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¨ fer1, Roland Hartig2, Fariba Sedehizade1, Tobias Welte3 and Georg Reiser1
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.
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involving activation of
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- leading to gression of proliferating LXF-289 cells, marked arrest of the cells in the S phase. This inhibi- tion is mediated by P2Y receptors through signaling pathways 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
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 (P2X1)7) and eight subtypes of the P2Y receptors (P2Y1,2,4,6,11,12,13,14) 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]).
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- inhibition at tration-dependent, with half-maximal 18.6 ± 1.9 lm (n ¼ 5) and 19.8 ± 0.6 lm (n ¼ 5), respectively (Fig. 1). Significant inhibition was already
tumorigenesis.
In view of
in vivo antitumor activity of
100
it will be important
) l o r t n o c
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 the antineoplastic action of extracellular nucleotides [6] and the signifi- cant intraperitoneally injected ATP against several aggressive carcinomas in tumor-bearing mice [7,15], to understand how the different P2 receptor subtypes contribute to the regulation of cancer cell prolifer- ation.
80
f o %
60
( n o i t a r o p r o c n
i
U d r B
40
Control
ATP
ADP
UTP
UDP
2-MeSADP
Ado
20
10
20
40
60
100
nucleotide concentration [µM]
inhibition of LXF-289 cell pro- Fig. 1. Concentration-dependent liferation by different nuccleotides. Increasing concentrations (10–100 lM) of the different nucleotides were added to proliferating incorporation was LXF-289 cells, and bromodeoxyuridine (BrdU) measured after 12 h. Values are means ± SD of n ¼ 4–7 experi- ments run in triplicate.
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 P2X5 receptors stimulated the differentiation of skin cancer cells with subsequent inhibition of proliferation [16], and induction of cell death was mediated via P2X7 receptors in skin cancer and prostate cancer cells [16,17]. The formation of the lytic pore of the P2X7 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 lines from colon [14], endometrium [11], tumor cell 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-
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Inhibition of proliferation of LXF-289 lung tumor cells. Table 1. 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 lM of the different nucleotides or nuc- leotide analogs listed below before the addition of 10 lM 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 (%)
expression
next
We
the
receptors
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 P2Y1,12 and P2Y11 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. of P2Y examined in (P2Y1,2,4,6,11,12,13) and P2X (P2X3,4,7) 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 P2Y2, P2Y6, P2Y11 and P2Y13 and for the P2X4 recep- tor, whereas expression of the P2Y1, P2Y4, P2Y12, P2X3 and P2X7 receptors could not be detected (Fig. 2).
ATP ADP ATPcS Bz-ATP UTP UDP a,b-MeATP b,c-MeATP 2-MeSADP 2-MeSATP UMP AMP
55.0 ± 5.1 54.7 ± 4.6 59.5 ± 3.8 10.8 ± 5.1 22.5 ± 3.6 25.6 ± 2.3 5.6 ± 4.1 6.8 ± 5.7 5.0 ± 1.6 5.9 ± 1.1 7.3 ± 2.3 11.7 ± 5.9
Inhibition of growth and cell cycle progression of LXF-289 cells
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
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%
GAPDH
P2Y1
P2Y4
P2Y11
P2X4
P2Y13 P2Y12 P2X3
P2X7
P2Y2
P2Y6
812
855
630
442
420
Fig. 2. Expression of P2 receptors in LXF-289 cells. RT-PCR analysis was performed using primers for P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12 and P2Y13 receptors and the P2X receptors P2X3, P2X4 and P2X7 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.
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A
80
ADP
control
ATP
(± 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.
60
) l a t o t
f o %
(
40
s l l e c
20
e s a h p - S
0
10 µM
20 µM
50 µM
100 µM
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.
B
a
c
b
control
16
ATP sensitizes LXF-289 cells to the activity of anticancer drugs
G0-G1: 65.74% S-Phase: 26.27%
80
0
20 µM ATP
16
) l e n n a h c / s t
G0-G1: 40.40% S-Phase: 48.39%
80
n u o c (
s
l l
0
e c
16
100 µM ATP G0-G1: 19.91% S-Phase: 77.97%
80
0
80
0
20
40
60
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 lM) 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 lM 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.
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].
the pathways
Signal transduction pathways involved in nucleotide-mediated inhibition of proliferation of LXF-289 cells
coupled receptors (GPCRs). We therefore investigated found in GPCR-mediated which of attenuation of cell proliferation, including phospho- lipase C (PLC), protein kinase C (PKC) and extra- cellular Ca2+ 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
Different mechanisms and signal transduction path- ways transduce control of proliferation by G protein-
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A
120
+ Cisplatin
+ ATP-Cisplatin
+ Etoposide
+ ATP-Etoposide
100
Control + ATP
) l a s a b
80
f o %
(
60
40
n o i t a r o p r o c n
i
20
U d r
B
Inhibition of proliferation of LXF-289 lung tumor cells. Table 2. 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 lM 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 lM 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.
