Báo cáo khoa học: Adenine nucleotides inhibit proliferation of the human lung adenocarcinoma cell line LXF-289 by activation of nuclear factor jB1 and mitogen-activated protein kinase pathways doc

Adenine nucleotides inhibit proliferation of the human
lung adenocarcinoma cell line LXF-289 by activation of
nuclear factor jB1 and mitogen-activated protein kinase
pathways
Rainer Scha
¨
fer
1
, Roland Hartig
2
, Fariba Sedehizade
1
, Tobias Welte
3
and Georg Reiser
1
1 Institut fu
¨
r Neurobiochemie, Otto-von-Guericke-Universita
¨
t, Medizinische Fakulta
¨
t, Magdeburg, Germany
2 Institut fu
¨
r Immunologie, Otto-von-Guericke-Universita
¨
t, Medizinische Fakulta
¨
t, Magdeburg, Germany
3 Klinik fu

¨
r Pneumologie, Medizinische Hochschule Hannover, Germany
Keywords
cell cycle progression; mitogen-activated
protein kinase; nuclear factor-jB1; P2Y
receptors; phosphatidylinositol-3-kinase;
protein kinase C
Correspondence
G. Reiser, Otto-von-Guericke-Universita
¨
t
Magdeburg, Medizinische Fakulta
¨
t, Institut
fu
¨
r Neurobiochemie, Leipziger Str. 44;
39120 Magdeburg, Germany
Fax: +49 391 6713097
Tel: +49 391 6713088
E-mail: georg.reiser@medizin.
uni-magdeburg.de
(Received 4 February 2006, revised 12 June
2006, accepted 16 June 2006)
doi:10.1111/j.1742-4658.2006.05384.x
Extracellular nucleotides have a profound role in the regulation of the pro-
liferation of diseased tissue. We studied how extracellular nucleotides regu-
late the proliferation of LXF-289 cells, the adenocarcinoma-derived cell
line from human lung bronchial tumor. ATP and ADP strongly inhibited
LXF-289 cell proliferation. The nucleotide potency profile was ATP ¼

ADP ¼ ATPcS > > UTP, UDP, whereas a,b-methylene-ATP, b,c-methy-
lene-ATP, 2¢,3¢-O-(4-benzoylbenzoyl)-ATP, AMP and UMP were inactive.
The nucleotide potency profile and the total blockade of the ATP-mediated
inhibitory effect by the phospholipase C inhibitor U-73122 clearly show
that P2Y receptors, but not P2X receptors, control LXF-289 cell prolifer-
ation. Treatment of proliferating LXF-289 cells with 100 lm ATP or ADP
induced significant reduction of cell number and massive accumulation of
cells in the S phase. Arrest in S phase is also indicated by the enhancement
of the antiproliferative effect of ATP by coapplication of the cytostatic
drugs cisplatin, paclitaxel and etoposide. Inhibition of LXF-289 cell prolif-
eration by ATP was completely reversed by inhibitors of extracellular sig-
nal related kinase-activating kinase ⁄ extracellular signal related kinase 1 ⁄ 2
(PD98059, U0126), p38 mitogen-activated protein kinase (SB203508), phos-
phatidylinositol-3-kinase (wortmannin), and nuclear factor jB1 (SN50).
Western blot analysis revealed transient activation of p38 mitogen-activated
protein kinase, extracellular signal-related kinase 1 ⁄ 2, and nuclear factor
jB1 and possibly new formation of p50 from its precursor p105. ATP-
induced attenuation of LXF-289 cell proliferation was accompanied by
transient translocation of p50 nuclear factor jB1 and extracellular signal-
related kinase 1⁄ 2 to the nucleus in a similar time period. In summary,
inhibition of LXF-289 cell proliferation is mediated via P2Y receptors by
activation of multiple mitogen-activated protein kinase pathways and
nuclear factor jB1, arresting the cells in the S phase.
Abbreviations
a,b-MeATP, a,b-methylene adenosine 5¢-triphosphate; b,c-MeATP, b,c-methylene adenosine 5¢-triphosphate; BrdU, bromodeoxyuridine;
Bz-ATP, 2¢,3¢-O-(4-benzoylbenzoyl)adenosine 5¢-triphosphate; CaMKII, calcium ⁄ calmodulin-dependent protein kinase; ERK, extracellular signal-
regulated kinase; GPCR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; MEK1 ⁄ 2, ERK-activating kinase; 2MeS-ADP,
2-methylthioadenosine 5¢-diphosphate; 2MeS-ATP, 2-methylthioadenosine 5¢-triphosphate; NF-jB, nuclear factor jB; NSCLC, nonsmall cell
lung cancer; PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; PLC, phospholipase C.
3756 FEBS Journal 273 (2006) 3756–3767 ª 2006 The Authors Journal compilation ª 2006 FEBS

There is growing evidence that extracellular nucleotides
can regulate the proliferation of numerous tumorigenic
or nontumorigenic tissues. The nucleotide-activated
purinergic P2 receptor family comprises the ionotropic
P2X receptor ion channels and the metabotropic, G
protein-coupled P2Y receptors [1–3]. Seven subtypes of
the P2X receptors (P2X
1)7
) and eight subtypes of the
P2Y receptors (P2Y
1,2,4,6,11,12,13,14
) have been identified
and functionally characterized [4,5]. The regulatory
function of extracellular nucleotides on cell growth,
the role of nucleotides as effectors of neoplastic
transformation and the ubiquitous expression of the
purinergic receptors led to the investigation of the
therapeutic potential of nucleotides and P2 receptors
in clinical trials targeting various diseases (for review
see [6]).
ATP is already known to inhibit the growth of var-
ious tumors by activating specific P2 receptors. Inhibi-
tion of cancer growth by adenine nucleotides was first
described by Rapaport [7]. In vitro extracellular nucle-
otides exert a strong antineoplastic effect on breast,
ovarian [8], skin [9], prostate [10], endometrium [11],
esophagus [12], intestinal [13] and colorectal carcinoma
[14], suggesting that extracellular nucleotides can sup-
press tumorigenesis. In view of the antineoplastic
action of extracellular nucleotides [6] and the signifi-

