- Email: [email protected]
The cytoplasmic mucin in Paget cells of extramammary Paget 's disease
Acta histochem. 69, 50-56 (1981)
Depftrtment of Dermatology, Knrume University, School of Medicine, Kurume, Japan
The cytoplasmic mucin in PAGET cells of extramammary PAGET'S disease By YOICHIRO SASAI, HIDEMASA KAJI, MAKIKO NA'rSUAK! and TAKESHI NAKAMA With 4 figures (Received September 15, 1980)
Summary The cytoplasmic mucin in the PAGET cells of extra mammary PAGET'S disease was examined with a battery of histochemical techniques. The staining methods used were ale ian blue, azure A and periodic acid-Schiff. In a further attempt to identify various polyanions, staining was carried out with alcian blue containing various concentrations of electrolyte. Methylation, saponification, borohydride reduction, acid hydrolysis, and digestion with diastase, sialidase, chondroitinase ABC, or nucleases were also employed. The results obtained suggest that the cytoplasmic mucin in the PAGET cells is sialomucin without side-chain substituent.
Introduction Extramammary P AGE'r'S disease, occurring in the ano-genital region or the axilla, is characterized by large pale-staining cells, viz. PAGE'r cells, scattered throughout the epidermis. In a paper entitled "PAGE'r'S disease affecting the scrotum and penis", CROCKER (1889) was the first to describe extramammary PAGET'S disease. He considered the disease to start from the sweat and sebaceous glands and hair follicles. Since his description, many theories concerning its pathogenesis and histogenesis have been proposed: 1. The disease begins in an underlying carcinoma of glandular structures in the skin, and extends by continuity into the epidermis (DUBREILH 1901). 2. The disease results from metastasis to the epidermis from an underlying adenocarcinoma (WEINER 1937). 3. The disease arises simultaneously in both the epidermis and the epithclial structures of adnexa (HELWIG and GRAHAM 1963). 4. The disease arises in the epidermis and extends from there into epithelial structures of adnexa and into the dermis (WOODRUFF 1955). 5. The disease results from an extracutaneous contiguous adenocarcinoma that extends into the epidermis (TAKESHITA et al. 1978). Recently, JONES ct al. (1979), studying 55 cases of extramammary PAGET'S disease, suggested that pluripotential germinative cells within the epidermis go awry in their
The cytoplasmic muein in PAGET cells
51
attempt to form apocrine structure as they did in embryonal life, and that the faulty result is PAGET cells of extramammary PAGET'S disease. The mucous secretion pattern of variolls mucous membranes in malignancy is well known to differ from that in normal state. Some workers indicated that this change is accompanied by variations in the proportions of sialic acid derivatives (ROGERS et al. 1978). Early histochemical studies in ours suggested the presence of sialomllcin in PAGET cells (SASAI and TANAHASHI 1969). The present study was designed to analyze precisely the sialolllucin by histochemical methods.
Materials and Methods Biopsy skill speeimens were relllovpd from ten patients with extramammary PAGET'S disease. Eaeh speeimcll was fixed in 10 % neutr'al·buffered formalin for 24 h. After being dehydrated and paraffin·embedded in the usual fashion, they were ('ut at 6 pm in thickness. In addition to routine staining methods, the following techniques were employed. 1. Alcian blue. Aleian blue was dissolved to make OJJ5 % solution in 0.05 M acetate buffer of pH = 5.H. The solution was distributed in several dye baths and to these magnesium ehloride or zirconium oxyehloride was added sequentially so as to give molarities from 0.025 to 2.0. Sections were stained for 12 h in respeetive dye baths, then rinsed three times for 5 min in baths containing the buffer and the same eoneentrations of the same eledrolytes as used for staining. After rinsing. the sections were washed three times with distilled water in 3·minute baths and dehydrated. cleared and mounted. 2. Azure A. SeetiOlLH were stained for 30 min with 0.05 % azure A in 0.1 M phosphate·HCI or phosphate· citrate buffer of a spleet('d pH. After being blotted dry, the sections were cleared in an a('ptone·xylene mixture. 