Subcellular localization of beta-catenin in malignant cell lines and squamous cell carcinomas of the...

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Subcellular localization of beta-catenin in

malignant cell lines and squamous cell

carcinomas of the oral cavity

A. Gasparoni1,2

A. Chaves1

L. Fonzi2

G. K. Johnson1

G. B. Schneider1

C. A. Squier1

1Dows Institute for Dental Research, College ofDentistry, University of Iowa, IA, USA and2Dipartimento di Scienze Biomediche,Universita0 di Siena, Italy

Correspondence to:Alberto GasparoniN439E Dows Institute for Dental Research,College of Dentistry, University of Iowa,Iowa City, IA 52242, USAe-mail: [email protected]

Accepted for publication January 16, 2002

Copyright � Blackwell Munksgaard 2002

J Oral Pathol Med . ISSN 0904-2512

Printed in Denmark . All rights reserved

AbstractBackground: Beta-catenin, an E-cadherin-associated proteininvolved in cell–cell adhesion and signaling, has been hypothe-sized to translocate to the nucleus and activate transcription inseveral human cancers, including oral squamous cell carcinomas(OSCC).Methods: In the present study, we analyzed the subcellularlocalization of beta-catenin in cultures of human oral normaland malignant (cell lines SCC15 and SCC25) keratinocytesand in 24 frozen samples of oral squamous cell carcinomasby a double-staining technique for nucleic acids and beta-catenin.Growth potential, as assessed by cell count at different timeperiods, was established for normal, SCC15 and SCC25 celllines; oral squamous cell carcinomas were classified accordingto the histopathological and malignancy indexes.Results: Beta-catenin localized at the plasma membrane in thenormal and SCC15 cells, not in the SCC25 cells, where itlocalized mostly in the perinuclear and nuclear areas. In thegrowth assays, SCC25 cell lines proliferated faster than innormal and SCC15 cells over a period of 6 days (cell numberswere significantly different, P<0.0001). Carcinoma sectionsshowed a combination of membranous, cytoplasmic and, infew invading epithelial islands of two tumors, nuclear localizationof beta-catenin.Conclusions: In oral squamous cell carcinomas, nuclear beta-catenin staining was observed only within invading islands of twocarcinomas deep in the underlying connective tissue. On thebasis of this study, we conclude that intranuclear beta-catenindoes not appear to be a common finding in oral squamous cellcarcinomas and that a clear association between intranuclearbeta-catenin and histopathological and malignancy indexes in vivocould not be established.

Key words: beta-catenin; confocal laser scanning microscopy;growth assay; oral squamous cell carcinomas; SCC15; SCC25

J Oral Pathol Med 2002: 31: 385–94

Beta-catenin is known to serve both a cell–cell adhesive function,

by providing the intracytoplasmic bond between the E-cadherin

tail and other components of the desmosomal and tight junc-

tions (1–3), and an effective signaling path in developing

embryos and tumors. As a signaling molecule in developing

embryos, overexpressed beta-catenin has been shown to

385

induce secondary axis formation in the frog Xenopus laevis (4),

and tumor formation and alteration in hair morphogenesis in the

adult mouse squamous stratified epithelia (5). The known

mechanism for signaling transduction in cancer involves the

adenomatous polypi coli (APC) gene, mutated in familial poly-

posis and in many colon cancers, which associates with beta-

catenin and GSK3b in a complex necessary for beta-catenin

phosphorylation and degradation (6–8). A mutation in the APC

gene or GSK3b or beta-catenin phosphorylation sites may cause

an increase in beta-catenin cytoplasmic levels, which in turn

facilitates binding with T-cell factor 1–4 (9) and lymphoid enhan-

cer factor (Lef-1), and induces nuclear translocation (10, 11).

Within the nucleus, the translocated complex causes activation of

transcription, whose target genes identified so far include the

urokinase-type plasminogen activator receptor (12) and c-myc

(13). High levels of stable beta-catenin have also been found to be

associated with proliferative potential in human keratinocytes

(14).

Mutations of beta-catenin causing increased cytoplasmic accu-

mulation have been shown in gastric carcinomas and gastric

cancer cell lines (15, 16). Increased cytoplasmic levels of beta-

catenin, consequent to alteration in phosphorylation pathways

either due to beta-catenin or APC mutation, have been observed

in breast carcinoma and breast cancer cells (17–19).