cant in vivo antitumor activity of intraperitoneally
injected ATP against several aggressive carcinomas in
tumor-bearing mice [7,15], it will be important to
understand how the different P2 receptor subtypes
contribute to the regulation of cancer cell prolifer-
ation.
In the majority of cell lines, P2Y receptors mediate
the inhibition of growth or proliferation, whereas only
a few cases have been reported where control of prolif-
eration had been claimed to be mediated by P2X
receptors. Activation of P2X
5
receptors stimulated the
differentiation of skin cancer cells with subsequent
inhibition of proliferation [16], and induction of cell
death was mediated via P2X
7
receptors in skin cancer
and prostate cancer cells [16,17]. The formation of the
lytic pore of the P2X
7
receptor is dependent on coordi-
nated signaling involving the p38 mitogen-activated
protein kinase (MAPK) pathway and caspase activa-
tion [18]. Several mechanisms have been found for the
inhibitory action of ATP on cancer cell growth. In
tumor cell lines from colon [14], endometrium [11],
and esophagus [12], activation of P2Y receptors caused
inhibition of proliferation by induction of cell cycle
arrest and ⁄ or apoptosis. However, the pathways

involved in P2Y receptor-mediated attenuation of cell
proliferation are not known. Therefore, we here
explored which pathways are involved in the antineo-
plastic action of extracellular nucleotides on the
human lung adenocarcinoma cell line LXF-289, which
is derived from solid lung adenocarcinoma. Extracellu-
lar nucleotides strongly attenuated the cell cycle pro-
gression of proliferating LXF-289 cells, leading to
marked arrest of the cells in the S phase. This inhibi-
tion is mediated by P2Y receptors through signaling
pathways involving activation of the transcription
factor nuclear factor jB1 (NF-jB1) and the MAPKs
extracellular signal-related kinase 1 ⁄ 2 (ERK1 ⁄ 2) and
p38. Our findings underline the importance of extracel-
lular nucleotides in the specific modulation of cell
proliferation.
Results
Extracellular nucleotides attenuate LXF-289 cell
proliferation via P2Y receptors
Single pulses of ATP and ADP equipotently inhibited
LXF-289 cell proliferation. The effect was concen-
tration-dependent, with half-maximal inhibition at
18.6 ± 1.9 lm (n ¼ 5) and 19.8 ± 0.6 lm (n ¼ 5),
respectively (Fig. 1). Significant inhibition was already
nucleotide concentration [µM]
10 20 40 60 100
)lortn
o
cfo
%(noitaroprocniUdr

B
20
40
60
80
100
Control ATP ADP UTP
UDP 2-MeSADP Ado
Fig. 1. Concentration-dependent inhibition of LXF-289 cell pro-
liferation by different nuccleotides. Increasing concentrations
(10–100 l
M) of the different nucleotides were added to proliferating
LXF-289 cells, and bromodeoxyuridine (BrdU) incorporation was
measured after 12 h. Values are means ± SD of n ¼ 4–7 experi-
ments run in triplicate.
R. Scha
¨
fer et al. ATP and ADP inhibit lung cell proliferation
FEBS Journal 273 (2006) 3756–3767 ª 2006 The Authors Journal compilation ª 2006 FEBS 3757
seen at 10 lm ATP (18.2 ± 4.9%; P < 0.01) or ADP
(18.5 ± 3.5%; P < 0.01), which exhibited a reduction
of 55% at 100 lm concentration. The pyrimidine nu-
cleotides UTP and UDP reduced the proliferation at
100 lm only by 23–26% (Table 1). ATPcS inhibited
the proliferation of LXF-289 cells as potently as
ADP and ATP (Table 1). Other nucleotides, such as
a,b-methylene-ATP (a,b-MeATP) and b,c-MeATP,
which preferentially activate P2X receptors, and AMP
or UMP displayed no activity (Table 1). Moreover,
adenosine, which is known to tightly regulate the pro-

liferation of tumor cells [19], had no effect on the pro-
liferation of LXF-289 cells up to concentrations of
100 lm (Fig. 1). Likewise, 2-methylthio-ADP (2-MeS-
ADP), 2-methylthio-ATP (2-MeSATP) and 2¢,3¢-O-(4-
benzoylbenzoyl)-ATP (Bz-ATP), which most potently
activate human P2Y
1,12
and P2Y
11
receptors, respect-
ively [20,21], did not influence the proliferation of
LXF-289 cells (Table 1). Thus, the pharmacologic pro-
file of the different nucleotides or nucleotide analogs
tested strongly indicates that P2Y, but not P2X, recep-
tors attenuate LXF-289 cell proliferation.
We next examined the expression of P2Y
(P2Y
1,2,4,6,11,12,13
) and P2X (P2X
3,4,7
) receptors in
LXF-289 cells by RT-PCR using primers specific for
the human isoforms. There was evidence for the
expression of mRNA for the P2Y receptor subtypes
P2Y
2
, P2Y
6
, P2Y
11

and P2Y
13
and for the P2X
4
recep-
tor, whereas expression of the P2Y
1
, P2Y
4
, P2Y
12
,
P2X
3
and P2X
7
receptors could not be detected
(Fig. 2).
Inhibition of growth and cell cycle progression
of LXF-289 cells
We confirmed that the attenuation of DNA synthesis
by ATP or ADP was indeed due to growth inhibition
of LXF-289 cells. Treatment of proliferating LXF-289
cells with 100 lm ATP or ADP reduced the cell num-
ber. We observed reductions by 19.1 ± 3.8% (± SD;
n ¼ 3) and 20.8 ± 4.4% (± SD; n ¼ 3) after 24 h,
and by 54.0 ± 4.3% (± SD; n ¼ 3) and 54.1 ± 2.6%
Table 1. Inhibition of proliferation of LXF-289 lung tumor cells.
Effects of P2 receptor agonists. The influence of the different P2
receptor agonists is shown as percentage inhibition of the prolifer-

ation of LXF-289 cells. Bromodeoxyuridine (BrdU) incorporation into
proliferating LXF-289 cells. LXF-289 cells in 96-well plates were
incubated for 1 h with 100 l
M of the different nucleotides or nuc-
leotide analogs listed below before the addition of 10 l
M BrdU.
BrdU incorporation was determined by chemiluminescence as des-
cribed in Experimental procedures, following incubation for 12 h.
Proliferation in the absence of nucleotides is taken as the reference
value. Inhibition of that value in percentage is given as means ± SD
of 3–6 independent experiments run in triplicate. Bz-ATP, 2¢,3¢-O-(4-
benzoylbenzoyl)adenosine 5¢-triphosphate; 2MeS-ADP, 2-methyl-
thioadenosine 5¢-diphosphate; 2MeS-ATP, 2-methylthioadenosine
5¢-triphosphate.
P2 receptor agonist Inhibition of proliferation (%)
ATP 55.0 ± 5.1
ADP 54.7 ± 4.6
ATPcS 59.5 ± 3.8
Bz-ATP 10.8 ± 5.1
UTP 22.5 ± 3.6
UDP 25.6 ± 2.3
a,b-MeATP 5.6 ± 4.1
b,c-MeATP 6.8 ± 5.7
2-MeSADP 5.0 ± 1.6
2-MeSATP 5.9 ± 1.1
UMP 7.3 ± 2.3
AMP 11.7 ± 5.9
P2Y
1
P2Y

