component of intracellular microtubular structures that are essential for a variety of
normal cell functions, including cell division. The lethal effects of
the Vinca alkaloids are commonly attributed to their tubulinbinding properties (23). Historically, the primary target of tubulinbinding agents was thought to be the mitotic spindle, the disrup
tion of which prevents centriole migration (2, 8, 21). However,
other microtubule structures, such as the kinetochore microtubules, are also involved in the mitotic process. The transient
VCR-induced G2 block observed in these and other published
CANCER

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RESEARCH

VOL. 43

Vincristine Schedule Dependence

studies (25) may reflect the disruption or prevention of formation
of microtubular structures associated with very early stages of
mitosis. Microtubules are also involved in the maintenance of cell
shape and locomotion; disruption of the cell cytoskeleton may
account for reported observations of interphase cell lysis by
tubulin-binding agents (12, 13, 20).
Total cell tubulin content normally doubles between cell divi
sions, increasing in sigmoidal fashion as cells progress through
the cell cycle (4, 7). Peak rates of tubulin synthesis occur during
a phase of the cell cycle that overlaps S and G2 (4). Using
fluorescent antitubulin antibody to determine cell tubulin content,
we have shown by flow cytometry that the cell tubulin content
distribution in Sarcoma 180 cells spans a 2-fold range and
exhibits a prominent postmitotic peak and a saddle shape, much
like the DMA histogram (15). This would imply that cell tubulin
content increases in parallel with cell DMA content as cells
progress through the cell cycle; like cell DMA content, cell tubulin
content doubles between cell divisions and halves at mitosis.
It is conceivable that the increase in sensitivity to the lethal
effects of VCR during the course of cell cycle progression in
Sarcoma 180 cells might be related to the increase in cell tubulin
content during the course of cell cycle progression. That is, cells
exposed to VCR before they have synthesized a full complement
of tubulin might, after VCR removal, go on to synthesize a
sufficient amount of new, VCR-free tubulin to form the microtu
bular structures that are required for mitosis. On the other hand,
cells in which tubulin synthesis had nearly been completed at the
time of VCR exposure would not synthesize enough new tubulin
for normal cell division, even if the drug were removed prior to
mitosis. Additional studies will be required to explore this possi
bility.

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