In oral squamous cell carcinomas (OSCC), beta-catenin

staining has been shown to decrease when compared to normal

tissue. In both frozen and formalin-fixed/paraffin-embedded

OSCC, beta-catenin staining was found decreased (or even

lost in few cases) in the less differentiated tumors, usually

accompanied by low levels of E- and P-cadherin, alpha- and

gamma-catenins and beta-1 integrins (20, 21). This suggested a

close correlation between differentiation, invasive properties of

cancerous cells and the expression of their membranous

components, including members of the cadherin, integrin, and

catenin families. When membranous and cytoplasmic beta-cate-

nin were evaluated in OSCC in relation to tumor differentiation

stage, the results showed a significant association between loss of

beta-catenin membranous staining and loss of cell differentiation

(22).

The main goal of the present study was to analyze beta-catenin

localization (i.e. membranous, cytoplasmic, and nuclear) in cul-

tured human oral keratinocytes (normal gingival cells as controls,

and the malignant SCC15 and SCC25 cell lines) in relation to their

growth capability, as measured by the cell count at different time

points. Secondly, we analyzed frozen samples of OSCC to see

whether beta-catenin nuclear localization was associated with

malignancy or differentiation grade.

Materials and methods

Cell cultures

Six normal oral keratinocyte strains (from six individuals, num-

bered as 196, 287, 290, 291, 294, and 296 on the basis of the

chronological order of preparation) were used for all the experi-

mental procedures, immunocytochemical staining, and growth

curve assays. Normal keratinocyte cultures were prepared

according to the previously reported procedures (23) as follows:

gingival tissue samples were obtained from six healthy, non-

smoking patients, aged 12–50 years, undergoing crown-lengthen-

ing procedures according to the guidelines of the Institutional

Review Board (College of Dentistry, University of Iowa). Speci-

mens were washed in Dulbecco’s phosphate buffered saline,

containing penicillin (200 IU/ml), streptomycin (200 mg/ml)

and amphotericin B (5 mg/ml), and then placed overnight in

dispase (Grade II, Boheringer, Mannheim, Germany) at 48C

(24). After separation of the epithelial tissue from the connective

tissue, epithelial tissue was disaggregated by incubation in 0.25%

trypsin for 15 min with gentle pipetting. Isolated epithelial cells

were then seeded into T25 tissue culture flasks (Corning Costar

Co., Cambridge, MA) containing a feeder layer of mytomycin-

treated 3T3 murine fibroblasts (NIH, Bethesda, MD) (25). The

cultures were grown in a medium consisting of three parts of

Dulbecco’s modified Eagle’s media (DMEM)þ one part of Ham’s

F12 medium and 10% fetal bovine serum (FBS), supplemented

with hydrocortisone (400 ng/ml), cholera toxin (0.1 nM), epider-

mal growth factor (10 ng/ml), penicillin (100 IU/ml), streptomy-

cin (100 mg/ml), amphotericin B (2.5mg/ml), insulin (4.5 mg/ml),

and adenine (16.38 mg/ml). After the cells reached 70–80% con-

fluence, the feeder layer was removed by incubation in 0.012%

trypsin (Gibco, Grand Island, NY) for 2 min at 378C, keratinocytes

washed in phosphate buffered saline (PBS; Sigma Chemical, St.

Louis, MO) and released from the flask by incubation in 0.025%

trypsin�0.1 mM ethylenediaminotetraacetic acid (EDTA) in PBS

for 2 min at 378C. Keratinocytes were then counted in a hemo-

cytometer and seeded (4.3� 104 cells per well) in an eight-well

glass slide (Nunc, Fisher) for immunocytochemical analysis.

Cells were maintained in the absence of feeder layer in

0.15 mM calcium serum-free modified MCDB medium (KGM,

Clonetics Corp., San Diego, CA) containing 0.5 mg/ml hydrocor-

tisone, 5.0 mg/ml insulin, 7.5 mg/ml bovine pituitary extract,

0.1 mg/ml human recombinant epidermal growth factor,

50 mg/ml gentamicin, and 50 ng/ml amphotericin B.

SCC15 and SCC25 cells were cultured in T25 flasks containing

45% Ham’s F12 medium, 45% DMEM and 10% FBS, supplemented

Gasparoni et al.

