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The α subunit of S100 protein is present in tumor cells of human malignant melanoma, but not in schwannoma
Brain Research, 294 (1984) 381-384 Elsevier
381
BRE 20085
The a subunit of $100 protein is present in tumor cells of human malignant melanoma, but not in schwannoma TOSHIAKI ISOBE 1, KAYOKO ICHIMORI l , TAKASHI NAKAJIMA2 and TSUNEO OKUYAMA ~
1Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Setagaya-ku, Tokyo 158 and 2pathology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104 (Japan) (Accepted November 15th, 1983)
Key words: S 100 protein - - subunit distribution - - malignant melanoma - - schwannoma - - affinity chromatography - high performance liquid chromatography
The subunits of SI00 protein were isolated from surgically resected tissues of human malignant melanoma and schwannoma by means of an affinity chromatography followed by high performance liquid chromatography. The melanoma tissue gave rise to the a and fl subunits in almost equal quantities while the schwannoma tissue yielded only the fl subunit, indicating that the S100 subunits were distributed differently between these tumor tissues. This finding suggests that the cellular distribution of S10(Jsubunits varies, so that the cells containing S100 protein can be classified into several types on the basis of the subunit composition. $100 protein is a low molecular weight (mol.wt. 21,000) acidic calcium binding protein which is evolutionary related to calmodulin and other m e m b e r s of the calcium binding protein family 4,6,s. S100 protein, purified from various m a m m a l i a n brain tissues including human brain, is a mixture of at least 3 similar components, S100a 0, S100a, and S100b protein 2,7, with a subunit composition of aa (S100a0), aft (S100a), and tiff (S100b) 3,5,7. The amino acid sequence of the a subunit (93 residues) and the fl subunit (91 residues) are highly homologous to each other, by sharing 54 identical residues and 23 replacements compatible to single nucleotide-base substitutions 4,6. S100 protein appears to be a r e m a r k a b l y conservative protein as d e m o n s t r a t e d by comparative sequence studies of S100b protein s, as well as by the conservative immunological response among a wide variety of vertebrates j4,22. Extensive immunohistochemical studies using antibodies p r e p a r e d with a mixture of S100 protein have shown that $100 protein is localized in glial elements and Schwann cells of the nervous system 14.22, as well as in some non-neuroectodermal ceils such as Langerhans cells of the skin 1,15,16, interdigitating reticulum cells of the lymph
node a°,15,21, fat cells u, chondrocytes20, and in their related tumor cellsl7.1s. However, it is not known how each of the $100 protein is distributed and localized in those cells because such antibodies lack the specificity to distinguish one molecular species from the other. We report here that t u m o r cells of human schwannoma contain only the fl subunit of SI00 protein, while those of malignant m e l a n o m a contain both a and fl subunits. This finding suggests that there are differences in the cellular distribution of S100 subunits, and that $100 protein-positive cells can be classified into several types based on the subunits they contain. The tissues of malignant m e l a n o m a (amelanotic type) and schwannoma used in this study were surgically removed specimens (2 g wet weight each), and were typical in containing abundant S100 proteinpositive cells as assayed by i m m u n o p e r o x i d a s e stainingl9 of formalin-fixed and p a r a f f i n - e m b e d d e d sections (Fig. 1). We have p r e p a r e d soluble extracts of these tissues by homogenization in two volumes of 20 mM Tris-HCl (pH 7.5) containing 0.15 M NaCI, 4 mM MgCI2, and 1 mM CaCI 2 followed by centrifugation. Then the extracts were first applied to an af-
