β-catenin nuclear expression correlates with cyclin D1 overexpression in sporadic desmoid tumours

Original paper

b-catenin nuclear expression correlates with cyclin D1overexpression in sporadic desmoid tumours

Tsuyoshi Saito1, Yoshinao Oda1, Kazuhiro Tanaka2, Shuichi Matsuda2, Sadafumi Tamiya1, Yukihide Iwamoto2

and Masazumi Tsuneyoshi1*1 Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan2 Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

*Correspondence to:Masazumi Tsuneyoshi,Department of AnatomicPathology (Second Departmentof Pathology), PathologicalSciences, Graduate School ofMedical Sciences, KyushuUniversity, Maidashi 3-1-1,Higashi-ku, Fukuoka 812-8582,Japan.E-mail: [email protected]

Received: 13 December 2000

Revised: 31 January 2001

Accepted: 3 April 2001

Published online: 8 August 2001

Abstract

The immunohistochemical expression of b-catenin, cyclin D1, Ki-67 and PCNA was Examined in

38 cases of sporadic extra-abdominal or abdominal-wall desmoid tumours without familial

adenomatous polyposis (FAP), to evaluate the hypothesis that the accumulated b-catenin within

the nuclei could affect the regulation of the cyclin D1 gene. There was a statistically significant

correlation between b-catenin accumulation and cyclin D1 overexpression ( p=0.029). Each group

with b-catenin accumulation or cyclin D1 overexpression showed a higher PCNA-LI than those

without, the difference being statistically significant (p=0.007, p=0.004, respectively).

Differential PCR was also performed to detect amplification of the cyclin D1 gene and

mutational analysis was undertaken for exon 3 of the b-catenin gene. Amplification of the cyclin

D1 gene was observed in 13 out of 22 cases (59.1%). There were nine-point mutations in 7 out of

18 cases (38.9%). The distribution of b-catenin mutation fell within a wide range, from codon 21

to codon 67. In conclusion, b-catenin nuclear expression correlated with cyclin D1 overexpression

in sporadic desmoid tumours, which could be an in vivo model system for the APC-b-catenin-Tcf

pathway. In addition, b-catenin mutations in desmoid tumours occurred at an unusually wide

range of sites within the gene. Copyright # 2001 John Wiley & Sons, Ltd.

Keywords: desmoid tumour; b-catenin; mutation; cyclin D1; Ki-67; PCNA

Introduction

It has been reported that b-catenin is a multifunctional

protein involved in the wingless/Wnt signal transduc-

tion pathway, in addition to being a cell–cell adhesion

regulator when binding to E-cadherin adhesion mole-

cules [1,2]. The binding of b-catenin to adenomatous

polyposis coli (APC) protein requires phosphorylation

of b-catenin by GSK (glycogen synthase kinase)-3b on

serine/threonine residues, all of which are encoded in

exon 3 of the gene. In colorectal cancers, mutations of

APC or b-catenin result in stabilization of b-catenin

and a significant accumulation of this protein within

the cytoplasm [3]. Furthermore, increased b-catenin

may translocate to the nuclei and could serve as a

transcriptional factor by binding to the T-cell factor/

lymphoid enhancing factor (Tcf–Lef) family [3]. More-

over, we recently demonstrated that the accumulated

b-catenin within the nuclei of tumour cells served as an

oncoprotein in synovial sarcoma, increasing its pro-

liferative activity as assessed by Ki-67, thus resulting in

a poor overall survival rate [4]. However, it was still

not clear how the accumulated b-catenin within the

nuclei could act as an oncoprotein in the APC-b-

catenin-Tcf pathway.Desmoid tumour is an infiltrative and locally

aggressive lesion characterized by florid fibroblastic

proliferation. Desmoid tumours are also known to

have a high frequency of APC and b-catenin mutations

[5–9]. Li et al. [10] demonstrated that APC truncating

mutations provide aggressive fibromatosis cells with a

proliferative advantage through b-catenin and sug-

gested that b-catenin acted to transactivate transcrip-

tion. Desmoid tumors could therefore be thought of as

providing an in vivo model system for the APC-b-

catenin-Tcf pathway.Recently, some of the targeted genes of the APC-b-

catenin-Tcf pathway have been identified [11–14]; one

of the candidates is thought to be the cyclin D1 gene

[14]. Indeed, more recently, Tetsu et al. [14] demon-

strated that abnormal levels of b-catenin may con-

tribute to neoplastic transformation by causing the

accumulation of cyclin D1 in colon carcinoma cells.

