p53 status does not affect sensitivity of human ovarian cancer cell lines to paclitaxel

Abstract

Nine human ovarian cancer cell lines that express wild-type (wt) or mutated p53 were used to evaluate the cytotoxicity induced by paclitaxel. The IC50 calculated in the five mutated p53-expressing cell lines was not different from the four wt p53-expressing cell lines. The introduction of wt p53, by using a temperature-sensitive mutant murine p53 or the human p53 under the control of a tetracycline-dependent promoter, did not change the cytotoxicity of paclitaxel as compared to mock-transfected cells. By using for each cell line the paclitaxel IC50, we found that these concentrations were sufficient to induce an increase in p53 levels in all of the four wt p53-expressing cells, whereas in the mutated p53-expressing cells, the levels were unaffected. This increase in p53 levels led to an increase in the mRNA and protein levels of p53 downstream genes (WAF1, GADD45, and bax). In none of the cell lines examined was paclitaxel able to induce apoptosis, evaluated by terminal deoxynucleotidyl transferase-mediated nick end labeling staining and filter binding assay at concentrations closed to the IC50. By increasing the concentration of paclitaxel in the filter binding assay, we could see fragmentation of DNA in the different cell lines. We conclude that the presence of p53 is not a determinant for the cytotoxicity induced by paclitaxel in human ovarian cancer cell lines. Differences in the activation of p53 downstream genes could be observed in wt versus mutated p53-expressing cells, but this does not account either for a differential induction of apoptosis or for a change in cytotoxicity induced by paclitaxel.

Full text

1997;57:870-874. Published online March 1, 1997.Cancer Res
Domizia Debernardis, Eva Graniela Siré, Paola De Feudis, et al.
Cancer Cell Lines to Paclitaxel
p53 Status Does Not Affect Sensitivity of Human Ovarian
Updated Version
http://cancerres.aacrjournals.org/content/57/5/870
Access the most recent version of this article at:
Citing Articles
http://cancerres.aacrjournals.org/content/57/5/870#related-urls
This article has been cited by 21 HighWire-hosted articles. Access the articles at:
E-mail alerts
related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
.pubs@aacr.orgDepartment at
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
.permissions@aacr.orgDepartment at
To request permission to re-use all or part of this article, contact the AACR Publications
American Association for Cancer Research Copyright © 1997
on July 12, 2011cancerres.aacrjournals.orgDownloaded from

ICANCERRESEARCH57. 870-874. March I. 9971
ABSTRACT
Nine human ovarian cancer cell lines that express wild-type (wt) or
mutated p53 were used to evaluate the cytotoxicity induced by paclitaxel.
The IC50calculated in the five mutated p53-expressing cell lines was not
different from thefour wt p53-expressingcell lines. The introductionof wt
PS3, by using a temperature-sensitive mutant murine p53 or the human
p53 under the control of a tetracycline-dependent promoter, did not
change the cytotoxicity of paclitaxel as compared to mock-transfected
cells. By using for each cell line the paclitaxel IC50,we found that these
concentrations were sufficient to induce an increase in p53 levels in all of
the four wt p53-expressing cells, whereas in the mutated p53-expressing
cells, the levels were unaffected. This increase in p53 levels led to an
increase in the mRNA and protein levels ofp53 downstreamgenes (WAFJ,
GADD45,andbox). In noneof the cell linesexaminedwaspaclitaxelable
to induce apoptosis, evaluated by terminal deoxynucleotidyl transferase
mediated nick end labeling staining and filter binding assay at concentra
tions closed to the IC@.By increasingthe concentrationofpaclitaxel in the
filter binding assay, we could see fragmentation of DNA in the different
cell lines.
We conclude that the presence of p53 is not a determinant for the
cytotoxicity induced by paclitaxel in human ovarian cancer cell lines.
Differences in the activation of p53 downstream genes could be observed
in wt versusmutated p53-expressingcells,but this doesnot accounteither
for a differential induction of apoptosis or for a change in cytotoxicity
induced by paclitaxel.
INTRODUCTION
Paclitaxel is one of the most promising agents for the treatment of
ovarian and breast cancer (I). In ovarian cancer, in particular, pacli
taxel has been reported to be effective in patients refractory to stand
ard chemotherapy (1, 2). The drug does not exert its antitumor activity
by interacting with DNA but rather by binding tubulin and stabilizing
microtubule formation; this results in a block of the cell cycle at the
G2-M phase transition, thus preventing completion of mitosis (3). In
some cell types, the paclitaxel-induced G2-M block resulted in acti
vation of apoptosis (4—6).