386 J Oral Pathol Med 31: 385–94

with hydrocortisone (400 ng/ml), cholera toxin (2.5 mg/ml), peni-

cillin (100 IU/ml), streptomycin (100 mg/ml), and amphotericin B

(2.5 mg/ml) (26). Upon reaching 70–80% confluence, SCC cultures

were detached from the flask and seeded in a T75 flask or in an

eight-well glass slide, as previously described for normal cells.

Cultures were grown for 4–10 days with changes in the medium

every 1–2 days at 378C in a humidified environment containing 5%

CO2 until confluence. Experiments (single and double labels and

growth curve) were carried out in triplicate for each cell strain and

cell line.

Immunocytochemistry

Slides containing normal and malignant cell cultures were washed

three times with PBS for 5 min each, fixed at 48C in 20% dimethyl-

sulfoxide (DMSO)�80% methanol for 20 min, washed again, and

blocked for 1 h in 5% bovine serum albumin (BSA) in PBS. Then,

the slides were incubated with a solution of 200 ml of polyclonal

rabbit antibody anti-human E-cadherin (Santa Cruz Biotechnol-

ogy, Santa Cruz, CA) or monoclonal mouse antibody IgG1 against

recombinant chicken beta-catenin (Sigma Chemical, St. Louis,

MO) (27), respectively, diluted 1:500 and 1:1000 in 5% BSA/PBS

for 1.5 h. The slides were then washed again in PBS and incubated

either with tetramethylrhodamine (TRITC)-labeled donkey anti-

rabbit IgG (Sigma Chemical, St. Louis, MO) diluted 1:1000 or

TRITC-labeled goat anti-mouse IgG diluted 1:300 in BSA/PBS

(Sigma Chemical, St. Louis, MO) for 1 h at room temperature.

To assess subcellular localization of beta-catenin, double-stain-

ing procedures were performed with a cytoplasmic and nuclear

marker. Keratinocytes were preincubated with 0.01% Tween-20 in

PBS for 15 min, washed and then exposed to a mixture composed

of antibodies against cytokeratin (CK) 14 (mouse IgM, diluted

1:400) and beta-catenin. This was followed by incubation with a

mixture of goat FITC-labeled anti-mouse IgM/goat TRITC-

labeled anti-mouse IgG (diluted 1:400 and 1:1000, respectively).

These slides were examined at high contrast (power of laser

beam: 30%; black gain: �20; iris: 6.2). The green and red channels

were maintained separately, saved as tagged image file formats

(TIFF) and merged using a dedicated software (Confocal Assis-

tant). Another set of slides was then stained with a mixture of

200 ml of propidium iodide (PI) 1:5000 in PBS and 200ml of the

same beta-catenin antibody solution. After washing, the slides

were incubated with a mixture of 1:400 in 5% BSA/PBS FITC-

labeled goat anti-mouse IgG for 1 h and then washed and covered

as previously described.

Positive controls were slides with cultures of oral normal

keratinocytes; negative controls were run by omitting the primary

antibody. All the keratinocyte cultures were examined by confocal

scanning laser microscopy (CSLM, Bio-Rad 1024 MRC) with

appropriate filters for FITC and TRITC fluorochromes. In sin-

gle-label experiments, observations were carried out applying the

same values of black level, gain, and laser beam intensity. Speci-

mens were sealed using Gel/Mount (Biomeda Corp., Foster City,

CA) and coverslipped for microscopic examination.

Growth curve

We assessed the growth capability of normal and SCC cell

cultures with a growth assay. Approximately 4� 104 normal (cell

strains labeled numerically according to the order of isolation: 290

and 291) and malignant (SCC15 and SCC25) cells were seeded in

a 24-well plate (Costar, Corning Inc., Corning, NY) in triplicate

samples. At days 1, 2, 4 and 6, cells were washed with PBS,

incubated with 500ml of 0.025 trypsin�0.1 mM EDTA in PBS for

15 min, detached and resuspended in 10 ml total of Isoton liquid

and counted in a Coulter Counter ZM (Coulter Electronics,

Hialech, FL). The total numbers of cells were calculated and

analyzed by two-way ANOVA; differences (at a level of P< 0.05)

were identified by Tukey’s test.

