Journal of Dermatological

Science, 2 (1991) 18-23

18

Elsevier

DESC 00056

Expression of glutathione S-transferase-z Katsumi ‘Department

Hanada ‘, Hiroyasu of Dermatology

Ishikawa ‘, Katsuto Kiyomi S at0 2

in malignant skin tumors Tamai ‘, Isao Hashimoto

*

and

and 2Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki, Japan

(Received

Key words: Glutathione

19 February

1990; accepted

S-transferase-x;

Malignant

16 July 1990)

skin tumor; Immunohistochemistry

Abstract

GST-rr has been known to be markedly increased in human (pre) neoplasms of several organs. In this paper, the significance of immunohistochemical detection of GST-7t in human malignant tumors of the skin was studied. In specimens from 40 patients with various skin cancers, malignant melamoma, Paget’s disease and undifferentiated squamous cell carcinoma showed strong reactivity in GST-n staining. The reactions were negative or weak in Bowen’s disease, basal cell epithelioma and solar keratosis. In normal melanocytes, eccrine, apocrine, and breast gland cells stained positively but not in keratinocytes, sebaceus gland and tibroblasts. While immunohistochemical detection of GST-?r in the skin was not specific for malignancies, it contributed to aid the distinction of squamous cell carcinoma from other keratinocytic tumors. GST-A might provide potentially useful information on chemosensitivity of skin cancer, and might serve as a biomarker of disease activity.

Introduction

Glutathione S-transferase plays an important role in detoxification, either by conjugating glutathione with electrophilic substances, or by direct binding with carcinogenic dyes [ 1,2]. Recent reports revealed that cells to resistant anticarcinogenic agents increased levels of GST activity [ 3,4,5]. A placental form of GST has been isolated from both rat (GST-p) and human placentas (GST-rc), which shows considerable Correspondence to: K. Hanada, Department of Dermatology, Hirosaki University School of Medicine, Hirosaki, Japan. Abbreviations: GST-R: glutathione S-transferase-n; CEA: carcinoembryonic antigen; DAB: 3-amino-9-etylcarbazole; AEC: 3-amino-9-etylcarbazole; ABC: avidin-biotin-complex.

0923-181 l/91/$03.50

0 1991 Elsevier Science Publishers

interspecific homology of amino acid sequence [6,7]. GST-p was significantly increased in rat livers containing neoplastic or preneoplastic lesions induced by chemical carcinogens [ 6,8]. In those studies, GST-p was detectable before an increase in r-glutamyl transpeptidase (r-GPT) activity was apparent [9]. Thus, GST-p is estimated to be one of the earliest markers for ‘initiated cells ’ in involved liver tissue. In human hepatic tumors, a marked increase of GST-rc was recognized [lo], and in subsequent studies, positive immunohistochemical detection of GST-rc was demonstrated in a number of human carcinomas including gastric [ 111, colonic [ 121, and uterine cervix carcinoma [ 131 by immunostaining. Quantitative analysis of GST-x was also done

B.V. (Biomedical

Division)

19

[14,15,16]. High levels of total GST or GST-71 RNA contents, or GST-n enzymic activity were recognized in other human tumors such as lung, ovary, head and neck, and breast cancers. The present paper concerns the expression of GST-rc in human normal skin and cutaneous tumors, and the significance of GST-x as a marker for carcinogenesis. Materials and Methods Materials The materials in this study consisted of 61 skin biopsy specimens obtained from 21 controls and from 40 patients with skin cancer. Control speciTABLE

mens included six specimens of normal skin obtained from trunk (1) sole (2), and axilla (3), 6 specimens of nevocellular nevus (4) of compound type and 2 of intradermal type, 7 specimens of solar keratosis and 2 specimens of keratoacanthoma. The classification of each group of skin cancers shown in Table I was made by clinical and histological examinations. The diagnoses of both Paget’s disease and malignant melanoma were made by following examinations of CEA, SlOO protein and histochemistry. The group labelled Bowen’s disease included to socalled Pagetoid Bowen’s disease [ 171, in which CEA staining was negative. Basal squamous cell epithelioma (intermediary type) is contained in

I

GST-x staining in skin cancers Strongly positive

Weakly positive

Positive total (%)

(%)

Bowen’s disease (8)”

0

2

2 (25)b

6 (75)b

Paget’s disease (8) mammary genital perianal axillary

8 4 2 1 1

0

8 (100)

0

Basal cell epithelioma (10) solid type keratotic type superficial type adenoid type basal sqamous cell epithelioma