A possible determinant of the activity of anticancer agents is the
tumor suppressor protein p53. This protein has been reported to act as
a guardian of the genome, and its levels rapidly increase after treat
ment of cells with DNA-damaging agents (7—9).The rise in p53
protein levels, which is primarily because of a stabilization of the
protein itself, activates a cascade of genes that in turn bring about cell
cycle arrest or apoptosis, depending on the cell type. For many
Received 8/I 2/96; accepted 1/2/97.
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.
I This work was partially supported by Consiglio Nazionale delle Ricerche Progetto
Finalizato Applicazioni Cliniche della Ricerca Oncologia Grants 95.00557-PF39,
95.00559-PF39, and 95.00447-PF39. The generous contribution of the Italian Association
for Cancer Research is gratefully acknowledged. F. V. is a visiting scientist from the
Institute of Cytology, Russian Academy of Science, St. Petersburg, Russia. D. D. received
a fellowship from Associazione Italiana per Ia Ricerca sul Cancro. P. D. F. is a “fellow
Famiglie Belloni e Guglielmetti.―
2 To whom requests for reprints should be addressed. Fax: 39-2-3546277: E-mail:
broggini@irfmn.mnegri.it.
anticancer agents, the presence of a wild-type p.53 gene in tumors has
been associated with an increase in drug activity compared with
tumors harboring mutations in the p53 gene (7, 9). There are data,
however, showing that expression of wild-type p53 protein allows
more complete repair of DNA damage to occur by blocking cells
either in the G1 phase or in the G2 phase of the cell cycle (10, 11).
It has been reported recently that in different cell lines in culture
(HeLa and fibroblast-derived cell lines), the disruption of the wild
type p53 function resulted in an increased sensitivity of cells to
paclitaxel treatment compared to cells expressing wild-type p53 (12).
In an ovarian cancer cell line expressing wild-type p53, the disruption
of p53 by transfection with the E6 protein of human papillomavirus
type 16 led to a decrease in sensitivity to paclitaxel with a correspond
ing decrease in apoptosis induced by DNA-damaging agents (13). In
another ovarian cancer cell line not expressing p53, the introduction of
a wild-type p53 did not change the sensitivity of these cells to
paclitaxel (14). In addition, paclitaxel has been reported to increase
the levels of p53 in some cell types (15) but not in others (14).
In the present study, we report the cytotoxicity induced by pacli
taxel in different ovarian cancer cell lines with a different p53 status.
The cytotoxicity was studied in parallel with the changes in the
protein levels of p53 as well as in the changes in mRNA and protein
levels of the genes p21/WAFI, GADD45, and bax, which are directly
activated by p53 and represent the downstream effector of the p53
gene. In addition, we evaluated the induction of apoptosis after
treatment with paclitaxel.
MATERIALS AND METHODS
Cell Lines and Treatment. Nine humanovariancancercell lines (four
expressing wild-type p53: PA-I, IGROV-l, A2780, and A2774, and five
expressing no or mutated p53: OVCAR-3, OVCAR-5, OVCAR-8, SW626,
and SKOV-3) were used. Cell lines were obtained from American Type
Culture Collection (Rockville, MD) except for A2774, kindly obtained from
Dr. Ferrini (Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy), and
OVCAR-5, OVCAR-8, and IGROV-l, kindly obtained from Dr. Pommier
(National Cancer Institute, Bethesda, MD). The cell lines were maintained in
RPMI 1640 supplemented with 10% FCS.
Clones were obtained from the p53 null line SKOV3 upon transfection with
the temperature-sensitive murine p53 (SK23a; Ref. 16) or with the human
wild-type p53 under the control of a tetracycline-dependent promoter (SKT4).
In both cases, cotransfection with plasmids containing the neomycin resistance
gene was used to allow selection in G41 8 containing medium (500 .tg/ml).