However, it remains unclear whether b-catenin accu-

mulation could affect the regulation of the cyclin D1

gene in vivo.In this study, to test the hypothesis that the

accumulated b-catenin within the nuclei could affect

the regulation of the cyclin D1 gene, we examined the

immunohistochemical expression of b-catenin, cyclin

D1, Ki-67 and PCNA in sporadic extra-abdominal or

abdominal-wall desmoid tumours without familial

adenomatous polyposis (FAP), combined with some

molecular analyses.

Journal of PathologyJ Pathol 2001; 195: 222–228.DOI: 10.1002 /path.942

Copyright # 2001 John Wiley & Sons, Ltd.

Materials and methods

Materials

Thirty-eight formalin-fixed, paraffin-embedded cases ofsporadic (28 extra-abdominal and 10 of abdominal-wall) desmoid tumours without FAP were selectedfrom among the collection of soft-tissue tumoursregistered in the Department of Anatomic Pathology,Pathological Sciences, Graduate School of MedicalSciences Kyushu University, Japan.

Immunohistochemistry

Immunohistochemistry was performed using anti-b-catenin mouse monoclonal antibody (mAb) (clone 14,Transduction Laboratories, Lexington KY; 1 : 200),anti-cyclin D1 mouse mAb (clone P2D11F11,Novocastra; 1 : 25), anti-PCNA mouse mAb (clonePC10, Dakopatts, Copenhagen, Denmark; 1 : 100) andanti-Ki-67 mouse mAb (clone MIB-1, Immunotech,Marseille, France; 1 : 100). For evaluating b-cateninstaining, sections were considered to demonstratewidespread nuclear staining if more than 75% of thetumour cells showed nuclear staining [4,15]. Forevaluating the immunohistochemistry of cyclin D1,sections were considered as demonstrating over-expression if more than 5% of the cells were positivelystained [16–18]. The MIB-1-labelling index (LI) andPCNA-LI were expressed as previously described [4].

DNA extraction

Genomic DNA was purified from formalin-fixedparaffin-embedded material as previously described[4]. Genomic DNA was also extracted, using a DNAextraction kit, Sepa Gene, from an HeLa cell linepreviously described as lacking any amplification of thecyclin D1 gene, as a standard positive control fordifferential PCR [19].

Differential PCR

The primer sequences for the cyclin D1 gene and thedopamine-D2-receptor gene, used as an internal con-trol, have been previously described [20]. The PCRprotocol included initial denaturing at 96uC for fiveminutes, followed by 40 cycles of one minute each at96uC, 50uC and 72uC, and after the final cycle ofamplification, the extension was continued for anadditional seven minutes at 72uC. To avoid any lossof assay accuracy and to determine the appropriatenumber of PCR cycles, different cycles of PCRamplification were performed using DNA from HeLacells. The cyclin D1/dopamine receptor ratios werealmost constant at 35 to 40 cycles, so all clinicalspecimens were analysed using 40 cycles. Followingamplification, the products were electrophoresedthrough 3.0% agarose gel with ethidium bromide at100 V for 50 minutes. The UV-illuminated gels werephotographed using Polaroid film. The intensities ofthe DNA products were quantified using a National

Institutes of Health (NIH) image version 1.56. Thelevel of cyclin D1 amplification was determined bycomparing the ratio of the intensities of the cyclin D1and dopamine-D2-receptor PCR products for each ofthe samples with positive HeLa cells. Samples showingmore than two-fold amplification were judged asamplified.

