fCANCERRESEARCH 46, 2088-2095, April 19861
Decreased Numbers of Spindle and Cytoplasmic Microtubules in Hamster Embryo
Cells Treated with a Carcinogen, Diethylstilbestrol'
Robert W. Tucker2 and J. Carl Barrett
The Johns Hopkins Oncology Center. Baltimore, Maryland 21205 (R. W. T.J. and National Institute of Environmental Health Sciences, Research Triangle Park,
North Carolina, 27709 (J. C. B.J
ABSTRACT
Diethylstilbestrol
(DES)
has been shown to induce neoplastic
hans
formation in the absence of measurable mutations at specific loci in
Syrian hamster embryocells. It has been proposedthat DES inducescell
transformation
via the production of aneuploidy. In the present study we
document that concentrations of DES that cause aneuploidy also produce
abnormal or arrested mitotic spindles. Thus, DES may disrupt spindle
microtubules
and produce aneuploidy that results
expression and eventual neoplastic transformation.
in disordered
gene
INTRODUCTION
DES,3 a synthetic estrogen, is carcinogenic in both humans
and animals (1). The mechanism of this carcinogenic effect is
unknown and may differ from other known carcinogens since
DES has not been shown conclusively to be activated to an
electrophilic agent that can directly induce DNA damage (2).
Treatment of Syrian hamster embryo cells in culture with DES
induces morphological transformation of the cells. These al
tered cells are preneoplastic and ultimately progress to neo
plastic transformation (3—5).The observations that cell trans
formation can occur without measurable effects on structural
chromosome aberrations (6), mutations at two specific genetic
loci (3),or unscheduled DNA synthesis (7) support the hypoth
esis that the carcinogenic effects of DES may occur without
direct DNA damage. It has been reported recently (6) that
treatment of Syrian hamster embryo cells with DES may cause
neoplastic transformation by means of chromosomal aneu
ploidy. The lines of evidence (6, 8) for this hypothesis are the
following: (a) DES induces significant loss or gain of 1 or 2
chromosomes at nontoxic doses; (b) DES induces aneuploidy
and cell transformation with parallel dose-response curves; (c)
the induction of aneuploidy correlates with the production of
cell transformation by DES-related compounds; (d) both aneu
ploidy and cell transformation are induced by DES more fre
quently in mitotic than in S-phase cells; and (e) neoplastic cell
lines induced by DES are aneuploid with a near diploid chro
mosome number. In addition to DES, other known carcinogens
also induce aneuploidy (8, 9). The possible significance of
numerical chromosome changes in cancer has been discussed
previously (6, 10).
If changes in chromosome number are important in carci
nogenesis, agents like colchicine and Colcemid that induce
nondisjunction (1 1—14)should also cause aneuploidy and cell
transformation. In fact, Colcemid induces both morphological
and neoplastic transformation of Syrian hamster embryo cells
in culture (15), anchorage independence in rat fibroblasts (16),
altered foci in mouse skin cultures (I 7), and an increased
frequency of transformation by polyoma virus (18). In each of
these conditions, colchicine or Colcemid also produce either
aneuploidy or polyploidy. At high concentrations Colcemid
completely disrupts microtubule organization, inhibits normal
mitosis, and produces polyploid cells (19, 20). Lower doses of
Colcemid, which do not completely inhibit microtubule polym
erization, may induce nondisjunction of only a few chromo
somes, resulting primarily in aneuploid cells that are near
diploid (1 1—15).A similar dose dependence of chromosome
effects was observed in DES-treated cells (6). Low doses of
DES (0.001—1.0 @tg/ml),which do not affect cell growth, induce
cell transformation and aneuploid cells with a near diploid
chromosome number. Higher doses of DES (10—20 zg/ml)
cause a reversible inhibition of cell growth and induce poly
ploidy without an increase in cell transformation (6).
It has been proposed that DES has colchicine-like activity in
human and rodent cells in culture (21, 22). However, direct
evidence of an effect of DES on microtubules in cultured cells
has not been reported previously. In the present study we
describe evidence that DES alters microtubule organization in
a dose-dependent manner and that the types of abnormalities
observed at different doses correlate with the concentration
dependent effects on morphological cell transformation. These
results are consistent with the hypothesis that DES causes
chromosomal nondisjunction, aneuploidy, and cell transfor
mation via a decrease in the number of spindle microtubules.