3. Periodic (tcid·Schzjj (PAS). Seetions were stained aeeording to the method of LILLIE (1965). In ROme seetions, a two·step PAS method was carried out as suggested by SCOTT and DORLING (19(i9). 4. Acetylation. 8pc:tiollH weI''' imIl1PJ',;e(1 in a 2: 3 mixture of acetic anhydride/pyridine for (\ h at 22°C. 5. lvIethylat-ion. :Sections were placed in 0.1 N hydrochloric aeid in absolute methanol for 4 h at 60 DC. fl. Saponijication. :Sections were inllnerRpd in 0.1 % potassium hydroxide in 70 % ethanol for 30 min at 25 DC. i. Acid hydrolysis. Sections were illllnersed in 0.1 N sulfuric acid for 1 h at SO DC. H. llorohydride reduction. 8petiollS were treated with 0.1 % sodium borohydride in 1 % disodium hydrogen phosphate fo!' 30 min. 9. Diastase digestion. 8eetions were incubated for 1 hour at 37 DC in malt·diastase dissolved in 0.2 M phosphate buffer of pH ~ G.O, in a coneentration of 1 mg per m!. 10. Sialidase digestion. :Section,; were treated for 24 h at 37 DC with sialidase prepared as a 1: 4 dilution of sialidase to whieh was add"d 10 %, by volume, of 1 % ealeiulll chloride solution. 11. Chonlil'oitillitse .~BC digestion. 8eetiol1s were treated foJ' 2 h at 37 DC in chondr'oitinase ABC dissolved ill 0.0025 .M tris·HCI buffer of pH c_ S.O, in a concentration of 5 U per 0.3 m!. 12. Bibonucle(tse digestion. Hibonu<"iease was dissolved in distilled water in a concentration of 0.5 mg per 111!. The water was preadjusted to pH = fl.S with 0,1 N sodium hydroxide. Sections were treated for 8 h at 37 DC. 13. De8o,~yribonuclease digestion. Desoxyribonuelease was dissolved in 0.01 M phosphate buffer of pH = 7.0 in a concentration of 0.2 mg pe!' ml, and magnesium chloride was added as an activator in a COlH'('ntration of 0.003 M. Sections were treated for 6 h at 37 DC. 4*
52
Y.
SASA!
et al.
::\Ialt-diastase and sialidase were obtained from Sigma Chemical Co., St. Louis, Missouri, U.S.A., and chondroitinase ABC, ribonuclease and desoxyribonuclease from Seikagakn Kogyo Co., Tokyo, Japan.
Results Each specimen from the patients showed essentially the same histopathological features. The epidermis ,vas normal in thickness, or moderately acanthotic. PAGET cells were scattered through the epidermis (Fig. I). They were largerthan adjacent keratinoeytes and displayed a vacuolated or olear cytoplasm with indefinite cell membrane. The oval or rounded nuclei were usually vesicular, occasionally hyperehromatic, and moderately pleomorphic. No invasion into the dermis was eneountered; however, the cells extended downward in the epitheli9J lining of hair follicles and of eccrine ducts. The blood capillaries were dilated and congested, lined by swollen endothelial cells, and surrounded by a dense infiltrate of plasma cells with occasional lymphocytes and polymorphonuolear leukocytes. The cytoplasm of the PAGET cells showed an affinity for alcian blue (Fig. 2). When the sections were immersed in alcian blue solutions containing magnesium chloride from 0.0025 to 0.2 M, no variation in the affinity for alcian blue was found. The affinity markedly reduced in the presence of magnesium chloride at 0.25 M, and completely disappeared at 0.45 M. Methylation for 4 h at 60°C abolished the affinity. When methylation was followed by saponification for 30 min at 25 DC, the affinity was partly noticed in the presence of magnesium chloride at 0.2 1\1 or less. When zirconium oxychloride was added to alcian blue solution, the affinity markedly reduced at 0.025 M. On the other hand, the cytoplasm showed an azurophilia at pH = 3.0 or higher. At pH = 3.0 or higher, it was metachromatic. Also, the cytoplasm of the PAGET cells stained deep red with standard PAS procedure (Fig. 3), and faint with two-step procedure. The reaction was completely blocked by acetylation or borohydride treatment, and weakened by acid hydrolYRis. When the sections were treated with potassium hydroxide following borohydride reduction, the reaction was not noticed. Malt-diastase, chondroitinase ABC, ribonuclease or desoxyribonuclease digestion did not change significantly staining with any of the methods used. Treatment with sialidase for 24 h weakened the PAS reactivity (Fig. 4) and the alcianophilia in the presence of magnesium chloride at 0.2 M or less.
