[CANCER RESEARCH 58. .1633-3640. Augusl 15. 1998]
Paclitaxel-induced Apoptosis Is Associated with Expression and Activation of c-Mos
Gene Product in Human Ovarian Carcinoma SKOV3 Cells1
Yi-He Ling,2 Yandan Yang, Carmen Tornos, Balraj Singh, and Roman Perez-Soler
Departments i>f Thoracic/Head and Neck Medical Oncology, Section of Experimental
University of Texas M. D. Anderson Cancer Center, Houston. Texas 77030
Therapy ¡Y-H.L. R. P-S.I, Pathology [C. T.I. and Molecular Pathology ¡Y.Y., B. S.I, The
ABSTRACT
The c-Mos gene product is a component of the cytostatic factor and, as
such, stabilizes the maturation-promoting
factor causing cell-cycle block
ade at metaphase II in unfertilized eggs. The potential role of c-Mos in
regulating cell-cycle progression and cell death in somatic cells remains
unknown. We studied whether paclitaxel-induced M-phase arrest and
apoptosis are associated with c-Mos gene expression and activation in
SKOV3 ovarian carcinoma cells. The first cellular effect observed with
continuous exposure to 50 ng/ml paclitaxel (ID50) was mitotic arrest with
an increase in the accumulation of cyclin Bl and stimulation of cdc2/cyclin
Bl kinase in a time-dependent manner during a 36-h incubation. DNA
fragmentation determined by agarose gel electrophoresis and quantitation
of [ 'HJthvmidine-prelabeled genomic DNA was a later event, first detected
at 24 h and peaking at 48 h (later time points were not studied). Induction
of the c-Mos gene expression and activation were determined by Western
blot analysis, immunoprecipitation using a polyclonal anti-mos antibody,
reverse transcription-PCR assay, and 32P-ATP incorporation into c-Mos
protein or the substrate of glutathione
5-transferase
mitogen-activated
protein kinase kinase, respectively. Both induction and activation were
clearly detected after 24 h of exposure to paclitaxel concentrations of >50
ng/ml, coinciding with drug-induced apoptosis. Mitogen-activated protein
kinase activation preceded c-Mos gene induction. Paclitaxel-induced cMos gene expression was completely abrogated by cycloheximide and
actinomycin D. Mos gene expression was also induced in SKOV3 cells that
were treated with vinblastine but not in those that were treated with
camptothecin, etoposide, or cisplatin. We concluded that tubulin-disturbing agents induce c-Mos gene expression and activation in SKOV3 ovarian
carcinoma cells and that such an effect occurs after mitotic blockade and
coincides with drug-induced apoptosis.
INTRODUCTION
c-Mos, a p39 proto-oncogenic protein, was originally isolated as the
homologue of the v-mos oncogene of Moloney murine sarcoma virus.
c-Mos is a serine-threonine protein kinase that phosphorylates a
number of intracellular substrates (1-3). c-Mos is an active compo
nent of CSF,3 stabilizes MPF, and leads to the arrest of oocyte
maturation at the metaphase of meiosis II (4, 5). Recently, several
reports have shown that c-Mos is implicated in the direct activation of
MAP kinase pathways and in stimulation of cdc2/cyclin B kinase
activity in vitro and in vivo (6-8). In addition, c-Mos has also been
implicated in the reorganization of microtubules that leads to the
formation of the spindle and spindle pole (9). Although the c-Mos
protein is highly expressed in germ cells, it has also been reported to
be expressed in somatic cells. Recently, Gao et al. (10) demonstrated
Received 9/29/97; accepled 6/17/98.
The costs of publication of this article were defrayed in part by 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.
1This work was supported in part by NIH Grant CA50270 and a grant from the Texas
Higher Education Commission.
- To whom requests for reprints should be addressed, at Department of Thoracic/Head
and Neck Medical Oncology. Box 080. The University of Texas M. D. Anderson Cancer
Center, 1515 Holcombe Boulevard. Houston. TX 77030.
1 The abbreviations used are: CSF. cytostatic factor; Act D, actinomycin D; CHX,
cycloheximide; MAP, mitogen-activated protein: GST-MEK1. glutathione 5-transferaseMAP kinase kinase: MPF. maturation promoting factor; MTT. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium
bromide: PMSF. Phenylmethylsufonyl
fluoride: TE, Tris
EDTA; RT. reverse transcription: RT-PCR, reverse transcription-PCR.
c-Mos gene transcription in NIH 3T3 cells using a luciferase reporter
system. The c-Mos protein was found to accumulate at M phase but
at very low levels. Interestingly, Fukasawa et al. ( 11) reported that the
transfection of the c-Mos gene and overexpression of c-Mos protein
led to programmed cell death in S phase in Swiss 3T3 cells, suggest
ing that c-Mos protein may trigger the apoptotic pathways. Further
more, there is evidence that cells with high c-Mos expression arrest at
G-.-M phase and acquire an M phase-like phenotype such as rounding-up and detachment (12).
