Expression pattern of S100 calcium-binding proteins in human tumors

hi t . J. Cancer: 68, 325-332 (1996) G 1996 Wilcy-Liss, Inc.

Publication of the International Unlon Against Cancer Publication de I’Union Internationale Contre le Cancel

EXPRESSION PATTERN OF SlOO CALCIUM-BINDING PROTEINS IN HUMAN TUMORS Evelyn C. ILG, Beat W. SCIIAFER and Claus W. ~ I E I Z M A N N ’ Division of Clinical Chemistiy, Department of Pediam’cs, University of Zurich, Zirrich, Switzerland.

The SlOO &’+-binding proteins recently became of major interest because of their differential expression in neoplastic tissues, their involvement in metastatic processes, and the clustered organization of at least 10 SlOO genes on human chromosome I q2 I, a region fre uently rearranged In several tumors. As a first attempt towar% a specific and differentiated immunohistochemical classification of human tumors, we produced, purified and characterized a number of human recornbinant SlOO proteins and raised specific polyclonal antibodies. Their distinct cellular and intracellular localization was exarn- ined by immunohistochemical methods in normal and cancerogenic human tissues and cell lines. S I OOA I and S I OOA2 can be detected in a few normal tissues only, whereas S I OOA4, S I OOA6, and SIOOB are expressed at higher levels in cancer tissues. In the future, these SlOO antibodies will potentially be of great value in cancer diagnosis and therapy. G 1996 Wiley-Liss, Inc.

Calcium rcgulates a variety of biological proccsscs as second messenger. Ca?’-signals are transduced into intraccllular rc- sponses via interaction with Ca2+-binding protcins characterized by a common structural motif, the EF-hand (reviewed in HciLmann and Braun, 1995). This large protein family includes the SlOO proteins which have recently become of major interest because of their differential expression in neoplastic tissues and involvement in metastatic proccsscs (reviewed in Schafcr and HeiLmann, 1996). Some 17 differcnt proteins have been assigned to the SlOO protein family and at least 10 SlOO genes are found clustered on chromosome lq21 (Engelkampet al., 1993). This led to the introduction of a ncw nomenclature uscd throughout this study (Schafer et al., 1995). The chromo- somal region lq21 is frcqucntly rearranged (Gendler et al., 1990), and this might bc responsible for the differential regulation of SlOO protcins in tumor cells.

Antibodics against SlOO proteins are uscd worldwide in many hospitals for the immunohistochcmical classification of tumors in children and adults, including tumors of benign or malignant neuroendocrinc origin (Barrctt and Scully, 1994), melanomas (Hua Bei Guo et al., 1995), thyroid carcinoma (Nagasaka et al., 1987), renal carcinoma, cystic teratomas, Langcrhans-cell histiocytosis, sustentacular cclls of pheochro- mocytomas, tumors of sweat-gland origin, pleomorphic adeno- mas of the salivary gland, bronchoalveolar carcinomas of the lung, and various lesions of cartilage (Kahn et al., 1983). In fact, SlOO antibodies wcrc among the first markcrs for melanocytic tumors (Ben-Izhak et al., 1994) and arc able to detect small numbers of metastatic cells not dctcctablc by conven- tional histology (Cochran et al., 1993). Howcvcr, the commercially available antisera uscd in those studies were directed against a mixture of bovine brain SlOO proteins. Depending on thc manufacturer, the antibodies react with SlOOAl and/or SlOOB and against some novel SlOO proteins characterized in this study.

For a spccific and more differentiated immunohistochemical staining of various tumor tissues, we produced, purified and characterized a number of human recombinant SlOO proteins and were able to raise specific polyclonal antibodies against individual human SlOO proteins. In this study we included S100A4, thought to play an important role in thc acquisition of a metastatic phenotype (Davies et al., 1993), S100A6, which is ovcrcxpressed in many tumor cells (Wcterman ef al., 1992; Pedrocchi et al., 19946). and S100A2, which is generally

down-regulated in tumor cells (Lee et af., 1992). Thc distinct cellular and intraccllular localization of these SlOO proteins was examined by immunohistochcmical methods in normal and tumorigenic human tissues and in human tumor-cell lines. The combincd results demonstrate that we obtained a palette of well-defined and specific antibodies against human SlOO proteins, dircctly applicable to routine tumor analysis.

