[CANCER RESEARCH 54, 4355-4361.
Augusl 15. 1W4]
Paclitaxel Inhibits Progression of Mitotic Cells to G! Phase by Interference with
Spindle Formation without Affecting Other Microtubule
Functions during Anaphase and Telephase
Byron H. Long1 and Craig R. Fairchild
Department of Experimental
Therapeutics. Oncology Drug Discovery. Bristol-Myers Squibb Pharmaceutical
ABSTRACT
Very low concentrations of paclitaxel, a clinically active anticancer
agent isolated from the bark of the Pacific yew tree, were found to produce
micronuclei in human colon carcinoma cells, suggesting inhibition of
mitotic spindle assembly or function. The possibility that paclitaxel acts at
the level of the mitotic spindle was investigated by evaluating its ability to
inhibit the progression of mitotic cells to G, phase. Paclitaxel inhibited
mitotic progression with a median inhibitory concentration of 4 ini, a
concentration equivalent to the median cytotoxic concentration, without
arresting cells in mitosis. A direct correlation was shown to exist between
the cytotoxic potency and ability to inhibit mitotic progression for ana
logues of paclitaxel and antimicrotubule agents but not for the topoisomerase II-active agents etoposide and teniposide. After release from the
Research Institute. Princeton. New Jersey 08543-4000
and microtubules toward microtubule assembly (13-18). Paclitaxel
preferentially and reversibly binds to microtubules, rather than
tubulin dimers, at sites distinct from the binding sites of GTP,
colchicine, vinblastine, or podophyllotoxin.
Once bound, pacli
taxel induces tubulin polymerization to form microtubules, even in
the absence of GTP (15-18). Microtubules formed in the presence
of paclitaxel are stable to conditions that would cause microtubules
formed by GTP to disassemble, such as treatment with 4 HIM
calcium ions or low temperatures (15-18, 23).
The most visible effect of paclitaxel on cells is the formation of
microtubule bundles in interphase cells and spindle asters during
mitosis, which are visualized by indirect immunofluorescence (2133). Micromolar concentrations of the drug are generally used to
nocodazole block, cells synchronized in mitosis remained sensitive to very
cause pronounced bundling of microtubules in cells. Like other milow concentrations of paclitaxel for <30 min, the time required for spindle
crotubule-active agents or antimicrotubule agents known originally as
formation, yet remained sensitive to vinblastine for >90 min. This result
spindle poisons, paclitaxel arrests cell cycling in mitosis (23, 34).
indicates that very low concentrations of paclitaxel inhibit formation of
Other mechanisms of action have been proposed for paclitaxel as
mitotic spindles in cells without affecting function of preformed spindles
and without arresting cells in mitosis. Continuous exposure to low 11:1110- well. Paclitaxel inhibits the transition from G0 phase to S phase in
serum-starved fibroblasts stimulated by addition of growth factors,
molar concentrations of paclitaxel for more than one cell cycle resulted in
cells with DNA contents >4C and as much as 8C. These results support a
suggesting that its effect on the interphase cytoskeleton may disrupt
hypothesis that, by not being capable of segregating sister chromatids,
normal functions of the cell membrane, transmembrane signaling,
paclitaxel-treated cells eventually reform nuclear membranes around in
intracellular transport, or locomotion (35-41). This effect on locomo
dividual or clusters of chromosomes, revert to G, phase cells containing
tion has been expanded to include an observed inhibition of the
4C DNA, and enter S phase, resulting in cells with as much as 8C DNA
invasiveness of a metastatic variant of PC-3 human prostatic tumor
content. It is proposed that this is the primary cytotoxic mechanism of
cells (39). Recent findings also suggest that paclitaxel may play a role
paclitaxel.
in modulating either the interactions of growth factors with their
receptors on the cell surface or the resulting intracellular signaling.