Cytotoxicity was evaluated by the 3-[4,5-dimethylthiazol-2-yll-2,5-diphe
nyltetrazolium assay in 96-well plates (Nunc) at different times after treatment
with different concentrations of paclitaxel (Taxol, obtained from Bristol
Meyers, dissolved in DMSO at a concentration of 500 j.LMand stored at
—20°C).Concentrations inhibiting the growth by 50% (IC50s) were calculated
at 72 h of recovery in drug-free medium after paclitaxel treatment (24 h).
Northern Blot Analysis. Total RNA was extracted from untreated or
paclitaxel-treated cells (after 6 or 24 h treatment) with the guanidine/cesium
chloride gradient method (17). After fractionation through 1% agarose-form
aldehyde gels, RNA was blotted on nylon membranes (GeneScreen Plus;
DuPont) and hybridized with cDNAs encoding box (kindly supplied by Dr.
Korsmeyer, St. Louis, MI), WAFJ, and GADD45. Each cDNA was 32P-labeled
using a Rediprime kit (Amersham, United Kingdom). Hybridizations were
870
p53 Status Does Not Affect Sensitivity of Human Ovarian Cancer Cell Lines
to Paclitaxel'
Domizia Debernardis, Eva Gramela Sire, Paola De Feudis, Faina Vikhanskaya, Monica Valenti, Patrizia Russo,
Silvio Parodi, Maurizio D'Incalci, and Massimo Broggini2
Molecular Pharmacology Unit. LCP, Department of Oncology. Istituto di Ricerche Farmacologiche “MarioNegri. “via Eritrea, 62 20157 Milan fE. G. S. P. D. F. M. D.. M. B.J,
and Department of Experimental Oncology. Istituto Nazionale per Ia Ricerca sul Cancro. 16132 Genova ID. D.. F. V.. M. V.. P. R.. S. P.1. halv
American Association for Cancer Research Copyright © 1997
on July 12, 2011cancerres.aacrjournals.orgDownloaded from

TableI PaclitaxelIC5@,s
in the different human ovariancancer
celllinesp53
status―IC,0
(nM)―OVCAR-3mut
(248 R toQ)4.0OVCAR-5mut
(insertion 3 bp at224)6.0OVCAR-8mut
(deletion126—132)7.0SW626mut
(262 G toV)3.5SKOV-3del4.6A2774wt6.3A278Owt387PA-lwt3.5IGROV-lwt8.2SK4T
+tetrtdel62dSK4T
—tetra'@wt45dSK23a
37CCmut
(135 C toV)7.2SK23a
32CCwt9.2SKN
37CCdcl7.2SKN
32Cdel7.0
100
10
p53 AND SENSITIVITY OF OVARIAN CANCER CELLS TO PACLITAXEL
performed in 50% formamide, 10%dextran sulfate, 1%SDS, and 1MNaC1at HOURS AFTER
42°Cfor 16h, followed by two 10-mmwashes at room temperature with 2X PACLITAXEL 0 6 24
SSC(150 mMsodiumchloride,15mMsodiumcitrate)andonewashfor30 mm
at 65°Cin 2X SSC-l% SDS. GADD45and WAFJcDNAs were obtained by
PCR, as described previously (16). Each filter was hybridized with cs-actin
cDNA to normalize for RNA loading.
OVCAR-5 —,-,.,. A2780
OVCAR-8dL. @-@@@ PA-i
SW626:::@i@:::@ A2774
SKOV-3
Fig. 2. Westem blotting showing the change in p53 levels in the different human
ovarian cancer cell lines after 6 and 24 h treatment with paclitaxel at the IC3@sreported
in Table I . Red ponceau staining of the nitrocellulose filter was used to normalize for
loading.
WesternBlot Analysis. Totalcell extractswerepreparedfromuntreatedor
paclitaxel-treated cells, after 6 or 24 h treatment, according to standard pro
cedures (18). Twenty @.tgof proteins for each sample were electrophoresed
through 12% polyacrylamide-SDS gels and electroblotted onto nitrocellulose
membrane (Schleicher & Schuell, Dassel, Germany) in transfer buffer (50 mrvi
Tris, 100mMglycine, 0.01% SDS, and 20% methanol) for 2 h at 50 V. Filters
were stained with Ponceau Red, hybridized with monoclonal antibody against
p53 (clone DO-l; Santa Cruz Biotechnology, Santa Cruz, CA), and detected
with the enhanced chemiluminescence (ECL) system after the addition of
antirabbit or antimouse IgG (Santa Cruz Biotechnology). The experiments
were repeated in all of the cell lines at last twice.