Correspondence: T. Isobe, Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Fukasawa 2-1-1, Setagaya-ku, Tokyo 158, Japan. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
382
280 nmT, 9. Amino acid compositional analysis and double diffusion immunochemical analysis using an anti-S100 serum were also performed for the peaks collected from H P L C to confirm the identification: The results are shown in Fig. 2. The protein fraction, obtained from the melanoma tissue, gave rise to 3 major peaks at retention times of 52.5, 61.0, and 64.0 min, which corresponded to calmodulin, a subunit, andfl subunit of $t00 protein~ re-
[0.16 I
1.28
Q (molignont melonomo)
0,%
; 0~12
o
0.32
-
0
o
Fig. l. a: immunohistochemical demonstration of $100 protein in malignant melanoma developed in the rectum. The cytoplasm of polygonal amelanotic melanoma cells is covered by diffuse immunoreaction product for SI00 protein. (× 150, counter-stained by hematoxylin.) b: mediastinal solitary schwannoma is weakly positive for S100 protein immunohistochemistry. (150, counter stained by hematoxylin.) finity column which is specific for $100 proteins and calmodulin ~2. Namely, the tissue extracts were applied to a column (1 × 5 cm) of N-l-naphthylethylenediamine-coupled Sepharose 4B equilibrated with 20 mM Tris-HCl (pH 7.5) containing 0.15 M NaCI, 4 m M MgC12, and 1 mM CaCI 2, and after washing the column with the same buffer adsorbed S100 proteins and calmodulin were eluted with a buffer composed of 20 m M Tris-HCl (pH 7.5) containing 0.15 M NaC1, 4 m M MgCI2, and 4 mM E G T A (ethylene glycol bis (fl-aminoethyl ether)N,N'-tetra acetic acid). The isolated protein mixture was then subjected to high performance liquid chromatography (HPLC) on a Hitachi-gel 3013 resin, where the S100 subunits and calmodulin are separated and identified in terms of retention time and characteristic ultraviolet absorption in the effluent monitoring at 210 and
-
o RETENTION IME (mtn)
o,~
o,~i
T o.32
o.Q,
/
......................................................,"..., ~ .~ ,,' ,' ,- ' - ~ 0
0 20
40
60
80
RETENTIO~ITIME (mln)
Fig. 2. Identification of S100 subunits and calmodulin in tumor tissues of human malignant melanoma (a) and schwannoma (b), The mixtures of S100 proteins and catmodulin, isolated from 2 g each of the tissues by affinity chromatography, were analyzed by high performance liquid chromatography under the following conditions 7.9. Column: Hitachi-Gel 3013.0.46 × 25 cm. Elution: linear gradient of acetonitrile (20-75% v/v) in 0.1% trifluoroacetic acid. Flow rate: 0.5 ml/min. Temperature: 40 °C. Detection: UV 210 nm and 28(1 nm.
383 subunits showed amino acid compositions very simi-
TABLE I Amino acid compositions of a and fl subunit isolated from human neoplastic tissues Amino acid
Lys His Arg Asx Thr Set GIx Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Trp~
Melanoma
Schwannoma
a subunit
fl subunit
fl subunit
8.6 2.0 0.8 12.7 3.9 4.3 15.0 0.0 6.3 6.8 + 6.8 1.7 1.8 9.8 1.9 4.9 0.9
8.3 4.3 1.5 10.3 3.2 4.1 19.0 0.0 3.6 5.8 + 5.7 3.2 3.2 7.8 1.3 6.9 0.0
8.6 (8)b 4.9 (5) 1.4 (1) 10.0 (10) 3.0 (3) 3.6 (4) 20.2 (19) 0.1) (01 4.1 (4) 5.11 (5) + (2) 6.0 (7) 3.1 (4) 3.0 (3) 7.9 (8) 1.1 (1) 6.5 (7) 11.0 (0)
(9p (2) (01 (131 (4) (5) (15) (0) (6) (8) (1/ (8) (2) (11 (I1) (2) (5) (1)
The numbers of residues computed from the amino acid sequence of bovine brain cxsubunit6, b The numbers of residues computed from the amino acid sequence of human brain fl subunitT.'L Determined spectrophotometrically. spectively (Fig. 2a). On the other hand, the protein fraction from the schwannoma tissue resulted in major peaks of calmodulin (52.5 rain) and the fl subunit of S100 protein (64.0 rain), but no significant peak was detected at a position of the a subunit (61.0 rain; indicated by an arrow in Fig. 2b), The isolated S100