INTRODUCTION
For example, paclitaxel increases the amount of tumor growth factor
Paclitaxel (formulated as Taxol®)is an exciting new cancer chemRNA and tumor growth factor being released by macrophages, a
property
mimicking the effect of endotoxic bacterial lipopolysacchamotherapeutic drug with antitumor activity against ovarian, breast,
ride on these cells (40, 41).
and lung carcinomas (for recent reviews, see Refs. 1-4). This com
A major concern with essentially all of the above described effects
pound is extracted from the bark of the Pacific yew tree Taxus
of paclitaxel is the high (generally micromolar) concentrations used to
brevifolia, as well as from the needles and stems of this and other
Taxus species, and its chemical structure has been identified (5-8). Its produce the described effects. Paclitaxel usually is cytotoxic or cytostatic at concentrations ranging from 1 to 20 nM (5, 6, 9, 10). The
activity as an antitumor agent was recognized in early preclinical
possibility that paclitaxel produces its cytotoxicity by action at the
research involving in vivo treatment of P388 murine leukemia in mice
level of the mitotic spindle is only assumed from observations of its
(5, 6, 9-12). Interest in this compound stems not only from its clinical
ability
to arrest cycling cells in mitosis (15, 34). We describe here a
activity against poorly responsive solid tumors but also from its
novel procedure for evaluating the effects of agents that may act on
unique mechanism of action (1-4, 13, 14).
mitotic spindle formation or function and, using this assay, report that
The primary target responsible for the cytotoxic properties of
very low concentrations of paclitaxel inhibit human colon carcinoma
paclitaxel appears to be the microtubule, based on two observations:
cells chemically synchronized in mitosis from progressing to G,
the induction of tubulin assembly in vitro (15-22) and the induction
of tubulin assembly and the formation of microtubule bundles in phase. Furthermore, this inhibition occurs only during the time when
mitotic spindles are being formed and does not occur once the spin
cells (21-27). However, unlike other microtubule active agents,
dles are formed.
such as colchicine and the Vinca alkaloids, which induce micro
tubule disassembly, paclitaxel promotes microtubule assembly by
shifting the dynamic equilibrium existing between tubulin dimers
MATERIALS AND METHODS
Received 7/30/93: accepted 6/17/94.
The costs of publication of this article were defrayed in part hy the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
' To whom requests for reprints should he addressed, at Department of Experimental
Therapeutics (K.21I4E). Bristol-Myers Squibb, Co.. P.O. Box 4000. Princeton, NJ
08543-4(KX).
Chemicals. Paclitaxel and all analogues were provided by Dr. Vittorio
Farina (Central Chemistry, Pharmaceutical Research Institute, Bristol-Myers
Squibb. Inc.. Wallingford, CT) and were all at least 98% pure, as determined
by spectroscopic (nuclear magnetic resonance, mass spectroscopy) and ana
lytical methods. VP-16,2 VM-26, vinblastine, and colchicine were also ob
tained as pure, unformulated compounds from Research Compounds (Propri-
4355
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PROGRESSION
etary Information
Department,
Pharmaceutical
Research
INHIBITION
Institute,
OF M TO G, BY PAC'LlTAXIiL
Bristol-
Myers Squibb).
Cell Culture. Human colon carcinoma (HCT116) cells (42) were main
tained in McCoy's 5A medium (modified; GIBCO) containing 10% fetal
bovine serum (heat inactivated; GIBCO).
Cytotoxicity Evaluation. Cytotoxicities of paclitaxel, analogues of paclitaxel, and other cytotoxic agents were assessed using HCT116 cells and a
colorimetrie assay for number based on the metabolic conversion
of
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2Htetraxolium hydroxide to a reduced form that absorbs light at 450 nm. Cells
were plated at 4000 cells/well in 96-well microtiter plates, and drugs were
added with serial dilution. The cells were incubated at 37°C for 72 h,
at which time 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetraxolium
hydroxide (Polysciences, Warrington, PA) was added.
Following a 3-h incubation, the differences in /4450 of the medium were
measured with a spectrophotometer.
A proportional relationship has been
shown to exist between the number of live cells in each well and the resulting
increase in absorption at 450 nm. Results are expressed as the IC50 values.