Paclitaxel, an alkaloid extracted from the pacific yew Taxus brevifolio, is a c-Most effective anticancer agent in the treatment of breast,
ovarian, lung, and head and neck cancers (13, 14). Paclitaxel, as a
promoter of tubulin polymerization, changes the dynamic equilibrium
of assembling and disassembling of microtubules, disrupts the forma
tion of the normal spindle at metaphase, and causes the blockade of
dividing cells (15). We have demonstrated previously that paclitaxel
induces M-phase arrest, accumulation of cyclin Bl, and stimulation of
cdc2/cyclin B l kinase activity in KB cells. We have also shown that
the activation of cdc2/cyclin B kinase precedes drug-induced apopto
sis (16). Because the c-Mos gene has been reported to be involved in
events leading to M-phase arrest and programmed cell death, we were
interested in determining whether paclitaxel-induced mitotic blockade
and apoptosis were associated with c-Mos gene overexpression and
activation of its kinase activity. The results presented here indicate
that the treatment of SKOV3 cells with paclitaxel markedly induces
the expression of p39 c-Mos protein and activation of mos kinase.
More importantly, time course and concentration-response
studies
showed that the appearance of p39 c-Mos protein coincides with
drug-induced DNA fragmentation, thus suggesting that either c-Mos
gene expression or c-Mos protein accumulation may be involved in
the paclitaxel-induced apoptotic pathway.
MATERIALS
AND METHODS
Chemicals. Paclitaxel was purchased from Sigma Chemical Co. (St. Louis.
MO). A stock solution of Taxol (paclitaxel; 1 mg/ml) was prepared with
DMSO. stored at -20°C, and diluted with Ca2 +. Mg-^-free PBS (pH 7.4) to
the required concentrations before each experiment. Cisplatin was purchased
from Bristol-Myers Squibb Co. (Princeton, NJ). Vinblastine. etoposide, camp
tothecin. Act D. and cycloheximide were obtained from Sigma Chemical Co.
GST-MEK1 was prepared as described previously (17). Monoclonal anti-cdc2
and anti-cyclin Bl antibodies were the products of Calbiochem (Cambridge.
MA). erk2 antibody conjugated to agarose beads was purchased from Santa
Cruz Biotechnology. Polyclonal anti-mos (37-55) antibody were produced in
rabbits by injection of the corresponding synthetic peptides as described
previously (18).
Cell Culture and Treatment. Human ovarian adenocarcinoma SKOV3
cells were purchased from American Type Culture Collection (Rockville. MD)
and grown in DMEM supplemented with 10% fetal bovine serum and 250 mM
L-glutamine. Cells were maintained at 37°Cin a humidified atmosphere of 5%
CO2:95% air incubator. Exponentially growing cells were treated with pacli
taxel or other agents or the same volume of medium as a control. To determine
cytotoxicity, cells (1 x IO5 cells/ml) were seeded in 96-microwell plates and
incubated at 37°Covernight. Cells were exposed to different concentrations of
paclitaxel for 3 h. After removal of the drug, cells were washed twice with cold
Ca2+ and Mg2+-free PBS and reincubated in drug-free fresh medium for 3
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INDUCTION
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days. In other experiments, cells were continuously exposed to paclitaxel for
72 h. The surviving cells were determined by MTT dye reduction assay and the
ID50 value, defined as the concentration resulting in 50% cell killing, was
calculated as previously described (19).
Determination of DNA Fragmentation. To determine the laddering of
DNA fragmentation, cells were exposed to 50 ng/ml paclitaxel at 37°C.At the
indicated time points, cells were sampled from the culture and incubated in 0.5
ml of lysis buffer containing 10 mM Tris-HCl (pH 8.0), 200 mM NaCl, and
0.2% Triton X-100 at room temperature for 30 min. After centrifugation at
12,000 X g for 15 min, the supernatant fraction was collected, and
DNA was precipitated with 100 mM NaCl and an equal volume of
at -20°C overnight. DNA was dissolved in TE buffer containing
RNase and incubated at 60°Cfor 60 min. After electrophoresis in
fragmented
isopropanol
20 units of
1% agarose
gel, DNA was stained with ethidium bromide, and the resulting DNA frag
mentation pattern was visualized by UV illumination. To quantitatively deter
mine the DNA cleavage, cells were prelabeled with 1 /xCi [MC]thymidine for
24 h and then chased in fresh medium containing 1 mM cold thymidine
supplemented with 10% fetal bovine serum for 2 h. The prelabeled cells were
exposed to paclitaxel at 37°Cfor different periods of time. Cells were taken
from the culture and incubated in 0.5 ml lysis buffer at room temperature for
30 min. After centrifugation at 12,000 X g, for 15 min, the fractions of
supernatant and pellet were collected. The radioactivity in each fraction was
determined by liquid scintillation counting. The percentage of DNA cleavage
was calculated with the following formula:
' DNA cleavage
=
cpm in supernatant
cpm in supernatant
Western Blot Analysis.