Polymerase chain reaction–single-strandconformation polymorphism (PCR-SSCP) andmutational analysis of b-catenin gene

A genomic PCR fragment including the entire regionof exon 3 was amplified using a previously describedpair of intronic primers at 58uC of annealing tempera-ture [21]. Human genomic DNA (CLONTECH) wasused as a positive control for each PCR and for thesubsequent reactions. SSCP was performed as pre-viously described [4]. To increase the quantity ofmutant DNA prior to sequencing, extra bands whichseemed to be aberrantly migrating were excised fromthe SSCP gel, and re-amplified for 25 cycles under thesame conditions. The samples were analysed forsequencing after the subsequent reaction.

Statistical analysis

Fisher’s exact test was performed to assess thecorrelation among the various immunohistochemicalresults. A p-value <0.05 was considered significant.

Results

Immunohistochemistry

In all cases, positive staining for b-catenin wasrecognized in the nuclei and cytoplasm of the fibro-blastic tumour cells; b-catenin-positive cells appearedto be uniformly distributed throughout the sections,although the proportion of positive cells varied(Figures 1A, 2A). Nineteen cases (50%) were judgedas showing widespread nuclear staining. There was nostatistically significant correlation between the wide-spread nuclear staining of b-catenin and the tumourlocation (abdominal wall or extra-abdominal). On theother hand, cyclin D1-positive cells also appeared to beevenly scattered throughout the section (Figures 1B,2B), and 27 cases (71.1%) showed overexpression ofcyclin D1. Among 19 cases with widespread nuclearstaining of b-catenin, 17 showed overexpression ofcyclin D1. The remaining two cases were abdominal-wall desmoid tumours. Extra-abdominal desmoidtumours had a tendency to show cyclin D1 over-expression more frequently than abdominal-wall des-moid tumours, although this was not statisticallysignificant ( p=0.08). There was a statistically sign-ificant correlation between widespread nuclear stainingof b-catenin and cyclin D1 overexpression ( p=0.029,Table 1). With the exception of a small number ofcases, Ki-67-positive tumour cells either could not bedetected, or else were very few (Figures 1C, 2C),

b-catenin in sporadic desmoid tumours 223

Copyright # 2001 John Wiley & Sons, Ltd. J Pathol 2001; 195: 222–228.

although MIB-1-LI ranged from 0 to 28.6 (mean: 3.0).

There was no correlation between MIB-1-LI and b-

catenin accumulation or cyclin D1 overexpression. On

the other hand, all cases demonstrated a broad

spectrum of immunoreactivity for PCNA (Figures 1D,

2D), ranging from 2.5 to 67.4 (mean: 31.8), and most

Figure 1. Immunohistochemical staining of b-catenin, cyclin D1, Ki-67 and PCNA in a desmoid tumour with b-catenin mutation atcodon 45. Most of the tumour cells throughout the lesion are positively stained for b-catenin (A: top left), and more than 50% of thetumour cell nuclei are positively stained for cyclin D1 (B: top right). Tumour cells positively stained for Ki-67 are scatteredthroughout the lesion (C: bottom left); in this case, the MIB-1-LI is 12.2. Most of the tumour cells throughout the lesion arepositively stained for PCNA (D: bottom right); in this case, the PCNA-LI is 67.4. Original magnifications, r57.5 (A–D)

Figure 2. Immunohistochemical staining of b-catenin, cyclin D1, Ki-67 and PCNA in a desmoid tumour without b-catenin mutation.Only a few tumour cells are positively stained for b-catenin (A: top left) and cyclin D1 (B: top right). Tumour cells positively stainedfor Ki-67 are almost undetectable (C: bottom left); in this case, the MIB-1-LI is 0.1. Only a few tumour cells are positively stained forPCNA (D: bottom right); in this case, the PCNA-LI is 2.5. Original magnifications, r57.5 (A–D)

224 T. Saito et al.


cases were stained homogeneously for PCNA. Eachgroup with widespread nuclear staining of b-catenin orcyclin D1 overexpression showed a higher PCNA-LIthan those without, and the differences were statisti-cally significant ( p=0.007, p=0.004, respectively:Tables 2 and 3).

