doi:10.1006/cyto.2001.1008, available online at http://www.idealibrary.

com on

OVEREXPRESSION OF IL-6 BUT NOT IL-8

INCREASES PACLITAXEL RESISTANCE OF
U-2OS HUMAN OSTEOSARCOMA CELLS1
Z. Duan, D. E. Lamendola, R. T. Penson, K. M. Kronish, M. V. Seiden2*

The cytokines IL-6, initially recognized as a regulator of immune and inflammatory response
and IL-8, a potential regulator of angiogenesis, also regulate the growth of many tumor cells.
Human cancer cells selected for multidrug resistance to common chemotherapeutic agents
demonstrate increased expression of IL-6 and IL-8. To determine whether IL-6 or IL-8
overexpression contributes directly to the drug resistant phenotype, IL-6 or IL-8 cDNA were
introduced into the paclitaxel sensitive human osteosarcoma cell line U-2OS using the
pIRESneo bicistronic expression vector. Interleukin-6 and IL-8 transfectants were selected for
either high IL-6 or IL-8 secretion and evaluated in drug resistance assays. Two IL-6 and two
IL-8 secreting clones express IL-6 or IL-8 levels of 10 ng/ml and 1 ng/ml in culture, while
parental U-2OS and pIRESneo vector transfected control cells express IL-6 and IL-8 levels of
0.005 ng/ml and 0.1 ng/ml, respectively. MTT cytotoxicity with IL-6 transfected cells demon-
strates a five-fold increase in resistance to paclitaxel and a four-fold increase in resistance to
doxorubicin as compared to U-2OS. There are no changes in mitoxantrone or topotecan
resistance in the IL-6 transfectants as compared to parental U-2OS. Northern analysis of IL-6
transfectants demonstrates that the resistant phenotype is not related to increased levels of
MDR-1, MRP-1, or LRP. Western analysis also confirms that P-glycoprotein levels are not
altered in IL-6 transfectants. Further supporting an MDR-1 independent mechanism of drug
resistance, verapamil cannot reverse paclitaxel resistance in transfected cells, findings further
supported by rhodamine 123 exclusion data. Treatment of IL-6 transfected cells with paclitaxel,
compared with drug-sensitive parental U-2OS, shows U-2OSIL-6 are significantly more resistant
to apoptosis induced by paclitaxel and exhibit decreased proteolytic activation of caspase-3. In
contrast U-2OSIL-8 transfectants demonstrate no appreciable increase in paclitaxel resistance
when compared with parental cells. In summary, while both IL-6 and IL-8 are overexpressed in
paclitaxel resistant cell lines, only IL-6 has the potential to contribute directly to paclitaxel and
doxorubicin resistance in U-2OS. This resistance is through a non-MDR-1 pathway.
 2002 Elsevier Science Ltd. All rights reserved.

From the Department of Hematology/Oncology, Massachusetts General Hospital, Boston, MA, USA
1
Work is supported in part by a grant from the Massachusetts Department of Public Health.
Correspondence to: 2Michael V. Seiden, M.D., Ph.D., Massachusetts General Hospital, Cox 640, Department of Hematology/Oncology, Boston,
MA 02114, USA. E-mail: mseiden@partners.org
*Dr. Seiden is the John Dalton Clinical Scholar.
3
Abbreviations used: MDR-1, multidrug-resistance-1 gene; IL-6, interleukin-6; IL-8, interleukin-8; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; IC50, concentration which inhibits growth by 50%; MRP, multidrug resistance-associated protein; LRP, lung
cancer resistance protein; Rh123, rhodamine 123; ABC, ATP-binding cassette family; FBS, fetal bovine serum; EDTA, disodium
ethylenediaminetetra-acetate; pCMV, human cytomegalovirus immediate early promoter/enhancer; PCR, polymerase chain reaction; PBS,
phosphate buffered saline; SDS, sodium dodecyl sulfate
1043–4666/02/$-see front matter  2002 Elsevier Science Ltd. All rights reserved.
KEY WORDS: drug resistance/IL-6/paclitaxel/ovarian cancer