+ cpm in pellet
20 ftCi [-y-32P]ATP. After incubation at 30°Cfor 15 min, the reaction was
stopped by adding 4 X Laemmli sampling buffer. After running 10% SDSPAGE, the 32P incorporation into histone HI was determined by either autoradiography or counting cpm in histone HI. To determine MAP kinase
activity, cell lysates were prepared and immunoprecipitated by anti-erk2 con
jugated to agarose beads. The kinase activity was determined in a reaction
mixture containing 30 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 2 mM MnCl2, 1
mM EGTA, 1 mM DTT, 1 mM sodium orthovanadate, 20 mM ß-glycerophosphate, 0.2 mg/ml myelin basic protein, 50 ¡JLM
ATP, and 10 /xCi [y-i2P]A.TP
at 30°Cfor 15 min. The reaction was stopped by the addition of 4 X Laemmli
sampling buffer and boiled at 100°Cfor 5 min. 32P-incorporated myelin basic
protein was separated by 12% SDS-PAGE and measured by either autoradiog
raphy or counting radioactivity in myelin basic protein. To determine c-Mos
kinase activity, cell lysates were prepared and c-Mos protein was immunopre
cipitated by polyclonal anti-mos antibody as described above. The c-Mos
kinase was assessed by either 32P incorporation into c-Mos protein itself or into
the substrate of GST-MEK1 as described by Maxwell et al. (20) and Yang et
al. (17). Briefly, c-Mos protein kinase was determined in the reaction mixture
containing 2 mM DTT, 1 mM sodium PP¡,7.5 mM ß-glycerophosphate, l ¿IM
ATP, and 5 fxCi [•y-12P]ATP
or in the reaction mixture containing 20 mM
HEPES
7.5), fig
150GST-MEK1,
mM NaCl, 7 mM
MgCU, 7and
mM5MnCU,
1 mM DTT, After
1 mM
sodium (pH
PP¡,10
1 /XMATP,
p,Ci"[7-32P]ATP.
incubation at 30°Cfor 15 min, the reaction was stopped by adding 4 x Lae
mmli sampling buffer and was boiled at 100°Cfor 5 min. After centrifugation
at 14,000 rpm for 5 min, the supernatant fraction was collected and subjected
to electrophoresis in 10% SDS-PAGE. The bands of 32P-phosphorylated
X 100
c-Mos and GST-MEK1
were visualized by autoradiography
as described by
Liu et al. (21).
c-Mos Gene Expression by RT-PCR Assay. SKOV3 cells were exposed
to 50 ng/ml paclitaxel at 37°Cfor the indicated periods of time and the total
RNA was extracted by using RNAzol according to the manufacturer's protocol
SKOV3 cells were treated at 37°Cwith pacli
taxel or different types of anticancer agents for different periods of time.
After washing twice with cold Ca2+ and Mg2+-free PBS solution (pH 7.4),
cells were lysed with lysis buffer containing 50 mM Tris-HCl (pH 7.5),
0.1% Triton X-100, 1% SDS, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1
mM PMSF, 1 mM DTT, 10 /xg/ml leupeptin, and 10 fig/ml aprotinin. The
protein content was determined by a Bio-Rad DC protein assay kit (Her
cules, CA). An equal amount of lysate (50 fig of protein) from control cells
and treated cells was subjected to electrophoresis
in a 0.1% SDS-10%
acrylamide gel. After electrophoresis, protein blots were transferred onto a
nitrocellulose membrane. The p39 c-Mos gene product, cyclin Bl, and erk2
were probed with polyclonal anti-mos (37-55) antibody and monoclonal
anti-cyclin Bl and anti-erk2 antibodies and were visualized with antirabbit
or antimouse IgG coupled to horseradish peroxidase using enhanced chemiluminescence according to the manufacture's recommendation (Amersham,
Arlington Heights, IL).
Metabolic
Labeling and Immunoprecipitation.
For labeling with
[155]methionine, SKOV3 cells—exposed to paclitaxel for different periods of
(Biotecx Laboratories Inc. Houston, TX). cDNA was synthesized from the
isolated RNA by RT as described by Wei et al. (22). The PCR primers were
designed based on the human c-Mos cDNA (23) and ß-actincDNA (accession
number M10277) obtained from the genebank Sequence Data Library through
the Genetic Computer Group Genetic Database (Version 7). The primers
designed for amplification of c-Mos gene are 5'-AGCAGGTGTGCTTGCTGCAG-3' (sense) and 5'-GAGGAAGAGCAGGCCGTTCA-3'
(antisense),
and the primers for ß-actingene are 5'-ACACTGTGCCCATCTACGAGG-3'
(sense) and 5'-AGGGGCCGGACTCGTCATACT-3'
(antisense). All of the
primers were synthesized by Life Technologies (Grand Island, NY). Each PCR
was performed in a 50-fil reaction mixture containing 5 juil of RT reaction
mixture, 50 mM Tris-HCl (pH 9.0), 1% Triton X-100, 1.2 mM MgCU, 0.1 mw
of each dNTP, 2.5 units of Taq polymerase (Promega Biotech, Madison, WI),
0.1 JU.Mß-actin primers, and 2 ¿J.M
c-Mos primers. Thermal cycling was
performed in a DNA amplifier by using the following profile: (a) an initial
denaturation at 95°Cfor 5 min; (b) 30 cycles of denaturation at 95°Cfor 30 s,
annealing at 59°Cfor 30 s, and extension at 72°Cfor 45 s; and (c) final
elongation at 72°Cfor 10 min. The PCR products were electrophoresed in a
time—were incubated in a methionine-free medium containing 10% dialyzed
PCS and 0.2 mCi of trans-label (ICN, Costa Mesa, CA) for 2 h. After labeling,
cells were lysed with 1 ml of lysis buffer at 4°C for 30 min, and the
2% agarose gel and stained with 0.5 ng/ml ethidium bromide, and the sizes of
supernatants were collected by centrifugation at 12,000 X g for 5 min. The cell
cDNA products were determined by comparison with a 100-bp ladder of DNA
extracts were incubated in 30 n\ of normal rabbit serum for 15 min. After
size markers.