4
P2Y
11
P2Y
13
P2X
4
GAPDH
P2Y
2
P2Y
6
P2Y
12
P2X
3
P2X
7
630
442
420
812
855
Fig. 2. Expression of P2 receptors in LXF-289 cells. RT-PCR analysis was performed using primers for P2Y
1
, P2Y
2
, P2Y
4
, P2Y

6
, P2Y
11
, P2Y
12
and P2Y
13
receptors and the P2X receptors P2X
3
, P2X
4
and P2X
7
in LXF-289 cells. PCR products were separated in a 1% agarose gel and
visualized with ethidium bromide. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Sizes of RT-PCR products of the different P2 recep-
tors are indicated.
ATP and ADP inhibit lung cell proliferation R. Scha
¨
fer et al.
3758 FEBS Journal 273 (2006) 3756–3767 ª 2006 The Authors Journal compilation ª 2006 FEBS
(± SD; n ¼ 3) after 48 h, respectively. These results
confirm the identical potency of ATP and ADP in the
inhibition of LXF-289 cell proliferation.
Cell cycle analysis revealed that inhibition of prolif-
eration of LXF-289 cells by ATP and ADP was medi-
ated by retardation of cell cycle progression. Flow
cytometry analysis of subconfluent LXF-289 cell cul-
tures showed that treatment with ATP or ADP (10–
100 lm) for 24 h caused a concentration-dependent
increase of cells in the S phase. With 100 lm ATP and

ADP, 74.6 ± 3.2% (n ¼ 3) and 66.4 ± 1.3% (n ¼ 3)
of the cells, respectively, were arrested in S phase
(Fig. 3A). Figure 3B illustrates the changes of the cell
cycle distribution induced by two concentrations of
ATP in LXF-289 cells.
ATP sensitizes LXF-289 cells to the activity of
anticancer drugs
Extracellular nucleotides caused accumulation of LXF-
289 cells in the S phase, where cells are especially sensi-
tive to anticancer drugs. Therefore, we used cisplatin,
etoposide and paclitaxel to investigate the effects of
these anticancer drugs on LXF-289 cells in the presence
of ATP. These anticancer substances inhibit cell cycle
progression by different mechanisms and form the basis
of current chemotherapeutic regimens in lung cancer
treatment. Cisplatin, etoposide and paclitaxel dose-
dependently inhibited the proliferation of LXF-289 cells
with IC50 values of 13.8 ± 1.3 lm (n ¼ 3), 19.4 ±
2.2 lm (n ¼ 3), and 16.2 ± 1.4 nm (n ¼ 3), respectively
(Fig. 4). The simultaneous addition of 100 lm ATP
enhanced the antiproliferative potency of cisplatin
3-fold, of etoposide 2-fold and of paclitaxel 2.5-fold,
with resulting IC50 values of 4.7 ± 0.8 lm, 9.3 ±
2.3 lm and 6.8 ± 1.2 nm (Fig. 4). The data suggest an
additive effect of the anticancer drugs and ATP on pro-
liferation, and support our conclusion that in LXF-289
cells the ATP-mediated arrest of cell cycle progression
targets the S phase. We did not find any significant
induction of DNA fragmentation in LXF-289 cells or
an increase in the number of dead cells under our assay

conditions (data not shown). Moreover, the concentra-
tions of cisplatin and etoposide used do not induce
significant apoptosis in other human lung cancer cells
such as A549 cells, as reported by others [22,23].
Signal transduction pathways involved in
nucleotide-mediated inhibition of proliferation
of LXF-289 cells
Different mechanisms and signal transduction path-
ways transduce control of proliferation by G protein-
coupled receptors (GPCRs). We therefore investigated
which of the pathways found in GPCR-mediated
attenuation of cell proliferation, including phospho-
lipase C (PLC), protein kinase C (PKC) and extra-
cellular Ca
2+
influx, are involved in the control of
LXF-289 cell proliferation by P2Y receptors.
At a concentration of 10 lm, the PLC inhibitor
U-73122 completely reversed the effect of ATP on
10 µM 20 µM 50 µM 100 µM
)latotfo%(sl
l
ecesahp-S
0
20
A
B
40
60
80

0
80
20 40 60
control
G0-G1: 65.74%
S-Phase:
26.27%
100 µM ATP
G0-G1: 19.91%
S-Phase:
77.97%
20 µM ATP
G0-G1: 40.40%
S-Phase:
48.39%
0
80
16
0
80
16
0
80
16
)len
n
ahc
/stnuoc(sllec
a
b

c
control ATP
ADP
Fig. 3. Effect of ATP and ADP on cell cycle progression of LXF-289
cells. (A) Accumulation of LXF-289 cells in the S phase. LXF-289
cells were incubated with ATP or ADP (10–100 l
M) for 24 h, and
the distribution of cells in the different cell cycle phases was ana-
lyzed by fluorescence-activated cell sorting (FACS). (B) Differences
in the cell cycle distribution of LXF-289 cells. Depicted are the per-
centages of the cells in the G0 ⁄ G1, S and G2 ⁄ M phases of the cell
cycle after treatment with 20 l
M or 100 lM ATP for 24 h. The gat-
ing used for the quantification of the cells in the G0 ⁄ G1 (a), S (b)
and G2-M (c) phases is depicted by the respective bar.
R. Scha
¨
fer et al. ATP and ADP inhibit lung cell proliferation
FEBS Journal 273 (2006) 3756–3767 ª 2006 The Authors Journal compilation ª 2006 FEBS 3759
LXF-289 cell proliferation, whereas the inactive analog
U-73343 had no effect (Table 2). Blockade of receptor-
operated Ca
2+
channels with SKF-96365 dose-depend-
ently reduced the inhibitory effect of ATP (data not
shown). A reduction of 80% was seen at 50 lm
(Table 2). This result and the pharmacologic profile
obtained for inhibition of proliferation clearly show
that P2Y receptors mediate the attenuation of LXF-
289 cell proliferation.