MATERIAL A N D METHODS Cloning of human SIOOAI, SIOOA2, and SlOOB into a prokaiyotic expression system

Coding regions of full-length S100A1, SlOOA2 and SlOOB clones werc amplificd by PCR, using specific oligonucleotides containing an NdeI site (N-terminal oligos) and a Hind111 or BamHI site (C-terminal oligos). The N-terminal NdcI site was introduced using the start site of translation. The amplified DNA was cut with the corresponding restriction cnzymcs and introduced into thc expression vector pGEMEX (Promega, Madison, WI). Plasmids from transformed Escherichia coli strain TG-1 were sequenced by the dideoxynucleotidc chain termination method with an A.L.F. DNA Sequenccr (Pharma- cia LKB, Uppsala, Sweden) to check for correct open reading frames. The plasmids were then transformed into the E. coli strain BL-21 Lys S for bacterial expression.

By sequencing we found a discrepancy in the S1OOA2 cDNA compared to the database, which was confirmed by sequencing genomic DNA: AAC + AGC (mutation at position 222). The translated protcin therefore contains a serine instead of an aspara- gine at position 62, leading to an altered molecular weight, which needs to be taken into account in mass spectrometry.

Oligonucleotides were synthesized on a Gcnc Assembler DNA synthesizer (Pharmacia LKB). The primers uscd to amplify the cDNA for cloning into pGEMEX wcrc as follows:

S1 OOAl-5: 5’-AGTCATATGGGCTCTGAGCTGGAG-3’

S lOOA 1-3: S‘-AGTAAGCITGGTGAGGTGGAAGCAGG-3‘

S100A2-5: 5’-AGCCATATGATGTGCAGTTCTCTGGA-3’ S 100A2-3: 5’-AGCAAGCTTCCTGGGCCCAAGAGATC-3’

S100B-5: 5’-GAAGCATATGTCTGAGCTGGAGAAGG-3’

S 1OOB-3: 5 ’-GCGGATCCTGTCTGCC‘ITGCATG-3’

’To whom correspondence and reprint requests should be sent, at the Division of Clinical Chemistry, Department of Pediatrics, Stein- wiesstr. 75, CH-8032 Zurich. Fax: 41 1 266 71 69.

Ahhreviurions: BSA, bovine serum albumin; DTT, dithiothreitol; EGTA, ethylene glycol bis (P-aminoethy1)-N,N,N’,N’-tetraacetate; FITC, fluorescein-5-isothiocyanate; FCS, fetal calf serum; HPLC, high-performance liquid chromatography; IPTG, isopro yl p D thiogalactopyranoside; NP-40, ethylphenylpolyethylene gl co , PAGE $Irrylamide gel electrophoresis; PBS, hos hate-bdered saline:

C , polymerasc chain reaction: PMSF, pl?enykkthanesulfonyl fluo- ride; TRITC, tetramethylrhodamine-5-(and-6)-isothiocyanate; SDS, sodium dodecyl sulfate.

f - - -

Received: May 18, 1996 and in revised form July 15, 1996.

326 ILG E7'A.L.

The inserted restriction sites for NdeI, HindIII, and BamHI are underlined.

htrification and characterization of recombinant human SIOOAI, SlOOA2, SlOOA4, SIOOA6, and SlOOB by mass spectrometry, gel electrophoresis, and isoelectnc focusing

Recombinant SlOO proteins were purified and their molecular mass was analyzed by electrospray ionization mass spectrometry as described by Pedrocchi et al. (1994~). Tricine SDS- PAGE (15%) under reducing conditions was performed as described (Fohr et aL, 1993). In taurinc SDS-PAGE (non- reducing, 5-15%; reducing, 11%) the trailing ion is replaced (gel buffer, 600 mM Tris-HCI, pH 8.6; cathode buffer, 100 mM Tris-HC1, 100 mM taurine, pH 8.4; anode buffer, 200 mM Tris-HCI, pH 8.4). For reducing conditions 150 mM DTT was added to the sample buffer. Proteins were visualized by Coomassie blue staining (0.25% brilliant blue R-250 in 50% methanol, 8% acetic acid).