addition of 30 ¿¿I
of protein G plus/protein A agarose, the cell extracts were
incubated in an ice bath for 30 min. After removal of the agarose beads by
centrifugation at 800 x g for 5 min, the extracts were reincubated with l ¿ig RESULTS
of anti-mos antibody and 50 /j.1 of protein G plus/protein A agarose beads at
4°Covernight. After incubation, the agarose beads were collected by microBecause paclitaxel is very effective in the treatment of ovarian cancer
centrifugation and washed three times with lysis buffer. After boiling for 5
min, the precipitated protein was subjected to electrophoresis in a 0.1%
SDS-10% acrylamide gel and visualized by autoradiography. Specific peptides
that compete with the c-Mos protein in binding to the anti-mos antibody were
provided by one of the authors (B.S.) and used in control samples.
Determination of cdc2/cyclin Bl, MAP, and c-Mos Kinase Activity. To
determine cdc2/cyclin B l kinase, cells were incubated in 0.5 ml of lysis buffer
as described above without 1% SDS at 4°Cfor 30 min. After centrifugation at
1,200 rpm at 4°Cfor 10 min, the supernatant fraction was collected, and the
cdc2/cyclin B complex was immunoprecipitated
with anti-cdc2 and anti-cyclin
Bl monoclonal antibodies and protein G plus/protein A agarose. The kinase
activity was determined in the reaction mixture containing 50 mM Tris-HCl
(pH 7.4), 10 mM MgCl,, 1 mM DTT, 500 jig/ml histone HI, 10 /AMATP, and
(13), we used a human ovarian cancer cell line, SKOV3, as a model to
determine whether paclitaxel treatment could cause inhibition of cell
proliferation, induction of c-Mos gene expression, and induction of
apoptosis. First, exponentially growing SKOV3 cells were plated in
96-microwell plates at 37°C,cultured overnight, and then treated with
various concentrations of paclitaxel for 3 h or 72 h. After a 3-day
incubation, the percent surviving cells were determined by MTT assay as
described in "Materials and Methods." As shown in Fig. 1, treatment with
paclitaxel for either 3 h or 72 h resulted in a striking inhibition of SKOV3
cell growth. The ID50 values, defined as the concentration resulting in
50% cell growth inhibition, are 233 ±23 ng/ml for a 3-h exposure and
42 ±7 ng/ml for a 72-h exposure.
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INDUCTION
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20, the pattern of paclitaxel-induced DNA fragmentation observed
with this method was similar to that observed with agarose gel
electrophoresis.
It has been known that MPF, a complex of p34cdc2 and cyclin B, is
responsible for the entry into and the exit from M-phase by the
activation and inactivation of p34cdc2 and the accumulation and deg
radation of cyclin B (24). We have demonstrated previously that
paclitaxel blocks cell-cycle progression at M phase associated with
<5
but not proportional to an increase in the accumulation of cyclin B l
content and the stimulation of cdc2/cyclin Bl kinase in KB cells (16).
In this work, we also determined the effect of paclitaxel on cyclin B l
expression and cdc2/cyclin Bl kinase in SKOV3 cells. Cells were
exposed to 50 ng/ml paclitaxel for the indicated times, aliquots were
taken from the culture, and the levels of cyclin B1 and cdc2/cyclin B l
kinase were determined as described in "Materials and Methods." The
60-
40-
Cyclin B1 amount in SKOV3 cells was clearly related to the time of
paclitaxel exposures and increased by ~2.7-8.6-fold between 3 h and
3 h Exposure
20-
72 h Exposure
.001
.01
10
Taxol (u,g/ml)
Fig. 1. The effect of pacHtaxe! on cell growth. Exponentially growing SKOV3 cells
were exposed to different concentrations of paclitaxel for 3 h. After withdrawal of the
drug, cells were washed twice with cold Ca2* and Mg2 +-free PBS and reincubated in
fresh medium for 3 days. In other experiments, cells were continuously exposed to
different concentrations of paclitaxel for 72 h. The cell survival curve was performed as
described in "Materials and Methods." Each point is the mean ±SD of three independent
experiments.
Next, we determined whether paclitaxel at the ID50 could induce
programmed cell death. SKOV3 cells were treated with 50 ng/ml
paclitaxel for the indicated times and then taken from culture to
determine the drug-induced DNA fragmentation by agarose gel electrophoresis. As shown in Fig. 2A, DNA fragmentation was not de
tected until 24 h of drug exposure and was directly related to the drug
exposure time. DNA fragmentation was also quantitatively assessed
by prelabeling genomic DNA with [14C]thymidine. As shown in Fig.
36 h compared with the amount at time 0. Similarly, the relative
activity of cdc2/cyclin Bl kinase was also dramatically stimulated in
a time-dependent manner, reaching its peak at 36 h and being about
35-fold higher than at time 0 (Fig. 3).
There is increasing evidence that expression of the c-Mos gene is
implicated in the events of mitosis or meiosis in somatic and germ cell
systems, and overexpression of c-Mos oncogene has been reported to
result in the induction of apoptosis in Swiss 3T3 cells in S phase.