Fluorescence Microscopy. HCT116 cells grown on coverslips were incu
bated in medium containing different concentrations of paclitaxel for 48 h, then
were washed free of medium in Dulbecco's phosphate-buffered saline, and
were fixed for 5 min at room temperature with 4% paraformaldehyde in 0.08
M sodium phosphate buffer at pH 7.4. The fixed cells were permeabilized by
placing slides in 100% ethanol for 2 min at room temperature. Following
overnight rehydration in Dulbecco's phosphate-buffered saline, the cells were
indirectly immunofluorescently
stained by incubation at 37°Cwith mouse
monoclonal anti-a tubulin (clone DMIA, diluted I/SIX); Sigma) and goat
anti-mouse IgG coupled with fluorescein (Cappel, diluted 1/10). Visualization
was accomplished using a Nikon Optiphot-2-EF-D/DIC
(Garden City, NY).
Cell Cycle Arrest. Cells grown in 25-cm2 tissue culture flasks were incu
bated with different concentrations of nocodazole (Sigma) or paclitaxel (Cen
tral Chemistry, Bristol-Myers Squibb) for 24 h. Cells were fixed in 70%
methanol, treated with 1 mg/ml bovine pancreas ribonuclease A (type III-A;
Sigma) at 4°overnight, and stained with 50 /¿g/mlpropidium iodide (Sigma)
for 30 min. Cell cycle analysis was conducted with a Coulter Epics Profile II
(fluorescence flow cytometer, Hialeah. FI_).
Inhibition of Mitotic Progression. HCT116 cells were incubated with 0.2
/j.g/ml nocodazole for 15 h to synchronize the cells in early M phase. Mitotic
cells shaken off the surface of each flask and harvested by centrifugation were
washed free of nocodazole by resuspension in cold medium and centrifugation.
For evaluation of the ability of nocodazole-arrested mitotic cells to progress to
G, phase, the cells were added immediately to fresh, warm, drug-free medium
for continued incubation at the designated temperature. Aliquots were either
directly removed or removed after designated incubation times and were fixed,
stained, and analyzed for DNA content as described above. For inhibition of
mitotic progression, mitotic HCT116 cells were washed free of nocodazole and
added immediately to fresh medium containing different dilutions of pacli
taxel, analogues, or other reference compounds. After incubation at 37°Cfor
6 h, the cells were fixed, stained with propidium iodide, and evaluated for
DNA content by flow cytometry. Assessment of sensitivity windows for
inhibition of mitotic progression was accomplished by adding mitotic HCT116
cells either immediately to fresh medium containing different dilutions of
vinblastine or paclitaxel or immediately to fresh, warm, drug-free medium for
continued incubation at 37°C.Aliquots of mitotic cells subjected to incubation
in drug-free medium were removed at designated
times and were added to
fresh medium containing different dilutions of vinblastine or paclitaxel. Cells
incubated with either vinblastine or paclitaxel were allowed to incubate for a
total of 6 h after removal from nocodazole before being fixed, stained, and
analyzed for DNA content, as described above.
RESULTS
Formation of Multiple Micronuclei in Cells by Paclitaxel. When
HCT116 cells, a human colon carcinoma cell line, were continuously
exposed to different concentrations of paclitaxel for 48 h, tubulin
2 The abbrevialions used are: VP-16, eloposide; VM-26; teniposide; IC51I,concentra
bundling, observed by indirect immunofluorescence staining, was
very pronounced in the cells exposed to micromolar concentrations of
paclitaxel and was observed in cells exposed to concentrations as low
as 0.1 /J.Mfor 48 h. Most striking, however, was the observation of
numerous multinucleated cells containing micronuclei at all concen
trations of paclitaxel <0.1 JJ.Mbut most obviously at 10 nM (Fig. 1).
The presence of multiple micronuclei suggests that the mitotic spindle
failed to segregate the sister chromatids properly, resulting in the
reformation of nuclear membranes around groups of tetraploid chro
mosomes and the failure of daughter cells to form without the tight
clustering of segregated chromosomes around the individual poles.
This effect, which was first reported by Brues and Jackson in 1937
(43), would be fatal to dividing cells and would not be relevant to
nondividing cells.
