Original Paper Urol Int 1992;48:20-24

Mako to Satoha Yasuo Fukushib Sadafwni Kawamuraa Chikara Ohyamaa Seiichi Saitoa Seiichi Orikasaa Edward Nudlemanc Sen-itiroh Hakamoric

Glycolipid Expression in Prostatic Tissue and Analysis of the Antigen Recognized by Antiprostatic Monoclonal Antibody APG1

Department of Urology, Tohoku University, School of Medicine, and Kidney Center Sendaisyakaihoken Hospital, Sendai, Japan; The Biomembrane Institute, Seattle Wash., USA

KeyWords

Abstract

Mouse monoclonal antibody Prostate carcinoma Glycolipid

The expression patterns of glycolipid from prostatic hyperplasia, prostatic cancer and normal prostate tissue were observed. A further analysis of antigen recognized by mouse monoclonal antibody APG1, which was gained by immunizing glycolipids extracted from human prostate cancer, was also per­ formed. In cancer tissue, both of the lactosyl and globoside series glycolipids were found to be generally reduced, although in the ganglioside series, GM3 and GD3 were not reduced and only the glycolipids with longer chains than GD2 were found to be reduced. These results indicated that the inhibition of sugar chain elongation, but not sialylation, was the main synthetic change occurring with carcinogenesis of the human prostate. APG1 reacted with only two bands near GM2 and GD2 of the ganglioside fraction on a thin-layer chromatography plate, but it did not react with any of the known gangliosides of the ganglioside series including GM2 and GD2. Histochemically, APGl showed intense reaction only in frozen tissue sections of human prostate, and the reactivity decreased with the increasing grade of cancer. Therefore, this antigen was considered to be a prostate-specific and differentiated antigen reacting with nonganglioseries gangliosides.

Glycolipids, which are the trace constituents of the cell surface membrane, have attracted attention as important substances involved in the development and differentia­

Received: July 27, 1990 Accpctcd after revision: December 19. 1990

tion of cells, tissues and organs, and in the receptor func­ tion of physiologically active substances and cell interac­ tion [1], They have also been shown to exhibit various changes in the process of carcinogenesis [2, 3], We have reported some remarkable changes in glycolipids in tis-

Makoto Satoh, MD Department of Urology Tohoku University School of Medicine 1-1 Seiryomachi, Sendai 980 (Japan)

© 1992 S. Karger AG. Basel 0042-1138/92/0481-0020 $2.75/0

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Introduction

Table 1. Summary o f HP TLC pattern

Case

Stage (grade)

PCI

D1 (solid)

PC2

PC3 PC4

PC5 PC 6

Materials and Methods Materials Glycolipids were extracted from the following surgical speci­ mens: 3 prostatic hyperplasia rich in glands, 6 prostatic cancer (4 stage C and 2 stage D 1; 1 cribriform, 1 fused glands and 4 solid) and 1 normal prostate tissue resected in total cystectomy for bladder can­ cer. Immunostaining by APG1, anti-prostate-specific acid phospha­ tase (anti-PSAP) and anti-prostate-specific antigen (anti-PSA) was performed in 20 formalin-fixed paraffin-embedded sections (simple glands 2, cribriform/fused glands 10, solid/trabecular 8) and 14 fro­ zen sections (2 stage A, 5 stage B. 4 stage C, 3 stage D; simple glands 2, cribriform/fused glands 6, solid/trabecular 6) o f surgical or biopsy specimens of prostatic cancer. Histopathologic grading was made according to the WHO system, and clinical stage was classified based on Whitemore’s classification. Methods Extraction o f Glycolipids. Tissues were homogenized and ex­ tracted with 20 volumes of chloroform-methanol mixtures (2 : 1, 1: 1, 1:2 v/v). The same procedure was also performed with an isopropanol-hexane-water mixture (55:25:20 v/v/v). The extract was dried in a rotary evaporator and divided into the upper and lower phases by the partition of Folch et al. [8], The upper phase was dialyzed against distilled water for 2 days and subjected to diethylaminoethanol-Sephadex A25 column (Pharmacia) chroma­ tography to separate neutral glycolipids and gangliosides [9], The ganglioside fraction was further dialyzed against distilled water for 2 days. The lower phase was freed from phospholipid contamination by acetylation [10]. Acetylated glycolipids were deacetylated with 0.25 % sodium metoxide: chloroform-methanol (CM) (2:1), and neu­ tralized with Dowex 50 W-X 8 (Bio-Rad). Each glycolipid sample was dissolved in CM (2:1) and placed on a high-performance thin-layer chromatography (HPTLC) plate (Baker, Phillipsburg, N.J., USA) using a microsyringe in amounts derived from the same wet weights of tissue. The upper-phase neutral and ganglioside glycolipids were run together with C:M:0.5% CaCL = 60:40:9 (v:v:v). and the lower-