Accordingly, we used SKOV3 cells as a model to determine whether
paclitaxel affects c-Mos gene expression and to understand the rela
tionship between the stimulation of cdc2/cyclin B l kinase, induction
of apoptosis, and alterations in c-Mos gene expression. First, expo
nentially growing cells were treated with 50 ng/ml paclitaxel and
sampled from culture at the indicated time points. The c-Mos gene
product was determined by Western blot analysis using a polyclonal
anti-mos antibody. As shown in Fig. 4/4, the c-Mos gene product (39
kDa protein) was not detected in control cells or in cells treated with
50 ng/ml paclitaxel during the first 3-12 h of drug incubation but was
clearly detectable in cells treated with paclitaxel for 24 h and 36 h,
suggesting that the c-Mos gene is not expressed in untreated SKOV3
cells and only prolonged exposure to paclitaxel is able to induce its
expression. To confirm these findings, we also used KB cells as a
B
55 -
45 -
Fig. 2. Paclitaxel-induced DNA cleavage. In A,
SKOV3 cells were exposed at 37°Cto 50 ng/ml
paclitaxel for the indicated time. The fragmented
DNA was determined by 1% agarose gel electro
phoresis as described in "Materials and Methods."
In B, cells were prelabeled with [14C]thymidine for
.g
«j
35-
0)
24 h and then chased in fresh medium for 3 h. The
prelabeled cells were exposed at 37°Cto 50 ng/ml
I
paclitaxel. At the indicated time points, cells were
taken from the culture and lysed with lysis buffer.
After centrifugation, the I4C-DNA in fractions of
25 -
supernatant and pellet was determined by liquid
scintillation counting, and DNA fragmentation (%)
was calculated as described in "Materials and
Methods." Each point is the mean of two independ
15 -
ent experiments.
M O 3 6 12 24 36 48 (h)
Drug Exposure Time
10
20
30
40
Drug Exposure Time (h)
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50
INDUCTION
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OF C-MOS GENE BY PACLITAXEL
c-Mos gene product (404 bp) was clearly seen in cells treated with the
drug for 24 or 36 h, respectively. Taken together, these results indicate
that prolonged paclitaxel treatment leads to the induction of c-Mos
gene expression in SKOV3 cells.
Cyclin B1
0
6
12 24 36 (h)
A
MW(kDa)
B
123Histone H1
03
6
12
85-
24 36 (h)
50Mos-
40 n
34<U
29-
30 -
0
3
6
0
10
50
12 24 36 (h)
m
B
10
MW(kDa)
12312
24
85-
36
Drug Exposure Time (h)
50Mos-
Fig. 3. The effect of paclilaxel on cyclin B1 accumulation and cdc2/cyclin B l kinase
activity. SKOV3 cells were exposed to 50 ng/ml paclitaxel for the indicated time; the
cyclin Bl content and the activity of cdc2/cyclin Bl kinase were determined as described
in "Materials and Methods." A. the increase in cyclin Bl accumulation by paclitaxel
treatment. B. the stimulation of cdc2/cyclin B1 kinase by paclitaxel: lop, "P incorporation
3429-
into histone HI determined by autoradiography; bottom, the relative activity of cdc2/
cyclin BI kinase determined by "P incorporation into histone HI at each time point
compared wilh that at time 0.
model to determine paclitaxel-induced c-Mos gene expression and
obtained the same results (data not shown). Next, we investigated
c-Mos protein expression in SKOV3 cells after a 24-h incubation in
the presence of paclitaxel at different concentrations. The results
demonstrated that at 10 ng/ml, paclitaxel does not induce c-Mos gene
expression, but at >50 ng/ml, paclitaxel clearly and markedly induces
c-Mos gene expression, although the degree of induction was only
slightly increased with increasing paclitaxel concentrations (Fig. 4ß).
To further confirm the induction of c-Mos gene expression by
paclitaxel, we labeled SKOV3 cells with [35S]methionine and used
anti-mos antibody to immunoprecipitate [155]methionine-labeled c-
Fig. 4. The induction of c-Mos gene expression by paclitaxel. A, time course study of
paclitaxel-induced c-Mos gene expression. SKOV3 cells were treated with 50 ng/ml
paclitaxel for the indicated time, cell lysate (50 /xg of protein) in each sample was
subjected to 10% SDS/PAGE, and the c-Mos protein was probed with polyclonal anti-mos
antibody with ECL detection. B, concentration response study of paclitaxel-induced
c-Mos gene expression. Cells were treated with different concentrations of paclitaxel for
24 h, and then an equal amount of lysate (50 fig of protein) was taken from each sample
for Western blot analysis.