1 Coccia, D., Michetti, F. and Donato, R., Immunochemical and immunocytochemical localization of S100 antigen in normal human skin, Nature (Lond.), 294 (1981) 85-87. 2 Isobe, T.. Nakajima, T. and Okuyama, T., Reinvestigation of extremely acidic proteins in bovine brain, Biochim. biophys. Acta, 494 (1977) 222-232. 3 Isobe, T., Tsugita, A. and Okuyama, T., The amino acid sequence and the subunit structure of bovine brain S100 protein (PAP I-b), J. Neurochem., 30 (1978) 921-923. 4 Isobe, T. and Okuyama, T., The amino acid sequence of S100 protein (PAP I-b protein) and its relation to the calcium-binding proteins. Europ. J. Biochem., 89 (1978) 379-388. 5 Isobe, T., Ishioka, N. and Okuyama, T., Structural relation ot two S-100 proteins in bovine brain: subunit composition of S-100a protein, Europ. J. Biochem.. 115 (1981) 469-474. 6 lsobe, T. and Okuyama, T., The amino acid sequence of the c~subunit in bovine brain S100a protein, Europ. J. Biochem., 116 (1981) 7%86. 7 Isobe. T., Ishioka, N., Masuda, T., Takahashi, Y., Ganno,
lar to the equivalent subunits purified from normal brain tissue 4,6-8 (Table I), suggesting that these neoplastic tissues produced S100 subunits that were structurally identical to those of the normal tissue. In the case of schwannoma tissue, another protein peak having a retention time of 58.0 rain was observed more clearly than in the m e l a n o m a tissue (Fig. 2b), but this was different from the S100 subunits or calmodulin in terms of amino acid composition. We have examined two cases of each type of tumor with essentially the same results. These results show that the S100 subunits are distributed differently between these neoplastic tissues, where tumor cells of malignant m e l a n o m a contain both ct and fl subunits in almost equal quantities, while schwannoma cells contain only the fl subunit, or S100b protein. This indicates that there are cellular differences in the distribution of S100 subunits, so that cells containing S100 protein can be classified into several types on the basis of subunit composition. Our finding, that the tumor cells of malignant melanoma are clearly distinguishable from schwannoma in containing the (z subunit, also implies that neoplastic melanocytes are rather less closely related ontogenetically to Schwann cells than previously thoughfl 3, even though both cell types are derived from neural crest cells. Thus, the analysis of S100 subunits appears to be a clue to elucidate the origin of cells when the cells contain S100 protein.
S. and Okuyama, T., A rapid separation ot S100 subunits by high performance liquid chromatography: the subunit compositions of S100 proteins, Biochem. Int., 6 (19831 419-426. 8 lsobe, T., Ishioka, N., Kocha, T. and Okuyama, T., Chemical structure and molecular evolution of S100 proteins. In H. Peeters (Ed.), Protides of the Biological Fluids', Vol. 30, Pergamon, Oxford, 1983, pp. 21-24. 9 lsobe, T., Kurosu, Y. and Okuyama, T., High performance liquid chromatography ¢~fpeptides on macroreticular polystyrene resins. In H. Peeters (Ed.), Protides ((the Biological Fluids, Vol. 30, Pergamon, Oxford, 1983, pp. 727-730. 10 Iwanaga, T., Fujita, T., Masuda, T. and Takahashi. Y., S100 protein-immunoreactive cells in the lymph node and spleen of the rat, Arch. histol, jap., 45 (1982) 393-397. 11 Kato, K., Suzuki, F. and Nakajima, T., S100 protein in adipose tissue, Int. J. Biochem., 15 (1983) 6(19-613. 12 Kocha, T., Kadoya, T., Isobe, T. and Okuyama, T., Isolation of calmodulin and S100 protein by affinity chromatography on a N-l-naphthylethylenediamine-coupled Sepha-
384 rose, Bull. Jap. biochem. Soc., 54 (1982) 535 (in Japanese). 13 Mishima, Y. and Matsunaka, M., Pagetoid premalignant melanosis and malanoma: differentiation from Hutchinson's melanotic fleckle, J. invest. Dermatol., 65 (1975) 434-440. 14 Moore, B. W., Chemistry and biology of two proteins, SI00 and 14-3-2, specific to the nervous system, Int. Rev. Neurobiol., 15 (1972) 215-225. 15 Nakajima, T., Watanabe, S., Sato, Y., Shimosato, Y., Motoi, M. and Lennert, K., S100 protein in Langerhans cells, interdigitating reticulum cells and histiocytosis X cells. Gann, 73 (1982) 429-432. 16 Nakajima, T., Sato, Y., Watanabe, S., Shirnosato, Y., Ishihara, K. and Isobe, T., Immunoelectron microscopic demonstration of S100 protein in epidermal Langerhans celt, Biomed. Res., 3 (1982) 226-231. 17 Nakajima, T., Watanabe, S., Sato, Y., Kameya, T., Shimosato, Y. and Ishihara, K., Immunohistochemical demonstration of S100 protein in malignant melanoma and pigmented nevus, and its diagnostic application, Cancer, 50 (1982) 912-918. 18 Nakajima, T., Kameya, ;F., Watanabe, S., Hirota, T.,