234 CYTOKINE, Vol. 17. No. 5 (7 March), 2002: pp 234–242


Drug resistance to currently available chemo-
therapeutic drugs is a major barrier limiting the efficacy
of cancer treatment. Paclitaxel is an antimitotic agent,
which stabilizes the assembly of microtubules by pre-
venting depolymerization, thus arresting cell progres-
sion through mitosis. Several potential mechanisms of
paclitaxel resistance have been reported in cultured cell
lines, including overexpression of MDR-13,1 alter-
ations of the
-tubulin subunit2 and differing expres-
sion of
tubulin isotypes3. Data on how these or other
mechanisms account for the majority of clinical drug
resistance, especially in solid tumors, is still poorly
understood. In an attempt to more broadly define the
spectrum of transcriptional changes associated with
acquired paclitaxel resistance, cDNA array has been
used to evaluate paclitaxel sensitive and resistant
SKOV-3 ovarian cancer cells and this analysis has
identified overexpression of several cytokines and
chemokines including IL-6 and IL-8.4 In addition,
evaluation of several additional paclitaxel resistant cell
lines demonstrated that many of these lines secreted
excessive IL-6 and IL-8 as compared to the paclitaxel
sensitive parental line. This in vitro data has been
supported in clinical studies that have demonstrated
high levels of IL-6 and IL-8 in the ascites and serum of
ovarian cancer patients.5,6
Interleukin-6 was initially described as a cytokine
important in the terminal differentiation of B-cells and
in the support of both normal and malignant plasma
cells. Subsequently, IL-6 has had several additional
activities described, including supporting host immu-
nological defense mechanisms as well as modulating
growth and differentiation in various malignancies.7
Several studies have shown that IL-6 is a growth
stimulatory factor for various tumors such as
myeloma, AIDS-associated Kaposi sarcoma, and cer-
tain T- and B-cell lymphomas.8–10 High serum IL-6
levels have been associated with poor prognosis in
several solid and hematopoietic neoplasms.11–13 In
addition, IL-6 is an important autocrine factor and
may be required for optimal cell growth of ovarian
cancer cells, although it may not directly enhance cell
proliferation.14 Finally, induction of apoptosis by
transforming growth factor
1, wild type p53 and
cytotoxic agents is suppressed by IL-6.15–17 Although
paclitaxel resistant ovarian and breast cancer cell lines
have been shown to secrete high levels of IL-6, little is
known about a possible correlation or association
between IL-6 expression and the resistance of tumor to
anticancer chemotherapeutic agents.
Interleukin-8 is a member of the CXC chemokine
family. This cytokine was initially shown to selectively
stimulate chemotactic activity for neutrophils and lym-
phocytes.18 Recent studies have found that IL-8 is
multifunctional: it can induce haptotactic migration
and proliferation of keratinocytes and melanoma
IL-6 overexpression confers paclitaxel resistance / 235
cells.19,20 Interleukin-8 is also known as a pro-
angiogenic factor for numerous carcinomas. Further-
more, increased expression of IL-8 has been reported in
aggressive cases of ovarian cancer and in ovarian
cancer cell lines exposed to paclitaxel.21 As with IL-6,
little is known about IL-8’s role in drug resistance.
To determine the contribution of IL-6 and IL-8 to
the regulation of drug resistance, a paclitaxel sensitive
cell line, U-2OS, was transfected with a plasmid con-
taining either the full-length coding region of human
IL-6 or IL-8 to determine whether overexpression of
these genes can increase paclitaxel resistance in this cell
line.
RESULTS
Expression of IL-6 or IL-8 in U-2OS
transfectants
After transfection of pIRESIL-6 or pIRESIL-8 into
U-2OS, two G418 resistant clones from each cytokine
transfection experiment were isolated and used for
subsequent study (see Fig. 1). As determined by
ELISA, these clones continuously demonstrated high
level secretion of their respective cytokine as compared
with parental U-2OS and control U-2OSpIRES cells
(Fig. 1).
Effect of IL-6 or IL-8 transfection on in vitro
growth of transfected cells
As IL-6 is reportedly a growth factor and IL-8 a
pro-angiogenic factor for various tumor cells, we ana-
lysed whether transfection with the cytokines stimu-
lates the in vitro growth of the transfectants compared