Human surgical samples

Thirty-three frozen specimens of oral squamous carcinoma and

five specimens from dysplastic lesions were kindly provided by Dr

Nilton Herter (Head and Neck Surgery Department, Santa Rita

Hospital, Porto Alegre, Brazil). Of the tumor biopsies, nine were

eliminated because of unacceptable condition of the sample (four

cases), lack of clinical data (four cases), or because they were

metastatic tumors (one case). The 24 remaining carcinomas were

classified according to the histopathological grade (28) and

malignancy index (29) using sections of formalin-fixed/paraf-

fin-embedded hematoxylin–eosin-stained samples. The histo-

pathological grade was assessed by Dr R. Furian (Pathology

Department, Fundacao Faculdade Federal de Ciencias Medicas

de Porto Alegre, Porto Alegre, Brazil). The malignancy index has

been shown to provide better prognostic value and interobserver

agreement than the classification based on the proportion of

differentiated cells alone (29, 30). Two investigators (A.G. and

G.S.), one of whom (G.S.) was blinded as to the tumor biopsy,

graded malignancy independently. According to the methodology

proposed by Anneroth & Hansen (31) and modified by Bryne et al.

(29, 30), the parameters considered were: degree of keratiniza-

tion, nuclear polymorphism, number of mitoses, pattern of inva-

sion, and host–tissue relationship (leukocyte infiltration). Each

J Oral Pathol Med 31: 385–94 387

Subcellular localization of beta-catenin

parameter was assigned a value 1–4. The malignancy indexes

recorded by the two investigators were averaged (range of values:

10.6–18; mean: 13.88; median: 14.3) and classified into three

groups (22) as follows – group 1: <12; group 2: 12.1–14; group

3: >14.1.

Immunohistochemistry

Biopsies were snap-frozen in liquid nitrogen (�808C) and

embedded in OCT compound (Miles Inc., Elkhart, IN) for sec-

tioning in a cryostat. Positive controls were six biopsies of normal

gingival tissue discarded during crown-lengthening procedures at

the Department of Periodontics, University of Iowa; negative

controls were run as in cell cultures.

For each biopsy sample (normal tissue, dysplastic lesion,

and tumor biopsies), 10–12-mm sections were cut, fixed in cold

methanol for 20 min, washed and incubated with 0.01% Tween-20

in PBS for 15 min before incubation with the anti-beta-catenin/PI

mixture already described for cell culture staining. Two investi-

gators (A.G. and A.C.) independently assessed beta-catenin

nuclear, membranous and cytoplasmic staining; interobserver

agreement was assessed with the Kendall ranked correlation

coefficient.

Results

Cell cultures and immunocytochemistry

E-cadherin antibody staining was localized at the cytoplasmic

membrane in all keratinocytes, normal and SCC lines; the staining

appeared slightly decreased in intensity in SCC15 and SCC25 cell

lines (not shown). Similarly, due to either single staining (not

shown) or double staining with CK14 or PI and beta-catenin, beta-

catenin localized at the cytoplasmic membrane in normal

(Figs. 1A and D–F) and SCC15 cells (Figs. 1B and G–I). The latter

showed less intense membranous staining, sometimes resem-

bling a desmosomal-type junction (Fig. 1I). Most SCC25 cells

showed decreased membranous staining and more perinuclear

or nuclear staining (Figs. 1C and J–L), demonstrated by red dots

inside the nucleus (Fig. 1C) in double stains for CK14 and beta-

catenin. Decrease in membranous staining and increase in cyto-

plasmic and nuclear staining in the SCC25 cells was confirmed by

observing the cells stained for beta-catenin and PI (Figs. 1J–1L),

where areas of bright yellow color inside the nucleus suggested

colocalization of PI and beta-catenin.

Growth curve

Of the two normal cell strains (290, 291), the former increased in

number more rapidly than the latter, although this difference was

not significant (Fig. 2). Both cell strains increased in number

significantly less than the SCC15 cell line (P< 0.0001) at day 6. At

the same time point (day 6), SCC25 cells increased in number

significantly more than SCC15 cells and normal strains, and this

difference was significant (P< 0.0001).

Human surgical samples and immunohistochemistry

Characteristics of patients, tumors’ anatomical site and main

findings for membranous, cytoplasmic and nuclear beta-catenin

staining are shown in Table 1; it also reports the histopatho-

logical groups and the groups individuated by malignancy

grades.

The overall agreement between the two investigators for

membranous, cytoplasmic and nuclear staining in OSCC tissue

specimens was 0.65, 0.53 and 0.57, respectively (Kendall tau

correlation coefficient). Frozen sections of the upper layers of

normal (Fig. 3A) and differentiated areas of carcinoma tissue

(Fig. 3B, tumor 18) showed membranous staining for beta-catenin

throughout the epithelium, with PI uniformly staining the nuclei.