MATERIALS
AND METhODS
Cell Culture and Chemicals.Syrian hamster embryo cell cultures were
established from I 3-day gestation fetuses collected aseptically by cc
sarian
section
from
inbred
Syrian
hamsters,
strain
LSH/ssLAK
(Lakeview Hamster Colony, Newlield, NJ). Pools of primary cultures
from litter mates were stored at liquid nitrogen temperature. Secondary
cultures were initiated from the frozen stocks, and all experiments were
performed
with tertiary
or later
cultures.
All cultures
were free of
Mycoplasmacontaminationas testedby Microbiological Associates
(Bethesda, MD). The cell culture medium used was IBR Dulbecco's
modified Eagle's reinforced medium (Biolabs, Northbrook, IL) supple
mented with 0.37% NaHCO3 and 10% fetal bovine serum (GIBCO).
Cells were transferred by gentle trypsinization with 0.1% trypsin (1:250;
GIBCO) for 5 mm at 3TC. DES and hydroxyurea were obtained from
Sigma Chemical Company (St. Louis, MO). Stock solutions (10 mg/
ml or 37 mM) of DES were made in dimethyl sulfoxide, so that the
highest concentration of dimethyl sulfoxide used in the experiments
was 0.1%.
Antibody.The antitubulin antibody has been characterized and de
scribed previously (23, 24). Briefly,vinblastine-induced tubulin crystals
from sea urchin eggs were used in both the primary injection and a
second injection I month later. After the second injection, the rabbit's
serum contained antitubulin antibody, as demonstrated by double im
munodiffusion and immunoelectrophoresis
tals, sperm tail axonemes,
and embryonic
against tubulin paracrys
chick brain tubulin.
Immunofluorescence. The methods used here for fixing and staining
cells for indirect
immunofluorescence
has been described
previously
(25). Briefly, cells on glass coverslips were fixed for 30 mm with 10%
Received 10/I 5/84; revised 7/I 1/85, 1/7/86; accepted 1/7/86.
I Work
was
partly
supported
by
NIH
Grant
GM25606.
2Towhomrequests
forreprints
should
beaddressed,
atTheJohnsHopkins
Oncology Center, 600 North Wolfe Street, Baltimore, MD 21205.
3 The
abbreviation
used
is:
DES.
formalin (Baker) in phosphate-buffered saline, permeabilized with cold
acetone for 7 mm, air-dried, incubated with rabbit antitubulin anti
serum, washed
in phosphate-buffered
saline, and finally stained
with a
1:60dilution ofrhodamine-conjugated goat anti-rabbit globulin (Cappel
diethylstilbestrol.
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DES EFFECT ON SPINDLE
AND CYTOPLASMIC
MICROTUBULES
Laboratories).
The wet coverslips were mounted in 90% glycerol and
viewed in a Leitz epifluorescence
microscope (Ortholux II). The fluo
rescent images were photographed
using 35-mm Tri-X film (Kodak)
and developed in Acufine.
Experimental Conditions. Cells were synchronized
using hydroxyurea
as described previously (6). Three h after release of cells from the G1-S
block, varying concentrations
(0.001—10 @zg/ml)of DES were added to
triplicate cultures. After 3.5 h exposure to DES, cultures were fixed
spindles (Fig. 1). Multiple focal planes were observed to ensure
that these spindles were not elongated normal spindles viewed
end-on. These structures resemble C-metaphases observed in
cells treated with Colcemid (26). Occasionally, DES (I sg/ml)
also produced a mitotic spindle, the microtubules ofwhich were
normal length but were curved so that the spindle shape was
distorted (Fig. 3, Part 6).
Lower concentrations of DES (0.001—0.1 @tg/ml)produced
and processed for indirect immunofluorescence.
In order to determine the frequency of changes in mitotic microtu
more subtle changes in the distribution of spindle microtubules.
bules, 200 consecutive cells on each coverslip were scored as normal or
There were two kinds of morphologically abnormal spindles
abnormal, and every abnormal cell was photographed
for later detailed
observed. The first was a shortened spindle that was approxi
analysis. For each concentration
of DES the percentage of cells with
mately 75% as long and wide as metaphase spindles in untreated
an abnormal
microtubule
pattern was determined
from the photo
cells (Fig. 4, Part 2, compared to Fig. 1, Part 3). The second
graphs.