MW(kDa)
i
123-
Mos protein. As shown in Fig. 5, no p39 c-Mos protein band was seen
in control cells and cells treated with paclitaxel for 6-12 h, but the
[•"Slmethionine-labeled c-Mos protein band was clearly detected in
cells treated with paclitaxel for 24 and 36 h. Incubation with specific
peptides that compete with the c-Mos protein in binding to the
polyclonal anti-mos antibody resulted in the abolishment of the p39
band (Lanes 6 and 7), indicating that the p39 protein detected by the
polyclonal anti-mos antibody is the c-Mos gene product. Furthermore,
we also used RT-PCR assay to determine paclitaxel-induced c-Mos
gene expression at mRNA level. As shown in Fig. 6, ß-actin(621 bp),
as an internal control, was equally expressed in control cells and cells
treated with paclitaxel for different times. No c-Mos gene expression
was detected in cells treated with paclitaxel for 0-12 h, whereas the
100500 1,000 (ng/ml)
8550-
3429-
-Mos
Fig. 5. The effect of paclitaxel on c-Mos protein synthesis. SKOV3 cells were treated
with 50 ng/ml paclitaxel for the indicated time points. Cells were labeled with [155]methionine for 2 h, the labeled cells were lysed. and c-Mos protein was immunoprecipitated
as described in "Materials and Methods." Lanes 1-5. cells treated with paclitaxel for 0. 6,
12. 24. and 36 h, respectively. Lanes 6 and 7, cells treated with paclitaxel for 24 and 36 h,
respectively, in the presence of specific competing peptides for anti-mos antibody.
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INDUCTION
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ß-actin(621bp)
c-mos (404 bp)
M O 6
10 ng/ml. Because the key substrate of c-Mos kinase is MAP kinase/
ERK kinase MEK (26, 27), we also used GST-MEKI as a substrate to
assess c-Mos kinase activity in cells treated with paclitaxel. As shown
in Fig. IB, the bands corresponding to GST-MEKI phosphorylated by
c-Mos kinase were observed in cells treated with paclitaxel at 50
ng/ml for 24-36 h but not in control cells or cells treated with
paclitaxel for 6 to 12 h. A concentration response study showed that
GST-MEKI phosphorylation by c-Mos kinase occurred after a 24-h
exposure to >50 ng/ml paclitaxel. All of the data indicate that the
activation of c-Mos protein kinase correlated with the induction of
c-Mos gene expression. Interestingly, the induction of c-Mos gene
expression and the activation of c-Mos protein kinase by paclitaxel
coincide with drug-induced DNA fragmentation, suggesting that c-
12 24 36 (h)
Fig. 6. The effect of paclitaxel on c-Mos gene expression at mRNA level. SKOV3 cells
were Ireated with 50 ng/ml paclitaxel for the indicated time. Total RNA was extracted.
cDNA was prepared, and PCR for amplification of ß-aclinand c-Mos gene was preformed
as described in "Materials and Methods."
The c-Mos gene product is a serine-threonine protein kinase that
phosphorylates a number of intracellular proteins that are involved in
the regulation of mitosis and meiosis (1,2, 25). Therefore, we were
interested in determining the effect of paclitaxel on the activation of
c-Mos protein kinase in SKOV3 cells. Cells were treated with 50
ng/ml paclitaxel for different times and then lysed with lysis buffer.
The c-Mos protein was immunoprecipitated with polyclonal anti-mos
antibody, and the c-Mos protein kinase activity was determined by
[y-32P]ATP incorporation into c-Mos protein. As shown in Fig. 7/4,
the autophosphorylated p39 protein band was observed in cells treated
with paclitaxel for 24-36 h. In addition, we also determined the
activity of the c-Mos protein kinase in SKOV3 cells after 24-h
exposure to different concentrations of paclitaxel. The autophospho
rylated c-Mos protein was noted in cells treated with paclitaxel at
50-1,000 ng/ml but not in controls or cells treated with paclitaxel at
Mos gene expression and activation may be involved in triggering cell
apoptotic pathways.
Because recent studies have demonstrated that c-Mos can activate
MAP kinase in both oocytes and somatic cells (2), we were interested
in understanding whether paclitaxel-induced c-Mos gene expression is
linked to an increase in MAP kinase expression and activation.
SKOV3 cells were exposed to 50 ng/ml for the indicated times,
aliquots of cells were sampled from culture, and the amount of erk2
and erk2 kinase activity were determined either by Western blot
analysis or by [•y-32/']ATP
incorporation into myelin basic protein as
described in "Materials and Methods," respectively. The results pre
sented in Fig. 8/4 show that paclitaxel did not markedly induce the
expression of erk2 in SKOV3 cells. In contrast, as shown in Fig. 8ß,
paclitaxel rapidly and strikingly stimulated erk2 kinase activity com
pared with baseline, i.e., erk2 kinase activity was increased by ~33.5-fold at 6-12 h and by ~5-fold at 24-36 h. These results are
consistent with reports by others (28, 29).
To understand whether paclitaxel-induced
expression
of c-Mos
protein could be dependent on protein and RNA synthesis, we treated
B
-.9
GST-MEK1-
MosFig. 7. Paclitaxel-induced activation of c-Mos ki
nase activity. SKOV3 cells were treated with 50 ng/ml
paclitaxel for the indicated time or with different con
centrations of paclitaxel for 24 h. The c-Mos protein in
each sample was immunoprecipitated by polyclonal
anti-mos antibody and protein A/plus protein G-conjugated agarose. The c-Mos kinase activity was deter
mined by "P incorporation into c-Mos (A) or into
GST-MEKI (ß)as described in "Materials and Meth
ods." A (pan /) and B (pan I), time course study of
c-Mos kinase activity induced by paclitaxel. A (part 21
and B (part 2i. concentration course study of c-Mos
kinase activity induced by paclitaxel.