Cell Cycle Arrest by Nocodazole and Paclitaxel. To test this
possibility, a system was devised to evaluate the effects of very low
concentrations of paclitaxel on spindle formation and function. One
means of evaluating the formation of functioning mitotic spindles in
cells would be to observe the ability of mitotic cells to convert to G,
phase daughter cells. This could be accomplished with cells chemi
cally synchronized in M phase with a readily reversible agent and
evaluation of the effects of paclitaxel on the progression to G, phase
after removal from the arresting agent. The antimicrotubule agent
nocodazole is an ideal synchronizing agent by virtue of its relatively
rapid release from tubulin and exit from cells when the surrounding
drug-containing medium is replaced with drug-free medium. This
rapid release from cells results in the equally rapid formation of
symmetric mitotic spindles and normal cell division (44, 45).
The ability of nocodazole and paclitaxel to arrest cells in mitosis
was determined by flow cytometry. More than 90% of the HCT116
cells were found in mitosis after 24 h of incubation with nocodazole
concentrations >0.16 JU.M(Fig. 2A). The effect of paclitaxel on
HCT116 cells is shown in Fig. 2B. Paclitaxel also arrested cells in
mitosis, with the number approaching 90% at the higher concentra
tions and an EC50 of approximately 25 nM, which is >6-fold higher
than the median cytotoxic concentration of 4 nM (see below). In fact,
no increase in mitotic cells over control levels was observed when the
cells were exposed to 4 nM paclitaxel, suggesting that mitotic arrest
may not be relevant to the mechanism of action of paclitaxel. Visual
microscopic inspection of the cells exposed to paclitaxel confirmed
the flow cytometry results.
Microscopic observation of HCT116 cells continuously exposed to
10 nM paclitaxel revealed that a large percentage of the cell population
appeared as mitotic cells between 12 and 18 h, this number decreased
with the appearance of cells containing multiple micronuclei after 24
h, and cell number began to decrease between 36 and 48 h of
continuous exposure.3 Interestingly, approximately 5% of the cells
exposed to 8-16 nM paclitaxel for 24 h contained >4C DNA content,
which increased to 25% after 48 h. Essentially none of the untreated
or nocodazole-treated cells had DNA contents greater than 4C, as
determined by flow cytometry.3 One possible explanation for this
observation is that these cells did not arrest in mitosis but reverted
back to G, phase with a 4C content and then proceeded on to S phase
with an additional round of DNA synthesis.
Progression of Mitotic Cells to G, Phase. Once nocodazole syn
chronized mitotic cells are incubated in drug-free medium at 37°C,
they progress toward G, phase with a peak population of G, cells
appearing after 6 h, after which the percentage decreases as cells begin
to enter S phase and a substantial increase in S phase cells appears
after 10 h of incubation (Fig. 3). The effect of incubation temperature
tion of compound required to inhibit cell proliferation (i.e., AA50) to 50% of thai of
untreated control cells; EC,,,, medium effective concentration.
^ B. H. Long and F. Y. Lee, manuscript in preparation.
4356
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PROGRESSION
INHIBITION
OF M TO 0,
BY PACL1TAXEL
b
Fig. 1. Light microscopy of HCT116 cells incu
bated without and with paclilaxcl for 48 h. «and <•,
without paclitaxel: ft and/, with 10 nu paclitaxel; c
and g. with 30 nMpaclilaxel: d and h, 100 nMpacli
taxel. Visible microscopy («-</)and indirect immunofluorescent microscopy of microtubules within cells
(c-h).
'
-
on mitotic progression was investigated since it is well known that
microtubule function is substantially more sensitive to reduced tem
perature than are enzyme reactions. Incubation of cells at 30°Cslowed
their appearance as G, phase cells to 9-10 h without an appreciable
increase in S phase cells over the time course of the study (results not
shown).