abnormality was an irregular or asymmetrical spindle. Instead
of a normal spindle with smooth, regular contours of microtu
RESULTS
bules converging on the poles and a similar distribution of
microtubules in the two half-spindles (Fig. 1, Part 3), irregular
Normal Microtubule Distribution. Mitotic and interphase
or
asymmetrical spindles were observed (Fig. 4, Parts 1 and 3
cells in both unsynchronized and partially synchronized cultures
to
6). Some of the spindles were of normal length but had a
were examined by indirect immunofluorescence with antitubu
different distribution of microtubules in the two half-spindles
lin antibody. The normal patterns of microtubule distribution
(Fig. 4, Parts 1, 4, and 5). Often spindles were both asymmet
are shown in Fig. 1. The full complement of cytoplasmic
rical and shortened in pole-to-pole length (Fig. 4, Parts 2 and
microtubules in the interphase cell (Fig. 1, Part 1) disappeared
3). An occasional spindle in treated cells was shortened and
as centrosomes separated in prophase (Fig. 1, Part 2); later,
appeared to lack precise focusing of microtubules at two inde
metaphase (Fig. I, Part 3) and anaphase (Fig. 1, Part 4) spindles
were formed. Telophase and cell division then began (Fig. 1, pendent poles (Fig. 4, Part 6). No attempt was made to make
subclassifications of these abnormalities and all were classified
Part 5) and produced (Fig. 1, Part 6) complete chromosome
by us as asymmetrical spindles.
separation to two daughter cells. To understand the possible
The dose dependence of DES effects on the distribution of
role of microtubule changes in the effect of DES on cell growth,
microtubules was quantitated (Table 1). None of the abnormal
cell transformation, and chromosomal abnormalities, we next
ities detected in DES-treated cells (asymmetrical, shortened,
studied whether these normal microtubule patterns were
small,
or absent spindles) were observed in over 200 control
changed by different concentrations of DES.
cells examined. As alredy suggested, the type of alterations in
DES and Cytoplasmic Microtubules in Interphase Cells. Cells
treated with high concentrations of DES (10 @ig/ml)had a the treated cells varied with the dose of DES. At high doses (10
@tg/ml),no spindle microtubules were observed in 84% of the
marked decrease in the number of cytoplasmic microtubules.
cells;
only microtubule organizing centers were stained with
Microtubules were polymerized around organizing centers but
were fewer and did not extend completely to the periphery of antitubulin antibody. In cells treated with DES (3 @tg/ml),45%
of the cells had a small spindle resembling C-metaphase arrest
the cell (Fig. 2, Part 1). An intermediate concentration of DES
(3.0 @g/ml)produced fewer microtubules that still extended to (26). Treatment with DES (1.0 @g/ml)caused 10% of the cells
the periphery of the cell (Fig. 2, Part 2). In contrast, no to have a shortened and/or asymmetrical spindle. Lower doses
of DES produced asymmetrical spindles in less than 10% of
detectable changes in the distribution of cytoplasmic microtu
bules were observed in cells treated with DES (0.01 to 1.0 @sg/ the cells in a dose-dependent manner.
As with interphase microtubules, the effect ofDES on spindle
ml) or with 0.1 % dimethyl sulfoxide for 3 h (Fig. 2, Parts 3 to
microtubules was fully reversible. For example, 84% of cells
6). These dose-dependent effects were quantitated in over 200
treated with DES (10 sg/ml for 3 h) lost their spindle micro
cells at each dose of DES (Table 1). One hundred % of inter
tubules but recovered normal spindles 30 mm after washing out
phase cells treated with DES (10 @g/ml)had very few cyto
DES.
plasmic microtubules; lower concentrations had no effect except
It should also be noted that the effect of DES seems to be
for a slight decrease in the number of microtubules in a few
much more pronounced on spindle as compared to cytoplasmic
cells treated with DES (3 .tg/ml).
microtubules (Table 1). For example, in cells treated with DES
These marked changes in the distribution of cytoplasmic
(10 @sg/ml),cytoplasmic microtubules in interphase cells were
microtubules were fully reversible. Normal microtubule distri
bution was observed in cells treated with DES (10 @ig/ml)for 3 never completely absent, whereas in 84% of mitotic cells there
were no detectable spindle microtubules.
h, then washed three times with medium, and allowed to recover
for 1 to 3 h.
