Roles of ERK, PI3 kinase, and PLC-γ pathways induced by overexpression of translationally controlled tumor protein in HeLa cells

Archives of Biochemistry and Biophysics 485 (2009) 82–87

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Roles of ERK, PI3 kinase, and PLC-c pathways induced by overexpression of translationally controlled tumor protein in HeLa cells Moonhee Kim 1, Jaehoon Jung 1, Kyunglim Lee * College of Pharmacy, Center for Cell Signaling and Drug Discovery Research, Ewha Womans University, 11-1 Daehyun-dong, Sudaemoon-gu, Seoul, Republic of Korea

a r t i c l e

i n f o

Article history: Received 15 December 2008 and in revised form 30 January 2009 Available online 11 February 2009 Keywords: EGFR ERK Na,K-ATPase Ouabain PI3K PLC-c TCTP

a b s t r a c t We reported previously that translationally controlled tumor protein (TCTP) is a cytoplasmic repressor of Na,K-ATPase in HeLa cells. In the current study, we showed that TCTP overexpression using adenovirus as vehicle, induced partial inhibition of Na,K-ATPase; phosphorylation of EGFR tyrosine residues 845, 992, 1068, and 1148; activation of Ras/Raf/ERK pathway; activation of PI3K/Akt pathway; and phosphorylation of PLC-c in HeLa cells. Specific inhibition of PI3K/Akt pathway in contrast to the inhibition of ERK, significantly decreased TCTP overexpression-induced survival signal. Inhibition of PLC-c pathway significantly decreased TCTP overexpression-induced cell migration but inhibition of ERK had less effect. These results suggest that TCTP plays a key physiological role in cell survival through Akt pathway and migration through PLC-c pathway. Ó 2009 Elsevier Inc. All rights reserved.

Introduction Na,K-ATPase maintains normal gradients of Na+ and K+ across the plasma membranes of nearly all animal cells [1–3]. For several decades, attention has been directed at the structure and function of Na,K-ATPase as an ion pump, and more recently at its role as a signal transducer [4–6]. Na,K-ATPase responds to extracellular stimuli such as ouabain and low potassium, and transduces signals through protein–protein interactions. Nontoxic concentrations of ouabain in cardiac myocytes result in the interaction of Na,K-ATPase with Src, Src-induced transactivation of EGFR2 and the activation of Ras/Raf/MEK/ERK cascade [7–9]. It has been suggested that ouabain-induced signaling is not limited to cardiac myocytes and that there might be different downstream consequences of such signaling in various cell types [10–13]. Translationally controlled tumor protein (TCTP), also known as histamine-releasing factor and fortilin, is considered to be a house keeping protein because of its ubiquitous expression, high degree of conservation between species, and its presence in both tumor and normal cells [14]. TCTP has been implicated in human allergic response [15], apoptotic regulation [16–17] and in various cancer-

* Corresponding author. Fax: +82 3277 2851. E-mail address: [email protected] (K. Lee). 1 These authors contributed equally to this work. 2 Abbreviations used: EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran4-one; PI3K, phosphatidylinositol 3-kinase; PLC-c, phospholipase C-gamma; Raf, Raf1 kinase; TCTP, translationally controlled tumor protein. 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.02.002

related functions [18–19], but the exact biological role of this protein remains elusive. In a previous study, we showed that TCTP binds to the third cytoplasmic loop of Na,K-ATPase and inhibits Na,K-ATPase in HeLa cells [20]. We hypothesized that Na,K-ATPase inhibition by TCTP might also act as signal transducer similar to inhibition by ouabain. To test this hypothesis, we employed a modified adenoviral expression system to achieve higher expressions of TCTP and examined the signal transduction pathways and physiological events induced by the overexpression of TCTP. Materials and methods Cell culture HeLa cells were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco’s minimal essential media (DMEM) of 10% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin. Cells were cultured at 37 °C in a humidified 5% CO2 atmosphere incubator. Generation of adenoviruses Adenoviruses were generated and maintained as previously described [21]. Adenoviruses were purified using cesium chloride and concentrated to minimize any potential effects of cytosol or media from the HEK293 cells in which they were amplified. For adenoviral expression, subconfluent dishes of HeLa cells were infected with 1 moi/cell for 2 h in DMEM containing 10% serum at 37 °C