to parental U-2OS cells. Cell growth was not affected
by IL-6 transfection of U-2OS cells, whereas IL-8
modestly stimulates growth compared with parental
U-2OS (Fig. 2).
Drug resistance in U-2OSIL-6 and U-2OSIL-8 cells
As measured by MTT assay22, U-2OSIL-6a and
U-2OSIL-6b cells were relatively resistant to paclitaxel
and doxorubicin, whereas there was no significant
change in relative resistance to topotecan or mitox-
antrone (Fig. 3). The IC50 values of both clones
U-2OSIL-6a and U-2OSIL-6b are five-fold higher for
paclitaxel and four-fold higher for doxorubicin as
compared to the control U-2OSpIRES line suggesting
that IL-6 overexpression confers a moderate level of
drug resistance. Although the IC50 of U-2OSIL-8 cells is
minimally higher than controls (Fig. 4), this is attribu-
table to the increased growth rate of the transfected
cells, not resistance. Therefore IL-6, but not IL-8
transfection into U-2OS cells generates moderate
resistance to paclitaxel and doxorubicin.
236 / Duan et al.
10.00
Cytokine concentration (ng/mL)
1.00
0.10
0.01
U-2OS
Figure 1. ELISA evaluating IL-6 and IL-8 secretion from U-2OS, U-2OSpIRES, U-2OSIL-6a, U-2OSIL-6b, U-2OSIL-8a and U-2OSIL-8b.
Regulation of paclitaxel resistance in U-2OSIL-6
cells
Cross-resistance to both paclitaxel and doxoru-
bicin suggests a multi-drug resistant phenotype poss-
ibly explained by export through known membrane
bound pumps. To evaluate this possibility the expres-
sion of MDR-1, MRP and LRP was measured by
northern blot analysis. This analysis demonstrates that
there was no difference in expression of these genes in
U-2OS, U-2OSpIRES, U-2OSIL-6a and U-2OSIL-6b (Fig.
5). To further exclude MDR-1 as a mechanism of
paclitaxel resistance in IL-6 transfectants, expression of
the MDR-1 protein, P-glycoprotein was evaluated by
western analysis comparing U-2OS, U-2OSpIRES,
U-2OSIL-6a, and U-2OSIL-6b. Western analysis demon-
strates P-glycoprotein expression is not increased (Fig.
6). Finally, to evaluate MDR-1 for possible changes in
export efficiency both verapamil inhibition studies and
rhodamine 123 exclusion studies23,24 were performed.
MTT assays were performed with verapamil, an
MDR-1 inhibitor, included in the culture medium.
Verapamil did not alter U-2OSIL-6 resistance to pacli-
taxel (Fig. 7). Furthermore, rhodamine 123 efflux
studies supported the hypothesis that paclitaxel resist-
ance in U-2OSIL-6 was not secondary to MDR-1 (data
not shown). Therefore, IL-6 associated paclitaxel
resistance is not due to changes in quantity or function
of MDR-1.
Caspase-3 activation in paclitaxel treated
U-2OSIL-6
Caspase-3 plays a direct role in proteolytic cleav-
age of cellular proteins responsible for progression to
CYTOKINE, Vol. 17, No. 5 (7 March, 2002: 234–242)
IL-6
IL-8
U-2OSpIRES U-2OSIL-6a U-2OSIL-6b U-2OSIL-8a U-2OSIL-8b
apoptosis. To test whether paclitaxel resistance in IL-6
transfected cells may have reduced caspase-3 activity
with paclitaxel exposure, we measured caspase-3 activi-
ties in these cells after exposure to paclitaxel. The
results showed that there was a significantly reduced
level of caspase-3 in U-2OSIL-6 as compared with
parental U-2OS and control U-2OSpIRES (Fig. 8).
DISCUSSION
Interleukin-6 and IL-8 have been reported as
overexpressed in paclitaxel and doxorubicin resistant
cell lines.4 In addition, several investigations have
reported that in cancer patients elevated serum IL-6 or
IL-8 levels are correlated with advanced disease status
and shortened survival time.11–13,25,26 Mizutani and
colleagues demonstrate that a combination treatment
of cisplatin and anti-IL-6 antibody can overcome cis-
platin resistance in human renal carcinoma cells.27 In
the present study, IL-6 transfection into U-2OS cells
resulted in a paclitaxel and doxorubicin resistant phe-
notype whereas IL-8 transfection did not apparently
change paclitaxel sensitivity in U-2OS. Conclusions
regarding the potential role of IL-8 in paclitaxel resist-
ance are limited by the relatively modest overexpres-
sion of IL-8 in the U-2OSIL-8 transfectants. Indeed,
since IL-8 may have complex physiologic roles in
angiogenesis, further evaluation of this chemokine in in
vivo models is warranted. Interleukin-6 transfected
U-2OS cells showed an IC50 five-fold higher than
parental U-2OS and control U-2OSpIRES in paclitaxel
 IL-6 overexpression confers paclitaxel resistance / 237