DES and Spindle Microtubules in Mitotic Cells. High concen
DISCUSSION
trations of DES (10 @tg/ml)had a dramatic effect on the
microtubule distribution in the mitotic spindle (Fig. 3, Parts 1
The results in this paper document that the concentrations
and 2). Spindle microtubules were essentially absent, and only of DES that have induced both aneuploidy and neoplastic
transformation in other studies (6) of Syrian hamster embryo
microtubule organizing centers were detectable. Lower concen
trations of DES produced mitotic spindles with microtubules
cells also inhibit the formation of mitotic spindles in the same
cell line. We will discuss how DES might produce changes in
present, but in an abnormal distribution.
Intermediate concentrations of DES (1 to 3 jsg/ml) produced
microtubules that influence the induction of aneuploidy and
a large percentage (45%) of cells with a small spindle (Fig. 3, transformation.
Parts 3 to 5) characterized by a compact area of microtubules
DES and Microtubule Polymerization. DES appears to cause
the longest diameter of which was less than 50% that of normal
a net decrease in microtubule polymerization. In both mitotic
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DES EFFECT ON SPINDLE AND CYTOPLASMIC MICROTUBULES
I
I
0
0
0
0
0
0
Fig. I . Normal patterns of mitotic and cytoplasmic microtubules. 1, cytoplasmic microtubules distributed throughout an interphase cell. 2, separation of
centrosomes or microtubule-organizing centers (arrows) occurring at prophase. Mitotic spindle development includes metaphase spindle (3), onset of anaphase (4),
and early telophase (5). After late telophase (6), cell completes division into two daughter cells. Bar, 10 @m.
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@
..
DES EFFECT ON SPINDLE
AND CYTOPLASMIC
MICROTUBULES
.. !:.
:
‘-
I
@‘
•14@
@
(@‘•
4
L@
@..
‘p@
@•
@::‘
9,4%
;‘.‘
‘@w'@
6
@.
Fig. 2. Fewer cytoplasmic microtubules are found in cells treated with high concentrations of DES. Cells treated with DES (10 @g/ml)(1) had a dramatic decrease
in the distribution of cytoplasmic microtubules; a few microtubules still emanated from microtubule organizing centers (black arrows). but most did not extend
completely to the periphery of the cell (white arrows); in 2, DES (3.0 ,@g/ml)produced a slight reduction in the number of microtubules that still extended from
cytocenter to cell periphery. No change in cytoplasmic microtubules was observed in cells treated with DES (1.0 @g/ml)(3), DES (0.1 @g/ml)(4). DES (0.01 @g/ml)
(5). or in control cells (6). Bar, 10 Mm.
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DES EFFECF
ON SPINDLE
AND CYTOPLASMIC
MICROTUBULES
Table I EffectsofDES
on microtubule polymerization inhamster embryo
cellsCells
mitotic spindle.4 In contrast,
withabnormalDES
suggests
concen
mitoticCells
abnormaltrationspindle―interphase(@g/ml)(%)Spindle
that DES interacts
study used indirect immunofluorescent
with
some steroid hormones
abnormalitymicrotubules
(%)0<1None00.0012.5Asymmetrical―00.013Asymmetrical00.15.5Asymmetrical0I
.010Asymmetrical
and
shortened03.045Small―0'10.084No
microtubules1100
aCells (100—200)
were treated with each dose of DES for 1.5—3
h prior to
fixation.
spindle
morphology
with
two
half-spindles
that
are
not
equal
One
evidence to show that
localized at the pericen
to include DES.
An effect of DES on tubulin dimers could conceivably explain
our results showing increased sensitivity ofspindle as compared
to cytoplasmic microtubules. Tubulin dimer flux into spindle
microtubules is known to be more rapid than into cytoplasmic
microtubules (33). Indeed, drugs like colchicine (34) and no
codazole (28) that inhibit tubulin dimer addition also affect
microtubules in mitotic spindles more than those in the cyto
plasm
and symmetrical.
(e.g., estradiol)
information
material.
triolar material (32), but these results have not been confirmed
or extended
b Asymmetrical
only fragmentary
with pericentriolar
of an interphase
cell. Thus,
both in vitro and in vivo
results suggest that DES decreased the number of microtubules
less than 75% that of normal spindle.
by inhibiting the polymerization of tubulin dimer into micro
d Small spindle with pole-to-pole
(6 Mm) and metaphase (3 Mm) diameter less
tubules.
than 50% that of normal spindle. Appears similar to C-metaphase spindle.
e Some
cells
had decreased
numbers
of cytoplasmic
microtubules,
all of which
DES, Microtubules, and Neoplastic Transformation. Col
extended to the cell periphery.