In the upper layers, membranous beta-catenin appeared

decreased in some dysplastic lesions and some carcinomas

(not shown); complete loss of membranous beta-catenin staining

in the upper layers was evident only in few tumors. Invading

epithelial islands showed alternatively cytoplasmic (Fig. 3C,

tumor 19) or membranous (Fig. 3D, tumor 8) staining. In most

tumors, cytoplasmic staining was usually observed with

decreased intensity of staining; areas with complete loss of

staining were rarely observed (tumors 8, 12, 18, 19, 22 and 24;

not shown). In localized regions of two tumors, alternating pat-

terns of membranous, cytoplasmic and nuclear staining for beta-

catenin were observed, where colocalization of PI and beta-cate-

nin in the nucleus was demonstrated by the color shift within the

same optical field from green to red and yellow (Figs. 3E and F,

tumor 18).

Discussion

In this study, we have demonstrated beta-catenin localization

by a recombinant chicken beta-catenin antibody (27) at the


Gasparoni et al.

Fig. 1. (A–C) Cultured oral keratinocytes stained with CK14 and beta-catenin: (A) normal keratinocytes (196); (B) SCC15 cell line; (C) SCC25 cell

line. Note the membranous staining in normal and SCC15 cells (arrowheads in A and B) and little membranous staining in SCC25 cells, with

perinuclear (asterisks in C) and nuclear staining (arrows in C). Magnifications: 630�, oil immersion; 400�, H2O immersion; 630�, oil immersion. (D–

L) Cultured oral keratinocytes stained with PI and beta-catenin: (D–F) normal keratinocytes (294); (G–I) SCC15 cells; ( J–L) SCC25 cells. Beta-catenin

nuclear staining appears increased in SCC25 cells, as observed by the yellow color shift (arrows in L). Arrowheads indicate membranous staining,

arrows indicate nuclear staining, and asterisks indicate cytoplasmic and perinuclear staining. Magnifications: (D–F) 400�, H2O immersion; (G–L)

630�, oil immersion.



membranous levels in normal cultured oral keratinocytes and

SCC15 cell lines. Membranous beta-catenin decreased progres-

sively in SCC15 and SCC25 cells; staining appeared membranous

in normal and SCC15 cells and mostly cytoplasmic and nuclear in

SCC25 cells. Beta-catenin levels appeared to decrease in SCC15

and SCC25 cells compared to normal, as evaluated by staining

intensity. To show evidence of beta-catenin nuclear staining,

we used a double-staining technique (with an antibody for a

Fig. 2. Growth assay for normal keratino-

cytes, SCC15 and SCC25 cell lines. Over a

period of 6 days, SCC25 cells increased in

number more than SCC15 cells and SCC15

cells increased more than normal cells

(strains 290 and 291). No significant differ-

ence was observed between the normal cell

strains 290 and 291. The difference among

cell numbers at day 6 for normal, SCC15 and

SCC25 cells was significant (P< 0.0001).

Table 1. Patients’ characteristics and main findings on beta-catenin staining pattern in normal tissue, dysplastic lesions, and squamous cell

carcinomas of the oral cavity, with the corresponding histopathological and malignancy groups

Biopsy

type

Original case

number

Histopathological

grade

Malignancy

group

Lesion

site Sex Age Smoking Drinking

M. beta-

catenin

C. beta-

catenin

N. beta-

catenin

Normal 1 – – AGP F 39 S D þþ 0 0tissue 2 – – AGP M 35 S D þþ 0 0(cases 1–6) 3 – – LMG M 55 NS ND þþ 0 0

4 – – RMT F 62 NS D þþ 0 05 – – AGP M 53 MS ND þþ 0 06 – – LMG F 49 MS D þþ 0 0

Dysplasia 1 – – BT M 56 NS NI þ 0 0(cases 1–5) 2 – – FM M 62 S D þ 0 0

3 – – RTM M 52 S D þ 0 04 – – RTM M 58 S D þ 0 05 – – APA M 55 S ND þ 0 0

Squamous 2 I 1 FM M 52 S D þ þ 0cell 8 I 1 VT, FM F 61 S D þ þ 0carcinomas 10 I 1 BT, FM F 63 S D þ þ 0(cases 1–24) 20 I 1 LBT M 58 S D þ þ 0

