Immunohistological analysis of P53 expression in human skin tumors

Journal of Dermatological Science, 4 (1992) 69-15 0 1992 Elsevier Science Publishers B.V. All rights reserved.

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69

00153

Immunohistological

analysis of P53 expression in human skin tumors

Yoshio Urano, Hajimu Oura, Akihiko Sakaki, Hiroaki Nagae, Kazuya Matsumoto, Kosaku Fukuhara, Tetsuo Nagae, Seiji Arase, Yoshiro Ninomiya, Hideki Nakanishi, Fumio Shigemi and Katsuyuki Takeda Department of Dermatology, School of Medicine, The University of Tokushima, Tokushima, Japan (Received

25 November

Key words:

1991; accepted

p53 expression:

Skin tumor;

20 April

1992)

Immunohistology

Abstract The ~53 expression ies, PAb421

the ~53 expression noma,

in various

and PAb1801.

was observed

one proliferating

cancer.

None

skin tumors

was immunohistologically

The p53 expression

in 10 of 26 squamous

trichilemmal

off four cases

was not detected

cyst, one malignant

of Bowen’s

disease

(SCC

evaluated in the normal

cell carcinomas proliferating

(SCCs)

trichilemmal

in situ) showed

nuclear

using

two mouse

epidermal

monoclonal

cells. Nuclear

from

24 patients,

tumor

and in one metastatic

staining.

anti-p53

staining

in one undifferentiated

In the SCCs,

antibod-

suggested

carcinoma

five of 20 primary

that carci-

of breast lesions,

three of four recurrent lesions and both of two metastatic lesions had positive nuclei. There was one case of SCC in which a primary lesion was negative but a recurrent lesion was positive. Thus, p53 expression was more frequently observed in SCCs at more clinically tected

advanced

stages.

This may suggest

in any of the following

cases:

that ~53 has some relevance

two cases

of seborrheic

keratosis,

epitheliomas, two mammary Paget’s disease, three genital Paget’s six lymphomas, two leukemia cutis and two angiosarcomas.

introduction

The ~53 protein, which is a 53 000 Da nuclear protein, was first identified as a host protein bound to SV40 large T antigen [ 1,2] and also as a transformation-related antigen [ 31. It is expressed in very low amounts in non-transformed cells but in relatively large amounts in various types of malignantly transformed cells [4]. BeCorresporldence to: Dr Yoshio

Urano,

tology, School of Medicine, Tokushima 770, Japan.

The

Department

University

of Derma-

of Tokushima,

to progression

one eccrine

disease,

of SCC.

poroma,

one sebaceous

Nuclear

staining

one keratoacanthoma,

carcinoma,

four malignant

was not de11 basal

cell

melanomas,

cause of its ability to cooperate with the mutated rus oncogene in transforming primary cells, p53 was initially defined as a member of the dominant nuclear oncogene family [5-71. Recent studies have demonstrated that this cooperation between p53 and rots in transformation requires mutant forms of the ~53 gene [8]. Further, it has been more recently shown that the wild-type ~53 inhibits the ability of adenovirus ElA plus MS, or myc plus YQS,or mutant p53 plus rus, to transform primary cells in vitro [9,10]. These suggest that the normal ~53 acts as a suppressor of transformation and that loss of the ~53 function is ad-