C (solid) D1 (solid)

CMH

CDH





4

-

-

-







C (cribriform)

CTH

Glob

GD3

GD2



-

4

-

4

-

4

4 —

4

4

-

4

-

4

C (solid)

!

1

4

4

C (fused glands)

!

1

4

4

4

PC = Prostate cancer; CMH = ceramide monohexoside; CDH = ceramide dihexoside; CTH = ceramide trihexoside; Glob = globoside; 4 = decrease; — = no change.

Table 2. Relationship between tumor grade and A PG 1 reactivity on frozen sections

Grade

APG1

PSA

PSAP

wel mod por

2/ 2 ( 100)

2/ 2 ( 100)

2/6 (33) 1/6(17)

3/6 (50) 2/6(33)

2/ 2 ( 100) 6/ 6 ( 100) 3/6 (50)

wel = Simple glands; mod = cribriform/fused glands; por = solid/trabecular. Figures in parentheses indicate percentages.

phase glycolipids were run with C:M:watcr = 60:25:4 (v:v:v). OrcinolH 2SO4 was used as the staining reagent. Immunostaining. Paraffin-embedded specimens and frozen spec­ imens were immunologically stained with anti-PSAP MoAb, antiPSA MoAb (Biogencx Laboratories) and APG1 by the avidin-biotin complex method. HPTLC Radioimmunostaining. The standard gangliosides of GM3. GD3, GM2, GD2, GDI a, G T lb, G D lb (Sigma, Bachem, Funakoshi), disialyl paragloboside, disialyl galactosyl globoside (pro­ vided by the Biomembrane Institute) and ganglioside fraction ex­ tracted from the tissue of prostatic hyperplasia were run on the same HPTLC plate. After the plate had ben run it was cut into two halves, one half being treated with orcinol H 2SO 4 for staining. The other half was reacted with APG 1 overnight, and incubated with second anti­ body, and then with l 25I-protein A, and autoradiographed.

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sues of testicular and renal cancer [4-6], We have also prepared mouse monoclonal antibody (MoAb) APG1 by immunizing glycolipids extracted from human prostate cancer, which reacted with the ganglioside fraction ex­ tracted from the prostate gland [7], Prostatic cancer shows characteristic pathologic fea­ tures such as hormone dependence and a high tendency of bone metastasis. Though the diagnostic value of several tumor markers has been evaluated, there have been no reports on changes in glycolipids and markers that recog­ nize sugar chains. In this study, we observed the patterns of glycolipid expression in the tissues of prostate cancer, normal prostate gland and prostatic hyperplasia, and fur­ ther analyses of the biochemical properties and histologic features of APG1 reacting antigen were performed.

Fig. 1. HPTLC pattern o f glycoplipids extracted from prostatic tissue. A = Type A red blood cells; 0 = type 0 red blood cells; PH = prostatic hyperplasia; N = normal prostate; PC = prostate cancer. GD2 and gangliosides with longer chain reduced in all 6 PC cases.