3 I 2 SP M 68 S D þþ 0 07 II 2 RTM M 63 S D þþ 0 05 I 2 RMT M 65 S D þ 0 09 I 2 BT, FM F 63 S D þþ 0 0

11 I 2 FM M 65 S D þ þ 012 I 2 VT, LBT M 47 S D þþ þ 016 I 2 VT M 65 S D þ 0 04 I 3 VT F 78 NS ND þþ 0 06 I 3 LBT M 63 S D þ þ 01 II 3 SP, APA M 48 S D 0/þ 0 0

13 I 3 SP, RMT M 70 S D þ 0 014 I 3 RMT, BT M 73 S D þþ 0 015 III 3 RMT M 59 S D þþ 0 017 III 3 VT M 59 S D þ 0 018 III 3 VT, LBT M 56 S D þþ þ þ19 I 3 VT M 47 S D þ þ 021 II 3 RMT M 73 S D þ 0/þ 022 I 3 SP M 70 S D þ 0/þ 023 II 3 VT F 58 S ND þ þ 024 III 3 VT M 52 S D 0/þ 0/þ þ

APA, anterior pillar amygdala; AGP, anterior gingival palate; BT, base of tongue; FM, floor of mouth; LBT, lateral border of tongue; LMG, lower mandibular gingiva; RMT,retromolar trigon; SP, soft palate; VT, ventral tongue; S, smoker; MS, moderate smoker; NS, non-smoker; D, drinker; ND, non-drinker; NI, no information available; M.,membranous staining; C., cytoplasmic staining; N., nuclear staining; 0/þ, weak positive staining; 0, no stain detectable; þ, stain detectable; þþ, stain of the highestintensity.


Gasparoni et al.

cytoplasmic antigen, CK14, and a nucleic acid marker, PI, with the

beta-catenin antibody). The first double staining (CK14/beta-

catenin) allowed an increase in the beta-catenin signal, reducing

the background (cytoplasmic signal); the second staining (PI/

beta-catenin) showed colocalization of the nucleic acid marker

and beta-catenin in the nuclei of SCC25 cells and in oral carci-

nomas. PI is a nucleic acid dye that after binding to DNA or RNA

fluoresces when irradiated by a 488-nm wavelength laser beam

(32, 33).

In our assay, we also considered E-cadherin staining, as it

binds beta-catenin (1–3). The decrease of cadherin staining in

SCC cells as compared to normal cells has been previously

Fig. 3. Normal tissue and oral squamous

cell carcinoma (OSCC) sections stained with

PI and beta-catenin. Beta-catenin is mostly

membranous in normal tissue (A) and

membranous (B: tumor 18; D: tumor 8) and

cytoplasmic (C: tumor 19) in OSCCs. Invad-

ing islands of less differentiated OSCCs show

alternate patterns of membranous, cytoplas-

mic and nuclear staining (E and F; F is a

detailed version of E: tumor 18). Magnifica-

tions: (A) 400�, H2O immersion; (B, D, F)

630�, oil immersion; (C) 40�; (E) 200�.



demonstrated (34). The reduction in membranous E-cadherin

staining in SCC15 and SCC25 cell lines, but not complete loss of

staining, suggests that loss of membranous E-cadherin may not

be the only cause of beta-catenin cytoplasmic localization in

SCC25 cells (35). Instead, the increased beta-catenin cytoplasmic

levels might be due to a mutation either in the APC gene, as

described in colon cancers (11) and breast cancer cell lines (19),

or in the beta-catenin phosphorylation sites (15, 16, 18), rather

than to a non-functional E-cadherin (35). The cytoplasmic and

nuclear localization of beta-catenin in SCC cultures secondary

to beta-catenin gene alteration is consistent with findings in

human hepatocellular carcinomas (36). In contrast to a human

cancer breast cell line, DU4475, where beta-catenin levels were

shown to increase (19), beta-catenin levels in both SCC cells and

oral squamous cell carcinomas appeared, as evaluated by the

intensity of staining analysis, to be lower in our and in previously

reported (20–22) immunocytochemical assays. In our experience,

Western blot analysis of pellet and supernatant of normal and SCC

cells confirmed that beta-catenin steady-state levels in SCC25

cells decrease as compared to normal and SCC15 cells (not

shown).