Discussion All polyanions are pl"ecipitated by such quaternary ammoniul1l salts as cetylpyridiniulll chloride or aleian blue, and tho water-insoluble complexes are soluble in salt solutions, if the concentration of the salt is maintaim·d above a cel·tain level (SCOTT 19(0). Aeeording to a model experiment (SARAl 1971; SASAl et al. 1978), hyaluronic acid loses an affinity for alcian blue in the presence of magnesium ehleJl'ide at 0.1 M, chondroitin sulfate Band heparitin sulfate at 0.7 M, chcmdroitill sulfates A and C at O.S M, and hepar'in at 1.0 M. \\'hell zirconium oxyehloride is used UK an added salt, the affinity of aeid mncopolysat'{·harides for alcian blue disappE'ars at 0.05 M (hyalurmic acid),
The cytoplasmic mucin in
PAGET
Fig. 1. Extramammal'Y PAGET'S disease. Hematoxylin and eosin stain X 75. tered through epidermis.
Fig. 2. Extramammary PAGET's disease. Alcian blue stain showed an affinity for ale ian blue.
X
53
cells
PAGET
cells are scat·
150. The cytoplasm of the
PAGET
cells
54
Y.
SASAI
et a l.
Fig. 3. Extramammary 1'AGET'S d isease. PAS stain strongly reactive to l' AS.
Fig. 4. Extramammary 1'AGET'S disease. PAS stain wea kened the PAS reactivity.
X
150. The cytoplaRm of the
X
1'AGET
cells is
150. 'Treat ment with sialidasn for 24 h
The cytoplasmic mucin in PAGE'l' cells
55
0.25 M (ehondroitin wlfate A), 0.3 :l\i (heparitin sulfate), and 0.4 M (ehondroitin sulfates Band C and hpparin). All carboxyl grpupoJ' phosphate group· containing polyanions are in an undifferen· tiat('d group at about the same low criti('al salt ('oncpntration level as hyaluronic acid. The pattern of the (,J'itieal ,mIt ('oneentration of each polyanion in tissue seetions was similar to that in the model exp('rill1Pnt, although thp forll1Pl' was about 30 % highp!, than the latter. On the other hand, methy· latioll hydrolyzes sulfate groups, resulting in the' formation of frpe methyl esters, while it esterifies carboxyl and phosphatp groups. And, subseqm'nt saponifir'ation restores only thp latter two. In the present study, the eytoplasm of PAGET (,PUs showed an affinity for al('ian blup in the prcspn('e of magnpsiulll chloride at 0.4 M or Ipss. The affinity was bloekpd by methylation, and partly reo stored by saponification. Togpthel' with thp aZlll'ophilia at pH 3.0 or higher pH range, these results suggpst the prE'sE'nce of carboxyl gronp-('ontaining polyanioll in PAGET ('plls, A jY,sitive PAR readion llsllully indi,'atE's the prcsem'" of earbohydratp. Thporetically, all periodate-oxidizabk polysaccharides, whdher a('id 0[' neutral, should bp PAR-positive. In practieE', howevpr, acid mucopolysa('ch'lridps fail to give a positive roaction. RCO'l'T and HARBINSO:Y (HHi8, 1969) ,howed that the a('cess of ppriodate ions to oxidizablE' glycol grOllps of acid mucopolysaccharidE'S is redu('ed by the intense' negative elpf'trostati(' field of the poltanion, and that the enusing partial inhibition of oxidation ('an be largely or completely overcome by se('rE'ting the polyanionic charges with an inE'I't E'lE'ctrolytes. Thus, mu(,opolysaccharides ('ontaining 1: 4 linked ur(mic acids shoul
56
Y. SASAI et aI., The cytoplasmic mucin in PAGET cells