Preincubation of LXF-289 cells with the potent
PKC inhibitor Go
¨
-6983 concentration-dependently
abolished the antiproliferative effect of ATP (Table 2).
In addition, we investigated the involvement of the
cisplatin, etoposide (µM)
01 10 100
)
lasabfo%(noitarop
r
ocniUd
r
B
0
20
40
60
80
100
120
+ Cisplatin
+ ATP-Cisplatin
+ Etoposide
+ ATP-Etoposide
Control
+ ATP
paclitaxel (nM)
01 10 100
)

l
a
sa
b
fo
%(n
oitar
o
p
r
oc
n
iU
drB
0
20
40
60
80
100
120
+ Paclitaxel
+ ATP-Paclitaxel
Control
+ ATP
A
B
Fig. 4. Effect of anticancer drugs and ATP on proliferation of LXF-
289 cells. Bromodeoxyuridine (BrdU) incorporation into DNA was
measured in LXF-289 cells (control) or cells incubated with different

concentrations of the anticancer drugs cisplatin or etoposide (A) or
paclitaxel (B) in the absence (solid symbols) or presence (open
symbols) of 100 l
M ATP. Values (means ± SD of four or five inde-
pendent experiments run in triplicate) are expressed as percentage
of BrdU incorporation into DNA in cells without the addition of
either ATP or the anticancer drugs (basal proliferation ¼ 100% con-
trol). IC50 values were calculated using the
SIGMAPLOT curve-fitting
program (SPSS, Chicago, IL, USA).
Table 2. Inhibition of proliferation of LXF-289 lung tumor cells.
Effects of signaling pathway inhibitors on ATP-inhibited proliferation
(A) and of basal proliferation (B) of LXF-289 lung tumor cells. Prolif-
erating LXF-289 cells in 96-well plates were incubated for 1 h with
the various signal transduction pathway inhibitors listed below
before the addition of 100 l
M ATP (ATP) or medium (B). Bromode-
oxyuridine (BrdU) incorporation was then determined by chemilumi-
nescence as described in Experimental procedures following
incubation for 12 h. (A) Data give percentage reduction of the inhibi-
tory effect exerted by ATP on BrdU incorporation. The inhibition of
proliferation of LXF-289 cells by 100 l
M ATP (see value in Table 1)
is taken as 100%. (B) Basal incorporation of BrdU into proliferating
LXF-289 cells in the presence of the inhibitors for the kinases cal-
cium ⁄ calmodulin-dependent protein kinase (CaMKII), extracellular
signal-regulated kinase 1 ⁄ 2 (ERK1 ⁄ 2), p38 mitogen-activated pro-
tein kinase (MAPK), protein kinase C and PI3 kinase. All values are
means ± SD of 3–6 independent experiments run in triplicate.
(A)

Signaling
pathway inhibitors Concentration
Reduction of inhibitory
effect of ATP (%)
U-73122 10 l
M 101 ± 6.4
U-73343 10 l
M 8.1 ± 7.0
SKF-96365 50 l
M 79.8 ± 4.7
KN-62 25 l
M 98.6 ± 3.7
Wortmannin 0.1 l
M 93.9 ± 3.2
PD98059 20 l
M 92.8 ± 3.3
U0126 1 l
M 91.8 ± 4.1
SB203580 20 l
M 95.1 ± 2.9
Go
¨
6983 0.1 l
M 24.6 ± 4.1
Go
¨
6983 1.0 l
M 86.7 ± 2.8
NF-jB SN50 20 l
M 94.2 ± 5.3

Curcumin 20 l
M 97.1 ± 1.9
Sulindac sulfide 10 l
M 88.4 ± 9.0
(B)
Kinase
inhibitors Concentration
Reduction of basal
proliferation (%)
KN-62 25 l
M 5.6 ± 2.4
Wortmannin 0.1 l
M 8.7 ± 4.8
PD98059 20 l
M 8.8 ± 5.7
U0126 1 l
M 7.9 ± 5.1
SB203580 20 l
M 5.8 ± 4.2
Go
¨
6983 0.1 l
M 4.8 ± 2.3
Go
¨
6983 1.0 l
M 6.5 ± 2.8
ATP and ADP inhibit lung cell proliferation R. Scha
¨
fer et al.

3760 FEBS Journal 273 (2006) 3756–3767 ª 2006 The Authors Journal compilation ª 2006 FEBS
calcium ⁄ calmodulin-dependent kinase II (CaMKII),
which is also known to regulate cell cycle progression
and proliferation. Inhibition of CaMKII activity by
KN-62 (25 lm) completely reduced the inhibition by
ATP.
The inhibition of LXF-289 cell proliferation by
100 lm ATP was totally attenuated by blockers of
either ERK-activating kinase (MEK1 ⁄ 2) (20 lm
PD98059, 1 lm U0126), p38 MAPK (20 lm
SB203580) or phosphatidylinositol-3-kinase (PI3K)
(wortmannin), indicating that each pathway seems to
be involved in ATP-mediated inhibition of LXF-289
cell proliferation (Table 2). Inhibition of the MAPK
cascade enzymes MEK1 ⁄ 2 and p38 MAPK by their
selective inhibitors PD98059 ⁄ U0126 and SB203580,
respectively, antagonized the nucleotide-mediated inhi-
bition of cell proliferation in a concentration-depend-
ent manner (data not shown).
We further investigated whether the activation of
ERK1 ⁄ 2 and p38 MAPK may be part of the antipro-
liferative activity of ATP, as these kinases are import-
ant regulators of proliferation as well as apoptosis.
Western blotting revealed a time-dependent increase in
both ERK1 and ERK2 phosphorylation with 100 lm
ATP (Fig. 5A). ERK1 ⁄ 2 activation was maximal in
the cytosolic fraction after 10 min and had strongly
declined below the control level after 20 min. Concom-
itant with this rapid activation, a transient transloca-
tion of activated ERK1 ⁄ 2 into the nuclear fraction