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Immunoblotting and immunoprecipitation The cells were washed twice with phosphate-buffered saline (PBS) and lysed in a lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, and 2 mM phenylmethylsulfonyl fluoride) on ice. Proteins were solubilized by rotating the cell mixture in the lysis buffer, end over end for 10 min at 4 °C, and any insoluble material was removed by microcentrifugation for 10 min at 4 °C. The amount of the solubilized protein was assessed by Bradford assay (Biorad), and samples were diluted in SDS sample buffer. Equivalent amounts of protein were separated using SDS–PAGE, transferred to nitrocellulose, detected using monoclonal antibodies against, or a rabbit polyclonal antibody against EGFR (Biosource, CA), GFP (Santa Cruz Biotechnology, Santa Cruz, CA), phospho tyrosine site specific EGFR (Biosource), ERK (Cell Signaling Technology), phospho ERK (Cell Signaling Technology), Akt (Cell Signaling Technology), phospho Akt (Cell Signaling Technology), PLC-c (Cell Signaling Technology) and phospho PLC-c (Cell Signaling Technology), followed by probing with a horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibody. Detected proteins were visualized by enhanced chemiluminescence (ECL) and UV Products Imaging system, LAS 3000 (Fuji).

10 min. The cells were incubated with ice-cold lysis buffer (MLB) containing Mg2+, 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol, 25 mM NaF, 10 mM MgCl2, 1 mM EDTA, 1 mM Na3VO4, 10 lg/ml aprotinin, and 10 lg/ ml leupeptin. Active Ras was affinity precipitated by adding the Ras-binding domain (RBD) of Raf-1 conjugated agarose (Upstate) to the cell lysates (1 mg/ml) for 45 min at 4 °C with gentle agitation. Then the agarose beads were washed three times with MLB and pelleted at 10,000g for 10 s at 4 °C, eluted with 2 SDS sample buffer and separated on SDS–PAGE. The blots obtained were incubated with anti-Ras antibody (Upstate). Quantitative results of the positive bands were analyzed by Multi Gauge software version 2.3 (Fuji). Apoptosis assay Apoptosis assay was performed using Cell Counting Kit (CCK)-8 from Dojindo Laboratories (DOJINDO Laboratories). HeLa cells

Ad GFP

in 5% CO2, followed by a 24 h incubation in DMEM without serum for serum starvation. Mock infected cells were incubated with adenovirus expressing GFP. Recombinant protein expression from adenoviruses was verified by immunoblotting with anti-GFP antibody and/or indirect immunofluorescence.

Ad TCTP-GFP 0

1

8

16

24 hr TCTP-GFP

GFP 8 7 6 5 4 3 2 1 0

∗∗

∗∗

∗∗ ∗

Ad GFP

HeLa cells were incubated with ice-cold lysis buffer (20 mM Tris– HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 2 mM EGTA, 1% NP-40, 0.5% Sodium deoxycholate, NaF, Na3VO4, CompleteTM protease inhibitor cocktail tablets) for 30 min on ice and gently homogenized with Pyrex #7727-07. The cell lysates were centrifuged at 10,000g, at 4 °C for 10 min. The total cell lysates (1 mg/ml) were incubated overnight with EGFR antibody (Biosource) at 4 °C. Protein A or G-agarose (Boehringer Mannheim) was then added and the mixture incubated for an additional 4 h at 4 °C. Protein A or G-agarose bound immunecomplexes so obtained were pelletized and washed twice with icecold PBS. After washing, the immune-complexes were eluted with 2 SDS sample buffer and separated on 12% SDS–PAGE. Blots were incubated with phospho tyrosine antibody (Santacruz).

