693

p120 Catenin Translocation is Involved in Enhance- ment of Hepatoma Cellular Malignant Features

Huayi Huang 1

Chaozan Nong 1

Weisheng He ~

Ungxiao Guo ~

Shaoyun Nong 1

Uli Pan ~

Xiliang Zha 2

1 Department of Experimental Center, Guangxi Nationalities Hospital, Nanning 530001, China. 2 Department of Biochemistry and Molec- ular Biology, Fudan University College of Medicine, Shanghai 200032, China.

Correspondence to: Huayi Huang E-mail:huayi.huang@roswellspark.org

This work was supported by the National Natural Science Foundation of China (No. 30160096).

Received April 30, 2005, accepted May 20, 2005.

Chinese Journal of Clinical Oncology

E-mail: cocr@eyou.com Tel(Fax): 86-22-2352-2919

OBJECTIVE To investigate the relationship between p120 c~ translocation and

hepatocellular carcinoma cell malignant features and the relationship be-

tween p120 ~ and ~-catenin translocation in cell signaling.

METHODS Human hepatocellular carcinoma cells were over expressed

with p120 ~tn isoform 3A using a DNA transfection method. The effects of

transfection and expression of p120 c~ and its binding capacity to E-cad-

herin were examined using immunoprecipitation and immunoblotting meth-

ods. p120 ~ subcellular localization and its relation with ~-catenin were de-

tected using immunofluorescent microscopy, p120 c~ phosphorylation was

produced by EGF treatment. Cell adhesion, cell migration and cell prolifera-

tion were also examined in this study.

RESULTS We found that p120~ was increased after transfection

and the binding capacity of p120 c~ to E-cadherin was enhanced. Tyrosine

phosphorylation of p120 ~ increased after transfection and EGF treatment.

p120 c~ and l~-catenin cellular localization displayd a membrane and cyto-

plasmic expression pattern, but they translocated into the nucleus for relo-

calization after p120 c~ overexpression plus EGF stimulation. Cell adhesion

ability was increased and migration ability reduced after transfection without

EGF. Following transfection without EGF cellular proliferation was reduced,

but increased after EGF treatment.

CONCLUSION Our results suggest that p120 ~ plays an important role in

hepatocellular carcinoma cell adhesion, migration and proliferation. In addi-

tion there is a relationship between p120 ~ and [B-catenin subcellular local-

ization and signaling.

KEYWORDS: p120 ~, [3-catenin, h/rosine phosphorylation, translocation, hep- aroma.

p 120 ~ is a newly identified catenin family member. SO far, its role

in E-cadherin mediated cell-cell adhesion and signaling is not

well understood. Recently reports revealed that p120 ~n translocated in-

to the nucleus in some tumor cells like [3-eatenin, so it might play a

role as an oncogene affecting transcription of [3-eatenin. [~1 Other re-

ports showed that p 120 ~ acted as a modulator in E-cadherin mediated

cell-cell adhesion and signaling, so it might play a role as a tumor sup-

pressor gene. [71 Evidence has shown that p 120 ~ binds to a domain of

E-cadherin at the juxtamembrane region. This binding is crucial for

p120 ~ in cell adhesion and signaling, [8] but its exact role is uncertain.

In this study, we investigated some malignant features of the human

694Chinese Journal of Clinical Oncology 2005/Volume 2/Number 4

hepatoma cell line BEL-7404 after over expression of

p120 ~ isoform 3A to further disclose the role ofpl20 ~

in cell biology.

MATERIALS AND METHODS

Materials

Methods

Co//eu/ture

Cells were cultured in RPMI 1640 medium supple-

mented with 10% fetal bovine serum, 100 U/ml of

penicillin, 100 mg/ml of streptomycin, and grown un-

der a humidified atmosphere with 5% CO2 at 37 ~

B~tetia and cell line DH5o~ competent cells were kept in the laboratory of

Fermentation and Enzyme Biotechnology at the

Guangxi University Biotechnology Center. The

BEL-7404 human hepatoma cell line was purchased

from the Institute of Biochemistry and Cellular Biolo-

gy, Chinese Academy of Sciences in Shanghai.

e h e m i c ~ and antibodies

Anti-pl20% anti-E-cadherin, anti-[3-catenin and an-

ti-phosphotyrosine monoclonal antibodies were pur-

chased from B-D Transduction Laboratory (Lexington,

KY, USA). Goat anti-mouse IgG-FITC and goat an-

ti-mouse IgG-HRP were purchased from Sino-Ameri-

ca Biotechnology in Shanghai China. Fibronectin and

lipofectamin TM were obtained from GIBCO BRL Life

Technologies (GIBCO BRL, Grand Island, NY, USA).