was induced by ATP. The translocation was maximal
at 10 min and declined thereafter, with no active
ERK1 ⁄ 2 detectable in the nuclear fraction after 60 min
(Fig. 5A).
Exposure of LXF-289 cells to ATP also caused
rapid phosphorylation of p38 MAPK (Fig. 5B). Again,
as for ERK1 ⁄ 2, maximal activation was seen at 10 min
after addition of ATP. However, in contrast to
ERK1 ⁄ 2, no translocation of activated p38 MAPK to
the nucleus could be detected. Instead, after a rapid
decline of phosphorylated p38 MAPK down to unde-
tectable levels after 30 min, activated p38 MAPK reap-
peared at 60 min after addition of agonist (Fig. 5B).
These results further indicate the involvement of both
MAPKs, ERK1 ⁄ 2 and p38, in the signaling pathways,
consistent with the inhibition of the antiproliferative
activity of ATP by the MEK1 ⁄ 2 inhibitors PD98059
and UO126 and the p38 MAPK inhibitor SB20358
(Table 1). The differences in the phosphorylation of
ERK1 ⁄ 2 and p38 MAPK are not due to altered
expression of ERK1 ⁄ 2 and p38 MAPK, as western
blotting of total cell lysates did not reveal any change
of either kinase after treatment with 100 lm ATP for
up to 60 min (Fig. 5C).
Activation of the transcription factor NF-jB can
lead to induced tumor proliferation or to suppression
of tumor growth, resulting in apoptosis [24]. Therefore,
we also investigated the involvement of NF-jBin
nucleotide-mediated inhibition of LXF-289 cell pro-
liferation. Preincubation of LXF-289 cells with the

NF-jB1 inhibitory peptide NF-jB SN50 (20 lm) com-
pletely abrogated the inhibitory effect of 100 lm ATP
(Table 2). This suggests that inhibition of LXF-289 cell
proliferation by ATP is mediated through NF-jB. Fur-
ther evidence for the involvement of NF-jB in the
signaling pathway activated by P2Y receptors was
obtained by the use of nonsteroidal anti-inflammatory
drugs, which have been shown to inhibit activation of
kDa
0 10 20 30 60
0 10 20 30 60
Cytosol Nucleus
[min]
50
37
phospho-p38
phospho-ERK 1/2
50
37
A
B
C
0 10 30 60 [min]
Cell lysate
kD
ERK 1/2
37
p38
37
RelA (p65)

50
10
NF-κB1
75
50
p105
p50
D
E
0 10 20 30 60
0 10 20 30
Cytosol Nucleus
[min]
kD
Fig. 5. ATP-mediated activation and nuclear translocation of extra-
cellular signal-related kinase 1 ⁄ 2 (ERK1 ⁄ 2), p38 mitogen-activated
protein kinase (MAPK) and nuclear factor jB (NF-jB). LXF-289 cells
were incubated with 100 l
M ATP, and the incubation was stopped
by aspiration of the medium at the different time points indicated.
After preparation of subcellular fractions as described in Experimen-
tal procedures, proteins were separated by SDS ⁄ PAGE and trans-
ferred to nitrocellulose. Blots from cytosolic and nuclear fractions
were incubated with antibodies to (A) phospho-ERK1 ⁄ 2 (1 : 1000
dilution) and (B) phospho-p38 MAPK (1 : 1000 dilution), and with
polyclonal antibodies (1 : 1000 dilution) specific for (D) NF-jB1 (p50
and p105) and (E) RelA (p65). Total cell lysate (C) was incubated
with antibodies to total ERK1 ⁄ 2 and p38, respectively. Primary anti-
bodies were incubated at 4 °C overnight, and horseradish peroxi-
dase (HRP)-conjugated secondary antibody (1 : 20 000 dilution) was

incubated for 1.5 h at room temperature. The antibody reaction
was visualized with enhanced chemiluminescence. The positions of
the protein molecular mass markers are indicated in kilodaltons
(kDa). The experimental data shown are typical for three independ-
ent experiments performed with different passages of LXF-289
cells.
R. Scha
¨
fer et al. ATP and ADP inhibit lung cell proliferation
FEBS Journal 273 (2006) 3756–3767 ª 2006 The Authors Journal compilation ª 2006 FEBS 3761
NF-jB and cancer cell proliferation [25–27]. Curcumin
(20 lm) and sulindac sulfide (10 lm) totally abolished
the ATP-induced inhibition of LXF-289 cell prolifer-
ation (Table 2), without inhibiting LXF-289 cell prolif-
eration at this concentration in the absence of
nucleotides (data not shown). These results further
support the involvement of NF-jB in the P2Y recep-
tor-mediated control of LXF-289 cell proliferation.
Western blot analysis showed that incubation of
LXF-289 cells with 100 lm ATP caused a detectable
decrease of the p50 form of NF-jB1 in the cytosol after
10 min, which is still visible after 60 min (Fig. 5D).
Simultaneously with the decrease of p50 in the cytosol,
an increase of NF-jB1 p50 in the nuclear fraction
could be observed, starting after 10 min, which persis-
ted after 60 min of nucleotide exposure (Fig. 5D). Sign-
aling by ATP also influenced the formation of p50
from its precursor p105. Partial degradation of the
p105 form of NF-jB1, which has an inhibitory func-
tion in the cytosol, like the inhibitor jB(IjB)s, to the

p50 form is visible after 20–30 min (Fig. 5D). Thus,
ATP not only induced the translocation of NF-jB1,
but also possibly led to newly formed p50 from the pre-
cursor p105. In addition, ATP induced the disappear-
ance of p65 NF-jB from the cytosol after 20–30 min,
but, surprisingly, no translocation into the nucleus was
observed (Fig. 5E). These data suggest that NF-jB1
and p65 have different roles in the cell cycle regulation
of proliferating LXF-289 cells by nucleotides.
Discussion
Our data demonstrate an important role of extracellu-
lar nucleotides in the regulation of proliferation of
human lung tumor cells. Thus far, it has not been
found that extracellular nucleotide P2Y receptors
downregulate cell cycle progression in epithelial cells
via activation of NF-jB. Attenuation of LXF-289 cell
proliferation by ATP is mediated by the activation of
the MEK ⁄ ERK1 ⁄ 2, PI3K and p38 MAPK pathways.
Activation and translocation of the transcription fac-
tors NF-jB1 (p50) and RelA (p65) was also involved.
These pathways lead to a massive arrest of the cells in
the S phase. Activation of NF-jB by P2Y receptors
has only been found so far to be associated with the
stimulation of cell proliferation, such as in osteoclasts
[28] or A549 alveolar lung tumor cells [29].
The activity profile of the different nucleotides and
nucleotide analogs used, as well as the complete inhibi-
tion of the antiproliferative effect of ATP by the PLC
inhibitor U-73122, clearly indicate that P2Y receptors,
but not P2X receptors, mediate the inhibition of