Expression level (Fold Ratio)

Immunoprecipitation

0

1

8

16

24hr

Ad TCTP-GFP

120

HeLa cells (3.0  105 – 1  106 cells/ml) were inoculated into 24 wells and infected with adenoviral GFP and TCTP-GFP. After 24 h, the serum-starved cells were washed with Krebs-Ringer buffer (KRP, 140 mM NaCl, 5 mM KCl, 10 mM Na2HPO4, 1 mM MgSO4, 1.4 mM CaCl2, 2.5 mM glucose (pH 7.4)), and incubated at 37 °C for 15 min with or without 1 mM ouabain in 0.1% bovine serum albumin (BSA)-containing KRP buffer. 86Rb+ was used as a tracer to measure the uptake of K+. After 5–10 min incubation with 86Rb+ (2 lCi/ml), cellular 86Rb+-uptake was stopped by the addition of ice-chilled KRP buffer and the cells washed twice and lysed by adding 50 mM NaOH. The cell lysates were then counted after mixing with scintillation cocktail. Difference between 86Rb+-uptake in the presence and absence of ouabain was taken as ouabain-sensitive 86Rb+-uptake. Ras activity assay HeLa cells cultured in 100 mm dishes were infected with adenoviral GFP or TCTP-GFP for 24 h or treated with 1 lM ouabain for

Na,K- ATPase activity (% of control)

100

Rb uptake assay

∗

80

60

40

20

0

Ad GFP

Ad TCTP-GFP

Fig. 1. TCTP downregulates Na,K-ATPase in HeLa cells. (A) HeLa cells infected with adenoviral GFP and TCTP-GFP at 1 moi in time-dependent manner. Total cell extract (50 lg) was blotted with anti-GFP antibody. (B) HeLa cells infected with adenoviral GFP and GFP-TCTP. After 24 h, the cells were incubated with or without 1 mM ouabain in KRP buffer for 15 min at room temperature. 2 lCi/ml 86Rb+ was added and 86Rb+ uptake counted in triplicate. Difference between 86Rb+-uptake in the presence and absence of ouabain was taken as Ouabain-sensitive 86Rb+-uptake. Results shown are representative of three experiments or means ± SD of three experiments (*p < 0.05, **p < 0.01, significantly different from the Ad TCTP-GFP 0 h (A) or Ad GFP value (B)).

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were inoculated into 100 mm2 dishes and infected with adenoviral GFP and TCTP. After 24 h, HeLa cells (100,000 cells/ml) were seeded to 96 well plate and treated with 10 lM of MEK inhibitor, U0126 or 50 lM PI3K inhibitor, LY294002 for 30 min and then with 50 lM of etoposide for 18 h. 10 ll of CCK-8 solution were added to each well, and HeLa cells were incubated at 37 °C for 2 h. Cell viability was measured at 450 nm of the O.D. in each well.

plate in serum free media containing 10 lM of MEK inhibitor, U0126 or 10 lM of PLC inhibitor, U73122 for 18 h. To the lower well of the migration plate was added 500 ll of media containing 10% fetal bovine serum. After 18 h, the interior of the upper chamber was gently swabbed to remove non-migratory cells and the upper chamber was transferred to a clean well containing cell staining solution and incubated for 10 min at room temperature. After washing, the migrated cells were counted under a light microscope.

Cell migration assay Statistical analysis

Fold Increase (± S.D.)

Fold Increase (± S.D.)

Fold Increase (± S.D.)

Fold Increase (± S.D.)

Fold Increase (± S.D.)

Fold Increase (± S.D.)

∗∗ ∗∗ 16.98 23.91

1.00 0.11 0.61

1.00 0.04

∗∗ 2.11 0.02

1.64 0.96 0.07

1.00 0.05 0.52

0.31

∗∗ ∗∗ 23.25 22.48

1.00 0.17 1.26

1.12

∗∗ ∗∗ 10.90 11.85

1.00 0.10 0.33

∗ 3.39

1.00 0.03 0.15

∗∗ 22.07

0.66 1.21 0.03 0.02

∗∗ ∗∗ 9.77 14.20

0.17

∗

Ad TCTP-GFP

Ad TCTP-GFP

Ad GFP

Ouabain

EGF

Control

IP: EGFR

∗

Ad GFP

Control

EGFR

∗∗

Oubaain

Phospho Tyr (Fold Ratio)

pY

10 8 6 4 2 0

EGF

Data are presented as mean ± SD. Comparison of differences was made using the unpaired, two-tailed Student t-test. The differences were considered statistically significant when the P value was <0.05.