SCC25 cells have been shown to have higher colony-forming

efficiency than SCC15 cells (26). In our assay, SCC25 cells

increased in number significantly more than SCC15 cells, and

SCC15 cells increased significantly more than normal cells over a

period of 6 days; no significant difference was detected between

the cell numbers of normal cells. Our immunocytochemical

staining suggested that the cell cultures with the largest increase

in cell number (SCC25) have the highest levels of intranuclear

beta-catenin staining. This could be due to the association

between beta-catenin transcription activation and cell growth

potential, as suggested for normal keratinocytes (37). Our obser-

vations of sections of frozen normal and OSCC sections suggest

that, together with a decrease in membranous beta-catenin,

nuclear localization happens in a limited subset of the invading

cells of few tumors. The reasons of this observation should be

carefully considered: a likely correlation to the tumor differentia-

tion stage, as evaluated by the malignancy index (22), does not

seem to be the case, although our observations would suggest

that only the tumors with higher histopathological and malig-

nancy grades (group 3) showed clear cytoplasmic and nuclear

staining, where beta-catenin-positive nuclei were found in invad-

ing epithelial islands organized in circular, whirl-like shapes,

surrounded by other unstained nuclei. The areas with cyto-

plasmic and nuclear staining also appeared to be less differ-

entiated, while membranous beta-catenin was apparently

associated with more differentiated cells (Fig. 3B). Still, we cannot

rule out the possibility that epithelial islands with nuclear staining

could also be found deeper in the connective tissue in other

tumors, with lower malignancy scores. Whether for an external

stimulus, such as Wnt signaling (36), or internal events such as

mutation in the APC/beta-catenin pathway, our observations

suggest that nuclear localization of beta-catenin might occur as

a consequence of a random mutation in some, but not all, invading

cells. This interesting data would as well suggest that nuclear

beta-catenin might be directly associated not only with cell

dedifferentiation but also with tissue invasion by activating tissue

protease transcription in these cells. Alternatively or concur-

rently, transcription of genes directly related to proliferative

events, such as c-myc (13), might be involved. In both cases,

the agent leading to beta-catenin nuclear translocation should be

clarified.

The nuclear staining present only in a subset of invading cells

would be confirmed by the observation that most cultured SCC25

cells showed an increased nuclear staining, while SCC15, another

oral carcinoma cell line, showed less nuclear staining. This

observation should be confirmed by either Western blot analysis,

or immunoprecipitation assays of nuclear proteins, or by control-

ling the expression of genes directly related to the beta-catenin

transcription activation.

Another question concerned the observation of the positively

stained nuclear membrane: whether it is due to the cross-

reactivity of some components of the nuclear envelope with

the antibody or to the actual localization of beta-catenin at

this level. Lastly, if the levels of nuclear beta-catenin can

be different in different SCC cell lines, the reasons could be

different: on one hand, this difference might be explained by

the fact that keratinocytes would tend to oppose the increased

cytoplasmic levels of beta-catenin, for example by activating

p53 transcription (39), and that only the cells where selective

pressure is overcome and beta-catenin translocates to the

nucleus, can activate transcription of genes relative to tissue

invasion and proliferation.

The activation of c-myc by beta-catenin nuclear localization or

the interaction with Lef and Tcf factors (12, 38) might help to

understand beta-catenin role and its relevance to tissue invasion

and growth potential.

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Acknowledgements

Support for Dr Anna Cecilia Chaves was provided by the Brazilian Post-

graduate Federal Agency: CAPES (Coordenacao de Aperfeicoamento de

Pessoal de Nıvel Superior). Support for Dr Alberto Gasparoni was

provided by University of Siena and University of Iowa. Authors are

grateful to the following collaborators in this project. Dr Nilton Herter

(Head and Neck Surgery Department, Santa Rita Hospital, Porto Alegre,

Brazil) and Dr Roque Furian (Pathology Department of the Fundacao

Faculdade Federal de Ciencias Medicas de Porto Alegre, Brazil) for

providing frozen samples and hematoxylin–eosin-stained sections of oral

squamous cell carcinoma biopsies. Connie Organ (Dows Institute for

Dental Research, College of Dentistry, University of Iowa, Iowa City) for

help in providing cultures of oral normal keratinocytes, SCC15 and SCC25

and Xian Jin Xie (Dows Institute for Dental Research) for help in carrying

out statistical analysis.


Gasparoni et al.

Subcellular localization of beta-catenin in malignant cell lines and squamous cell carcinomas of the...

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