Inhibition of Mitotic Progression by Paclitaxel and Other Taxanes. Experiments were conducted to evaluate the effects of pacli
taxel on the ability of nocodazole-arrested HCT116 cells to progress
to G, phase after release from the nocodazole block. Mitotic cells
incubated with different concentrations of paclitaxel were inhibited
from progressing to G, phase 6 h after release from the nocodazole
block, with a median inhibitory concentration of 4 nw (Fig. 4). This
concentration is equivalent to the median cytotoxic concentration for
HCT116 cells after 72 h incubation with paclitaxel (Table 1).
It is possible that any cytotoxic agent could produce a similar
inhibition of mitotic cells progressing to G, phase. In order to inves
tigate this possibility, further characterization of the inhibition of
mitotic progression assay was accomplished by evaluating the effects
of other taxanes, antimicrotubule agents, and unrelated cytotoxic
agents on mitotic progression. Fig. 5 presents results of two such
experiments, and these results are summarized in Table 1. These
experiments included 10-acetyldocetaxel, 7-epipaclitaxel, 2'-O-methylpaclitaxel, and baccatin III as other active and inactive taxanes
(46-49); vinblastine and colchicine as other types of antimicrotubule
agents (50-54); VM-26 and VP-16 as unrelated cytotoxic agents that
act through a well-defined, alternative mechanism, namely, by action
on eukaryote topoisomerase II (for reviews, see Refs. 55-58); and
dimethylsulfoxide as a solvent control.
The results expressed as EC5(>values and summarized in Table 1
reveal that both VM-26 and VP-16, compounds having a mechanism
of action unrelated to that of paclitaxel (55-58), inhibited mitotic
progression at concentrations 59- and 41-fold higher than required
for cytotoxicity, whereas the concentrations for inhibiting mitotic
progression by paclitaxel, 7-epipaclitaxel, 10-acetyldocetaxel, 2'-Omethylpaclitaxel, baccatin III, colchicine, and vinblastine were at or
only slightly above the concentrations required for their respective
median cytotoxic effects. The close relationship inhibition of mitotic
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PROGRESSION
INHIBITION
OF M TO G, BY PACLITAXEL
Sensitivity Window for Inhibition of Mitotic Progression by
Paclitaxel. Although this assay distinguishes between cytotoxic
agents having mechanisms of action involving targets other than
microtubules and tubulin, it does not appear to distinguish between
tubulin-polymerizing activity of paclitaxel and those of classical an-
100
timicrotubule agents, such as the Vinca alkaloids. In this regard, it is
possible that paclitaxel inhibits the formation of mitotic spindles in
cells without affecting their function, once assembled. This effect is
different from classical antimicrotubule agents, which not only
prevent the assembly of tubulin into mitotic spindles but also facil
itate the disassembly of preformed mitotic spindles or other mi
crotubules functioning in the mechanism of cytokinesis and sepa
ration of daughter cells in anaphase.
To test this possibility, nocodazole-synchronized mitotic cells were
incubated for different durations up to 90 min before addition of either
paclitaxel or vinblastine and the progression to G, phase was assessed
(Fig. 7). Mitotic cells either treated directly with paclitaxel or allowed
to incubate at 37°Cfor 15 min were equally inhibited from progress
Si
(9
Concentration
(.uM)
ing to G, phase, with an EC5I>of 10 nM. Interestingly, mitotic cells
preincubated for 30 min in drug-free medium before exposure to
paclitaxel yielded an EC5()of 30 nM, and mitotic cells preincubated for
60 min yielded an ECSO>100 nM (Table 2). It should be pointed out
Concentration
(nM)
Fig. 2. Cell cycle arresi by nocodazole and paclitaxel. HCT116 cells were incubated
with nocodazole (.4) or paclitaxel (B) for 24 h, and the resulting cell populations were
analyzed for 2C and 4C DNA content by fluorescence-activated flow cytometry.
o
S
a
o.
o
O.