Ad TCTP-GFP

Ad GFP

Ouabain

EGF

Control

Cell migration was measured by CytoselectTM 24-Well Cell Migration Assay from Cell Biolabs (Cell Biolabs). HeLa cells were inoculated into 100 mm2 dishes and infected with adenoviral GFP and TCTP. After 24 h, 300 ll of infected HeLa cells (1.0  106 cells/ ml) were seeded into the upper chamber of the 24-well migration

-pY845

1.74

∗ 5.27

-pY992

0.61

∗ 7.55

-pY1068

1.77 0.13 0.76

-pY1086 1.48 0.13

1.96 0.42

∗∗ 5.04

-pY1148

1.29 0.13 0.37

-pY1173 1.17 0.06

1.27 0.04

0.92 0.08

Total EGFR Fig. 2. TCTP phosphorylates EGFR. HeLa cells were incubated with 50 ng/ml EGF for 5 min or 1 lM ouabain for 10 min after 24 h of serum starvation. For GFP or TCTP-GFP overexpression, HeLa cells were infected with adenoviral GFP or TCTP-GFP at 1 moi in DMEM containing serum for 2 h, followed by 24 h of serum starvation. (A) Total cell extracts (1 mg/ml) immunoprecipitated with total EGFR antibodies. The immune-complexes were blotted with anti-phospho tyrosine antibodies. (B) Total cell extracts (50 lg) blotted with phospho EGFR tyrosine residues 845, 992, 1068, 1086, 1148, 1172 and total EGFR antibodies. Quantification of EGFR phosphorylation was performed through normalization of total EGFR level (bottom panel of A and B). Results shown are representative of three experiments or means ± SD of three experiments (*p < 0.05, **p < 0.01, significantly different from the control value).

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TCTP-GFP was expressed weakly at 8 h after adenoviral infection but increased at 16 and 24 h. We performed the experiments described in this study 24 h after infection of TCTP adenovirus. Fig. 1B shows that the activity of Na,K-ATPase in TCTP-GFP overexpressing cells decreased to about 67% of control-GFP overexpressing HeLa cells.

Results and discussion TCTP overexpression inhibits Na,K-ATPase in HeLa cells

Ad GFP

Oubaain

We promoted high expression of TCTP in mammalian host cells, using the adenovirus overexpression system. Fig. 1A shows that

Ad TCTP-GFP 0

8

16

24 hr

IP: Raf-RBD

Ras TCTP-GFP

Ras Activity (Fold Ratio)

GFP 6

∗∗

4

∗∗

2

∗∗

Ad GFP

0

8

16

24 hr

Phospho Erk (Fold Ratio)

∗

-Erk

Total Erk

Total Erk

TCTP-GFP

TCTP-GFP

GFP

GFP

3.0

1.5

0.5

Ouabain

2.0

AdGFP

1.0

Ad TCTP-GFP

0.0

-

∗

4.0

∗

1.0

+

Ad TCTP-GFP

-Erk

2.5

∗

Ad GFP

PD98059

Genistein

2.0

Ouabain

Ad GFP

Ouabain

Ad TCTP-GFP

Ad TCTP-GFP

Ad TCTP-GFP

Ad GFP

Ouabain

Ad TCTP-GFP

Ad GFP

Ouabain

Oubaain

0

Genistein

∗∗

∗∗

Ouabain Ad GFP Ad TCTP-GFP

0.0

-

+

PD98059

Fig. 3. TCTP induces the activation of Ras/Raf/ERK. (A) HeLa cells were incubated with 1 lM ouabain for 10 min or infected with adenoviral GFP or GFP-TCTP at 1 moi. Total cell lysates (1 mg/ml) immunoprecipitated with Raf1-RBD agarose (45 min incubation) and blotted with anti-Ras antibody. (B) HeLa cells pretreated with 100 lM genistein for 1 h and 30 lM PD98059 for 1h, followed by a 15 min incubation of 1 lM ouabain. The cells were infected with adenoviral GFP or TCTP-GFP for 23 h, followed by 100 lM genistein and 30 lM PD98059 for another hour before the end of infection. Total cell extracts (50 lg) were blotted with anti-phospho ERK, total ERK. Quantification of ERK phosphorylation was performed through normalization of total ERK level (bottom panel). Results shown are representative of three experiments or means ± SD of three experiments (*p < 0.05, **p < 0.01, significantly different from the Ad TCTP-GFP 0 h (A) or indicated value (B)).