Evaluation of Apoptosis. The filter binding assay method described
previously (19) was used. Briefly, 5 X iO@ cells prelabeled with 0.02
MCi/ml[t4C]thymidine were loaded onto polyvinyl chloride filters, washed
with PBS, and lysed with 5 ml of a solution containing 0.2% sodium
sarkosyl, 2 M NaC1, and 0.04 M EDTA (pH 10.0). After washing with 5 ml
of 0.02 MEDTA (pH 10.0), radioactivity was measured in filters, loading
fractions, washes, lysis fractions, and EDTA washes. DNA fragmentation
was determined as the fraction of ‘4C-labeled DNA in the lysis fraction and
in the EDTA wash relatively to total intracellular 4C@@labeled DNA. Results
are expressed as the percentage of DNA fragmented in treated cells
compared to DNA fragmented in control untreated cells (background) using
the formula [(F —F0)/(l —F0)l times 100, where F and FOrepresent the
fraction of DNA fragmented in treated and control cells, respectively.
Results obtained with the filter binding assay were confirmed using the
TUNEL3 method (20).
RESULTS
We first examined paclitaxel-induced cytotoxicity in the different
human ovarian cancer cell lines. The calculated IC50s are reported in
Table 1, together with the status of the p53 gene for the different
ovarian cell lines.
Paclitaxel was active in all the cell lines except for one (A2780) at
very low concentrations (IC50s of approximately 10 n@ior below). The
presence of mutations and/or deletions in the p53 gene did not affect
the sensitivity to paclitaxel, which had comparable IC50s in all the cell
3The abbreviation used is: TUNEL. terminal deoxynucleotidyl transferase-mediated
nick end labeling.
I00
OVCAR-3
1000
a The p53 status was determined for each cell line by PCR amplifications and
sequencing of exons 5—8.In parentheses is reported the codon and the sequence changed
[for the mutated (mut) p53-expressing cell line]. wt, wild type.
b Concentrations inhibiting by 50% the growth of the cells were calculated 72 h after
cells were treated with paclitaxel for 24 h.
C Cells cultured in the presence (+) or absence (—) of I @zg/ml of tetracycline.
dCalculated at 24 h.
...- 37°C
Cl) -0- 32°C
-J
0
cr
I-
z
0
0
U-
0
1000
C',
—I
0
I-.
z
0
0
U-
0
PACLITAXELnM
Fig. 1. Cytotoxicity induced by paclitaxel in cell clones derived from the human
ovarian cancer cell line SKOV3 by transfecting the cells with plasmids containing a
neomycin-resistancegene (for selection)and either a temperature-sensitivemutant murine
p53 (SK23a) or a human p53 under the control of a tetracycline-sensitive promoter (SK4).
3-[4,5-Dimethylthiazol-2-yl]-2,5.diphenyltetrazolium (thiazolyl blue) test was used 72
and 24 h after treatment with paclitaxel, respectively, for SK23a and SK4 cells. Bars, SD.
100
SK23
PACLITAXEL nM
SK4
-.- + TETRACYCLINE
-0@- - TETRACYCLINE
871
0 6 24
@ IGROV-i
American Association for Cancer Research Copyright © 1997
on July 12, 2011cancerres.aacrjournals.orgDownloaded from

P53 AND SENSITIVITY OF OVARIAN CANCER CELLS TO PACLITAXEL
A
HOURS AFTER
PACLITAXEL
WAF-i
GADD45
SW626 OVCAR-3 OVCAR-5 OVCAR-8 SKOV-3
062401624016240 1 624 01 624
- . @Oi4iW
@@ I
bax@ @,_@4@ s*@ IMMmi.
I—-.'
B IGROV-1 A2780 PA-i A2774
HOURSAFTER o i 6 24 0 1 6 24 0 1 6 24 0 6 24
GADD45 @s
bax@ ..
WAF-1
a-actin
Fig. 3. mRNA expression of WAFI, GADD45, and
bax in mutated/null p53-expressing cells (A) and wild
type p53-expressing cells (B) at 0, 1, 6, and 24 h after
paclitaxel treatment with the IC50 concentrations re
ported in Table 1. For each cell line, hybridization of the
same filters with a-actin is shown.
cz-actin
lines examined. The only exception was represented by the wild-type
p53 expressing cell line A2780 in which paclitaxel IC50 was 387 nM,
much higher than that found in the other wild-type p53-expressing cell
lines or mutated p53-expressing cell lines.