cemid, a drug which binds to tubulin dimer and directly inhibits
tOnly microtubule organizing centers were detected.
polymerization of microtubules (34), also produced both chro
mosomal nondisjunction and neoplastic changes in hamster
embryo cells (15). The dose-response curves for cell transfor
and interphase cells after DES treatment, there were fewer mation and aneuploidy induction were similar, suggesting that
polymerized microtubules. High (10 @tg/mlor 37 @M)
doses of aneuploidy was involved in producing eventual neoplastic trans
DES prevented all or most microtubules from polymerizing
formation. These results suggest that Colcemid and DES may
from the centrosome, intermediate doses (1—3@ig/ml,or 3.7— both produce neoplastic transformation via a depolymerization
of some mitotic microtubules that produces nondisjunction and
11 @M)
allowed small spindles and more cytoplasmic microtu
aneuploidy (6, 15). The resulting change in chromosome num
bules to form, while low doses (0.1—0.001 @tg/ml,or 0.37 zM
ber may produce heritable genetic alteration in gene expression
3.7 nM) had little detectable effect on cytoplasmic microtubules
leading to neoplastic transformation (6). Agents such as DES
but still produced occasional asymmetrical or shortened spin
that do not inhibit cell growth and primarily inhibit spindle
dies. We interpret these changes as reflecting a decreasing
amount of microtubule polymerization as the DES dose is rather than cytoplasmic microtubules should be especially effi
cient at inducing aneuploidy and neoplastic change at nontoxic
increased from 0.001 to 10 @tg/ml.
doses. We would predict that agents such as taxol that poten
Spindle microtubules appear more sensitive than cytoplasmic
microtubules to DES. The highest concentrations of DES (10 tiate the polymerization of microtubules might also prevent
neoplastic transformation induced by DES or Colcemid. Taxol
and 3 @tg/ml)did not completely take away cytoplasmic micro
does indeed stabilize the intrinsically labile mitotic spindle in a
tubules, while the mitotic spindle was absent in cells treated
with DES (10 @g/ml)and more than 50% shorter with DES (3 taxol-requiring mutant Chinese hamster ovary cell so that di
@@g/ml).
Thus, the mechanism of the DES effect must relate to vision can occur (35). It is also possible that some doses of
taxol may induce selective stabilization of spindle microtubules
some intrinsic difference between the polymerization kinetics
and actually induce aneuploidy (36).
of spindle and cytoplasmic microtubules.
It is interesting to compare the concentration-dependent
Possible Mechanisms. While the mechanism of the inhibition
of microtubule polymerization by DES is unknown, there are a effects of DES on microtubules to other biological effects of
DES observed previously (4, 6). As summarized in Table 2,
number of logical possibilities: (a) DES might inhibit the in
high doses ofDES (10 @g/ml)completely blocked the formation
trinsic activity of pericentriolar material or centrosome to or
of spindle microtubules, dramatically affected cytoplasmic mi
ganize microtubules; (b) the synthesis, attachment, or modifi
crotubules, and totally inhibited cell growth as shown by the
cation (e.g., phosphorylation)
of the microtubule-associated
increased mitotic index. The cells which escaped this growth
proteins or other factors associated with microtubules might
inhibition were polyploid. At lower doses of DES (<I zg/ml),
be inhibited, thereby changing the stability of microtubules
no effect on cell growth or mitotic index was observed. This is
(27); (c) DES might bind to tubulin dimer and prevent its po
consistent with the lack of effect on cytoplasmic microtubules
lymerization into microtubules, similar to the action of colchi
and the subtle effects on spindle microtubules at this dose.
cine and nocodazole (28); and (d) DES may bind to intact mi
crotubules and prevent further assembly, similar to drugs like However, the observed spindle abnormalities may be sufficient
to explain chromosomal nondisjunction. Low doses of DES
vinblastine. Because DES did not produce mitotic spindles with
more than two poles (e.g., tripolar), DES probably does not could cause decreased polymerization of some, but not all
spindle microtubules, so that the subsequent anaphase and
act like isopropyl N-(3-chlorophenyl)carbamate,
a drug that di
metaphase abnormalities could lead to chromosome nondis
rectly inhibits the duplication or splitting of the centriole/cen
junction. Higher doses of DES cause complete block of micro
trosome (29).
tubule polymerization resulting in mitotic inhibition and poly
There is some evidence that DES affects pericentriolar ma
ploidy. These results are important in understanding the dose
terial, microtubule-organizing centers, and tubulin dimers. The
most convincing data (30, 31) document the binding of DES
4 B. R. Brinkley, A. Tousson, and M. M. Valdivia.
The kinetochore
of mam
(3—70@zg/ml)to tubulin dimer and the inhibition of microtubule
malian chromosomes: structure and function in normal mitosis and aneuploidy.
assembly in vitro. There is also morphological evidence that
Proceedings of the Symposium on Aneuploidy: Etiology and Mechanisms, March
DES alters the structure of kinetochores and centrioles in the
25—29.Washington, DC, 1985.