Anti-[3-tubulin and DAPI were procured from Sigma

(Sigma-Aldrich, St. Louis, MO, USA). ECL was pur-

chased from Amersham Biosciences (Amersham, Pis-

cataway, N J, USA).

Plnsmid

A p120 = isoform 3A plasmid and empty vector was a

generous gift from Dr. Albert B. Reynolds in the De-

partment of Tumor Cell Biology at Vanderbilt Univer-

sity, United States.

/nstruments

A CO2 incubator was a product of Harris. Microplate

Reader 550 was from Bio-Rad equipped with 490 nm

and 540 nm wavelength filters and the X-ray film and

auto film processor was produced of KODAK (Ko-

dak, Rochester, NY, USA). A confocal microscope

was a product of Leica (Germany).

Ampli~cation of plasmid DNA

Two ixl ofplasmid solution (about 2 Ixg of DNA) was

added into 80 Ixl of DH5oL competent cells, mixed and

heat shocked for 40 s. The solution was spreaded on a

LB plate containing ampicillin and incubated at 37 ~

overnight (about 16 h). Single colonies were picked

and seeded into 100 ml of LB media for another incu-

bation until the OD value reached larger than 1. DNA

purification was based on the method introduced in the

Molecular Cloning Manual. [91

Transfection

All procedures were based on the instruction of the

manufacturer. Colonies were selected using G418 for

stable expression.

p120 ~ phosphorylation

The method employed was based on that of Hazan and

Morton. tl0j In brief, the cells were grown in 60 mm

dishes until 100% confluent, and then starved for 36 h

by withdrawing serum. Then cells were treated with

200 ng/ml of EGF to enhance tyrosine phosphorylation

ofpl20 ~.

lmraunoprecipitafion and Western blotting Cells were broken using lysis buffer (40 mM Na2PO~,

pH 7.2; 250 mM NaC1; 50 mM NaF; 5 mM EDTA,

1% Triton X-100, 1% deoxycholate) for 20 min on ice.

Fifty ixg of protein was loaded into a SDA-PAGE gel

for protein resolution by Western blotting. For im-

munoprecipitation, 1,000 Ixg of total protein from each

cell lysate was immunoprecipitated with 10 p,g of an-

ti-E-cadherin or anti-pl20 = antibody. Protein G a-

garose (50 Ixl of 50 % slurry in PBS) was added. Sam-

ples were mixed by rotation for 1 h at 4 ~ The beads

were pelleted and washed 4 times, followed by the ad-

Huayi Huang et aL 695

dition of 50 txl of 2xSDS sample buffer and boiling for

5 min. Samples were kept on ice for 5 min and then

spun down, 30 Ixl of supernatant was loaded onto a 8%

SDS-PAGE gel and separated. Proteins were trans-

ferred to a PVDF membrane, the membrane was incu-

bated with mouse anti-phosphotyrosine (PY20) anti-

body at 4~ over night and then incubated with

HRP-conjugated anti-mouse antibody at room temper-

ature for 1 h. The membrane was exposed with ECL

using Kodak X-ray films and processed by Kodak film

processor.

Trnmtmofluorcsccnt microscopy Cells were grown on coverslips and fixed with 3%

formaldehyde, blocked with 3% BSA, then incubated

with primary antibodies for 1 h at room temperature.

The coverslips were washed with TBS-T and incubat-

ed with FITC-conjugated secondary antibody for 1 h at

room temperature and examined under a laser confocal

microscope or an Olympus microscope.

amined and counted.

Cell prolifcration assay

Cells (2 x l05 ) were seeded into 60 mm dishes and

grown for 7 days. The Trypan blue exclusion assay

was used in triplicate for viable cell counting.