In addition, the introduction of wild-type p53, either by using a
temperature-sensitive mutant p53 or a wild-type p53 gene under the
control of tetracycline in SKOV3 cells (which do not express p53) did
not result in a significant change in the IC50s (Fig. 1). In fact, in the
clone SK23a which expresses mutated p53 at 37°Cand wild-type p53
at 32°C,IC50s were 7. 1 and 9.4, respectively, at the two temperatures.
As shown in Table 1 and in data reported previously (14), the change
in temperature from 37°Cto 32°Cof the mock-transfected clone SKN
does not induce any change in the cytotoxicity induced by paclitaxel.
The sensitivity to paclitaxel of the clone SK4T was similar in the
presence of tetracycline (i.e., without expression of wild-type pS3) or
in the absence of tetracycline (i.e., with expression of wild-type p53),
again confirming that paclitaxel-induced cytotoxicity is not related to
p53 expression (note that these IC50s were calculated at 24 h instead
of 72 h because the removal of tetracycline for a longer period
resulted in complete arrest of the growth of control cells due to the
continuous presence of a wild-type p53.4
We analyzed the levels of p53 after 6 and 24 h treatment with
paclitaxel by using the respective IC5t)s reported in Table 1 for each
cell line. A typical experiment is reported in Fig. 2. Paclitaxel treat
ment caused an increase in the levels of p53 in cells expressing
wild-type PS3, although in IGROV-l only a slight induction was
observable. In mutated p53-expressing cell lines, p53 was not detect
able in SKOV3 and OVCAR-5, whereas p53 was even reduced in
OVCAR-8 and SW626.
The levels of mRNAs encoding WAFI, GADD45, and bax (three
genes which have been previously shown to be inducible by wild-type
PS3) were analyzed by Northern blotting 1, 6, and 24 h after paclitaxel
treatment using the same experimental conditions used for the West
ern blotting.
Fig. 3A shows the result obtained in the mutated (or null) p53-
expressing cell lines SW626, OVCAR-3, OVCAR-5, OVCAR-8, and
SKOV-3. In none of the cell lines was paclitaxel able to induce the
mRNA levels of WAF-1 and bax genes, whereas we observed a slight
increase in GADD45 expression 24 h after paclitaxel treatment in
SW626. We also observed in the five cell lines a different basal
expression of the three genes (compare, for example, GADD4S and
bax expression in OVCAR-3 and SKOV-3 cell lines).
A different picture was obtained when p53-expressing cell lines
were analyzed (Fig. 3B). WAF-I mRNA expression was increased
4 P. Dc Feudis and M. Broggini. manuscript in preparation.
872
@.1@
, T7@•w@@
-@
American Association for Cancer Research Copyright © 1997
on July 12, 2011cancerres.aacrjournals.orgDownloaded from

Table 2 DNA fragmentation induced by different paclitaxel concentrations in differe?It
human ovarian cancer cell lines
DNA fragmentation in cells treated for 72 h with paclitaxel is shown. Values
(mean ±SD oftwo independent experiments each consisting of three replicates) represent
the percentage of radioactivity recovered after cell lysis by using the filter binding assay
(19).
Paclitaxel concentration (nM)
CelllineI10100OVCAR-32.5
±2.012.7
±10.619.0 ±8.2OVCAR-56.0
±8.48. 1 ±II.41 8.4 ±8.7OVCAR-82.5
±0.19.1 ±1.131.8 ±0.15W626I
.9 ±I.64.9
±I.I5.5 ±0.7SKOV-32.0
±1.01.1 ±0.843.5
±25.8A27743.1
±1.17.8
±7.072.8 ±3.6A2780—1.1
±—0.1—1.6
±—0.21.6 ±0.7PA-I—0.6
±—0.14.8
±4.239.7 ±0.9IGROV-
I0.8 ±0.99.9
±3.446.4
±I.1SK23a
37'C2.7 ±0.11
1.1 ±3.624.7 ±3.6SK23a
32'C2.0 ±0.92.5
±0.64.0
±2.6
P53 AND SENSITIVITY OF OVARIAN CANCER CELLS TO PACLITAXEL
over controls in all the cell lines examined, although some differences
could be found (for example, in IGROV-l WAF-1 mRNA, expression
was increased as early as 1h after treatment, without further increase,
whereas in A2780, A2774, and PA-i, the maximum increase was
found at 24 h). GADD45 mRNA expression was activated by pacli
taxel in IGROV-1, A2774, and PA-i and to a much lower extent in
A2780. bax was only clearly increased over controls in PA-i ; it was
not induced in IGROV-l, where the basal mRNA level of this gene
was almost undetectable, whereas in the A2780 and A2774 cell lines,
only minor changes could be detected.