1000

U-2OS
900
U-2OSpIRES
U-2OSIL-6a
800
U-2OSIL-8a
Cell number ( 103 cells/well)

700

600

500

400

300

200

100

0
0 1 2 3 4 5 6 7 8 9
Culture time (days)
Figure 2. Growth assay evaluating effect of IL-6 or IL-8 overexpression on U-2OS, U-2OSpIRES, U-2OSIL-6a and U-2OSIL-8a.

A B
1.2 1.2 U-2OS
U-2OSpIRES
1.0 1.0 U-2OSIL-6a
U-2OSIL-6b
0.8 0.8
Absorbance

Absorbance

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0.00001 0.001 0.1 10 0.00001 0.001 0.1 10

C D
1.2 1.2

1.0 1.0

0.8 0.8
Absorbance

Absorbance

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0.00001 0.001 0.1 10 0.00001 0.001 0.1 10

Figure 3. MTT assay demonstrating that IL-6 overexpression generates a paclitaxel (A) and doxorubicin (B) resistant phenotype but does not
change relative resistance to topotecan (C) or mitoxantrone (D) in U-2OSIL-6a and U-2OSIL-6b as compared to parental U-2OS and vector control
transfected U-2OSpIRES.

cytotoxicity assays and a four-fold increase in doxoru- provide evidence that IL-6 may directly participate in
bicin cytotoxicity assays. The resistance conferred by the drug resistance phenotype.
IL-6 transfection and expression is independent to Multidrug-resistance-1 gene overexpression is the
changes in MDR-1 expression or activity. These results most widely studied mechanism of both taxane and
238 / Duan et al. CYTOKINE, Vol. 17, No. 5 (7 March, 2002: 234–242)

1.2

U-2OS
U-2OSpIRES
1.0 U-2OSIL-8a
U-2OSIL-8b

0.8
Asorbance

0.6

0.4

0.2

0.0
0.00001 0.0001 0.001 0.01 0.1

Figure 4. MTT assay demonstrating that IL-8 overexpression does not generate a paclitaxel resistant phenotype in U-2OS transfected cells.