Amplification of cyclin D1 gene

We were able to perform differential PCR successfullyin only 22 out of 38 cases, and we could detectamplification of the cyclin D1 gene in 13 of these(59.1%, Figure 3). There was no statistically significantassociation between tumour location and amplifica-tion of the cyclin D1 gene (p=0.39). Among 13 cases

with cyclin D1 gene amplification, nine showed over-expression of cyclin D1; eight of these also showedwidespread nuclear staining of b-catenin and theremaining one had b-catenin mutation. None of thefour cases with cyclin D1 gene amplification but with-out cyclin D1 overexpression showed widespreadnuclear staining of b-catenin. Moreover, among the eightcases with overexpression of cyclin D1 but withoutamplification of the cyclin D1 gene, six showed wide-spread nuclear staining of b-catenin, whereas the onecase without amplification of the cyclin D1 gene andwithout cyclin D1 overexpression did not show wide-spread nuclear staining of b-catenin (Table 4). How-ever, as a result, there was no statistically significantcorrelation between the amplification of the cyclin D1gene and cyclin D1 overexpression (p=0.360).

b-catenin gene mutation

We succeeded in obtaining PCR product in only 18 outof 38 cases. There were nine-point mutations in sevencases (38.9%, Figures 4 and 5), all of which weremissense mutations (Table 5), but no interstitial dele-tion was detected. There was no statistically significantdifference between tumour location and the incidenceof b-catenin mutation (p=0.39). All cases with b-catenin mutation showed overexpression of cyclin D1.

Table 1. Correlation between widespread nuclear stain-ing of b-catenin and cyclin D1 overexpression

Widespread nuclearstaining of b-catenin

(x) (+)

Cyclin D1 overexpression (x) 9 2 11

(+) 10 17 27

19 19 38

p=0.029

Table 2. Correlation between widespread nuclear stain-ing of b-catenin and PCNA-LI

PCNA-LI

Mean SD

Widespread nuclear staining of b-catenin (x) n=19 25.6 12.4

(+) n=19 37.9 14.1p=0.007

Table 3. Correlation between cyclin D1 overexpressionand PCNA-LI

PCNA-LI

Mean SD

Cyclin D1 overexpression (x) n=11 21.5 11.5(+) n=27 35.9 13.6

p=0.004

Figure 3. Differential PCR to detect cyclin D1 gene amplification in desmoid tumour. DNA product of cyclin D1 is upper 157bp,while DNA product of dopamine-D2-receptor is lower 128bp, respectively. Lane 1 shows differential PCR products in the controlHeLa cell line without amplification of the cyclin D1 gene. Lane 2 shows negative control. Cyclin D1 gene amplification is obvious inlanes 4, 5 and 6

Figure 4. Results of SSCP analysis of the b-catenin gene indesmoid tumours. Aberrantly migrating bands (arrows) can beobserved in lanes 2 (case 17), 3(case 19) and 7 (case 3)

Table 4. Correlation between amplification of cyclin D1gene and cyclin D1 overexpression

Amplification ofcyclin D1 gene

(x) (+)

Cyclin D1 overexpression (x) 1 4 5

(+) 8 9 17

9 13 22

p=0.360



In addition, all cases with b-catenin mutation showedamplification of the cyclin D1 gene, with the exceptionof one case which was not available for differentialPCR analysis. The distribution of b-catenin mutationin exon 3 observed in this study fell within a widerange, from codon 21 to codon 67.

Discussion

In this study, we have demonstrated for the first time a

statistically significant correlation between widespread

nuclear staining of b-catenin and cyclin D1 over-expression in desmoid tumours. However, widespread

nuclear staining of b-catenin or cyclin D1 over-expression did not affect proliferative activity as

assessed by MIB-1-LI. This finding may reflect the

essentially benign nature of desmoid tumors. On theother hand, PCNA has been known to be involved in

DNA repair when binding to cyclin D1 and p21, in

addition to DNA replication [22–24]. PCNA wasdiffusely expressed in sporadic desmoid tumours, and

interestingly, each group with widespread nuclearstaining of b-catenin or cyclin D1 overexpression

showed higher PCNA-LI than those without. These

findings may suggest that PCNA has an important rolein DNA repair and the inhibition of DNA replication

in sporadic desmoid tumours; PCNA may therefore be

expressed more strongly in cases with overexpressionof b-catenin and cyclin D1. Furthermore, the cases

with a higher MIB-1-LI and a higher local recurrencerate may have some abnormalities in the function of