LXF-289 lung cell proliferation. The inability of
2-MeSATP and Bz-ATP to influence the proliferation
of LXF-289 cells excludes the participation of the
P2Y
11
receptor subtype in the inhibition. Bz-ATP has
been shown to activate human P2Y
11
receptors,
whereas 2-MeSATP activates the human P2Y
11
recep-
tor with similar potency as ATP [20]. The phar-
macologic profile seen here for the inhibition of
proliferation best fits the characteristics of the subtypes
P2Y
2
and P2Y
13
, which were found to be expressed in
LXF-289 cells by RT-PCR. Thus, our data suggest
that these P2Y receptors are involved in the down-
regulation of LXF-289 cell proliferation. The P2Y
2
receptor has been found to be important for the
proliferation of stem cells and of human melanomas,
and to play a role during differentiation as well as dur-
ing development and aging [9,30–32]. However, our
finding here that ATP was much more potent than
UTP is not consistent with the equipotent activation

of the P2Y
2
receptor by ATP and UTP (reviewed in
[3]). Similarly, the inactivity of 2-MeSADP, which is
more potent than ADP at the human P2Y
13
receptor
[33], creates another conundrum. Similaryl, a nucleo-
tide activity profile, which was not consistent with the
pattern of P2Y receptors expressed, posed an enigma
in the work by Neary and coworkers, where the stimu-
lation of the ERK cascade in rat cortical astrocytes by
nucleotide analogs was studied [34].
The control of LXF-289 cell proliferation by purin-
ergic metabotropic P2Y receptors involves the activa-
tion of multiple pathways. One pathway activates the
transcription factor NF-jB, a major regulatory factor
of proliferation and apoptosis. Attenuation of LXF-
289 cell proliferation by extracellular ATP is mediated
by the induction of nuclear translocation of p50
NF-jB1 and possibly new formation of p50 from its
precursor p105. This unique activation of NF-jB
during attenuation of cell proliferation through P2Y
receptors has been found for the first time. Thus far,
activation of NF-jB through P2Y or P2X receptors
has been associated with stimulation of proliferation of
different cell types. This has been reported for lung
alveolar tumor cells [29], osteoclasts [28], T cells [35]
and human monocytes [36]. We also found a decrease
of p65 in the cytosolic fraction, but no corresponding

accumulation in the nuclear fraction, suggesting a dif-
ferent role and site of action for the NF-jB forms p50
and p65 in nucleotide-mediated growth control. Gen-
etic deletion studies have revealed an important role
for NF-jB1 in the regulation of the proliferation and
fate of neural progenitor cells [37]. Moreover, NF-jB1
p50 plays a central role in the pathogenesis of classical
Hodgkin’s lymphoma and in anaplastic large-cell
lymphomas, complexed with BCL-3 [38].
ATP and ADP inhibit lung cell proliferation R. Scha
¨
fer et al.
3762 FEBS Journal 273 (2006) 3756–3767 ª 2006 The Authors Journal compilation ª 2006 FEBS
In addition to the transcription factor NF-jB1,
extracellular ATP simultaneously activates the MAPK
signaling pathways of p38 MAPK and ERK1⁄ 2. The
simultaneous activation of both MAPK pathways is
indicated by the similar time dependence in the activa-
tion of ERK1 ⁄ 2 and p38 MAPK. Both pathways seem
to be involved in the downregulation of the prolifer-
ation of LXF-289 cells. This is indicated by the fact
that blockage of either pathway by the inhibitors for
p38 MAPK and ERK1 ⁄ 2 completely reversed the
inhibitory effect of ATP on LXF-289 cell proliferation.
Inhibitor blockage of either pathway completely
reversed the inhibition of LXF-289 cell proliferation
by ATP. Further indirect support for the involvement
of ERK1 ⁄ 2 is derived from the observed translocation
of ERK1 ⁄ 2 to the nucleus. The translocation of activa-
ted ERK1 ⁄ 2 to the nucleus is a prerequisite for the

control of cell cycle progression [39,40].
The significance of ERK and p38 phosphorylation
for the activation of specific gene products under these
conditions of downregulation of cell cycle progression
via P2Y receptors still has to be identified. There are
multiple targets (e.g. transcription factors E2F, ETS,
ATF and AP1, p27(KIP); HBP1, pRB) that can be
phosphorylated by ERK1 ⁄ 2 and ⁄ or p38 MAPK and
are possibly involved in the promotion of cell cycle
progression and the regulation of the cell cycle check-
points (for reviews see [41–45]). Recent advances indi-
cate a number of links between the activation of p38
kinase and the DNA checkpoint pathways and their
possible interaction in the modulation of cell cycle con-
trol and DNA mismatch repair [46–50]. Intriguingly,
another mechanism by which p38 MAPK may negat-
ively regulate the cell cycle is by activation of the mito-
tic spindle assembly checkpoint pathway that monitors
the correct formation of the spindle and attachment of
kinetochores [51,52].
There is extensive crosstalk between the ERK1 ⁄ 2-
activated, p38 MAPK-activated and NF-jB-activated
pathways. The simultaneous activation of ERK1⁄ 2,
p38 kinase and NF-jB1 has been described only for
inflammatory mediators, such as lipopolysaccharide or
tumor necrosis factor-a (reviewed in [53]), but not for
extracellular nucleotides. Thus, in addition to the con-
trol of lymphocyte or macrophage function, the simul-
taneous activation of these pathways may participate
in the attenuation of epithelial cell proliferation by

extracellular nucleotides. Moreover, in some systems
GPCRs couple to NF-jB through sequential activation
of conventional PKC isoforms and IjB kinase, leading
to degradation of IjB by the proteasome [54]. It is
possible that the P2Y receptors act through PKC or,
alternatively, through PI3K to activate NF-jB in lung
tumor cells. However, we do not know whether these
pathways converge at a single target, e.g. NF-jB, or
whether they interfere with the regulation of S phase
entry, S phase progression and ⁄ or exit from S phase to
G2 phase.
Until now, P2Y receptor-mediated inhibition of cell
proliferation by control of cell cycle progression has
been found only in esophageal cells [12]. Activation of
P2Y receptors by ATP and ADP inhibits LXF-289 cell
proliferation very likely through direct attenuation of
S phase progression. This is indicated by the massive
increase of cells in the S phase, the reduction in cell
proliferation, and the decrease in bromodeoxyuridine
(BrdU) incorporation into newly synthesized DNA. In
addition, the additive effect of ATP with that of paclit-
axel and etoposide also suggests that ATP inhibits
growth by interfering with cell cycle progression at the
S phase. Paclitaxel and etoposide exert a block of
NSCLC cells at the G2 ⁄ M and G2 phase, respectively
[22,23], whereas cisplatin causes an accumulation of
G0 ⁄ G1 cells [55].
The transient translocation of ERK1 ⁄ 2 and of p50
NF-jB1 to the nucleus suggests a role of both proteins
in the regulation of cell cycle progression by extracellu-