In the SK23a clone, the Northern analysis was performed at either
37°C(mutated p53) or at 32°C(wild-type pS3). The results of the
experiments, performed in parallel with a mock-transfected cell clone
(SKN), are reported in Fig. 4. In these clonal cell lines, paclitaxel was
unable to increase the levels of WAF-1, GADD4S, and bax in all of the
conditions tested. When SK23a cells were incubated at 32°C,there
was an increase in the basal levels of WAF-] and GADD4S mRNAs
due to the shift from the mutant to the wild-type form of p53 protein,
but also in these conditions, paclitaxel treatment did not further
increase these levels.
We also analyzed paclitaxel-induced apoptosis after the same
treatment conditions used for Northern and Western analysis. We
used the the filter binding assay and the TUNEL staining method
to evaluate the induction of apoptosis. Using the different pacli
taxel doses for each of the cell lines, we could see evidence of
DNA fragmentation using the filter binding assay (Table 2) only at
ioon@,whereasat 10n@i(i.e.,concentrationsmuchclosertothe
IC50), no significant apoptosis could be determined. The absence
of significant apoptosis in the different cell lines was confirmed by
TUNEL staining using the paclitaxel IC50s reported in Table 1 for
each cell line (data not shown).
37°C32°CDISCUSSIONHOURS
AFTER
PACL1TAXEL@ 1 6 24 0 1 6 24Paclitaxel
is one of the most promising anticancer agents forthe.
.,.:@@@ :@@ :@
4. 5 WAF 1 ‘@
•:,@@ .@@ . .therapy
of ovarian cancer, where it has shown activity also in tumors
refractory to cisplatin treatment (1 2) It is therefore important to
understand if there are cellular factors that can play a role as deter
.@
@ .@. *:@@@@@ GADD 45 •@ø@@
.
SKN ,, .,. @minant
of the response of ovarian cancer cells to paclitaxel treatment.
p53 is one of the proteins that plays a central role in the response
to anticancer agent treatment (8, 21). It has, in fact, been shown that
in different cell types, the presence of a wild-type p53 inducesa.
.@b@.•!@bax@@@@ ‘c@ ..sensitization
to DNA damaging agents (7 9) although more recent
evidence of a wild-type p53-induced chemoresistance has been de
,scribed
(10, 11). Paclitaxel, which does not interact directly with
DNA, was found to be able to activate p53 in some cell types, and this
increase has been associated mainly with its ability to activatethea-actinraf-1
cascade (15, 22, 23). In other cell types, including one human
ovarian cancer cell line, p53 did not increase after paclitaxel treatment
(14), and the presence of a wild-type p53 did not result in change in
sensitivity to paclitaxel in respect to cells expressing mutatedp53..@f
:@@
WAF1Recently,
the presence of wild-type p53 has been reported to decrease
the cytotoxicity of paclitaxel (compared to the same cell lines not
expressing wild type p53) and this was explained with a p53depend.
. ‘@. ——@—-—-.@-.-
:@* .@.@ .@ 1.
.. .@@ •:.,,..;.•@ GADO 45@@ (@
SK23a@ . .
@@ @a
v'...‘...@.@:..@ .. bax...@ .@ ,$
@@@@@ :@@@ :@ @ent
block in G@after treatment that would prevent the cells from
getting to G2-M, where paclitaxel is known to exert its activity (12).
In that work, however, no ovarian tumors were used. Another report,
however, showed that in a human ovarian cancer cell line, the dis
ruption of wlld type p53 did reduce the cytotoxicity induced by
paclitaxel (13).