PCNA.There were 10 cases with cyclin D1 overexpression

which did not demonstrate widespread nuclear stainingof b-catenin. Although we could only perform muta-

tional analysis for three of these ten cases, two hadb-catenin mutations; some of the remaining seven cases

may also have had b-catenin mutations, bearing in

mind that desmoid tumours have a rather highfrequency of such events. In addition, differential

PCR analysis was possible for five out of nine cases

without widespread nuclear staining of b-catenin andwithout cyclin D1 overexpression; four of these cases

showed amplification of the cyclin D1 gene. Further-more, all six cases with b-catenin mutation showed

overexpression of cyclin D1, together with amplifica-

tion of the cyclin D1 gene. One additional case was notavailable for differential PCR analysis. These results

strongly suggest that the cyclin D1 gene is frequently

amplified in desmoid tumours, but this alone cannotaccount for the immunohistochemically detectable

expression of cyclin D1. This further suggests that

a

b

Figure 5. Sequencing results for exon 3 of b-catenin in desmoidtumours. (A) Tumour sequences showing the substitution ATTfor ATC (arrow, left) at codon 35 and the substitution ATA forACA (arrow, centre) at codon 42 in case 17. Tumour sequenceshowing the substitution TTT for TCT (arrow, right) at codon45 in case 3. (B) Tumour sequences showing the substitutionGAA for GAG (arrow, left) at codon 65 and the substitutionAAA for GAA (arrow, right) at codon 67 in case 19

Table 5. Correlation of cyclin D1 overexpression and b-catenin mutation in sporadic desmoid tumours

Case

Age(years)/

sex Location Codon

Nucleotide

change

Amino acid

change

b-catenin

aberrant staining

Amplification

of cyclin D1 gene

Cyclin D1

overexpression

3 34/M Left thigh 45 TCT to TTT Ser to Phe >75% (+) (+)

17 31/F Abdominal wall 35 ATC to ATT Ile to Ile <75% (+) (+)

42 ACA to ATA Thr to Ile19 25/F Right upper arm 65 GAG to GAA Glu to Glu >75% (+) (+)

67 GAA to AAA Glu to Lys

24 12/F Chest wall 53 GAG to AAG Glu to Lys >75% (+) (+)32 68/M Right lower leg 21 GCT to ACT Ala to Thr <75% ND (+)

33 49/F Back 21 GCT to ACT Ala to Thr >75% (+) (+)

34 73/M Back 45 TCT to TTT Ser to Phe >75% (+) (+)

N D: No data.

226 T. Saito et al.


cyclin D1 expression is activated to some extent byb-catenin accumulation in sporadic desmoid tumours.The effect of this pathway is reinforced when combinedwith amplification of the cyclin D1 gene.

The reported values of the incidence of b-cateninmutation in sporadic desmoid tumours without FAPare quite similar [5,9]. In this study, we found b-cateninmutation in 7 out of 18 cases (38.9%), lower, thoughnot significantly, than previously reported values, [5,9]but the most striking difference concerned the muta-tional sites, which had a wider range than in previousstudies [5,8,9]. Previously, reports were confined tocodon 41 or codon 45 [5,8,9], whereas only two out ofour seven cases had a b-catenin mutation at codon 45,while the other five involved codons 21, 35, 42, 53, 65and 67. Furthermore, most of the previously reportedb-catenin point mutation sites in other human benignand malignant tumours were at codons 32 (Asp), 33(Ser), 34 (Gly), 37 (Ser), 41 (Thr) and 45 (Ser) [25–31].To our knowledge, only anaplastic thyroid carcinomascontain such a wide range of b-catenin mutations,falling between codon 17 (Asp) and codon 60 (Ser)[32], although mutations at codons 21, 35, 53, 65 and67 had not previously been reported. All of our caseswith b-catenin mutation showed nuclear expression ofb-catenin and overexpression of cyclin D1, althoughtwo did not show widespread nuclear staining ofb-catenin. However, the function of b-catenin mutationsother than those affecting serine/threonine residues ortheir neighbouring sites is still unclear, since thepossibility that APC gene mutations were involved inthese cases could not be completely ruled out, despitethe lower frequency of such mutations in sporadicdesmoid tumours [7].