lar nucleotides. Sustained activation of the ERK1 ⁄ 2
and PI3K pathways is not only necessary for cell cycle
progression into S phase but also regulates progression
during G2 ⁄ M phase by distinct cell cycle timing
requirements [39,56]. However, we observed only tran-
sient ERK1 ⁄ 2 activity in the nucleus for up to 60 min,
which is much shorter than the sustained activity for
ERK1 ⁄ 2, which persists from late S phase to G2 and
M phases [39]. Moreover, nothing is known about a
direct link of NF-jB1 with regulation of the S phase
checkpoint or S phase progression. Thus, for both pro-
teins, the mechanistic details of inhibition of cell cycle
progression remain unclear.
Different mechanisms can be targeted for attenuation
of cell cycle progression. Putative targets for nucleotide-
regulated cell cycle progression are either (a) the activa-
tion of cell cycle-regulated kinases, Cdc7, and cyclin
dependent kinase, which are required for the initiation
of DNA replication during S phase, or (b) the recruit-
ment of the initiation factor Cdc45, which is required
for the elongation phase of replication, or (c) the replica-
tion forks (reviewed in [57]). Especially in the last case,
extracellular nucleotides may perhaps act as a signal
causing replication forks to stall and halt cell cycle pro-
gression, similar to energy depletion or DNA damage
(see review [57]). However, this needs to be investigated
for extracellular nucleotides. This is very important to
understand, because the stabilization of stalled replica-
tion forks is crucial for maintaining cell viability [57].
R. Scha

¨
fer et al. ATP and ADP inhibit lung cell proliferation
FEBS Journal 273 (2006) 3756–3767 ª 2006 The Authors Journal compilation ª 2006 FEBS 3763
In summary, our results reveal that extracellular
nucleotides inhibit proliferation of LXF-289 cells by
regulation of cell cycle progression through activation
of several signal transduction pathways. The delay in
cell cyle progression through activation of P2Y recep-
tors is possibly mediated by activation of the NF-jB1
isoform of transcription factor NF-jB, the MAPKs
ERK1 ⁄ 2 and p38 and PI3K. Thus, extracellular nucle-
otides need to be added to the class of the critical reg-
ulators of cell cycle progression, so far consisting of
hormones, growth factors, and cytokines. Further
detailed investigations are needed to unravel the sub-
type of P2Y receptor mediating the observed response
and the functionality of the proteins in the signaling
mechanisms that lead to the attenuation of cell cycle
progression.
Experimental procedures
Cell culture and reagents
The human lung adenocarcinoma cell line LXF-289, estab-
lished from the solid lung tumor of a 63-year-old man, was
obtained from the German Collection of Microorganisms
and Cell Cultures (DSMZ, Braunschweig, Germany). LXF-
289 cells were cultured in Ham’s F-10 (Biochrom, Berlin,
Germany) supplemented with 10% FBS, 100 IUÆmL
)1
peni-
cillin, and 100 lgÆmL

)1
streptomycin in a 5% CO
2
incuba-
tor at 37 °C. Paclitaxel, etoposide and cisplatin were
purchased from Calbiochem (Bad Soden, Germany). The
PKC inhibitors Go
¨
6983 (Alexis, Gru
¨
nberg, Germany),
U-73122, U-73343, SB-203580, wortmannin (Sigma-Aldrich,
Taufkirchen, Germany), KN-62, PD-908059 (Bio-Trend,
Ko
¨
ln, Germany), SKF-96365 and NF-jB SN-50 (Calbio-
chem) were dissolved in dimethyl sulfoxide or in NaCl ⁄ P
i
to give stock concentrations of 10 or 100 mm.
Measurement of cell proliferation
Cell proliferation was measured by luminometric immuno-
assay based on BrdU incorporation during DNA synthesis
using a cell proliferation ELISA BrdU Kit (Roche, Mann-
heim, Germany) according to the manufacturer’s protocol.
The effect of extracellular nucleotides on BrdU incorpor-
ation was measured in LXF-289 cells, as described [29].
Cells were seeded on 96-well plates (5 · 10
3
cells per well)
and then incubated for 24 h. Cells were then incubated with

100 lm (standard conditions) of the different nucleotides or
nucleotide analogs for 1 h in a final volume of 100 lL per
well. Antagonists were added to the cells 1 h before the
addition of the nucleotides. Subsequently, 10 lL of BrdU-
labeling solution was added to each well and the cells were
incubated again for 12 h. BrdU labeling was determined as
previously described [29].
Cell growth was determined in subconfluent LXF-289
cells seeded into 12-well plates (5 · 10
4
cells per well;
500 lL per well). At 24 h after seeding, cells were treated
with 100 lm ATP or ADP for 1 or 2 days. Antagonists
were added to the cells 1 h before the addition of the nucleo-
tides. Cells were washed twice with NaCl ⁄ P
i
, and dispersed
using trypsin-EDTA, and suspended cells were counted
using a hemacytometer. The percentage inhibition of cell
proliferation by nucleotide treatment was calculated by
comparison with control cells. Trypan blue was used to
determine cell viability.
We ascertained in all cases that the inhibitor concentra-
tions used did not affect basal DNA synthesis. Therefore,
we tested the effect of the compounds U-73122, U-73343,
SB-203580, KN-62, PD-908059, SKF-96365 and NF-jB
SN-50 in a concentration range from 0.1 to 100 lm and the
inhibitors Go
¨
6983, U0126 and wortmannin in a concentra-

tion range from 0.01 to 10 lm. Maximal concentrations,
which did not influence basal DNA synthesis, were used in
the proliferation tests with the nucleotides. The concentra-
tion-dependent inhibition (1–100-fold of initial concentra-
tion) of the antiproliferative effect of ATP was tested for
the inhibitors U-73122, PD98059, SB203580, SKF-96365
and Go
¨
6983 (data not shown).
Cell cycle analysis
LXF-289 cells (5 · 10
4
cells per well) were seeded in 12-
well plates, cultured for 24 h and exposed to the respect-
ive nucleotide for another 24 h or 48 h. Trypsinized cells
were pelleted at 300 g, washed once with 0.5 mL of ice-
cold NaCl ⁄ P
i
and fixed in 75% ethanol at ) 20 °C for
60 min. Cells were treated with RNaseA (200 lgÆmL
)1
)
and propidium iodide (50 lgÆmL
)1
) in NaCl ⁄ P
i
at 25 °C
for 30 min, and this followed by flow cytometry (FAC-
Scan; Becton Dickinson, Franklin Lakes, NJ). Distribu-
tion of the cells in G