0
6
12 24 36 (h)
0
6
12 24
36 (h)
GST-MEK1-
Mos-
0 10 50 1005001,000(ng/ml)
0 10 50 1005001,000(ng/ml)
3637
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INDUCTION
AND ACTIVATION
OF c-MOS GENE BY PACLITAXEL
SKOV3 cells with 50 ng/ml paclitaxel, 50 jug/ml CHX, 0.5 /j.g/ml Act
D alone, with Taxol plus either CHX or Act D, or with the same
volume of medium as control. After a 24-h incubation, p39 c-Mos
protein was determined by Western blot analysis using polyclonal
anti-mos antibody. As shown in Fig. 9, no c-Mos gene product was
detected in the cells treated with CHX or Act D alone, and the
induction of c-Mos protein by paclitaxel was completely abrogated by
either CHX or Act D. These results suggest that paclitaxel-induced
mos gene expression requires RNA and protein synthesis, and these
results are consistent with the reports indicating that the induction of
apoptosis by paclitaxel is dependent on new protein and RNA syn
thesis (30).
Finally, we studied whether other types of anticancer agents could
also induce c-Mos gene expression. SK.OV3 cells were exposed to 100
ng/ml camptothecin, 1 fig/ml etoposide, 10 pig/ml cisplatin, 50 ng/ml
paclitaxel, and 100 ng/ml vinblastine. After a 24-h incubation, the
c-Mos protein in each cell sample was determined by Western blot
analysis. As shown in Fig. 10, no c-Mos gene product was detected in
cells treated with DNA damaging agents such as Top I inhibitor
camptothecin, Top II inhibitor etoposide, and DNA adduci agent
cisplatin; but p39 c-Mos protein was clearly detected in cells treated
with the antimicrotubule agents paclitaxel and vinblastine. We also
determined the accumulation of cyclin B l protein in cells treated with
different types of agents and found that only paclitaxel and vinblastine
markedly increased the cyclin B1 accumulation. Cyclin B l levels do
not increase in cells treated with other kinds of drugs as compared
with control cells.
ERK2
Mos-
Fig. 9. The effects of CHX and Act D on paclitaxel-induced c-Mos gene expression.
SKOV3 cells were treated at 37°Cwith either 50 ng/ml paclitaxel. 50 jig/ml CHX, or 0.5
fig/ml Act D alone; paclitaxel plus CHX or Act D: or the same volume of medium as a
control for 24 h. p39 c-Mos protein was detected with Western blot analysis as described
in "Materials and Methods."
DISCUSSION
Our results strongly suggest that c-Mos gene expression and acti
vation may play a role in paclitaxel-induced apoptosis. Existing lit
erature on the function of the c-Mos gene supports our findings.
In unfertilized oocytes, the cell cycle is blocked in metaphase of the
second meiotic division, and it has been shown that the activation of
CSF and MPF is implicated in the events leading to metaphase arrest
in meiosis. Although CSF has never been purified, the proto-oncogene
product p39 c-Mos is an active component of CSF and exerts its
function both temporally and spatially at a very specific place, i.e.,
during meiotic maturation of vertebrate oocytes (31). It has been
demonstrated that antimicrotubule agents such as the Vinca alkaloids
and taxanes prevent cell-cycle progression at M phase by disturbing
the microtubulin dynamic equilibrium between polymeralization and
depolymeralization. Because some events involved in M-phase block
B
ade by antimicrotubule agents in somatic cells may be similar to those
MBP
that prevent the exit of meiosis from metaphase in unfertilized oo
cytes, we investigated whether paclitaxel-induced M-phase arrest and
0 6 12 24 36 (h)
apoptosis are associated with the induction of c-Mos gene expression
and activation of its kinase activity. The results presented here dem
onstrate that paclitaxel at the ID50 causes a rapid increase in cyclin B l
protein accumulation and stimulation of cdc2/cyclin B l kinase activ
ity in association with mitotic arrest and that the c-Mos protein is
detected later, after 24-36 h of exposure to paclitaxel. In oocytes,
c-Mos protein is thought to be implicated in the regulation of M phase
by stabilizing and activating the cdc2/cyclin B complex (32). Some
reports demonstrate that the transition into meiotic M phase requires
c-Mos protein synthesis and that newly synthesized c-Mos protein can
stabilize and activate cdc2/cyclin B complex (33, 34). In contrast with
these studies, we found that paclitaxel-induced M-phase arrest and the
activation of cdc2/cyclin Bl complex in SKOV3 preceded the induc
tion of c-Mos gene expression. One possible explanation for these
surprising results is that the detection of c-Mos protein in SKOV3
cells requires a prolonged exposure to paclitaxel. Some reports have
0
6
12
24
36
demonstrated that transcription of the c-Mos gene in somatic cells is
Drug Exposure Time (h)
low and unstable (35). It is possible that a short exposure to paclitaxel
may induce c-Mos gene expression in association with mitotic arrest
Fig. 8. The effect of paclitaxel on MAP kinase expression and activation. SKOV3 cells
were treated with 50 ng/ml paclitaxel for the indicated time. The amount of erk2 was
and activation of cdc2/cyclin kinase activity but that the threshold for
detected by Western blot analysis (A). The erk2 protein was immunoprecipitated and
kinase activity was determined by 12P incorporation into myelin basic protein as described
detection of the protein is not reached until after 24 h because of its
in "Materials and Methods" (B).
poor stability.