0
(A)
0
1
10
100
cisplatin, etoposide (µM)
Signaling pathway inhibitors
Concentration
Reduction of inhibitory effect of ATP (%)
B
120
+ Paclitaxel
+ ATP-Paclitaxel
100
Control + ATP
) l a s a b
f
80
o %
(
n o
60
i t
40
a r o p r o c n
i
U-73122 U-73343 SKF-96365 KN-62 Wortmannin PD98059 U0126 SB203580 Go¨ 6983 Go¨ 6983 NF-jB SN50 Curcumin Sulindac sulfide
101 ± 6.4 8.1 ± 7.0 79.8 ± 4.7 98.6 ± 3.7 93.9 ± 3.2 92.8 ± 3.3 91.8 ± 4.1 95.1 ± 2.9 24.6 ± 4.1 86.7 ± 2.8 94.2 ± 5.3 97.1 ± 1.9 88.4 ± 9.0
10 lM 10 lM 50 lM 25 lM 0.1 lM 20 lM 1 lM 20 lM 0.1 lM 1.0 lM 20 lM 20 lM 10 lM
20
U d r
(B)
B
0
Kinase inhibitors
Concentration
Reduction of basal proliferation (%)
0
1
100
10 paclitaxel (nM)
5.6 ± 2.4 8.7 ± 4.8 8.8 ± 5.7 7.9 ± 5.1 5.8 ± 4.2 4.8 ± 2.3 6.5 ± 2.8
25 lM 0.1 lM 20 lM 1 lM 20 lM 0.1 lM 1.0 lM
KN-62 Wortmannin PD98059 U0126 SB203580 Go¨ 6983 Go¨ 6983
(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 in the absence (solid symbols) or presence (open symbols) of 100 lM 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).
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.
LXF-289 cell proliferation, whereas the inactive analog U-73343 had no effect (Table 2). Blockade of receptor- operated Ca2+ channels with SKF-96365 dose-depend- ently reduced the inhibitory effect of ATP (data not
Preincubation of LXF-289 cells with the potent concentration-dependently PKC inhibitor Go¨ -6983 abolished the antiproliferative effect of ATP (Table 2). In addition, we investigated the involvement of the
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Cytosol Nucleus
kDa
[min]
0 10 20 30 60
0 10 20 30 60
50
A
phospho-ERK 1/2
37
50
(CaMKII), calcium ⁄ calmodulin-dependent kinase II 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.
B
phospho-p38
37
Cell lysate
0 10 30 60 [min]
kD
kinase
(MEK1 ⁄ 2)
1 lm U0126),
ERK 1/2
C
37
p38
37
Cytosol Nucleus
[min]
0 10 20 30
0 10 20 30 60
kD
p105
10
NF-κB1
D
p50
50
75
RelA (p65)
E
The inhibition of LXF-289 cell proliferation by 100 lm ATP was totally attenuated by blockers of (20 lm either ERK-activating p38 MAPK (20 lm PD98059, 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).
50
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).
in contrast
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 lM 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 lysate (C) was incubated and p105) and (E) RelA (p65). Total cell with antibodies to total ERK1 ⁄ 2 and p38, respectively. Primary anti- bodies were incubated at 4 (cid:2)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.
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-jB in 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
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, 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).
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this
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- concentration in the absence of eration at 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
2-MeSATP and Bz-ATP to influence the proliferation of LXF-289 cells excludes the participation of the P2Y11 receptor subtype in the inhibition. Bz-ATP has receptors, been shown to activate human P2Y11 whereas 2-MeSATP activates the human P2Y11 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 P2Y2 and P2Y13, 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 P2Y2 for the receptor has been found to be important 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 P2Y2 receptor by ATP and UTP (reviewed in [3]). Similarly, the inactivity of 2-MeSADP, which is more potent than ADP at the human P2Y13 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].
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
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 translocation of p50 by the induction of nuclear 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 large-cell lymphoma and in anaplastic lymphomas, complexed with BCL-3 [38].
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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.
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].
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.
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].
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].
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
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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
Cell growth was determined in subconfluent LXF-289 cells seeded into 12-well plates (5 · 104 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 ⁄ Pi, 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.
Cell cycle analysis
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).
Measurement of cell proliferation
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% CO2 incuba- tor at 37 (cid:2)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 ⁄ Pi to give stock concentrations of 10 or 100 mm.
Determination of P2 receptor expression
LXF-289 cells (5 · 104 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 ⁄ Pi and fixed in 75% ethanol at ) 20 (cid:2)C for 60 min. Cells were treated with RNaseA (200 lgÆmL)1) and propidium iodide (50 lgÆmL)1) in NaCl ⁄ Pi at 25 (cid:2)C for 30 min, and this followed by flow cytometry (FAC- Scan; Becton Dickinson, Franklin Lakes, NJ). Distribu- tion of the cells in G1, S and G2 ⁄ M phases and apoptotic populations (sub-G1 phase) were analyzed by the modfit program (Verity Software House Inc., Topsham, ME, USA).
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Expression of mRNA for different P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13) and P2X (P2X3, P2X4, and P2X7) 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 P2Y1, P2Y2, P2Y4 and P2Y6 receptors were as described 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 · 103 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].
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and 7–27)
and
Statistical analyses
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 (cid:2)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).
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
(forward, positions
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.
Preparation of cell extracts and western blot analysis
References
[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 5¢-CGC CGA GCA TCC ACG TTG (reverse, positions 798–818) with 812 bp for AGC-3¢ 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), 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 P2X3,4,7 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¢ 148–168) and 5¢-TTG TTC TTG ATG AGC ACA GTG-3¢ (reverse, positions 660–680) with 533 bp for hP2X7 (accession no. NM002562).
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