70

vantageous for transformation. This idea is strongly supported by the findings that the p53 gene is often deleted in various types of human tumors and that the remaining (non-deleted) p53 alleles are mutated [ 1 l-131. It has been shown that mutant p53 proteins are much more metabolically stable than the normal protein [ 14,151. The increased expression of p53 at the protein level in transformed cells is largely due to the stabilization of the proteins [8,14,15]. This increased expression has made it possible immunohistologically to detect p53 proteins in human primary tumors [ 16,171. A previous study has shown that an immunohistological method provides strong evidence of mutation of the p53 gene [ 161. Although a number of studies on p53 have been done for various types of human tumors [ ll-13,16-221, few efforts have been made on skin tumors. In this paper we immunohistologically examined p53 expression at the protein level in human skin tumors using two kinds of mouse monoclonal anti-p53 antibodies, PAb42 1 and PAb 180 1, which recognize different epitopes of the p53 protein [23,24]. Materials and Methods Antibodies PAb421 and PAb1801 are mouse monoclonal anti-p53 antibodies. PAb421 reacts with an epitope located at the carboxy end of the p53 protein of a broad range of mammalian species including humans [ 23 1. PAb 180 1 preferentially recognizes an epitope near the amino end of the human p53 protein [24]. These antibodies were purchased from Oncogene Science, Inc. (Mineola, NY). Cells and specimens A SV40-transformed human lung fibroblast cell line, WI-38 VA13 sub2 RA, was supplied by Japanese Cancer Research Resources Bank. The cells were grown in Eagle’s minimal essential medium containing 12% fetal calf serum. Tumor specimens obtained from patients were quickly frozen in liquid nitrogen and stored at -70 “C

until use. These included specimens from: seborrheic keratosis, eccrine poroma, keratoacanthoma, basal cell epithelioma, Bowen’s disease, squamous cell carcinoma (SCC), proliferating trichilemmal cyst, malignant proliferating trichilemma1 tumor, malignant melanoma, mammary and genital Paget’s disease, sebaceous carcinoma, undifferentiated carcinoma, metastatic carcinoma, lymphoma, leukemia cutis and angiosarcoma. Four normal skin specimens were obtained from four patients undergoing plastic surgery. Immunostaining Cultured cells on a Lab-Tek chamber glass slide (Nunc, Naperville, IL) and frozen sections (6-urn thick) were fixed with acetone for 20 min at -20 ’ C and incubated for 60 min at room temperature either with PAb421 (1 pg/ml) or with PAb180 1 (5 ug/ml). They were then incubated with an affinity-purified, biotinylated sheep antibody against mouse IgG (Organon Teknika, West Chester, PA) for 30 min at a concentration of 5 ug/ml. Avidin-biotin-peroxidase complexes preformed using a VECTASTAIN ABC kit (Vector, ‘Burlingame, CA) were applied for 30 min to all the sections except melanoma. The bound peroxidase complexes were visualized by incubation with 0.04% 3,3’-diaminobenzidine tetrahydrochloride (DAB) (Nacalai Tesque, Kyoto, Japan) and O.OlSO,/,Hz02 in 10 mM imidazole (Nacalai Tesque) and 50 mM Tris-HCI, pH 7.4 [25,26]. Since the brown color of DAB-precipitate is similar to the tint of melanin pigment, indirect immunofluorescence using fluorescein-conjugated rabbit anti-mouse immunoglobulins (DAKOPATTS A/S, Glostrup, Denmark) was performed for melanoma sections to detect the p53 protein. Results Initially we examined immunohistologically the expression of p53 in the normal epidermis using PAb421 and two monoclonal antibodies, PAb180 1. PAb42 1 strongly stained the cytoplasm of the basal cell (Fig. la), while PAbl801 reacted

71

Fig. 1. Immunostaining

of the normal

monoclonal antibodies against PAb1801 stains the cytoplasms (d), react

epidermis

and

a SV40-transformed

cell line, WI-38

VA13

sub2

RA,

using

two mouse

~53, PAb421 and PAb1801. PAb421 reacts with the cytoplasm of the epidermal basal cell (a). of the epidermal basal and spinous cells (b). Both of the two antibodies, PAb421 (c) and PAb1801

with nuclei of the SV40-transformed

cells. Normal

mouse

IgG is used as a control

(e). (Magnification:

a,b

x

100; c,d,e

x 132).