10
Concentration
100
(nM)
Fig. 4. Inhibition of mitotic progression by paclitaxel. Mitotic HCT116 cells were
washed free of nocodazole and added immediately to fresh medium containing different
dilutions of paclitaxel. After incubation at 37°Cfor 6 h, the cells were fixed, stained with
Time (hr)
Fig. 3. Progression of nocodazole-arrested milotic HCT116 cells to G, and S phases
after replacement of medium containing nocodazole with drug-free medium. HCT116
cells incubated with 0.2 fig/ml nocodazole for 15 h to synchronize the cells in early M
phase were added immediately to fresh, warm, drug-free medium for continued incubation
at 37°C.Aliquots were either directly removed or removed after designated incubation
times and fixed, stained, and analyzed for DNA content by fluorescence-activated flow
cytometry. Point (bar), mean (±SD).
propidium iodide, and evaluated for DNA content by flow cytometry. Points (bars),
means (±SD) obtained from 6 separate experiments.
Table I Median concentrations for inhibition of mitolic progression by paclitaxel,
other taxanes, antimicrotubule agents, and other cytotoxic agents
Data were obtained from results shown in Fig. 5.
ofinhibitory
mitoticprogression(IC50,
of
mitoticprogression
tocytotoxicity1.02.02.51.01.40.41.2
CompoundPaclitaxel7-Epipaclitaxel2'-O-Melhylpaclitaxel10-AcetyldocetaxelBaccatin
(¿M)0.004
JIM)0.004
progression is demonstrated both by the ratio between these two
parameters shown in Table 1 and the proximity of points to a theo
retical diagonal line in a plot comparing potencies for inhibition of
mitotic progression versus cytotoxicity for each compound (Fig. 6).
Baccatin III, although described as inactive because it had no effect
on tubulin in vitro (46-49), may actually be cytotoxic by this mech
0.0010.002
±
0.0010.81
±
0.050.002
±
0.0000.35
±
IIIVinblastineColchicineVM-26VP0.010.005
±
0.0020.016
t
0.0010.17°1.3
±
anism, albeit at about l/100th the potency of paclitaxel. Concen
trations of dimethylsulfoxide up to 5% were without effect (results
not shown).
16Dimethyl
0.31.5%"Inhibition
±
sulfoxideCytotoxicitv(IC50,
' Historic data.
0.0020.004
±
0.00320.0020.50.0020.0210555%Ratio
±
4358
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PROGRESSION
O
INHIBITION
OF M TO G, BY PACLITAXEL
120
DISCUSSION
100-
From these observations, it is proposed that the primary effect of
paclitaxel is to interfere with the assembly of the mitotic spindle,
resulting in the failure of chromosomes to segregate. Without chro
mosome segregation, the HCT116 cells are inhibited from progressing
to G[ phase but apparently revert back to a pseudo G, phase after an
unknown period of time without arresting in mitosis. This reversion
results in cells containing multiple micronuclei formed by the decondensation of the resulting tetraploid chromosomes and nuclear mem
brane envelopment around chromosome clusters and individual chro
mosomes, which then have the propensity to progress to S phase.
These results also show that paclitaxel does not have an inhibiting
effect on spindle function once formed. The apparent inconsistent
results that HCT116 do not arrest in mitosis after exposure to 10 nM
paclitaxel for 24 h (Fig. 2) but are inhibited from progression to G,
80-
O
_o e
"S
40 -
S ¿
20-1
10-Ac-Docetaxel
•Paclitaxel
•Baccatin III
D 2'-O-melhylpaclitaxel
Sc S 60-
A
1 O'J
10"^
10"'
Concentration
10"
7-Epipaclilaxel
10
(uM)
0)
120-
O
O
Concentration
(uM)
Fig. 5. Inhibition of mitotic progression by paclitaxel, other taxanes, antimicrotubule
agents, VP-16, or VM-26. Mitotic HCT116 cells, prepared as described in Fig. 3, were
added immediately to fresh medium containing different dilutions of paclitaxel or other
cytotoxic agents, which were processed as described in Fig. 4. A, 10-acetyldocetaxel,
paclitaxel. baccatin III, 2'-O-methyipaclitaxel, and 7-epipaclitaxel; B, paclitaxel, vinblas-
,!¿C
(O O
20-
tine, colchicine, VM-26, and VP-16.