0

4

4 (40)

6 (60)

1 1

5 (100)

Squamous cell carcinoma grade 1 grade 2-3 Malignant ALM’ LMMd NM’ SSMf

melanoma (7)

Breast carcinoma, metastatic to the skin (2) breast cancer

(5)

Negative

4 1 1 1 4 4 7 3 2 1 1 2

7 (100)

0

2 (100)

0

a Number in parenthesis represents the total number of samples in each disease; b Number in parenthesis represents the percentage of the total number of samples; ’ ALM; acral lentigenous melanoma; d LMM; lentigo maligna melanoma; e NM; nodular melanoma; f SSM; superficial spreading melanoma.

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the group of basal cell epitheliomas. The biopsy specimens were fared in 10% formaldehyde and embedded in paraffin by routine processing.

Immunostaining GST-71 was purified from the human terminal placenta by S-hexylglutathione column chromatography followed by chromatofocusing, and antiGST-71 antibody was prepared in a rabbit, using methods described previously [lo]. Specificity of the antibody against GST-71 was confirmed by the technique of Ouchterlony double-immunodiffusion. The immunoperoxidase staining was performed using the Biotin-StreptAvidin, an amplified system of ABC method (STBAVIGENO, BioGenex Laboratories, USA). Briefly, primary antibody (purified rabbit anti GST-rc anti sera, 50 pg/ml) was added to each specimen and incubated at room temperature for 60 min. After washing by PBS buffer, they were incubated with the second antibody, biotinylated goat anti-rabbit IgG for 60 min. Finally, samples were treated with DAB solution to visualize the localization of GST-n, and counterstained with hematoxylin. In malignant melanoma and nevocellular nevus, AEC was used as a substrate chromogen because the brownish color of DAB could be confused with melanin pigment. A strongly dark brown (or strongly red) reaction was regarded as ‘strongly positive’ for GST-rc staining, while a light brown (or light red) reaction was regarded as ‘weakly positive’. As the staining intensity was affected by the staining techniques and other conditions, the total response in each sample was evaluated together with negative and positive controls. For negative control, the GST-71 immune serum was replaced with PBS buffer. As positive control, sections from a colonic cancer which was known to show a strongly positive reaction were used in every experiment.

demonstrated a slightly positive reaction. Epidermal melanocytes, dermal nevus cells, eccrine gland cells, apocrine gland cells, mammary gland cells, and Schwann’s cells were strongly stained. In all of these cells, immunoreactivity was seen predominantly in the cytoplasm. The ductal cells of secretory glands were positive only in the inner row. Nevus cells in the deep dermis tended to be low in the intensity of the reaction. Immunostaining of solar keratosis and keratoacanthoma revealed all negative response. The results of GST-7r staining in the skin cancers were summarized in Table I. Strongly positive staining was obtained in 52.5 y0 (21 cases) of the skin cancers. All of the Paget’s disease cases [8], all of the malignant melanoma cases [7], all of the metastatic carcinoma cases [2], and 4 of the five squamous cell carcinoma cases stained strongly. Paget’s cells were easily distinguishable from the adjacent intact epidermal cells, but showed no differences in staining intensity and in pattern among the 4 types of Paget’s disease. In squamous cell carcinoma, tumor lesions showed a diffusely positive pattern, except for the center of horn pearls with negative response. Scattered lesions in the dermis (grade 3-4 in Broders’ system) were well-defined (Fig. 1). Melanoma cells showed a strong positive staining, regardless of the differences of histological classifications, cell types and localizations. Their reaction was more

Results In the control skin, epidermal cells, sebaceous gland cells and tibroblasts showed a negative reaction. The upper epidermis in some specimens

Fig. 1. Squamous cell carcinioma stained with the antiGST-A antibody. Scattered tumor lesions in the dermis showed a well-defined positive reaction (arrows).