19
20
21
22
Sato. Y. and Shimosato. Y.. An tmmunoperoxidase study of S100 protein distribution in normal and neoplastic tissues. Amer. J. Surg. Path.. 6 (1982) 71.5--727. Sternberger. L. A.. Hardy, P. H lr Cuculis. J J. and Meyer H G.. The unlabelled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex I horseradish peroxidase-an.tihorseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cvtochem. 18 (1970) 315-333 Stefasson. K.. Wollmann. R L.. Moore. B. W and Arnason. B. G. W.. S100 protein in human chondroc~ tes. Nature (Lond. p, 295 (1982) 63--64 Takahashi. K.. Yamaguchi. H.. Ishizekl. J . Nakajima. T and Nakazato. Y.. Immunohistochemical and immunoelectronmicroscopic localization of S I00 protein in the interdigitating reticulum cells of the human lymph node, Virchows Arch. (Cell Pathology), 37 ( 1981 ) t25-135. Zomzely-Neurath. C. E. and Walker. W A.. Nervous system-specific protein: 14-3-2 protein, neuron-specific enolase. and SI00 protein. In R A. Bradshaw and D. M Schneider (Eds.), Proteins of the Nervous Svstern. f2nd edn. ~, Raven Press. New York. 19~0, pp. 30-57
381
BRE 20085
The a subunit of $100 protein is present in tumor cells of human malignant melanoma, but not in schwannoma TOSHIAKI ISOBE 1, KAYOKO ICHIMORI l , TAKASHI NAKAJIMA2 and TSUNEO OKUYAMA ~
1Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Setagaya-ku, Tokyo 158 and 2pathology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104 (Japan) (Accepted November 15th, 1983)
Key words: S 100 protein - - subunit distribution - - malignant melanoma - - schwannoma - - affinity chromatography - high performance liquid chromatography
The subunits of SI00 protein were isolated from surgically resected tissues of human malignant melanoma and schwannoma by means of an affinity chromatography followed by high performance liquid chromatography. The melanoma tissue gave rise to the a and fl subunits in almost equal quantities while the schwannoma tissue yielded only the fl subunit, indicating that the S100 subunits were distributed differently between these tumor tissues. This finding suggests that the cellular distribution of S10(Jsubunits varies, so that the cells containing S100 protein can be classified into several types on the basis of the subunit composition. $100 protein is a low molecular weight (mol.wt. 21,000) acidic calcium binding protein which is evolutionary related to calmodulin and other m e m b e r s of the calcium binding protein family 4,6,s. S100 protein, purified from various m a m m a l i a n brain tissues including human brain, is a mixture of at least 3 similar components, S100a 0, S100a, and S100b protein 2,7, with a subunit composition of aa (S100a0), aft (S100a), and tiff (S100b) 3,5,7. The amino acid sequence of the a subunit (93 residues) and the fl subunit (91 residues) are highly homologous to each other, by sharing 54 identical residues and 23 replacements compatible to single nucleotide-base substitutions 4,6. S100 protein appears to be a r e m a r k a b l y conservative protein as d e m o n s t r a t e d by comparative sequence studies of S100b protein s, as well as by the conservative immunological response among a wide variety of vertebrates j4,22. Extensive immunohistochemical studies using antibodies p r e p a r e d with a mixture of S100 protein have shown that $100 protein is localized in glial elements and Schwann cells of the nervous system 14.22, as well as in some non-neuroectodermal ceils such as Langerhans cells of the skin 1,15,16, interdigitating reticulum cells of the lymph