Literature CROCKER, H. R., Paget's disease affecting the scrotum and penis. Trans. Path. Soc. Lond. 40, 187 -191 (1889). CULLING, C. F. A., REID, P. E., CLAY, M. G., and DUNN, "V. L., The histochemical demonstration of O·acylated sialic acid in gastrointestinal mucins: their associations with the potassium hydroxide·periodic acid·Schiff effeet. ,J. Histochem. Cytochem. 22, 826-831 (1974). CULLING, C. F. A., REID, P. E., WORTH, A. ,J., and DUNN, "V. L., A new histochemical technique of use in the interpretation and diagnosis of adenocarcinoma and villous lesions in the large intestine. ,J. Clin. Pathol. 30, 1056-1067 (1977). DUBREUILH, W., Paget's disease of the vulva. Br..J. Dermatol. 13, 407 -418 (1901). FILIPE, M. I., and COOKE, K. B., Changes in composition of mucin in the mucosa adjacent to carcinoma of the colon as compared with the normal: a biochemical investigation. J. Clin. Pathol. 27,315-318 (1974). HELWIG, E. B., and GRAHA1\I, J. H., Anogenital extramammary Paget's disease, a clicopathological review. Caneer 16, 387 -403 (1963). JONES, R. E. jr., AUSTIN, C., and ACKERMAN, A. B., Extramammary Paget's disease. A critical examination. Am. ,J. Dermatopathol. I, 101-132 (1979). LILLIE, R. D., Histopathologic Technic and Practical Histochemistry, 3rd Ed., p. 198. McGraw Hill Book Co., New York 1965. REID, P. E., CULLING, C. F. A., and DUNN, W. L., Saponification-induced increase in the periodic acid-Schiff reaction in the gastrointestinal tract. Mechanism and distribution of the reactive substance. ,J. Histochem. Cytochem. 21, 473-482 (1973). ROGERS, C. M., COOKE, K. B., and FILIPE, M. I., The sialic acids of human large bowel mocosa: 0acylated variants in normal and malignant states. Gut 19, 587 - 592 (1978). SASAI, Y., Effect of tissue fixation on the histoehemical identification of acid mucopolysaccharides. Tohoku ,T. Exp. Med. 104, 85-91 (1971). NAGATA, M., INOKUCHI, K., and NAMBA, K., Histochemical differentiation between chondroitin 4-sulfate and chondroitin 6-sulfate. Kurume Med. ,T. 25, 45-51 (1978). and NATSUAKI, M., unpublished data. and TANAHASHI, Y., HistochemicaoLstudy of the alcianophilia of Paget cells in extramammary Pager's disease. Tohoku .J. Exp. Med. 97,289-295 (1969). SCOTT, ,J. E., Alipathic ammonium salts in the assay of acidic polysaccharides from tissue. In: Methods of Biochemical Analysis. Ed. D. Glick, Vol. 8, pp. 145 -197. Interscience Publishers, New York 1960. and DORLING, J., Periodate oxidation of acid polysaccharides. III. A PAS method for chondroitin sulfates and other glycosamino-glycuronans. Histochemie 19, 295-301 (1969). and HARBINSON, R. J., Periodate oxidation of acid polysaccharides inhibition by the electrostatic field of the substrate. Histochemie 14, 215-220 (1968). - Periodate oxidation of acid polysaccharides. II. Rates of oxidation of uronic acids in polyuronides and acid mueopolysaccharides. Histochemie 19,155-161 (1969). TAKESHITA, K., IZUMOI, S., EBl7CHI, M., YOSHIDA, M., KASHIMURA, A., MURAKAMI, T., KAGAWA, S., AOKI, N., and MIYAMOTO, H., A case of rectal carcinoma concomitant with Pageoid lesion in the perianal region. Gastroenterol. Jap. 13, 85-95 (1978). WEINER, H. A., Paget's disease of the skin and its relation to carcinoma of the apocrine swear glands. Am. J. Cancer 31,373-403 (1937). WOODRUFF, J. D., Paget's disease of the vulva; review report of two cases. Obstet. Gynecol. 5, 175-185 (1955).
Address: Dr. YOICHIRO SASAI, Department of Dermatology, Kurume University, School of Medicine, Kurume 830, Japan.