In conclusion, the most striking finding in this studywas that in sporadic desmoid tumours, b-cateninnuclear expression correlated with cyclin D1 over-expression, but not with cell proliferation as assessedby MIB-1-LI. This could be an in vivo model systemfor the APC-b-catenin-Tcf pathway. In addition, b-catenin mutations in desmoid tumours occurred at anunusually wide range of sites within the gene.

Acknowledgements

This work was supported in part by a Grant-in-Aid for Cancer

Research from the Fukuoka Cancer Society and Grants-in-Aid

for General Scientific Research from the Ministry of Education,

Science, Sports and Culture (09470052, 12670167) of Japan. We

are grateful to Miss M. Okano for her excellent technical

assistance. We thank Miss Katherine Miller (Royal English

Language Centre, Fukuoka, Japan) for revising the English used

in this article.

References

1. Brabletz T, Herrmann K, Jung A, Faller G, Kirchner T.

Expression of nuclear b-catenin and c-myc is correlated with

tumor size but not with proliferative activity of colorectal

adenomas. Am J Pathol 2000; 156: 865–870.

2. Hirohashi S. Inactivation of the E-cadherin-mediated cell

adhesion system in human cancers. Am J Pathol 1998; 153:

333–339.

3. Morin PJ, Sparks AB, Korinek V, et al. Activation of b-catenin-

Tcf signaling in colon cancer by mutations in b-catenin or APC.

Science 1997; 275: 1787–1790.

4. Saito T, Oda Y, Sakamoto A, et al. Prognostic value of the

preserved expression of the E-cadherin and catenin families of

adhesion molecules and of b-catenin mutations in synovial

sarcoma. J Pathol 2000; 192: 342–350.

5. Tejpar S, Nollet F, Li C, et al. Predominance of beta-catenin

mutations and beta-catenin dysregulation in sporadic aggressive

fibromatosis (desmoid tumor). Oncogene 1999; 18: 6615–6620.

6. Alman BA, Li C, Pajerski ME, Diaz-Cano S, Wolfe HJ.

Increased b-catenin protein and somatic APC mutations in

sporadic aggressive fibromatoses (desmoid tumor). Am J Pathol

1997; 151: 329–334.

7. Giarola M, Wells D, Mondini P, et al. Mutations of adenoma-

tous polyposis coli (APC) gene are uncommon in sporadic

desmoid tumors. Br J Cancer 1998; 78(5): 582–587.

8. Shitoh K, Konishi F, Iijima T, et al. A novel case of a sporadic

desmoid tumor with mutation of the beta catenin gene. J Clin

Pathol 1999; 52(9): 695–696.

9. Miyoshi K, Iwao K, Nawa G, Yoshikawa H, Ochi T, Nakamura

Y. Frequent mutation in the beta-catenin gene in desmoid

tumors from patients without familial adenomatous polyposis.

Oncol Res 1998; 10(11–12): 591–594.

10. Li C, Bapat B, Alman BA. Adenomatous polyposis coli gene

mutation alters proliferation through its beta-catenin-regulatory

function in aggressive fibromatosis (desmoid tumor). Am J Pathol

1998; 153: 709–714.

11. He TC, Sparks AB, Rago C, et al. Identification of c-myc as a

target of the APC pathway. Science 1998; 281: 1509–1512.

12. Brabletz T, Jung A, Dag S, Hlubek F, Kirchner T. b-catenin

regulates the expression of the matrix metalloproteinase-7 in

human colorectal cancer. Am J Pathol 1999; 155: 1033–1038.

13. Crawford HC, Fingleton BM, Rudolph-Owen LA, et al. The

metalloproteinase matrilysin is a target of b-catenin trans-

activation in intestinal tumor. Oncogene 1999; 18: 2883–2891.

14. Tetsu O, McCormick F. Beta-catenin regulates expression of

cyclin D1 in colon carcinoma cells. Nature 1999; 398: 422–426.

15. Hugh TJ, Dillon SA, Taylor BA, Pignatelli M, Poston GJ,

Kinsella AR. Cadherin–catenin expression in primary colorectal

cancer. Br J Cancer 1999; 80: 1046–1051.