Glycolipid Pattern o f Prostate Tissue In prostatic cancer, CDH and CMH were reduced in 2 and CTH was reduced in 4 of the 6 tissue specimens, as compared with those of the normal prostate or prostatic hyperplasia (fig. I, table 1). Globosides were also reduced in 4 of the 6 specimens. GD2 and longer sugar chains were clearly reduced in all 6 specimens. Immunostaining Paraffin-Embedded Sections. Weak staining by APG1 was observed in the normal and hyperplastic glands of most sections, but no staining was observed in the carci­ noma. The frequency of the positive staining for antiPSAP and anti-PSA was reduced with increasing grade as indicated by a number of reports [11, 12],

22

Frozen Sections. The positive staining rate of APG1 was 100% in simple glands, 33% in cribriform and fused glands and 17% in solid and trabecular types, showing a declining frequency of the positive staining with tumor grade (fig. 2, table 2). However, no relationship of the pos­ itive staining rate with stages was observed. HPTLC Radioimmunostaining by APG1 Autoradiography (fig. 3) showed reactions with only two bands of gangliosides extracted from the prostatic hyperplasia but not with standard gangliosides (GM3, GD3, GM2. GD2, GDI a, G Tlb, G Dlb. disialyl paragloboside, disialyl galactosyl globoside). The locations of these two positive bands on the HPTLC plate corre­ sponded with those of GM2 and GD2 gangliosides. but A PG1 did not react with standard GM2 or GD2.

Satoh/Fukushi/Kawamura/Ohyama/Saito/ Orikasa/N udleman/Hakamori

Glycolipid Expression in Prostatic Tissue

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Results

Fig. 2. Immunohistochemical staining of prostate carcinoma (cri­ briform type): positive staining of tumor cells by APG1. Frozen sec­ tion. X 100. Fig. 3. HPTLC immunostaining by APG1 of standard ganglioside and ganglioside extracted from prostatic hyperplasia (G D Ia, G D lb. G T lb not shown here). DS PG = Disialyl paragloboside; DS Gal Glob = disialyl galactosyl globoside; PH = ganglioside extracted from prostatic hypertrophy.

The usefulness of various markers for prostate cancer such as prostatic acid phosphatase (ACP), prostate-spe­ cific antigens (PSA) [13] and y -seminoprotein (ySU) [14] and some MoAbs that recognize prostate-specific anti­ gens [15, 16] have been reported. However, there are a few reports about MoAbs which recognize sugar chain structures of the prostatic cells [17]. In addition, there have been no reports concerning the expression pattern of glycolipid extracted from prostatic tissue. Major changes in glycolipids associated with tumorigenesis include general reductions of glycolipids with lon­ ger chains which was true in testicular tumor and renal tumor [4], The present study concerning the prostate also revealed general decreases in glycolipids with tumorigenesis. We could not find out any specific glycolipids remark­ ably increased in cancer tissue or prostatic hyperplasia.

Only in the ganglioside fraction could a characteristic change in the expression pattern of glycolipid be found, that is GM3 or GD3 did not decrease those amounts and only GD2 and gangliosides with longer chains were reduced. These findings indicate that, in prostatic cancer, suppression of sugar chain elongation, but not sialylation, is the major change occurring in the sugar chain synthetic process. APG1, which did not react with any other human organs than the prostate [7], reacted with only the ganglio­ side (GGL) fraction among glycolipids extracted from the prostate gland. Furthermore, this MoAb recognized only two bands near GD2 and GM2 on HPTLC. However, APG1 reacted with none of the glycolipids of the GGL series including GD2 and GM2 nor some ganglioside other than the GGL series. From these results, the antigen recognized by APG1 is considered to be an agent that pos­ sesses a new antigenic structure.