Depftrtment of Dermatology, Knrume University, School of Medicine, Kurume, Japan
The cytoplasmic mucin in PAGET cells of extramammary PAGET'S disease By YOICHIRO SASAI, HIDEMASA KAJI, MAKIKO NA'rSUAK! and TAKESHI NAKAMA With 4 figures (Received September 15, 1980)
Summary The cytoplasmic mucin in the PAGET cells of extra mammary PAGET'S disease was examined with a battery of histochemical techniques. The staining methods used were ale ian blue, azure A and periodic acid-Schiff. In a further attempt to identify various polyanions, staining was carried out with alcian blue containing various concentrations of electrolyte. Methylation, saponification, borohydride reduction, acid hydrolysis, and digestion with diastase, sialidase, chondroitinase ABC, or nucleases were also employed. The results obtained suggest that the cytoplasmic mucin in the PAGET cells is sialomucin without side-chain substituent.
Introduction Extramammary P AGE'r'S disease, occurring in the ano-genital region or the axilla, is characterized by large pale-staining cells, viz. PAGE'r cells, scattered throughout the epidermis. In a paper entitled "PAGE'r'S disease affecting the scrotum and penis", CROCKER (1889) was the first to describe extramammary PAGET'S disease. He considered the disease to start from the sweat and sebaceous glands and hair follicles. Since his description, many theories concerning its pathogenesis and histogenesis have been proposed: 1. The disease begins in an underlying carcinoma of glandular structures in the skin, and extends by continuity into the epidermis (DUBREILH 1901). 2. The disease results from metastasis to the epidermis from an underlying adenocarcinoma (WEINER 1937). 3. The disease arises simultaneously in both the epidermis and the epithclial structures of adnexa (HELWIG and GRAHAM 1963). 4. The disease arises in the epidermis and extends from there into epithelial structures of adnexa and into the dermis (WOODRUFF 1955). 5. The disease results from an extracutaneous contiguous adenocarcinoma that extends into the epidermis (TAKESHITA et al. 1978). Recently, JONES ct al. (1979), studying 55 cases of extramammary PAGET'S disease, suggested that pluripotential germinative cells within the epidermis go awry in their
The cytoplasmic muein in PAGET cells
51
attempt to form apocrine structure as they did in embryonal life, and that the faulty result is PAGET cells of extramammary PAGET'S disease. The mucous secretion pattern of variolls mucous membranes in malignancy is well known to differ from that in normal state. Some workers indicated that this change is accompanied by variations in the proportions of sialic acid derivatives (ROGERS et al. 1978). Early histochemical studies in ours suggested the presence of sialomllcin in PAGET cells (SASAI and TANAHASHI 1969). The present study was designed to analyze precisely the sialolllucin by histochemical methods.
Materials and Methods Biopsy skill speeimens were relllovpd from ten patients with extramammary PAGET'S disease. Eaeh speeimcll was fixed in 10 % neutr'al·buffered formalin for 24 h. After being dehydrated and paraffin·embedded in the usual fashion, they were ('ut at 6 pm in thickness. In addition to routine staining methods, the following techniques were employed. 1. Alcian blue. Aleian blue was dissolved to make OJJ5 % solution in 0.05 M acetate buffer of pH = 5.H. The solution was distributed in several dye baths and to these magnesium ehloride or zirconium oxyehloride was added sequentially so as to give molarities from 0.025 to 2.0. Sections were stained for 12 h in respeetive dye baths, then rinsed three times for 5 min in baths containing the buffer and the same eoneentrations of the same eledrolytes as used for staining. After rinsing. the sections were washed three times with distilled water in 3·minute baths and dehydrated. cleared and mounted. 2. Azure A. SeetiOlLH were stained for 30 min with 0.05 % azure A in 0.1 M phosphate·HCI or phosphate· citrate buffer of a spleet('d pH. After being blotted dry, the sections were cleared in an a('ptone·xylene mixture. 