marginally with the cytoplasm of the basal and spinous cells (Fig. lb). Thus, the staining mode was different between the two antibodies. Neither of these antibodies revealed a 53 000 Da band by a Western blot analysis of normal skin extracts (data not shown). Both antibodies reacted with nuclei of SV40-transformed human lung fibroblasts, WI-38 VA13 sub2 RA, (Fig. lc,d,e) and Western blot analysis, using PAbl80 1, revealed a 53 000 Da band in an extract of the cells (data not shown). Since p53 is a nuclear protein [27], these results suggest that the cytoplasmic staining of the normal epidermal cells by PAb42 1 and PAb 180 1 was not due to the presence of ~53 in the cytoplasm but due to cross-reactions with cytoplasmic proteins. It has been previously reported that

these two antibodies cross-react with cytoplasmic proteins in the epithelial cells of the human mammary gland [ 171. In this study, therefore, we regarded nuclear staining as a p53-positive reaction. Next, we examined the ~53 expression in various skin tumors using the two antibodies. Positive nuclear staining was observed in squamous cell carcinoma (SCC), undifferentiated carcinoma, proliferating trichilemmal cyst, malignant proliferating trichilemmal tumor and metastatic carcinoma of breast cancer (Table I), Nuclear positivity varied in tumor cells among positive cases from a majority to a few. The staining intensity of the nuclei also varied from faint to strong (Fig. 2).

72 TABLE

I

P53-positive

tumors Number

Squamous cell carcinoma Undifferentiated carcinoma Proliferating

proliferating

Metastatic

carcinoma

Number

24 (a metastatic cyst

trichilemmal

Malignant

of case examined

trichilemmal (breast

positive

with:

PAb 421

PAb

9

6

1801

lesion)

tumor

cancer)

C Fig. 2. Nuclear

staining

of squamous

PAb421 (a,c) and PAbl801 (cd). The staining intensity

cell carcinoma

(grade-l)

(a,b)

and malignant

proliferating

trichilemmal

tumor

(cd)

using

(b,d). Nuclear staining is observed in a majority of the cells (a,b) and in only small part of the cells varies from faint (c) to strong (a,b,d). Nuclei are counterstained with hematoxylin and arrows indicate positive

nuclei

The undifferentiated carcinoma and malignant proliferating trichilemmal tumor were stained with both PAb42 1 and PAb 180 1, whereas the proliferating trichilemmal cyst and metastatic breast cancer reacted only with PAb 180 1. Only one case

(d). (Magnification:

x

100).

was available in each of these four types of tumor (Table I). We examined 26 specimens of SCC from 24 patients (Table II); these included 20 primary lesions, four locally recurrent lesions and two

13 TABLE

II

Nuclear

reactivity

TABLE of SCC with anti-P53 Number

antibodies

positive/number

examined

III

P53negative (%)

tumors

Tumors

Number

of cases

examined PAb 421

PAb

1801 Benign tumors

Primary

lesions

5/20 (25)

3/19 (16)

Grade”

1

Eccrine poroma Seborrheic keratosis

2

Keratoacanthoma

1

1

2/11 (18)

l/l1

(9)

2

3/S

217

(29)

3

O/l

O/l

Semimalignant

4

O/O

O/O

Basal

314 (75)

114 (25)

Malignant tumors Bowen’s disease

2/2 (100)

212 (100)

Mammary Paget’s disease Genital Paget’s disease

6/25 (24)

Sebaceous Metastatic

Recurrent Metastatic

lesions lesions

lo/26

Total lesions a Grading

was

Schaumburg-Lever

(38)

(38)

performed

according

to

Lever

and

[ 351.

11

4

carcinoma carcinomas

1 2 origin

1

melanoma

4

Unknown

2

Angiosarcoma Lymphomas

6

Mycosis

fungoides

Adult T cell lymphoma/leukemia T cell lymphoma B cell lymphoma Leukemia Chronic Acute