anthracylin resistance. The MDR-1 gene product, Study of clinical materials from ovarian or breast
P-glycoprotein is an ATP-dependent, membrane- cancer patients treated with paclitaxel or doxorubicin
associated efflux transporter. It is highly expressed in has, in general, not confirmed the hypothesis that
various paclitaxel and doxorubicin resistant cell lines. MDR-1 is amplified or overexpressed. Indeed, the data
has been more consistent with the acquisition of non-
1 2 3 4
MDR based mechanisms of drug resistance. Multidrug
resistance-associated protein (MRP), in the same ATP-
binding-cassette (ABC) superfamily of proteins as
MDR-1 MDR-1, is overexpressed in many drug resistant
cells.28 Lung resistant protein (LRP), which was cloned
from a non-P-glycoprotein-expressing human multi-
drug resistant fibrosarcoma cell line, has also been
implicated in the multidrug resistant phenotype.29
However, there is currently no information to suggest
MRP that either MRP or LRP plays a significant role in
paclitaxel resistance. In toto, this data supports the
existence of an alternative pathway for IL-6 associated
paclitaxel chemoresistance.
Paclitaxel is a microtubule-stabilizing agent,
which can induce apoptosis with caspase-3 activation.
LRP Activation of caspase-3 by paclitaxel treatment was
reduced in IL-6 transfected cells as compared to their
parental cells consistent with a resistant phenotype.

1 2 3 4 5 6 7

ß-actin
P-glycoprotein

Figure 5. Northern analysis of MDR-1, MRP and LRP with a
Figure 6. Western analysis of P-glycoprotein expression.
actin control.
Lane 1 U-2OS; Lane 2 U-2OSpIRES; Lane 3 U-2OSIL-6a; Lane 4
Lane 1 U-2OS; Lane 2 U-2OSpIRES; Lane 3 U-2OSIL-6a; Lane 4 U-2OSIL-6b; Lane 5 and 6 SKOV-3, a negative control; Lane 7
U-2OSIL-6b. SKOV-3TR, a positive control.
 IL-6 overexpression confers paclitaxel resistance / 239

1.2

U-2OS
U-2OS/verapamil
1.0 U-2OSIL-6a
U-2OSIL-6a/verapamil

0.8
Asorbance

0.6

0.4

0.2

0.0
0.0001 0.001 0.01 0.1 1 10

Figure 7. MTT assay evaluating verapamil mediated cell growth on U-2OS and U-2OSIL-6a.

0.40

0.35

0.30

0.25
Absorbance

0.20

0.15

0.10

0.05

0.00
U-2OS U-2OSpIRES U-2OSIL-6a U-2OSIL-6b
Figure 8. Caspase-3 activation in U-2OS, U-2OSpIRES, U-2OSIL-6a, and U-2OSIL-6b at baseline and with paclitaxel treatment.