3. Periodic (tcid·Schzjj (PAS). Seetions were stained aeeording to the method of LILLIE (1965). In ROme seetions, a two·step PAS method was carried out as suggested by SCOTT and DORLING (19(i9). 4. Acetylation. 8pc:tiollH weI''' imIl1PJ',;e(1 in a 2: 3 mixture of acetic anhydride/pyridine for (\ h at 22°C. 5. lvIethylat-ion. :Sections were placed in 0.1 N hydrochloric aeid in absolute methanol for 4 h at 60 DC. fl. Saponijication. :Sections were inllnerRpd in 0.1 % potassium hydroxide in 70 % ethanol for 30 min at 25 DC. i. Acid hydrolysis. Sections were illllnersed in 0.1 N sulfuric acid for 1 h at SO DC. H. llorohydride reduction. 8petiollS were treated with 0.1 % sodium borohydride in 1 % disodium hydrogen phosphate fo!' 30 min. 9. Diastase digestion. 8eetions were incubated for 1 hour at 37 DC in malt·diastase dissolved in 0.2 M phosphate buffer of pH ~ G.O, in a coneentration of 1 mg per m!. 10. Sialidase digestion. :Section,; were treated for 24 h at 37 DC with sialidase prepared as a 1: 4 dilution of sialidase to whieh was add"d 10 %, by volume, of 1 % ealeiulll chloride solution. 11. Chonlil'oitillitse .~BC digestion. 8eetiol1s were treated foJ' 2 h at 37 DC in chondr'oitinase ABC dissolved ill 0.0025 .M tris·HCI buffer of pH c_ S.O, in a concentration of 5 U per 0.3 m!. 12. Bibonucle(tse digestion. Hibonu<"iease was dissolved in distilled water in a concentration of 0.5 mg per 111!. The water was preadjusted to pH = fl.S with 0,1 N sodium hydroxide. Sections were treated for 8 h at 37 DC. 13. De8o,~yribonuclease digestion. Desoxyribonuelease was dissolved in 0.01 M phosphate buffer of pH = 7.0 in a concentration of 0.2 mg pe!' ml, and magnesium chloride was added as an activator in a COlH'('ntration of 0.003 M. Sections were treated for 6 h at 37 DC. 4*
52
Y.
SASA!
et al.
::\Ialt-diastase and sialidase were obtained from Sigma Chemical Co., St. Louis, Missouri, U.S.A., and chondroitinase ABC, ribonuclease and desoxyribonuclease from Seikagakn Kogyo Co., Tokyo, Japan.
Results Each specimen from the patients showed essentially the same histopathological features. The epidermis ,vas normal in thickness, or moderately acanthotic. PAGET cells were scattered through the epidermis (Fig. I). They were largerthan adjacent keratinoeytes and displayed a vacuolated or olear cytoplasm with indefinite cell membrane. The oval or rounded nuclei were usually vesicular, occasionally hyperehromatic, and moderately pleomorphic. No invasion into the dermis was eneountered; however, the cells extended downward in the epitheli9J lining of hair follicles and of eccrine ducts. The blood capillaries were dilated and congested, lined by swollen endothelial cells, and surrounded by a dense infiltrate of plasma cells with occasional lymphocytes and polymorphonuolear leukocytes. The cytoplasm of the PAGET cells showed an affinity for alcian blue (Fig. 2). When the sections were immersed in alcian blue solutions containing magnesium chloride from 0.0025 to 0.2 M, no variation in the affinity for alcian blue was found. The affinity markedly reduced in the presence of magnesium chloride at 0.25 M, and completely disappeared at 0.45 M. Methylation for 4 h at 60°C abolished the affinity. When methylation was followed by saponification for 30 min at 25 DC, the affinity was partly noticed in the presence of magnesium chloride at 0.2 1\1 or less. When zirconium oxychloride was added to alcian blue solution, the affinity markedly reduced at 0.025 M. On the other hand, the cytoplasm showed an azurophilia at pH = 3.0 or higher. At pH = 3.0 or higher, it was metachromatic. Also, the cytoplasm of the PAGET cells stained deep red with standard PAS procedure (Fig. 3), and faint with two-step procedure. The reaction was completely blocked by acetylation or borohydride treatment, and weakened by acid hydrolYRis. When the sections were treated with potassium hydroxide following borohydride reduction, the reaction was not noticed. Malt-diastase, chondroitinase ABC, ribonuclease or desoxyribonuclease digestion did not change significantly staining with any of the methods used. Treatment with sialidase for 24 h weakened the PAS reactivity (Fig. 4) and the alcianophilia in the presence of magnesium chloride at 0.2 M or less.