node a°,15,21, fat cells u, chondrocytes20, and in their related tumor cellsl7.1s. However, it is not known how each of the $100 protein is distributed and localized in those cells because such antibodies lack the specificity to distinguish one molecular species from the other. We report here that t u m o r cells of human schwannoma contain only the fl subunit of SI00 protein, while those of malignant m e l a n o m a contain both a and fl subunits. This finding suggests that there are differences in the cellular distribution of S100 subunits, and that $100 protein-positive cells can be classified into several types based on the subunits they contain. The tissues of malignant m e l a n o m a (amelanotic type) and schwannoma used in this study were surgically removed specimens (2 g wet weight each), and were typical in containing abundant S100 proteinpositive cells as assayed by i m m u n o p e r o x i d a s e stainingl9 of formalin-fixed and p a r a f f i n - e m b e d d e d sections (Fig. 1). We have p r e p a r e d soluble extracts of these tissues by homogenization in two volumes of 20 mM Tris-HCl (pH 7.5) containing 0.15 M NaCI, 4 mM MgCI2, and 1 mM CaCI 2 followed by centrifugation. Then the extracts were first applied to an af-
Correspondence: T. Isobe, Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Fukasawa 2-1-1, Setagaya-ku, Tokyo 158, Japan. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
382
280 nmT, 9. Amino acid compositional analysis and double diffusion immunochemical analysis using an anti-S100 serum were also performed for the peaks collected from H P L C to confirm the identification: The results are shown in Fig. 2. The protein fraction, obtained from the melanoma tissue, gave rise to 3 major peaks at retention times of 52.5, 61.0, and 64.0 min, which corresponded to calmodulin, a subunit, andfl subunit of $t00 protein~ re-
[0.16 I
1.28
Q (molignont melonomo)
0,%
; 0~12
o
0.32
-
0
o
Fig. l. a: immunohistochemical demonstration of $100 protein in malignant melanoma developed in the rectum. The cytoplasm of polygonal amelanotic melanoma cells is covered by diffuse immunoreaction product for SI00 protein. (× 150, counter-stained by hematoxylin.) b: mediastinal solitary schwannoma is weakly positive for S100 protein immunohistochemistry. (150, counter stained by hematoxylin.) finity column which is specific for $100 proteins and calmodulin ~2. Namely, the tissue extracts were applied to a column (1 × 5 cm) of N-l-naphthylethylenediamine-coupled Sepharose 4B equilibrated with 20 mM Tris-HCl (pH 7.5) containing 0.15 M NaCI, 4 m M MgC12, and 1 mM CaCI 2, and after washing the column with the same buffer adsorbed S100 proteins and calmodulin were eluted with a buffer composed of 20 m M Tris-HCl (pH 7.5) containing 0.15 M NaC1, 4 m M MgCI2, and 4 mM E G T A (ethylene glycol bis (fl-aminoethyl ether)N,N'-tetra acetic acid). The isolated protein mixture was then subjected to high performance liquid chromatography (HPLC) on a Hitachi-gel 3013 resin, where the S100 subunits and calmodulin are separated and identified in terms of retention time and characteristic ultraviolet absorption in the effluent monitoring at 210 and
-
o RETENTION IME (mtn)
o,~
o,~i
T o.32
o.Q,
/
......................................................,"..., ~ .~ ,,' ,' ,- ' - ~ 0
0 20
40
60
80
RETENTIO~ITIME (mln)
Fig. 2. Identification of S100 subunits and calmodulin in tumor tissues of human malignant melanoma (a) and schwannoma (b), The mixtures of S100 proteins and catmodulin, isolated from 2 g each of the tissues by affinity chromatography, were analyzed by high performance liquid chromatography under the following conditions 7.9. Column: Hitachi-Gel 3013.0.46 × 25 cm. Elution: linear gradient of acetonitrile (20-75% v/v) in 0.1% trifluoroacetic acid. Flow rate: 0.5 ml/min. Temperature: 40 °C. Detection: UV 210 nm and 28(1 nm.