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Discussion

Immunohistologically, this antigen is revealed to be distributed in the normal prostate gland, prostatic hyper­ trophy and well-differentiated prostatic carcinoma cells. The intensity of positive staining decreased with increas­ ing grade of prostete cancer. Therefore, this antigen is considered to be a sugar chain antigen specific for the prostate gland which is present as a differentiated antigen. As changes in the intensity of this antigen associated with the degree of cell differentiation are more notable than those of PSA or PSAP, APG1 may be useful for evalua­ tion of the malignant potential of tumors and the progno­

sis of the patients. These results show that observation of the glycolipid expression pattern, especially of ganglioside, would offer us further information about biological features and a more precise diagnosis.

Acknowledgments The authors wish to thank Dr. R. Chiba for providing prostatic tissue, T. Masuko for technical assistance and Dr. A. Ohtani for pathologic assistance.

References

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7 Satoh M, Fukushi Y. Ohyama C. et al: Prostate specific monoclonal antibody gained by glyco­ lipid immunization. Jpn J Urol 1990:81:289295. 8 Folch J. Lees M, Sloane S: A simple method for isolation and purification of total lipids from animal tissues. J Biol Chem 1957:226:497509. 9 Yu RK, Ledeen RW: Gangliosides of human, bovine, and rabbit plasma. J Lipid Res 1972; 13:680-686. 10 Saito T. Hakomori S: Quantitative isolation of total glycosphingolipids from animal cells. J Lipid Res 1971:12:257-259. 11 Parkin L, Bvlsma G, Toree AV, et al: Acid phosphatase in carcinoma of the prostate in man. J Histochem Cytocem 1964:12:288—292. 12 Feiner HD. Gonzalez R: Carcinoma of the prostate with atypical immunohistological fea­ tures. Am J Surg Pathol 1986:10:765-769. 13 Wang MC, Valenzuela LA, Murphy GP. et al: Purification of a human prostate specific anti­ gen. Invest Urol 1979:17:159-163.

14 SchallerJ. Akiyama K, Tsuda R, et al: Isolation and characterization and aminoacid sequence of r seminoprotein, a glycoprotein from human seminal plasma. Eur J Biochem 1987; 170:111 —

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Glycolipid Expression in Prostatic Tissue

120. 15 Bazinet M, Cote RJ, Cordon CC. et al: Immu­ nohistochemical characterization of two monoclonal antibodies. P25.48 and P25.9I, which define a new prostate-specific antigen. Cancer Res 1988:48:6938-6942. 16 Starling JJ. SiegSM, Beckett ML, et al: Human prostate tissue antigens defined by murine monoclonal antibodies. Cancer Res 1986:46: 367-374. 17 Lindgren J. Blaszczyk M. Atkinson B. et al: Monoclonal antibody-defined antigens of hu­ man prostate cancer cell line PC3. Cancer Im­ munol Immunother 986:22:1-7.

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1 Hakomori S: Glycosphingolipids. Sci Am 1986;254:32-41. 2 Hakomori S: Glycosphingolipids tumor-cell membrane. Adv Cancer Res 1973;18:265— 315. 3 Hakomori S: Kannagi R: Glvcosphinogolipids as tumor-associated and differentiation mark­ ers - A guest editorial. J Natl Cancer Inst 1983: 71:231-251. 4 Ohyama C. Fukushi Y. Satoh M. et al: Changes in glycolipid expression in human testicular tumor. Int J Cancer 1990:45:1040-1044. 5 Fukushi Y. Nudleman E. Levery SB. et al: Novel fucolipids accumulating in human ade­ nocarcinoma. J Biol Chem 1984:259:10511— 10517. 6 Fukushi Y. Orikasa S, Shepard T. et al: Lex and dimeric Lex haptens and their sialylated anti­ gens during development of human kidney and tumors. J Urol 1985:135:1048-1059.

Glycolipid expression in prostatic tissue and analysis of the antigen recognized by antiprostatic monoclonal antibody APG1.

The expression patterns of glycolipid from prostatic hyperplasia, prostatic cancer and normal prostate tissue were observed. A further analysis of ant...
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