RESULTS

Effect of p120 d" isoform 3A transfection in BEL-7404

cells

Cells were stably transfected with p120 c"~ isoform 3A,

and the effect of transfection was determined by West-

ern blotting using mouse anti-pl20 ~ antibody. The re-

sults showed that increased p 120 ~ expression had been

detected in stably transfected cells (Fig. 1).

Ceil adhesion assayfHj

Cells were grown to 80% of confluence, starved by

withdrawing serum and grown for another 8 h. Then

the cells were treated with EGF and then detatched by

EDTA. The cells were seeded into fibronectin coated

96-well microplates containing 10,000 cells/well/100

~1. The plates were then incubated at atmosphere of

37~ in a 5% CO2 for 4 h. The cells were rinsed slight-

ly with warm medium to discard the unadherent cells,

0.4% EDTA/PBS was added followed by incubation at

37~ for 20 min to detach the cells. The detached cells

were collected in another microplate for counting.

Cell adhesion (%) = Number of adherent cells/Total

added cellsxl00.

1: Non-transfected 2: Transfected cells

F~.I. Effect of p120~isoform 3A transfection in BEL-7404 cells.

Capacity of p120 dn binding to E-cadherin after

lransfeclion of the cells with p120 c~ isoform 3A

In order to determine whether the capacity of p l20 ~

binding to E-cadherin strengthened, we immunopre-

cipitated E-cadherin and then immunobloted with an-

ti-pl20 ~. The results showed that transfection in-

Ceil migration assay l ~2j

Cells were seeded in fibronectin coated 6-well plates

and grown to 100% confluency. Using a 200 ~1 pipet

the bottom of each well was marked with a cross hori-

zontally and vertically. PBS was then used to slightly

rinse the wells in order to discard the loose cells. Medi-

um was added and the cells cultured for another 24 h.

Then the cells that migrated into the crosses were e x -

1: Non-transfected 2: Transfected cells

Fig. 2. Capacity of p120 | binding to E-cadherin after cell transfection

with p120 ̀= isoform 3A.

p120 n" phosphorylafion in transfected cells after EGF treatment

Cells were transfected with p120 ~ isoform 3A, and

696Chinese Journal of Clinical Oncology 2005/Volume 2~Number 4

then stimulated with 200 ng/ml of EGF for different

periods. The result showed that EGF increased p120 =

phosphorylation status (Fig.3).

p120 ~ and [3-catenin subcellular localization after cells were transfected with p120 ~ isoform 3A Fig.4 shows that p120 ~n and 13-catenin subcellular lo-

calizations were changed after cells were transfected

with p120 ~ isoform 3A. Both of the proteins were ex-

pressed in the cytosol and in the nucleus before trans-

fection, but after transfection the staining become

stronger at the level of the membrane.

p120 a" nuclear Itanslocation after the cells were transfected with p120 ~ isoform 3A and stimulated with EGF

Fig.5 shows that p120 ~ translocated into nucleus after

the cells were transfected with p120 = and stimulated

with EGF. Under these conditions membrane expres-

sion of the protein was reduced.

Cell adhesion ability changes after transfection Cell adhesion ability was enhanced after transfection

as shown in Table 1.

Table 1. Cell adhesion ability changes after transfection.

Groups Cell adhesion ( % )

Non-transfected 54.0 + 3.2

Transfected 67.0 + 3.0*

Transfected+EGF (20 rain) 39.0 -+ 3.1"*

Comparison with the group of non-transfected, * P>O.05 ;**P< 0.05.

Cell migration ability changes after ttansfection Cell migration ability was reduced after cells were

transfected with pl20=isoform 3A as shown in Fig.6.

Cell proliferation changes after transfedion Cell proliferation ability was reduced after cells were

transfected with p120 ~ isoform 3A, but increased with

EGF treatment as shown in Fig.7.