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TCTP induces EGFR phosphorylation

known to be involved in EGFR phosphorylation are tyr 845, 992, 1068, 1086, 1148 and 1173, and each of these tyrosine residues interacts with specific signaling molecules. As shown in Fig. 2B, EGF induced phosphorylation of all 6 residues as previously described [22–23]. However, ouabain induced EGFR phosphorylation at tyr 845, 1068, 1086 and 1148, while TCTP overexpression induced EGFR phosphorylation at tyr 845, 992, 1068, 1148 but not 1086 and 1173. Thus both ouabain and TCTP overexpression phosphorylated at tyr 845, 1068, 1148, but failed to phosphorylate at tyr 1172. Interestingly, although TCTP overexpression inhibited the Na,K-ATPase activity like ouabain, it induced phosphorylation at tyr 992 and did not induce phosphorylation of tyrosine residue 1086, unlike ouabain. Therefore, we think that there might be different physiological outcomes from Na,K-ATPase inhibition by TCTP and ouabain, respectively.

LY294002 -AKT AKT 1.3 1.2

∗

∗∗

0.9 0.8

Ad TCTP-GFP

Ad GFP

-PLCPLCAd GFP

Phospho PLC(Fold Ratio)

Ad TCTP-GFP

∗∗

4.0 3.0 2.0 1.0 0.0

U73122

20

+

2.5

+

+ +

∗∗

Ad GFP

Ad TCTP-GFP

∗∗

2 1.5 1 0.5 0

-

+ +

+

+ Ad TCTP-GFP

Ad GFP

-

U73122

6.0 5.0

40

Etoposide U0126 LY294002

∗∗

∗∗

∗∗

60

0

1.0

7.0

∗∗

Ad TCTP-GFP

1.1

LY294002

Ad TCTP-GFP

80

Cell Migration (RFU)

Phospho Akt (Fold Ratio)

Ad GFP

Ad GFP 100

Apoptotic cells (%)

Ad TCTP-GFP

Ad GFP

Ad TCTP-GFP

Ad GFP

EGF receptor (EGFR) is activated by extracellular stimuli including its cognate ligand and several non-EGFR binding growth factors and by intracellular stimuli such as transactivation mechanisms. Since it was reported that partial inhibition of Na,K-ATPase by ouabain transactivated EGFR through src kinase [4], we tested whether partial inhibition of Na,K-ATPase by TCTP overexpression also induces phosphorylation of EGFR. As can be seen in Fig. 2A, TCTP overexpression induced phosphorylation of EGFR in HeLa cells as EGF and ouabain did. EGFR contains various tyrosine phosphorylation sites and phosphorylation of each tyrosine residue activates the specific signaling pathway, including the Ras/Raf/MEK/ERK cascades, the PI3K/AKT pathway, the PLC/PKC pathways, and other pathways. The residues

Control

U0126

U73122

+

Fig. 4. TCTP induces anti-apoptotic signals through the PI3K/Akt pathway and cell migration signals through PLC-c pathway. HeLa cells were infected with adenoviral GFP or TCTP-GFP, followed by a incubation with indicated inhibitors. (A) Infected cells incubated with 50 lM LY 294002 for 18 h. Total cell lysate (50 lg) was blotted with antiphospho Akt and total Akt antibodies. (B) Infected cells were pretreated with 10 lM U0126 or 50 lM LY294002 for 30 min and treated with 50 lM of etoposide for 18 h, followed by 2 h incubation of CCK-8. Cell viability was measured at 450 nm of OD. (C) Infected cells incubated with 10 lM U73122 for 18 h. Total cell lysate (50 lg) was blotted with anti-phospho PLC-c and total PLC-c antibodies. (D) Infected cells incubated with 10 lM U0126 or 10 lM U73122 for 18 h. Stained migrating cells were counted under a light microscope. Quantification of Akt phosphorylation or PLC-c was performed through normalization of total Akt or PLC-c level (bottom panel of A and C). Results shown are representative of three experiments or means ± SD of three experiments (*p < 0.05, **p < 0.01, significantly different from the indicated value).