It
10"
10' '
10"
Vinblastine
.001
10 '
10-=
Concentration
(nM)
Paclitaxel Concentration
(nM)
10
Cytotoxicity
(Median Concentration,
\iM)
Fig. 7. Evaluation of a paclitaxel sensitivity window for inhibition of mitotic progres
sion. The existence of a window of sensitivity for paclitaxel was demonstrated by
preincubation of mitotic arrested HCT116 cells for different limes in drug-free fresh
medium before addition of aliquots of cells to medium containing different dilutions of
vinblastine (A ) or paclitaxel (B) for continued incubation at 37°Cfor a total of 6 h before
Fig. 6. Comparison of potencies of the compounds shown in Table 1 for their abilities
to inhibit mitotic progression plotted against their cytotoxic potencies.
being fixed, stained, and analyzed for DNA content.
that at least 80% of the cells preincubated for 90 min in drug-free
medium progressed to G[ phase in the presence of 3 JAMpaclitaxel.
Vinblastine did not produce a similar effect, in that preincubation of
mitotic cells for up to 90 min in drug-free medium had little effect on
the EC50 values for inhibiting progression of mitotic cells to G! phase
(Table 2). The small percentage of resistant cells seen after 60- and
90-min preincubation periods (Fig. 7/4) represent, in part, G, phase
cells appearing during preincubation (see Fig. 3).
Table 2 Median inhibitory concentration for inhibition of mitolic progression after
preincubation of mitotic arrested HCT116 cells in the absence of noeodazole for
different times
Data were obtained from results shown in Fig. 6.
Preincubation time before addition of inhibitor (min)
30
Compound
Paclitaxel
Vinblastine10
60
90
41053051605»30007
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PROGRESSION
INHIBITION
OF M TO G, BY PACLITAXEL
phase at this concentration (Fig. 4) can be explained by considering
the following: (a) 10 nM paclitaxel was observed to cause a slowing
of mitotic progression of an asynchronous cell population, as evi
denced by increased presence of mitotic cells between 12 and 18 h of
exposure;3 (b) the inhibition of mitotic progression observed in a
synchronized cell population is most likely a slowing of the appear
ance of G, phase cells rather than a true inhibition, per se. The overall
effect of this slowing is that the cell cycle clock continues in HCT116
cells and chromosome decondensation begins before chromosome
segregation and cytokinesis can occur.
De Brabander et al. (28-31) showed that paclitaxel treatment of
PtK2 cells resulted in the gradual disappearance of microtubules
emanating from the centrosomes of interphase cells and from centrosomes and kinetochores of mitotic cells. This disappearance was
followed by the appearance of microtubules unassociated with cen
trosomes, kinetochores. or other organdÃ-es (28-31). Cells pretreated
with nocodazole to depolymerize microtubules readily reformed mi
crotubules elongating from centrosomes in interphase cells and re
formed functional spindles in mitotic cells upon replacement of nocodazole-containing
medium with drug-free medium (59, 60).
However, nocodazole-pretreated cells exposed to paclitaxel in the
absence of nocodazole only formed free-floating microtubules,
whereas pretreated cells exposed to paclitaxel in the presence of
nocodazole-assembled centrosome organized microtubules. The pre
dominant conclusion from these observations was that paclitaxel
decreases the critical tubulin concentration within cells, causing tubulin to spontaneously polymerize and microtubules to grow from
these spontaneous centers rather than from centrosomes. Nocodazole
has the opposite effect, in that it raises the critical tubulin concentra
tion, causing disassembly of microtubules. Appropriate concentra
tions of nocodazole and paclitaxel counteract the influence each has
on the critical tubulin concentration, resulting in normal microtubule
production (28-31).
The results presented in Figure 7 indicate that a very short window
of sensitivity to paclitaxel exists in HCT116 cells immediately fol
lowing release from nocodazole-induced cell cycle arrest in mitosis.