Recently, IL-6 transfection into basal cell carcinoma
cells led to anti-apoptotic activity and tumorgenesis
through upregulation of Mcl-1, a Bcl-2 family member,
with enhanced cell survival with exposure to UV and
photodynamic therapies.30 These results are consistent
with previous data showing that cytokines, including
IL-6 and Interferon- can suppress apoptosis induced
by cytotoxic compounds or overexpression of wild-
type p53.16,17 The status of pro- and anti-apoptotic
factors in U-2OS as compared to the IL-6 transfectants
requires further study.
Although the magnitude of paclitaxel resistance
observed in U-2OSIL-6a and U-2OSIL-6b cell clones is
lower than in other in vitro models of drug resistance,
the modest levels observed cannot be discounted as
clinically irrelevant since these levels would be consist-
ent with clinically observed levels of drug resistance.
Indeed, most patients with ovarian carcinoma who
progress after cisplatin therapy have levels of acquired
cisplatin resistance of no greater than 2- to 3-fold.31
Overexpression of other proteins by transfection exper-
iments has been shown to induce drug resistance in the
240 / Duan et al.
same range as IL-6. For example, overexpression of
MDR-1 resulted in a 2- to 24-fold increase in resistance
to compounds including colchicine, doxorubicin, vin-
blastine and actinomycin D in LR73 cells.32 Overex-
pression of MRP in SW-1573 lung carcinoma cells
increases the resistance to doxorubicin, daunorubicin,
colchicine, VP-16 and vincristine 2.7- to 5.3-fold.33
Other growth factor signaling pathway components
such as c-H-ras, c-fos, Bcl-2 and c-erbB2 have been
transfected into cells in order to determine their con-
tribution to drug resistance.34,35 Benz and colleagues
demonstrated that transfection of c-erbB2 into the
MCF-7 cell line resulted in a 2–3-fold increase in
cisplatin resistance but had no effect on the sensitivity
of MCF-7 to 5-fluorouracil or doxorubicin.36
In summary, this study demonstrates that trans-
fection of IL-6 into U-2OS osteosarcoma cells pro-
duces paclitaxel and doxorubicin resistance, which is in
contrast to IL-8 transfection that has no effect on
relative paclitaxel resistance. This drug resistant phe-
notype is independent of MDR-1, MRP-1 and LRP
expression, which raises the possibility that clinical
maneuvres that down regulate IL-6 expression or sig-
naling through the IL-6 receptor may be useful in
modulating drug resistance to paclitaxel or doxoru-
bicin especially in tumors with exaggerated IL-6
production.
MATERIALS AND METHODS
Cell culture
U-2OS, a human osteosarcoma cell line was obtained
from the American Type Culture Collection (Rockville, MD,
USA). Cells were maintained in RPMI 1640 containing 10%
FBS, 100 units/ml penicillin, and 100 g/ml streptomycin, all
obtained from Gibco BRL (Grand Island, NY, USA).
pIRESIL-6 and pIRESIL-8 expression vector
construction
Clonetech’s (Palo Alto, CA, USA) mammalian expres-
sion vector pIRESneo contains the internal ribosomal entry
site (IRES) of the encephalomyocarditis virus (ECMV),
which permits the translation of two open reading frames
(ORF) from one mRNA. The expression cassette of pIRES-
neo contains the human cytomegalovirus major immediate
early promoter/ enhancer (pCMV) followed by a multiple
cloning site (MCS) and a synthetic intron known to enhance
the stability of the mRNA. Ribosomes can enter the bicis-
tronic mRNA either at the 5 end to translate the gene of
interest or at the ECMV IRES to translate the antibiotic
resistance marker. A 670 base pair cDNA fragment
containing the full ORF of human IL-6 was amplified by
reverse transcriptase-PCR from the RNA of SKOV-3TR, a
paclitaxel resistant cell line that highly overexpresses
IL-6. Polymerase chain reaction primers were: forward
5 -ATAAGAAATGCGGCCGCTCCAGGAGCCCAGC-3 the drug concentration required to inhibit A490 to 50% of the
to introduce a Not I site as italicized, and reverse:
CYTOKINE, Vol. 17, No. 5 (7 March, 2002: 234–242)
5 -GGTGGATCCAGGTGCCCATGCTACATT-3 to intro-
duce a BamH I site as italicized. A 343 base pair cDNA
fragment containing the full ORF of human IL-8 was
amplified from the RNA of SKOV-3TR. PCR primers for
IL-8 were: forward 5 -ATAAGAATGCGGCCGCTCTC
ACTGTGTGTAA-3 and reverse :5 -GGTGGATCC TTT
ATGAATTCTCAGCCCT-3 . The resulting IL-6 or IL-8
PCR product was cloned to pCR2.1 vector using Invitro-
gen’s Original TA Cloning Kit (Carlsbad, CA, USA). After
sequence confirmation, IL-6 or IL-8 was cut from the
pCR2.1 vector, purified, subcloned to the MCS of expres-
sion vector pIRESneo and subsequently sequenced to con-
firm the correct ORF. Expression of IL-6 or IL-8 cDNA was
under the control of the pCMV.
Transfection and production of stable cell lines
Transfections were performed using LipofectAmine Plus
reagents (Gibco BRL, Grand Island, NY, USA) as follows:
approximately 5105 U-2OS cells were plated into 90 mm
tissue culture dishes and cultured overnight. Prior to trans-
fection, the growth medium was replaced with serum free
RPMI 1640 and cultured for three hours. LipofectAmine
reagent containing 5 g of pIRESempty, pIRESIL-6 or
pIRESIL-8 was combined with Plus reagent and applied to
the cells. After culture for four hours, the media was replaced
with RPMI 1640 containing 10% FBS. G418 sulfate (Invit-
rogen, Carlsbad, CA, USA) selection (400 ug/ml) was started
at 24 hours post transfection. The selection medium was
changed every 2 days.
ELISA for human IL-6 and IL-8
IL-6 and IL-8 levels in culture supernatants were
measured using a quantitative immunoassay ELISA kit
according to the manufacturer’s instructions (Genzyme,
Boston, MA, USA).
In vitro growth assay
Five thousand cells per well of either U-2OS,
U-2OSpIRES, U-2OSIL-6 or U-2OSIL-8 transfectants were
plated in multiple 24 well plates. Cells were cultured in RPMI
1640 containing 400 g/ml G418 for the transfected cell lines.
On days 3, 5, 7 and 9 two wells of each cell line were
tripsinized, counted using trypan-blue (Sigma, St. Louis,
MO, USA) and averaged to obtain daily cell counts.
Cytotoxicity assay
In vitro cytotoxicity assays were performed by MTT
assay as previously described.22 MTT was obtained from
Sigma (St. Louis, MO, USA). Briefly, 2103 cells per well
were plated in 96–well plates. Culture medium was RPMI
1640 containing increasing concentrations of paclitaxel,
doxorubicin, topotecan or mitoxantrone (all obtained from
commercial sources). After culture for 7 days, 50 l of MTT
(5 mg/ml PBS) was added to each well and incubated for 4 h.
After dissolving the resulting formazan product with acid-
isopropanol, the absorbance (A490) was read on a BT 2000
Microkinetics Reader (Bio-Tek Instrument, Inc., Winooski,
VT, USA) at a wavelength of 490 nm. The IC50 is defined as
control value. The absorbance values were normalized