383 subunits showed amino acid compositions very simi-
TABLE I Amino acid compositions of a and fl subunit isolated from human neoplastic tissues Amino acid
Lys His Arg Asx Thr Set GIx Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Trp~
Melanoma
Schwannoma
a subunit
fl subunit
fl subunit
8.6 2.0 0.8 12.7 3.9 4.3 15.0 0.0 6.3 6.8 + 6.8 1.7 1.8 9.8 1.9 4.9 0.9
8.3 4.3 1.5 10.3 3.2 4.1 19.0 0.0 3.6 5.8 + 5.7 3.2 3.2 7.8 1.3 6.9 0.0
8.6 (8)b 4.9 (5) 1.4 (1) 10.0 (10) 3.0 (3) 3.6 (4) 20.2 (19) 0.1) (01 4.1 (4) 5.11 (5) + (2) 6.0 (7) 3.1 (4) 3.0 (3) 7.9 (8) 1.1 (1) 6.5 (7) 11.0 (0)
(9p (2) (01 (131 (4) (5) (15) (0) (6) (8) (1/ (8) (2) (11 (I1) (2) (5) (1)
The numbers of residues computed from the amino acid sequence of bovine brain cxsubunit6, b The numbers of residues computed from the amino acid sequence of human brain fl subunitT.'L Determined spectrophotometrically. spectively (Fig. 2a). On the other hand, the protein fraction from the schwannoma tissue resulted in major peaks of calmodulin (52.5 rain) and the fl subunit of S100 protein (64.0 rain), but no significant peak was detected at a position of the a subunit (61.0 rain; indicated by an arrow in Fig. 2b), The isolated S100
1 Coccia, D., Michetti, F. and Donato, R., Immunochemical and immunocytochemical localization of S100 antigen in normal human skin, Nature (Lond.), 294 (1981) 85-87. 2 Isobe, T.. Nakajima, T. and Okuyama, T., Reinvestigation of extremely acidic proteins in bovine brain, Biochim. biophys. Acta, 494 (1977) 222-232. 3 Isobe, T., Tsugita, A. and Okuyama, T., The amino acid sequence and the subunit structure of bovine brain S100 protein (PAP I-b), J. Neurochem., 30 (1978) 921-923. 4 Isobe, T. and Okuyama, T., The amino acid sequence of S100 protein (PAP I-b protein) and its relation to the calcium-binding proteins. Europ. J. Biochem., 89 (1978) 379-388. 5 Isobe, T., Ishioka, N. and Okuyama, T., Structural relation ot two S-100 proteins in bovine brain: subunit composition of S-100a protein, Europ. J. Biochem.. 115 (1981) 469-474. 6 lsobe, T. and Okuyama, T., The amino acid sequence of the c~subunit in bovine brain S100a protein, Europ. J. Biochem., 116 (1981) 7%86. 7 Isobe. T., Ishioka, N., Masuda, T., Takahashi, Y., Ganno,
lar to the equivalent subunits purified from normal brain tissue 4,6-8 (Table I), suggesting that these neoplastic tissues produced S100 subunits that were structurally identical to those of the normal tissue. In the case of schwannoma tissue, another protein peak having a retention time of 58.0 rain was observed more clearly than in the m e l a n o m a tissue (Fig. 2b), but this was different from the S100 subunits or calmodulin in terms of amino acid composition. We have examined two cases of each type of tumor with essentially the same results. These results show that the S100 subunits are distributed differently between these neoplastic tissues, where tumor cells of malignant m e l a n o m a contain both ct and fl subunits in almost equal quantities, while schwannoma cells contain only the fl subunit, or S100b protein. This indicates that there are cellular differences in the distribution of S100 subunits, so that cells containing S100 protein can be classified into several types on the basis of subunit composition. Our finding, that the tumor cells of malignant melanoma are clearly distinguishable from schwannoma in containing the (z subunit, also implies that neoplastic melanocytes are rather less closely related ontogenetically to Schwann cells than previously thoughfl 3, even though both cell types are derived from neural crest cells. Thus, the analysis of S100 subunits appears to be a clue to elucidate the origin of cells when the cells contain S100 protein.