Fig. 2. Malignant melanoma (superficial spreading melanoma). GST-K is strongly positive in the cytoplasm of both epidermal and dermal melanoma cells. Liberated individual tumor cells are easily detectable (arrows).

marked than that of nevus cells. Individual melanoma cells scattered in the epidermis or invading the dermis were easily detectable (Fig. 2). Two of 8 cases of Bowen’s diseases and 4 of 10 cases of basal cell epithelioma displayed a weak reaction for GST-7~. With regard to some clumped cells in Bowen’s disease, a strong reaction was seen. Negative staining was observed in 30% of all the cases of skin cancers, especially in Bowen’s disease (75%) and basal cell epithelioma (60%). Discussion GST is a cytoplasmic enzyme. Immunohistochemical localization of GST-n in normal and tumor cells with positive reactivity in the skin was demonstrated in the cytoplasm as shown in other organs [ 11,12,13]. Nuclear staining was not clear in normal skin nor in lesions, but its localization has been mentioned in other studies [ 18,191, implying a possible role for GST-n: in the detoxification of peroxidized DNA. GST represents at least three distinct gene families [ 2,7]. These have been conventionally divided into 3 groups: ‘acidic’, ‘neutral’ and ‘basic’ transferases. GST-rc is one of the basic forms. Of the isozymes of GST, GST-x has been shown to have a new role as a marker in certain cancers, suggesting the diagnostic usefulness of immunohistochemical detection [ 10,12,13,20]. In the present study, strong cyto-

plasmic staining was observed in about half of 40 specimens of skin cancers, mainly of Paget’s disease, squamous cell carcinoma, malignant melanoma and metastatic cancer of the skin. Both malignant melanoma and Paget’s cells were easily distinguishable from the surrounding intact epidermal cells, and the extended borders of the tumor growth were also easily detectable. Moreover, GST-71 staining could be helpful to distinguish Pagetoid Bowen disease, a variant type of Bowen disease [ 171, from Paget’s disease with different reactivities, because, in spite of the strong reaction of GST-7~ staining in Paget’s cells, characteristic tumor cells in Pagetoid Bowen disease having a close resemblance to the Paget’s cell were all negative. Developmentally, positively-staining cells in control skin tissues including nevocellular nevus, sweat glands or mammary glands, correspond to those in malignant tumors such as malignant melanoma, extra mammary Paget’s disease, mammary Paget’s or metastatic lesion of breast ‘cancer, respectively. Thus, similar properties of staining for GST-7~ in normal tissues are maintained in the corresponding skin tumors. ‘Therefore, for these tumors, it is difficult to estimate the malignancy by the staining of GST-7~. For squamous cell carcinoma, the usefulness of GST-7~ as a marker of malignancy is clear by the reason that the normal epidermis and benign epidermal tumor, even solar keratosis with a tendency towards squamous cell carcinoma in situ, showed a negative reaction. When classilied squamous cell carcinoma into several differentiated types, the intensity of GST-rc reaction was found to depend upon the degree of undifferentiation. Preneoplasic or early lesions of carcinoma in other organs including stomach [ 111, colon [ 121 and uterine cervix [ 131, exhibited positive staining and/or significant GST-x content, whereas cutaneous lesion of solar keratosis did not express a positive reaction. Negative staining for GST-7~ in solar keratosis neither contributes to early diagnosis of malignancy nor discriminates from Bowen’s disease because of negative responses.

22

The true meaning of the expression of GST-n in the skin remains obscure. Generally, the organs in which cancer-related GST-rc expression has been found, such as the skin, digestive tract, lung and uterus, are directly exposed to the exterior environment of the body. From the viewpoint of characteristic distribution, GST-x may play a significant role in detoxification of epoxides, indicating a possible role in detoxification of anticancer drugs. In fact, previous studies revealed a remarkable increase of GST-rc in certain cultured cells with multiple drug resistance [20], such as malignant melanoma cells [3], squamous cacinoma cells of the lung [ 41 and leukemia cells [ 51. GST-n positive tumors of the skin may be resistant to chemotherapy [20]. Quantitative analyses of GST-rc should be done in further studies not only in skin tissues of skin cancer but also in serum, especially in patients bearing metastatic lesions of the skin cancers, since it is known that the intracellular concentration of GST-rc was significantly higher in human malignant melanoma cell lines and solid materials than in non malignant nevus samples [3], and serum GST-71 has been shown to be a sensitive and reliable marker for internal malignancies [ 141.

Acknowledgment We thank Dr. Edward V. Maytin (Harvard Medical School) for valuable discussion.