Generally, replacement of nocodazole-containing medium with drugfree medium results in short microtubules typically appearing around
the centrosomes of mitotic cells within 5-10 min. This is followed by
preferential elongation of microtubules between the centrosomes and
kinetochores, resulting in the formation of normal metaphase spindles
within 40-60 min (59, 60). The fact that this sensitivity window is so
short in duration provides support for the hypothesis that paclitaxel
disrupts mitotic spindle assembly without having much effect on the
function of a preformed mitotic spindle apparatus and the subsequent
ability of cells to progress to G, phase. Results obtained with vinblastine where mitotic cells incubated in the absence of any drug for
as long as 90 min still retained sensitivity to vinblastine demonstrate
the importance of microtubule integrity beyond the first 90 min in
order for the nocodazole-blocked mitotic cells to successfully pro
gress to G | phase. Thus, concentrations of paclitaxel between 3 and 10
nM are not only cytotoxic to HCT116 human colon carcinoma cells
after a 72-h exposure but also inhibit mitotic cells from progressing to
G, phase after a 6-h exposure (Fig. 3). These low concentrations also
induce the formation of multiple micronuclei in an asynchronous cell
population after continued exposure (Fig. 1) without arresting cell
cycling in mitosis (Fig. 2). Furthermore, there exists a paclitaxel
sensitivity window for the inhibition of mitotic progression of <30
min, during which time the mitotic spindle is being formed.
Recently, Jordan et al. (61-63) proposed that the Vinca alkaloids,
podophyllotoxin, and nocodazole produce cytotoxicity by causing
aberrant organization of metaphase chromosomes as the result of
arresting cells in mitosis and that the observed mitotic arrest occurred
Cell Cycle
G,
S
DNA Content
2C
>2C
- S
G¡
4C
>2C
4C
62'
4C
>4C
8C
Fig. 8. Model for effects of paclitaxel on cell cycle progression. Asterisk denotes a new
cell cycle in which cytokinesis does not occur.
primarily by inhibiting the dynamics of spindle microtubules rather
than by depolymerizing the microtubules. These conclusions were
based on the observations that a greater correlation was observed
between cytotoxic potency and mitotic arrest than between cytotoxic
potency and cither microtubule depolymerization or spindle disorga
nization produced by either different Vinca alkaloids or different
chemotypes. Thus, it appears that antimicrotubule-active agents prod
uce cytotoxicity by interfering with the dynamics of spindle microtu
bules, as part of spindle function, whereas the tubulin-polymerizing
agent paclitaxel produces cytotoxicity by interfering with spindle
formation rather than spindle function.
It is possible that very low concentrations of paclitaxel could be
sufficient to have an effect on the critical tubulin concentration within
prophase cells, such that mitotic spindle formation would be inhibited
to the extent that normal chromosome segregation is impaired. The
cell cycle clock, which appears not be delayed in HCT116 cells by
these low paclitaxel concentrations, triggers chromosome deconden
sation and nuclear membrane envelopment of individual or groups of
unsegregated, tetraploid chromosomes, resulting in the formation of
micronuclei. These cells then proceed to S phase, yielding cells with
as much as 8C DNA content (see Fig. 8). It is proposed that this
process ultimately results in the selective cytotoxicity of proliferating
cells, which most likely die because of either a disproportionate
distribution of chromosomes in daughter cells from parent cells that
do eventually undergo cytokinesis or gene dose problems resulting
from tetraploidy and octaploidy.
The inhibition of mitotic progression also provides an assay for
biological function of an analogue or for assessing the metabolic
conversion of a prodrug to a biologically active compound. We have
shown that a very good correlation exists between the cytotoxic
potency and potency for inhibition of mitotic progression of a taxane
analogue (Table 1, Fig. 6), suggesting that the two parameters are
related.
ACKNOWLEDGMENTS
The authors wish to thank Drs. Anna Maria Casazza and Susan Horwitz for
the critical reading of the manuscript and for helpful discussions and encour
agement. The authors are also indebted to Kenneth Class. Robert Penhallow,
and Dr. Joseph B. Bolen for the flow cytometry results.
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Paclitaxel Inhibits Progression of Mitotic Cells to G1 Phase by
Interference with Spindle Formation without Affecting Other
Microtubule Functions during Anaphase and Telephase
Byron H. Long and Craig R. Fairchild
Cancer Res 1994;54:4355-4361.
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