Discussion All polyanions are pl"ecipitated by such quaternary ammoniul1l salts as cetylpyridiniulll chloride or aleian blue, and tho water-insoluble complexes are soluble in salt solutions, if the concentration of the salt is maintaim·d above a cel·tain level (SCOTT 19(0). Aeeording to a model experiment (SARAl 1971; SASAl et al. 1978), hyaluronic acid loses an affinity for alcian blue in the presence of magnesium ehleJl'ide at 0.1 M, chondroitin sulfate Band heparitin sulfate at 0.7 M, chcmdroitill sulfates A and C at O.S M, and hepar'in at 1.0 M. \\'hell zirconium oxyehloride is used UK an added salt, the affinity of aeid mncopolysat'{·harides for alcian blue disappE'ars at 0.05 M (hyalurmic acid),
The cytoplasmic mucin in
PAGET
Fig. 1. Extramammal'Y PAGET'S disease. Hematoxylin and eosin stain X 75. tered through epidermis.
Fig. 2. Extramammary PAGET's disease. Alcian blue stain showed an affinity for ale ian blue.
X
53
cells
PAGET
cells are scat·
150. The cytoplasm of the
PAGET
cells
54
Y.
SASAI
et a l.
Fig. 3. Extramammary 1'AGET'S d isease. PAS stain strongly reactive to l' AS.
Fig. 4. Extramammary 1'AGET'S disease. PAS stain wea kened the PAS reactivity.
X
150. The cytoplaRm of the
X
1'AGET
cells is
150. 'Treat ment with sialidasn for 24 h
The cytoplasmic mucin in PAGE'l' cells
55
0.25 M (ehondroitin wlfate A), 0.3 :l\i (heparitin sulfate), and 0.4 M (ehondroitin sulfates Band C and hpparin). All carboxyl grpupoJ' phosphate group· containing polyanions are in an undifferen· tiat('d group at about the same low criti('al salt ('oncpntration level as hyaluronic acid. The pattern of the (,J'itieal ,mIt ('oneentration of each polyanion in tissue seetions was similar to that in the model exp('rill1Pnt, although thp forll1Pl' was about 30 % highp!, than the latter. On the other hand, methy· latioll hydrolyzes sulfate groups, resulting in the' formation of frpe methyl esters, while it esterifies carboxyl and phosphatp groups. And, subseqm'nt saponifir'ation restores only thp latter two. In the present study, the eytoplasm of PAGET (,PUs showed an affinity for al('ian blup in the prcspn('e of magnpsiulll chloride at 0.4 M or Ipss. The affinity was bloekpd by methylation, and partly reo stored by saponification. Togpthel' with thp aZlll'ophilia at pH 3.0 or higher pH range, these results suggpst the prE'sE'nce of carboxyl gronp-('ontaining polyanioll in PAGET ('plls, A jY,sitive PAR readion llsllully indi,'atE's the prcsem'" of earbohydratp. Thporetically, all periodate-oxidizabk polysaccharides, whdher a('id 0[' neutral, should bp PAR-positive. In practieE', howevpr, acid mucopolysa('ch'lridps fail to give a positive roaction. RCO'l'T and HARBINSO:Y (HHi8, 1969) ,howed that the a('cess of ppriodate ions to oxidizablE' glycol grOllps of acid mucopolysaccharidE'S is redu('ed by the intense' negative elpf'trostati(' field of the poltanion, and that the enusing partial inhibition of oxidation ('an be largely or completely overcome by se('rE'ting the polyanionic charges with an inE'I't E'lE'ctrolytes. Thus, mu(,opolysaccharides ('ontaining 1: 4 linked ur(mic acids shoul
56
Y. SASAI et aI., The cytoplasmic mucin in PAGET cells
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Address: Dr. YOICHIRO SASAI, Department of Dermatology, Kurume University, School of Medicine, Kurume 830, Japan.