We here report that the presence of wild-type p53 does not change
the sensitivity to paclitaxel treatment by examining five mutant p53
and four wild-type p53-expressing human ovarian cancer cell lines.Incs-actinaddition,
in clones obtained by introduction of a wild-type p53 (from
cells not expressing p53), no differences could be observed in pacli
taxel-inducedcytotoxicity..
.
Fig. 4. mRNA expression of WAFI, GADD45, and bax in clones SKN and SK23a
treated with paclitaxel at 3TC or 32CC. mRNA was extracted 1, 6, and 24 h after treatmentAn
increase in the levels of p53 after paclitaxel treatment and
. . .
consequently an activation of the transcnption of downstreamgeneswith
paclitaxel at the IC5() reported in Table I.such
as WAFJ, GADD45, and bax could be observed in wild-type
873
American Association for Cancer Research Copyright © 1997
on July 12, 2011cancerres.aacrjournals.orgDownloaded from

P53 AND SENSITIVITY OF OVARIAN CANCER CELLS TO PACLITAXEL
p53-expressing cells. This increase, however, did not lead to a change
in cytotoxicity in respect to cells (expressing mutated p53) in which
paclitaxel treatment did not increase the levels of p53 downstream
genes. It is also interesting to note that the rise in p53 levels observed
in wild-type p53-expressing cells after paclitaxel treatment led to a
different kinetic of induction of the three examined genes, transcrip
tionally inducible by p53, in the different cell lines. In fact, for
example, in A2780 a clear induction of WAF-] was found, with
GADD45 and bax only slightly affected, whereas in PA-i, all of the
three genes were clearly induced.
In SKOV-3 cells transfected with wild-type p53 (SK23a), we could
not see an increase in p53 downstream genes after paciitaxel treat
ment, in agreement with data published previously (14). It could be
that the basal, high level of WAF-1 and GADD45 determined by the
introduction of wild-type p53 is already high enough that paclitaxel
treatment cannot induce a further increase. In the same system,
however, other drugs such as cisplatin and doxorubicin were able to
further increase WAFJ and GADD45 mRNA (14, 24). We also did not
observe apoptosis in all of the cell lines examined independently of
the presence of wild-type p53 at different times after paclitaxel
treatment, even if in other cell types paclitaxel was reported to activate
apoptosis (5, 23, 25—27).
In conclusion, our data show that in human ovarian cancer cell
lines, paclitaxel-induced cytotoxicity is independent of the pres
ence of a wild-type p53 and that this is not due to the inability of
paclitaxel to induce a functional p53 in wild-type p53-expressing
cells since a clear induction of p53 downstream genes after pacii
taxel treatment could be observed. The lack of induction of apop
tosis in all the cell lines used (independently on the presence of
PS3) could explain the similar cytotoxicity observed in the differ
ent ovarian cancer cell lines. These would imply that, at least in
ovarian cancer cell lines, p53 can be a determinant of the cellular
response to anticancer agents only when programmed cell death
can be activated.
REFERENCES
1. Gregory, R. E., and DeLisa, A. F. Padlitaxel: a new antineoplastic agent for refractory
ovarian cancer. Clin. Pharm., /2: 401—415, 1993.
2. McGuire, W. P. Taxol: a new drug with significant activity as a salvage therapy in
advanced epithelial ovarian carcinoma. Gynecol. Oncol., 51: 78—85, 1993.
3. Horwitz, S. B. Mechanism of action of taxol. Trends. Pharmacol. Sci., 13: 134—136,
1992.
4. Rose, W. C. Taxol: a review of its preclinical in s'ivo antitumor activity. Anticancer
Drugs, 3: 311—321,1992.
5. Donaldson, K. L., Goolsby, G. L., Kiener, P. A., and Wahl, A. F. Activation of
p34cdc2 coincident with Taxol-induced apoptosis. Cell Growth & Differ., 5: 1041—
1050, 1994.
6. Donaldson, K. L., Goolsby, G. L., and WahI, A. F. Cytotoxicity of the anticancer
agents cisplatin and Taxol during cell proliferation and the cell cycle. mt. J. Cancer,
57: 847—855,1994.
7. Kuerbitz, S. J., Plunkett, B. S., Walsh, W. V., and Kastan, M. B. Wild-type p53 is a
cell cycle checkpoint determinant following irradiation. Proc. Natl. Acad. Sci. USA,
89:7491—7495,1992.