C Shortened
spindle
with
pole-to-pole
(9
Mm)
and
metaphase
(4
Mm)
diameter
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DES EFFECT ON SPINDLE
AND CYTOPLASMIC
MICROTUBULES
4
“I
S
0
Fig. 3. High concentrations of DES produced marked changes in mitotic spindles. In cells treated with DES (10 Mg/mI) (1 and 2), no spindle microtubules were
present; only microtubule-organizing centers (arrows) stained with antitubulin antibody. In 3 and 4, DES (3.0 @g/ml)produced small spindles in 45% of treated cells.
In 5 and 6, DES (1.0 pg/mI) produced small or distorted spindles in 10% ofcells. Bar, 10 pm.
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DES EFFECT ON SPINDLE
AND CYTOPLASMIC
MICROTUBULES
Fig. 4. Low concentrations of DES produced abnormalities in morphology of mitotic spindles. In 1 and 2, DES (0.1 pg/mI) produced asymmetrical mitotic
spindles in 5.5% of treated cells. In 3 and 4, DES (0.01 pg/mI) caused 3% of mitotic spindles to be slightly abnormal in morphology. In 5 and 6, DES (0.001 pg/ml)
produced slightly asymmetrical mitotic spindles in 2.5% of treated cells. Bar, 10 pm.
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DES EFFECT ON SPINDLE
DESDES
Table 2 Summary ofchangesin
concen
tration
(%)0.001—0.1
(pg/mI)Alteration
MICROTUBULES
Syrian hamster embryo cells produced by different concentrations of
in mitotic
spindlesGrowth'Mitotic
morphology
Short spindle
Small central spindle
No spindleNo
I
3
10Abnormal
AND CYTOPLASMIC
index9 (%)Major
effect
No effect
Partially inhibited
Totally inhibited2—4
chromo
somal abnor
transformed
malitiesbMorphologicallycoloniesb
2—4
Nondisjunction
6—9
Polyploidy
35—45Nondisjunction
Polyploidy0.2—0.4
0.6
0.5
0.4
a Percentageof cells in mitosis 24 h after treatment with DES. Mitotic cells determinedin cell culturesfixed and then stainedwith hematoxylin (6).
b Measured
as
described
in
Refs.
4
and
6.
dependence of DES-induced morphological transformation of
these cells, which occurs most efficiently at doses (<1 @sg/ml)
that induce aneuploidy (6).
These resu'ts emphasize that disruption of spindle microtu
bules is one particular way of developing aneuploidy. Aneu
ploidy and neoplastic progression could be produced by any
agent that inhibits components of the mitotic spindle, including
centrosome, microtubules, microtubule-associated proteins, ac
tin and myosin, or regulatory systems such as calcium and
cyclic AMP. While it may be important that the agent acts
selectively on the mitotic spindle, any agent which inhibits
microtubule polymerization must be considered a possible car
cinogen. Thus, chemotherapy programs that involve drugs in
hibiting microtubules (vincristine, vindesine, vinblastine, po
dophyllotoxin, steganacin, and griseofulvin) may have some
carcinogenic potential, even though these agents do not bind to
DNA. One must now consider these new mechanisms of gen
erating preneoplastic or neoplastic cells when predicting the
carcinogenic potency of a new drug or an environmental chem
ical.
ACKNOWLEDGMENTS
We would like to thank R. Weddington for expert and diligent typing
of the manuscript,
help in preparation
and K. Meade-Cobun
and P. Lamb for technical
of the data. We would especially like to thank Dr.
John McLachlan for stimulating discussions.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1986 American Association for Cancer Research.
Decreased Numbers of Spindle and Cytoplasmic Microtubules
in Hamster Embryo Cells Treated with a Carcinogen,
Diethylstilbestrol
Robert W. Tucker and J. Carl Barrett
Cancer Res 1986;46:2088-2095.
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