assigning the value of the parent line in media without drug
to 1.0 and the value of the no-cell control to 0. Experiments
were performed in duplicate.
RNA isolation and northern blot hybridization
mRNA was prepared from trizol-extracted total RNA
using FastTrack mRNA Isolation Kit (Invitrogen, Carlsbad,
CA, USA). mRNA was separated by electrophoresis, trans-
ferred to Hybond N-plus membranes (Amersham Pharma-
cia, Piscataway, NJ, USA), and cross-linked by UV.
[32P]dCTP- (Dupont, Boston, MA, USA) labelled cDNA
probes were hybridized in Rapid-hyb buffer (Amersham
Pharmacia, Piscataway, NJ, USA) for 2 h and washed twice
at room temperature with 2.0SSC, 0.1% SDS for 15 min
and twice at 65C with 0.2SSC, 0.1% SDS for 15 min.
Blots were exposed to Kodak X-OMAT Blue film (Eastman
Kodak, Rochester, NY, USA) with an intensifying screen.
Protein isolation and western blot hybridization
Western blots were performed using an ECL+Plus Kit
(Amersham Pharmacia, Piscataway, NJ, USA). Trypsin-
EDTA washed cells were pelleted and solublized in Mam-
malian Protein Extraction Reagent M-PER (PIERCE,
Rockford, IL, USA). Equal aliquots of protein were resolved
using 6% SDS-polyacrylamide gel electrophoresis. The trans-
ferred nitrocellulose blot was blocked with 5% fat-free milk
powder in pH 7.6 TBS-T (20 mM Tris-HCl, 100 mM NaCl,
0.1% Tween 20) at room temperature for 2 h. The membrane
was then immunoblotted with monoclonal antibody C219
(Signet, Dedham, MA, USA) in TBS-T for 2 h at room
temperature. Detection was performed using the reagents
provided in the ECL+Plus kit.
Caspase-3 activation
Cells were cultured for 16 h in 0.1 M paclitaxel with
subsequent lysate analysis using Clonetech’s (Palo Alto,
CA, USA) ApoAlertCaspase-3 kit according to the
manufacturer’s instruction.
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