8. Lane, D. P. Cancer: p53, guardian of the genome. Nature (Lond.), 358: 15—16,1992.
9. Kastan, M. B., Onyekwere, 0., Sidransky, D., Vogelstein, B., and Craig, R. W.
Participation of p53 protein in the cellular response to DNA damage. Cancer Res., 51:
6304—6311,1991.
10. Waldman, T., Kinzler, K. W., and Vogelstein, B. p21 is necessary for the p53-
mediated G1 arrest in human cancer cells. Cancer Res., 55: 5187—5190,1995.
11. Enoch, T., and Norbury, C. Cellular responses to DNA damage: cell-cycle check
points, apoptosis and the roles of p53 and ATM. Trends Biochem. Sci., 20: 426—430,
1995.
12. WahI, A. F., Donaldson, K. L., Fairchild, C., Lee, F. Y., Foster, S. A., Demers, G. W.,
and Galloway, D. A. Loss of normal p53 function confers sensitization to Taxol by
increasing G2/M arrest and apoptosis. Nat. Med., 2: 72—79,1996.
13. Wu, G. S., and El-Deiry, W. S. p53 and chemosensitivity. Nat. Med., 2. 225—226,
1996.
14. Graniela Sire, E. A., Vikhanskaya, F., and Broggini. M. Sensitivity and cellular
response to different anticancer agents of a human ovarian cancer cell line expressing
wild-type, mutated or no p53. Ann. Oncol., 6: 589—593, 1995.
15. Tishier, R. B., Lamppu, D. M., Park, S., and Price, B. D. Microtubule-active drugs
Taxol, vinblastine, and nocodazole increase the levels of transcriptionally active p53.
Cancer Res., 55: 6021—6025, 1995.
16. Vikhanskaya, F., Erba, E., D'lncalci, M., and Broggini, M. Introduction of wild-type
p53 in a human ovarian cancer cell line not expressing endogenous p53. Nucleic
Acids Res., 22: 1012—1017,1994.
17. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. Isolation of
biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem
istry, 18: 5294—5299,1979.
18. Sambrook, J., Fritsch, E. F., and Maniatis, 1. Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.
19. Bertrand, R., Kohn, K. W., Solary, E., and Pommier, Y. Detection of apoptosis
associated DNA fragmentation using a rapid quantitative filter elution assay. Drug
Dcv. Res., 34: 138—144,1995.
20. Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. Identification of programmed cell
death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol., 119:
493—501,1992.
21. Lowe, S. W. Cancer therapy and p53. Curr. Opin. Oncol., 7: 547—553, 1995.
22. Blagoskionny, M. V., Schulte, T. W., Nguyen, P., Mimnaugh, E. G., Trepel, J., and
Neckers, L. Taxol induction of p2IWAF1 and p53 requires c-raf-l. Cancer Res., 55:
4623—4626,1995.
23. Blagosklonny, M. V., Schulte, T. W., Nguyen, P., Trepel, J., and Neckers, L.
Taxol-induced apoptosis and phosphorylation of bcl-2 protein involves c-ref-I and
represents a novel c-raf-l signal transduction pathway. Cancer Res., 56: 1851—1854,
1996.
24. Vikhanskaya, F., D'Incalci, M., and Broggini. M. Decreased cytotoxic effects of
doxorubicin in a human ovarian cancer-cell line expressing wild-type p53 and
WAFI/CIPI genes. tnt. J. Cancer, 6!: 397—401,1995.
25. Woods, C. M., Zhu, J., McQueney, P. A., Bollag, D., and Lazarides, E. Taxol-induced
mitotic block triggers rapid onset of a p53-independent apoptotic pathway. Mol.
Med.,1:506—526,1995.
26. Liu, Y., Bhalla, K., Hill, C., and Priest, D. G. Evidence for involvement of tyrosine
phosphorylation in taxol-induced apoptosis in a human ovarian tumor cell line.
Biochem. Pharmacol., 48: 1265—1272,1994.
27. Havrilesky, L. J., Elbendary, A., Hurteau, J. A., Whitaker, R. S., Rodriguez, G. C.,
and Berchuck, A. Chemotherapy-induced apoptosis in epithelial ovarian cancers.
Obstet. Gynecol., 85: 1007—1010,1995.
874
American Association for Cancer Research Copyright © 1997
on July 12, 2011cancerres.aacrjournals.orgDownloaded from