1
, S and G
2
⁄ M phases and apoptotic
populations (sub-G
1
phase) were analyzed by the modfit
program (Verity Software House Inc., Topsham, ME,
USA).
Determination of P2 receptor expression
Expression of mRNA for different P2Y (P2Y
1
, P2Y
2
, P2Y
4
,
P2Y
6
, P2Y
11
, P2Y
12
, and P2Y
13
) and P2X (P2X
3
, P2X
4
, and

P2X
7
) receptors in LXF-289 cells was determined by RT-
PCR. Total RNA was isolated from LXF-289 cells, as previ-
ously described [29], with the RNeasy kit (Qiagen, Hilden,
Germany). Possible genomic DNA contamination was
excluded in experiments by omitting the reverse transcrip-
tase in the PCR. Sets of specific oligonucleotide primers
were synthesized based on the published sequences for the
different P2Y receptors (see below). Primer sequences for
P2Y
1
, P2Y
2
, P2Y
4
and P2Y
6
receptors were as described
ATP and ADP inhibit lung cell proliferation R. Scha
¨
fer et al.
3764 FEBS Journal 273 (2006) 3756–3767 ª 2006 The Authors Journal compilation ª 2006 FEBS
[29]. Amplification was performed with 1 lL of cDNA,
for 30 cycles. The sequences for the primers were 5¢-
CGA GGT GCC AAG TCC TGC CCT-3¢ (forward, posi-
tions 7–27) and 5¢-CGC CGA GCA TCC ACG TTG
AGC-3¢ (reverse, positions 798–818) with 812 bp for
hP2Y11 (accession no. AF030335), 5¢-CCA GTC TGT
GCA CCA GAG ACT-3¢ (forward, positions 115–135) and

5¢-ATG CCA GACTAG ACC GAA CTC-3¢ (reverse, posi-
tions 615–635) with 520 bp for hP2Y12 (accession no.
AF313449), and 5¢-GGT GAC ACT GGA AGC AAT
GAA-3¢ (forward, positions 67–78) and 5¢-GAT GAT
CTT GAG GAA TCT GTC-3¢ (reverse, positions 437–457)
with 391 bp for hP2Y13 (accession no. NM176894). The
primers for the P2X
3,4,7
were 5¢-AGT CGG TGG TTG
TGA AGA GCT-3¢ (forward, positions 41–61) and 5¢-AAG
TTC TCA GCT TCC ATC ATG-3¢ (reverse, positions 492–
512) with 472 bp for hP2X3 (accession no. AB016608),
5¢-GCC TTC CTG TTC GAG TAC GAC-3¢ (forward, posi-
tions 1951–71) and 5¢-CGC ACC TGC CTG TTG AGA
CTC-3¢ (reverse, positions 2351–2371) with 421 bp for
hP2X4 (accession no. AF191093), and 5¢-GTC ACT CGG
ATC CAG AGC ATG-3¢ (forward, positions 148–168)
and 5¢-TTG TTC TTG ATG AGC ACA GTG-3¢ (reverse,
positions 660–680) with 533 bp for hP2X7 (accession no.
NM002562).
Preparation of cell extracts and western blot
analysis
LXF-289 cells, seeded in 55 mm dishes, were incubated
with 100 lm ATP at 37 °C for various times. Cells were
lysed with hypotonic buffer (20 mm Tris ⁄ HCl, pH 8.1,
1mm Mg
2+
, 0.05 mm Ca
2+
) containing an inhibitory cock-

tail for proteases and phosphatases (Complete EDTA;
Roche) and 2 mm NaVO
4
(hereafter named lysis buffer) by
incubation on ice for 15 min, dispersed by sonication and
then centrifuged at 1000 g for 10 min. For all centrifuga-
tions an Avanti 30 centrifuge from Beckman Coulter (Kre-
feld, Germany) was used. The resulting pellet, containing
nuclei and mitochondria, was resuspended in lysis buffer
containing 400 mm sucrose, layered on a sucrose cushion of
1.2 m sucrose in lysis buffer and centrifuged for 15 min at
5000 g to purify the nuclei. Purified nuclei were resuspend-
ed in lysis buffer containing 0.1% (v ⁄ v) Nonidet P-40,
2mm dithiothreitol and 0.4 m NaCl, incubated on ice for
30 min to extract the proteins, and then centrifuged at
12 000 g for 10 min to remove nonsolubilized debris. The
resulting supernatant (nuclear fraction) was stored at
) 80 °C for further investigations. The supernatant from
the first centrifugation (1000 g) was then centrifuged at
50 000 g for 45 min to prepare the cytosolic fraction (resul-
tant supernatant). The protein content was determined
using the BCA protein assay kit (Pierce, Rockford, IL,
USA). Protein fractions were subjected to SDS gel electro-
phoresis on 10% acrylamide gels and transferred to PVDF
membranes. To control the complete and even transfer of
proteins to the blotting membranes, membranes were
immersed in a Ponceau Red-containing solution and the
stained protein bands were scanned densitometrically before
processing with antibodies. Blots were incubated at 4 °C
overnight with anti-human phospho-ERK1 ⁄ 2 (Thr183,

Tyr185), anti-phospho-p38 MAPK (Cell Signaling Technol-
ogy, Beverley, MA, USA) or antibodies for NF-jB1 (p50,
p105) or RelA (p65). After incubation with horseradish per-
oxidase-conjugated anti-rabbit IgG (Dianova, Hamburg,
Germany) for 1.5 h at room temperature, the antibody
reaction was visualized by enhanced chemiluminescence
(Promega, Madison, WI, USA).
Statistical analyses
Experiments were conducted with cultures from different
seedings. The number of replica experiment is given in the
figure legends. Data were analyzed by Student’s t-tests for
two groups or anova for multiple groups.
Acknowledgements
We thank Annette Schulze for skillful technical assist-
ance in the experiments. The work was supported by
Deutsche Krebshilfe (project 10-1754) and Land Sach-
sen-Anhalt.
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