M. Kim et al. / Archives of Biochemistry and Biophysics 485 (2009) 82–87

TCTP overexpression activates Ras/Raf/Erk pathway Since it is known that phosphorylation of EGFR tyr 1068 residue, one of the tyr residues activated by TCTP overexpression, involves Ras/Raf/Erk molecules [23], we examined whether TCTP overexpression activates Ras/Raf/Erk pathway. TCTP overexpression induced Ras activation in time-dependent manner from 8 to 24 h post-infection when measured Ras activity using Raf-RBD agarose beads (Fig. 3A). TCTP overexpression induced ERK phosphorylation, detected using antibody against dual phosphorylation form of ERK, and this was blocked by the broad tyrosine kinase inhibitor, genistein and the MEK inhibitor, PD98059 (Fig. 3B). TCTP overexpression activates PI3K/Akt and PLC-c pathways Among the various downstream pathways of EGFR, we analyzed the phosphorylation of PI3K/Akt and PLC-c, the key signaling molecules along with Ras/Raf/Erk cascades. As shown in Fig. 4A, phosphorylation of Akt was resulted from TCTP overexpression as detected using antibody against phospho-Akt and this was blocked by PI3K specific inhibitor, LY294002. Next, we examined whether Akt phosphorylation induced by TCTP overexpression is associated with anti-apoptosis function. Fig. 4B demonstrates a decrease of apoptotic cells induced by Etoposide in TCTP overexpressing cells and this was blocked by PI3K inhibitor, LY294002, but not by MEK inhibitor, U0126. This suggests that TCTP overexpression in HeLa cells induces anti-apoptotic activity through PI3K/Akt pathway in HeLa cells. Phosphorylation of EGFR tyr 992 was reported to involve PLC-c pathways (23). Western blotting assay using antibodies against phospho-PLC-c and total PLC-c confirmed that phosphorylation of PLC-c was induced by TCTP overexpression and this was blocked by PLC-c inhibitor, U73122 (Fig. 4C). Since it was also reported that TCTP was overexpressed in some cancer cell lines (14), we tested if activation of PLC-c pathway by TCTP overexpression is involved in cell migration. Our studies demonstrated an increase in cell migration in TCTP overexpressing cells and that this was blocked by PLCc inhibitor, U73122, but not MEK inhibitor, U0126. This suggests that TCTP overexpression in HeLa cells induces cell migration through the PLC-c pathway (Fig. 4D). Akt is a major mediator of signals that protect cells from apoptosis and also a regulator of cell proliferation [24–25]. Specific inhibition of Akt, for example with LY249002, had an inhibiting effect on cell progression as well as apoptosis in tumor cells [26]. In the current study, overexpression of TCTP induced Akt phosphorylation and inhibition of Akt activation blocked TCTP overexpression-induced survival of HeLa cells, as expected, suggesting that Akt activation is involved in the anti-apoptotic function of TCTP overexpression. Cell motility, one of the defining characteristics of invasive tumors, enables tumors to migrate into adjacent tissues or transmigrate into basement membranes and extracellular matrices [27]. Invasive tumor cells exhibit dysregulated cell motility in response to extracellular signals from growth factors and cytokines, and PLC-c has a key function as a molecular switch in cell motility [28–29]. Indeed, TCTP overexpression activates PLC-

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c and PLC-c inhibitor, U73122, blocks an increase of cell migration in TCTP overexpressing HeLa cells. Our study suggests that antiapoptosis and cell migration induced by TCTP overexpression might be involved in tumorigenesis and that PI3K/Akt and PLC-c pathways might be the potential targets in the therapy of TCTP induced tumors. Acknowledgments This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund), the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (R012007-000-20263-0), and the NCRC program of MOST/KOSEF (R15-2006-020) through the Center for Cell Signalling and Drug Discovery at Ewha Womans University. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]

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