S. and Okuyama, T., A rapid separation ot S100 subunits by high performance liquid chromatography: the subunit compositions of S100 proteins, Biochem. Int., 6 (19831 419-426. 8 lsobe, T., Ishioka, N., Kocha, T. and Okuyama, T., Chemical structure and molecular evolution of S100 proteins. In H. Peeters (Ed.), Protides of the Biological Fluids', Vol. 30, Pergamon, Oxford, 1983, pp. 21-24. 9 lsobe, T., Kurosu, Y. and Okuyama, T., High performance liquid chromatography ¢~fpeptides on macroreticular polystyrene resins. In H. Peeters (Ed.), Protides ((the Biological Fluids, Vol. 30, Pergamon, Oxford, 1983, pp. 727-730. 10 Iwanaga, T., Fujita, T., Masuda, T. and Takahashi. Y., S100 protein-immunoreactive cells in the lymph node and spleen of the rat, Arch. histol, jap., 45 (1982) 393-397. 11 Kato, K., Suzuki, F. and Nakajima, T., S100 protein in adipose tissue, Int. J. Biochem., 15 (1983) 6(19-613. 12 Kocha, T., Kadoya, T., Isobe, T. and Okuyama, T., Isolation of calmodulin and S100 protein by affinity chromatography on a N-l-naphthylethylenediamine-coupled Sepha-
384 rose, Bull. Jap. biochem. Soc., 54 (1982) 535 (in Japanese). 13 Mishima, Y. and Matsunaka, M., Pagetoid premalignant melanosis and malanoma: differentiation from Hutchinson's melanotic fleckle, J. invest. Dermatol., 65 (1975) 434-440. 14 Moore, B. W., Chemistry and biology of two proteins, SI00 and 14-3-2, specific to the nervous system, Int. Rev. Neurobiol., 15 (1972) 215-225. 15 Nakajima, T., Watanabe, S., Sato, Y., Shimosato, Y., Motoi, M. and Lennert, K., S100 protein in Langerhans cells, interdigitating reticulum cells and histiocytosis X cells. Gann, 73 (1982) 429-432. 16 Nakajima, T., Sato, Y., Watanabe, S., Shirnosato, Y., Ishihara, K. and Isobe, T., Immunoelectron microscopic demonstration of S100 protein in epidermal Langerhans celt, Biomed. Res., 3 (1982) 226-231. 17 Nakajima, T., Watanabe, S., Sato, Y., Kameya, T., Shimosato, Y. and Ishihara, K., Immunohistochemical demonstration of S100 protein in malignant melanoma and pigmented nevus, and its diagnostic application, Cancer, 50 (1982) 912-918. 18 Nakajima, T., Kameya, ;F., Watanabe, S., Hirota, T.,
19
20
21
22
Sato. Y. and Shimosato. Y.. An tmmunoperoxidase study of S100 protein distribution in normal and neoplastic tissues. Amer. J. Surg. Path.. 6 (1982) 71.5--727. Sternberger. L. A.. Hardy, P. H lr Cuculis. J J. and Meyer H G.. The unlabelled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex I horseradish peroxidase-an.tihorseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cvtochem. 18 (1970) 315-333 Stefasson. K.. Wollmann. R L.. Moore. B. W and Arnason. B. G. W.. S100 protein in human chondroc~ tes. Nature (Lond. p, 295 (1982) 63--64 Takahashi. K.. Yamaguchi. H.. Ishizekl. J . Nakajima. T and Nakazato. Y.. Immunohistochemical and immunoelectronmicroscopic localization of S I00 protein in the interdigitating reticulum cells of the human lymph node, Virchows Arch. (Cell Pathology), 37 ( 1981 ) t25-135. Zomzely-Neurath. C. E. and Walker. W A.. Nervous system-specific protein: 14-3-2 protein, neuron-specific enolase. and SI00 protein. In R A. Bradshaw and D. M Schneider (Eds.), Proteins of the Nervous Svstern. f2nd edn. ~, Raven Press. New York. 19~0, pp. 30-57