16. Ishikawa T, Furihata M, Ohtsuki Y, Murakami H, Inoue A,

Ogoshi S. Cyclin D1 overexpression related to retinoblastoma

protein expression as a prognostic marker in human oesophageal

squamous cell carcinoma. Br J Cancer 1998; 77(1): 92–97.

17. Keum JS, Kong G, Yang SC, et al. Cyclin D1 overexpression is

an indicator of poor prognosis in resectable non-small cell lung

cancer. Br J Cancer 1999; 81(1): 127–132.

18. Kim SH, Lewis JJ, Brennan MF, Woodruff JM, Dudas M,

Cordon-Cardo C. Overexpression of cyclin D1 is associated with

poor prognosis in extremity soft-tissue sarcomas. Clin Cancer

Res 1998; 4(10): 2377–2382.

19. Gramlich TL, Fritsch CR, Maurer D, Eberle M, Gansler TS.

Differential polymerase chain reaction assay of cyclin D1 gene

amplification in esophageal carcinoma. Diagn Mol Pathol 1994;

3(4): 255–259.

20. Kyomoto R, Kumazawa H, Toda Y, et al. Cyclin-D1-gene

amplification is a more potent prognostic factor than its protein

over-expression in human head-and-neck squamous-cell carci-

noma. Int J Cancer 1997; 74: 576–581.

21. Iwao K, Nakamori S, Kameyama M, et al. Activation of the

b-catenin gene by interstitial deletions involving exon 3 in

primary colorectal carcinomas without adenomatous polyposis

coli mutations. Cancer Res 1998; 58: 1021–1026.

22. Xiong Y, Zhang H, Beach D. D type cyclins associate with

multiple protein kinases and the DNA replication and repair

factor PCNA. Cell 1992; 71: 505–514.

23. Toschi L, Bravo R. Changes in cyclin/proliferating cell nuclear

antigen distribution during DNA repair synthesis. J Cell Biol

1988; 107: 1623–1628.



24. Waga S, Hannon GJ, Beach D, Stillman B. The p21 inhibitor of

cyclin-dependent kinases control DNA replication by interaction

with PCNA. Nature 1994; 369: 574–578.

25. Chan EF, Gat U, McNiff JM, Fuchs E. A common human skin

tumour is caused by activating mutation in b-catenin. Nat Genet

1999; 21: 410–413.

26. Palacios J, Gamallo C. Mutation in the b-catenin gene

(CTNNB1) in endometrioid ovarian carcinomas. Cancer Res

1998; 58: 1344–1347.

27. Fukuchi T, Sakamoto M, Tsuda H, Maruyama K, Nozawa S,

Hirohashi S. b-catenin mutation in carcinoma of the uterine

endometrium. Cancer Res 1998; 58: 3526–3528.

28. Voeller HJ, Truica CI, Gelmann EP. b-catenin mutations in

human prostate cancer. Cancer Res 1998; 58: 2520–2523.

29. Miyoshi Y, Iwao K, Nagasawa Y, et al. Activation of the

b-catenin gene in primary hepatocellular carcinomas by somatic

alterations involving exon 3. Cancer Res 1998; 58: 2524–2527.

30. Coste AL, Romagnolo B, Biluart P, et al. Somatic mutations of

b-catenin gene are frequent in mouse and human hepatocellular

carcinomas. Proc Natl Acad Sci U S A 1998; 95: 8847–8851.

31. Koch A, Denkhaus D, Albrecht S, Leuschner I, von Schweinitz

D, Pietsch T. Childhood hepatoblastomas frequently carry a

mutated degradation targeting box of the b-catenin gene. Cancer

Res 1999; 59: 269–273.

32. Garcia-Rostan G, Tallini G, Herrero A, D’Aquila TG, Carcangiu

ML, Rimm DL. Frequent mutation and nuclear localization of

b-catenin in anaplastic thyroid carcinoma. Cancer Res 1999; 59:

1811–1815.

228 T. Saito et al.


Documents

β-catenin nuclear expression correlates with cyclin D1 overexpression in sporadic desmoid tumours