DISCUSSION

At present the role of p120 c~ in cell biology is not

clear, and different cells have different pattern of

p120 ~ isoform expression. Recent studies have shown

that p120 ~ plays an important role in E-cadherin-me-

diated epithelial cell-cell adhesion and signaling. One

potential function o fp l20 ~ is that it acts as a modula-

tor in the E-cadherin pathway, as it plays a "switch"

like role under different situations, f13] Obviously,

different levels of p120 ~ expression in tissues of

various origin or with different isoform expression

may influence this signaling cascade. Studies have

shown that there are 32 p120 ~ isoforms expressed in

different kinds of tissues, and these isoforms have their

origin from alternative splicing of the gene. B4-~63 It is

not yet clear as to what specific type of isoform is

expressed in different specific tissues. Research also

suggested that p 120 ~ expression and function might be

different under different stages of cell proliferation or

different phases of the cell cycle. [131

In general, liver cells have weak p120 = expression

and the isoform 2A is especially unstable. Our previ-

ous results have shown that the p120 = isoforms 1A

and 3A were expressed, i171 Cell adhesion ability in-

creased after the cells were overexpressed with the

p120 = isoform 3A, but migration ability reduced, sug-

gesting that the mechanism was due to enhanced

E-cadherin-catenin complex (CCC) formation and sta-

bility. Our results also suggested that p120 ~ is crucial

in E-cadherin mediated cell-cell adhesion, results

which are consistent with those of Hengel and

co-workers. [181

We also found that p120 ~ tyrosine phosphorylation

was significantly increased in response to EGF stimu-

lation after cellular transfection with p120 = isoform

3A, results which suggested that p120 ~ could easily be

phosphorylated in response to growth factors.

Cell proliferation reduced after cells were transfected

with p120 ~ isoform 3A, but opposite results were ob-

tained following transfection plus EGF treatment. We

also found that p120 an had significantly translocated in-

to nucleus with transfection plus EGF treatment, sug-

gesting that phosphorylation of p120 ~ disrupted the

CCC. Nuclear translocation of 120 = could be one of

the mechanisms that lead to features of cellular malig-

nancy.

ltuayi ltuang et aL 697

A: Non-transfected and non-EGF B: Transfected plus EGF 20 min C: Non-transfected plus EGF 20 min

Fig.3. p120 c~ tyrosine phosphorylation after cell tranfection with p120 | isoforrn 3A and stimulation with 200 ng/ml of EGF.

A, C: Pre-transfection B, D: Post-transfection

Fig.4. p120 = and I~-catenin subcellular localization changes after call transfection with p120 | isoform 3A. A and B show p120'~; C and D show

[3-cetenin..

A: Transfection without EGF B: Transfection with EGF 20 min

Fig.5. p120 ~" nuclear translocation after cell transfection with p120 = isoform 3A plus EGF stimulation.

A: Non-transfection B: Transfected

Fig.6. Cell migration ability changes after cell transfection with p120 =.

698 Chinese Journal of Clinical Oncology 2005/Volume 2/Number 4

Fig,7. Cell proliferation features after transfecti0n with p120 '= isoform

3A.

Other studies have revealed that with different cells

or with different cellular proliferation, nuclear translo-

cation o fp l20 ~ might play various roles in cell prolif-

eration and differentiation, so the role of p120 ~' as a

transcription modulator is not clear and it might act as

a "dual-direction modulator" in cells, t1~221

Our results also showed that there was a relationship

between p120 ~ and [3-catenin subcellular localization.

There was an abundance of p120 ~ expressed at the

membrane at the region of cell-cell contact, but weak

expression was seen in the cytosol after overexpres-

sion, with rare nuclear expression. Interestingly, in-

creased cytoplasmic and membrane levels of [3-catenin

could be seen after overexpression of p 120 ~ opposite

to the original expression pattern. It seems that p120 ~n

"recruited" 13-catenin from the nucleus to the cyto-

plasm and membrane, and cell biologic behaviour also

improved under these conditions. This opinion is based

on the results relating to cell adhesion.

From these results, we suggest the following: (1)

p 120 ~ is crucial in E-cadherin mediated cell-cell adhe-

sion; (2)there might be a competitive mechanism be-

tween pl20~and 13-catenin subcellular localization and

signaling; (3) p120 ~ nuclear translocation takes place

upon its tyrosine phosphorylation and its translocation

plays a role in cell biological function.

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