3638
0
6
12 24 36 (h)
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INDUCTION
AND ACTIVATION
OF C-MOS GENE BY PACLITAXEL
There is increasing evidence that c-Mos kinase may play a role in
the regulation of mitotic and apoptotic pathways in oocytes and
somatic cells by activating MAP kinase (2, 3), and recent studies
indicate that MAP kinase is rapidly activated by paclitaxel treatment
(28, 29). In this work, we found that paclitaxel-activated MAP kinase
preceded the induction of c-Mos gene expression, indicating that other
Cyclin B1-
factors or pathways are involved in the activation of MAP kinase in
cells exposed to paclitaxel. However, a contributory role of c-Mos in
activating MAP kinase cannot be ruled out because MAP kinase
activity continued to increase from 12 h to 24-36 h after exposure to
paclitaxel, coinciding with the induction of c-Mos.
Apoptosis is triggered by different stimuli including 7-irradiation
and a variety of chemotherapeutic agents (39). Shi et al. (40) dem
onstrated that inappropriate cell-cycle perturbation could induce apoptosis in some cell systems. King and Cidlowski (41) proposed the
possibility that mitosis and apoptosis may share similar pathways. We
observed that paclitaxel-induced DNA fragmentation coincides with
Con Tax CPT VP-16 CDDPVBL
B
Mos-
Con Tax CPT VP-16 CDDPVBL
Fig. 10. The effects of different antitumor agents on the induction of c-Mos gene
expression. SKOV3 cells were treated at 37°Cwith either 50 ng/ml paclitaxel, 100 ng/ml
camptothecin, 1 /ig/ml etoposide, 10 fig/ml cisplatin, 100 ng/ml vinblastine, or the same
volume of medium as a control for 24 h. Cyclin Bl and c-Mos protein were detected with
Western blot analysis as described in "Materials and Methods." A, cyclin Bl protein
expression. B, induction of c-Mos gene product.
O'Keefe et al. (36) demonstrated that microinjection of synthetic
mos gene mRNA into two-cell embryos prevents cleavage at metaphase. In addition, immunodepletion of endogenous p39 c-Mos gene
protein abrogated its cleavage-arrest effect in injected cells, suggest
ing that p39 c-Mos may be involved in catalysis of phosphorylation of
cyclins or may inhibit some enzymes that are required for degradation
of cyclins. Wang et al. (37) have presented a direct evidence that
c-Mos protein disrupts mitotic progression in somatic cells. They
microinjected bacterially expressed maltose-binding protein-xenopus
c-Mos fusion protein into PtKl cells and found mitosis arrest by
preventing progression of abnormal metaphase. In this work, we
demonstrate that paclitaxel treatment leads to the accumulation and
activation of c-Mos protein kinase. Therefore, accumulation of c-Mos
protein and activation of its kinase activity could enhance the paclitaxel-induced disruption of microtubule structures and functions. In
addition, c-Mos protein could also prevent cyclin B degradation and
enhance cdc2/cyclin B kinase activity, reenforcing the mitotic block
ade at metaphase. Zhou et al. (38) reported that the c-Mos gene
product is tightly associated with tubulin in unfertilized eggs and in
somatic cells transfected with the mos gene. The c-Mos protein, like
cdc2 and cyclin B, was localized in the mitotic spindle and spindle
pole and led to chromosome instability and nuclear condensation.
These observations suggest that the induction of c-Mos gene expres
sion may contribute to the disruption of structures and function of
microtubules.
mos protein accumulation and activation of its kinase, suggesting that
c-Mos could be a triggering factor of paclitaxel-induced apoptosis.
Sagata reported that cells expressing high levels of c-Mos gene
product undergo cell cycle arrest at G,-S or G2-M phases and even
tually die after rounding-up and detachment from monolayer (2, 12).
The rounding-up and detachment of cells by high c-Mos expression
are similar to what is seen in cells treated with paclitaxel, suggesting
that M-phase arrest and apoptotic induction by paclitaxel may be at
least in part mediated by c-Mos protein expression. Recently, several
reports have demonstrated that the c-Mos gene product directly stim
ulates MAP kinase and also activates Raf-1 kinase, a MAP kinase
kinase (42, 43). Blagosklonny et al. (44) reported that paclitaxelinduced apoptosis is caused by the phosphorylation of the Bcl-2
oncoprotein by activation of Raf-1 kinase and secondary loss of Bcl-2
mediated resistance to cell-death-promoting stimuli. Studies to link
c-Mos induction and activation with Raf-1 kinase activation after
paclitaxel treatment are presently being performed in our laboratory.
In addition, we plan to use c-Mos-antisense oligonucleitides or transfection of negative c-Mos gene constructs to further determine
whether c-Mos gene expression and activation are required for pacli
taxel-induced apoptosis.
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3640
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Paclitaxel-induced Apoptosis Is Associated with Expression
and Activation of c-Mos Gene Product in Human Ovarian
Carcinoma SKOV3 Cells
Yi-He Ling, Yandan Yang, Carmen Tornos, et al.
Cancer Res 1998;58:3633-3640.
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