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References Jakoby WB, Habig WH: Enzymic basis of detoxication. Edited by WB Jacoby, New York, Academic Press, 1980, vol. 2, pp 63-94. Mannervik B: The isozymes of glutathione transferase. Adv Enzymol Relat Areas Mol Biol 57: 357-417, 1985. Mannervik B, Castro VM, Danielson UH, Tahir MK, Hansson, J, Ringdorg U: Expression of class Pi glutathione transferase in human malignant melanoma. Carcinogenesis 8: 1929-1933, 1987. Teicher B, Holden S, Kelley MJ, Shea TC, Cucchi CA, Rosowsky A, Henner WD, Frei E: Characterization of human squamous cell carcinoma cell line resistant to

14

15

16

cis-diamminedichloro-platinum (II). Cancer Res 47: 388-393, 1987. Deffie AM, Alam T, Seneviratine C, Beenken SW, Batra JK, Shea TC, Henner WD, Goldenberg GJ: Multifactorial resistance to adriamycin. Relationship of DNA repair, glutathione transferase activity, drug efllux, and p-glycoprotein in cloned cell lines of adriamycin-sensitive and resistant P388 leukemia. Cancer Res 48: 3595-3605, 1988. Sato K, Kitahara A, Satoh K, Ishikawa T, Tatematsu M, Ito N: The placental form of glutathione S-transferase as a new marker protein for preneoplasia in rat chemical hepatocarcinogenesis. Gann 75: 199-202, 1984. Hydes DJ, Mantle TJ: Anomalous elecrtophoretic behaviour of the glutathione S-transferase Ya and Yk subunits isolated from man and rodents. Biochem 237: 731-740, 1986. Satoh K, Kitahara A, Soma Y: Purification, induction and distribution of placental glutathione transferase. Proc Nat1 Acad Sci USA 82: 3964-3968, 1985. Tatematsu M, Mera Y, Ito N, Satoh K, Sato K: Relative merits immunohistochemical demonstration of placental, A, B and C forms of glutathione S-transferase and histochemical demonstration of r-glutamyl transferase as markers of altered foci during liver carcinogenesis in rats. Carcinogenesis 6: 1621-1626, 1985. Soma Y, Satoh K, Sato K: Purification and subunitsubstructural and immunological characterization of five glutathione S-transferase in human liver, and the acidic form as a hepatic tumor marker. Biochem Biophys Acta 869: 247-288, 1986. Tsutsumi M, Sugisaki T, Makino T, Miyagi N, Nakatani K, Shiratori T, Takahashi S, Konishi Y: Oncofetal expression of glutathione S-transferase placental form in human stomach carcinoma. Gann 78: 631-633, 1987. Kodate C, Fukushi A, Narita T, Kudo H, Soma H: Human placental form of glutathione S-transferase (GST-x) as a new immuno-histochemical marker for human colonic carcinoma. Gann 77: 226-229, 1986. Shiratori Y, Soma Y, Maruyama H, Sato H, Takano A, Sato K: Immunohistochemical detection of the placental form of glutathione S-transferase in dysplastic and neoplastic human uterine cervix lesions. Cancer Res 47: 6806-6809, 1987. Niitsu, Y, Takahashi Y, Saito T, Hirata Y, Arisato N, Maruyama H, Kohgo Y: Serum glutathione S-transferase-rt as a tumor marker for gastrointestinal malignancies. Cancer 63: 317-323, 1989. Moskow JA, Fairchild CR, Madden MJ, Ramsom DT, Wieand HS, O’Brien E: Expression of anionic glutathione-S-tranferase and p-glycoprotein genes in human tissues and tumors. Cancer Res 49: 1422, 1989. Ilio CD, Boccio GD, Aceto A, Casaccia R, Mucilli F, Federici G: Elevation of glutathione transferase activity in human lung tumor. Carcinogenesis 9: 335-340, 1988.

23 17 Ackerman AB: Pagetoid Bowen’s disease vs. extramammary Paget’s disease, in the Differential diagnosis in The Dermatopathology. Edited by AB Ackerman, Nieven JM Grant-Kels, Lea and Febiger, Philadelphia, 1982, pp 126-129. 18 Bennett CF, Spector DL, Yeoman LC: Nonhistone protein BA is a gluathione S-transferase localized to interchromatinic regions of the cell nucleus. J Cell Biol 102: 600-609, 1986. 19 Ketterer B, Tan KH, Mayer DJ, Coles B: Glutathione

transferases. A possible role in the detoxification of DNA and lipid hydroperoxides in The Glutathione S-transferases and carcinogenesis. Edited by TJ Mantle, CB Pickett and JD Hayes. Taylor and Francis, London, 1987, pp. 149-163. 20 Eimoto H, Tsutsumi M, Nakajima A, Yamamoto K, Takashima Y, Maruyama H, Konishi Y: Expression of the glutathione S-transferase placental form in human lung carcinomas. Carcinogenesis 9: 2325-2327, 1988.