2 3 1 4

Bileduct Cervix Malignant

metastatic lesions. Of the 20 primary lesions, five showed positive nuclear staining with PAb421, and of these five three were also positive with PAb 180 1, while one was negative with it and the remaining one was not examined. None of the 15 cases negative with PAb421 was stained by PAbl801. Of the 20 primary lesions, 11 cases were classified into grade-l, eight into grade-2 and one into grade-3; no grade-4 carcinoma was found in the cases examined. Two of the 11 grade- 1 and three of the eight grade-2 carcinomas were p53-positive. The grade-3 carcinoma did not show nuclear staining. Of the four recurrent lesions, three showed nuclear positivity. Primary lesions were available from two of these four patients with a recurrent lesion; in one of the two, both primary and recurrent lesions were p53positive. In the other case, the primary lesion was p53-negative, while the recurrent lesion was p53positive. Both of the metastatic lesions showed positive nuclear staining. Primary lesions of the two metastatic cases were not available since they had been excised in other hospitals. Nuclear staining was not observed with either PAb42 1 or PA\, 1801 in any case of seborrheic keratosis, eccrine poroma, keratoacanthoma,

tumor

cell epithelioma

cutis B cell leukemia monocytic

leukemia

2 1 2 1 2 1 1

basal cell epithelioma, Bowen’s disease, mammary and genital Paget’s disease, malignant melanoma, lymphoma, leukemia cutis, angiosarcoma, sebaceous carcinoma or metastatic lesions from cervical carcinoma of the uterus and from the bile duct carcinoma (Table III). Discussion

Our immunohistological studies using two monoclonal antibodies against p53 revealed that ~53 was expressed rarely in the normal epidermal cells. Since the half-life of the normal p53 protein is very short and many kinds of normal cells contain much less ~53 than transformed cells [4,28,29], the protein may also not be detectable

in the normal epidermis. Increased expression was observed in a substantial fraction of SCCs of the skin and in the examined cases of undifferentiated carcinoma, proliferating trichilemmal cyst, malignant proliferating trichilemmal tumor and metastatic carcinoma of breast cancer. Some of the p53-positive tumors reacted with one of these antibodies but not with the other. Similar results in breast cancer, using the same antibodies, have been reported [ 171. Since the immunohistologically detected ~53 protein is most likely to be a mutant form [ 161 and these two antibodies recognize different epitopes [23,24], mutation of the ~53 gene in these tumors may be the cause of the discrepancy between the reactivities of PAb421 and PAb1801. It also may be possible that, since the two antibodies preferentially react with different electrophoreic forms of the ~53 protein [30], the presence of different forms of this protein in p53-positive tumors causes this discrepancy. ~53 expression was not observed in any of the four cases of Bowen’s disease (SCC in situ). However, it was found in five of the 20 primary SCCs, in three of four recurrent tumors and in both the metastatic tumors of SCC. Additionally, we found one patient with a p53-negative primary lesion and a p53-positive recurrent lesion. Thus, the increased expression of the ~53 protein was detected more frequently in the SCCs at more clinically advanced stages. This may suggest that ~53 proteins, which are likely to be mutant forms [ 161, have some relevance to progression of SCC. Since the two antibodies used here crossreacted with cytoplasmic proteins in the normal epidermis, we did not regard cytoplasmic staining without nuclear staining as a p53-positive reaction. Cytoplasmic staining was observed in most cases of seborrheic keratosis, SCC, Bowen’s disease and basal cell epithelioma, whereas it was not evident in lymphoma, leukemia cutis, malignant melanoma, mammary and genital Paget’s disease or angiosarcoma (data not shown). We cannot exclude the possibility that some, if not all, of the cytoplasmic-positive and nuclear-negative cells may be p53-positive. This is because mutant

~53 proteins are sometimes found in the cytoplasm as well [ 3 11. We found the increased expression of ~53 in two kinds of tumor showing trichilemmal keratinization, proliferating trichilemmal cyst and malignant proliferating trichilemmal tumor, although only one case of each tumor was examined. It will be interesting to see whether trichilemmal cyst shows increased ~53 expression, since malignant proliferating trichilemmal tumor and proliferating trichilemmal cyst have been known to develop from trichilemmal cyst [ 32-341. In this experiment, we examined slices from only a small part of each tumor because the tumor was too large to test the whole. Therefore, this method leaves some risks that p53-positive cells may exist in an unexamined part of a p53-negative tumor. Acknowledgements

We thank Japanese Cancer Research Resources Bank for supply of a cell line of WI-38 VA13 sub2 RA. We also thank T. Fujie for technical assistance.

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