Isoelectric focusing was performcd under native and denaturing/reducing (8 M urea and 1% 2-mercaptoethanol) conditions (Bollag and Edelstein, 1991). Carrier ampholytes, pH 3-10 and pH 4-6.5, were obtained from Pharmacia.

Production of polyclonal antibodies against human recombinant S100 proteins

Antiserum against S100A2 was raised in rabbits. Animals were immunized 4 times (at 3-weekly intervals) by S.C. injection of 500 pg of protein, followed by 250 pg of protcin for further boosts in an emulsion with Hunter's Titer Max (Cyt RX, Norcross, GA). Antisera against SIOOAI, S100A4 and S100A6 were raised in goats. Animals were immunized 6 times (at 4-weekly intervals) by injections of 1 mg of protein. The titers were determined with 50 ng and 1,000 ng of recombinant protein on Western blots. Antisera were diluted maximally: 1:5,000 (anti SlOOAI), 1:10,000 (anti SlOOA2), 1:1,000 (anti S100A4) and 1:15,000 (anti S100A6) with 50 ng, or 1:15,000 (anti SlOOAl), 1:75,000 (anti S100A2), 1:50,000 (anti S100A4), and 1:75,000 (anti S100A6) with 1,000 ng of protein.

For the immunolocalization of Sl00 proteins in human tissues, the polyclonal goat antibodies were purified by affinity chromatography. Recombinant S100A1, S100A4, and S100A6 proteins wcrc coupled to CNBr-activated sepharose 4B. Anti- sera diluted 1:lO in 10 mM Tris, pH 7.5, were loaded. The column was washed with 10 mM Tris, pH 7.5, followed by 0.5 M NaC1, 10 mM Tris, pH 7.5. Specific IgGs were eluted with 100 mM glycine, pH 2.5, and dialyzed against PBS.

For control incubations, affinity-purified antibodies were preabsorbed with the corresponding antigcns. To this end, 50 to 100 Fg of recombinant SlOOAl, S100A4 or S100A6 were added to 100 p1 undiluted affinity-purified antibody fraction and incubated for 3 hr at 4°C. After centrifugation at 14,00Og, the supernatant was taken for control stainings.

Polyclonal antisera against bovine brain Sl00 extracts were purchased from Dakopatts (Copenhagen, Denmark), Sigma (St. Louis), and Immunotech (Marseille, France).

Western blot analysis Total cell extracts were prepared as described previously

(Pedrocchi et al., 19946). Proteins were first separated on tricine SDS-PAGE, then blotted at 50 V for 1 hr onto 0.2 p n nitrocellulose (Schleicher and Schuell, Dassel, Germany), and finally cross-linked to the membrane by UV irradiation (480 mJ; Stratalinker, Stratagene, La Jolla, CA). Proteins were visualized with Ponceau S staining. The mcmbranc was blocked overnight in 3% BSA and 1% FCS and then incubated with antibodies against the SlOO proteins. The bands were visualized using a horseradish-peroxidax coupled secondary anti-

body diluted 1:1,500 (anti-goat: Sigma; anti-rabbit and mouse: BioRad, Richmond, CA).

Immunofluorescent staining The human mctastatic breast adenocarcinoma cell line

MDA-MB-231 (Garcia et al., 1992) and normal breast epithelial cell line HBL-100 from the ATCC (Rockville, MD) were grown on sterile glass cover-slips. For immunofluorescent staining, cells were washed with DMEM and fixed in 3.7% paraformaldehyde for 15 min at 37°C. After 2 washes with DMEM containing 5% horse serum, cells were permeabilized in ice-cold methanol for 10 min. The subsequent steps were performed in DMEM/horse serum. After blocking for 30 min, cells were incubated with the antibodies against the SlOO proteins (S100A1 diluted 1:300; S100A2, 1:300; S100A4, 1:lOO; SlOOA6, 1:200) for 2 hr at 37°C. Cells were then washed and incubated with an FITC coupled second antibody (Cappcl, Turnhout, Belgium; diluted 1:200) for 2 hr at 37°C. Cells were finally washed with PBS, pH 8.5. The cover-slips were mounted on glass slides, embedded with Mowiol (Hoechst, Frankfurt, Germany) and examined on a Zeiss fluorescence microscope equipped with the appropriate fluoresccnt filters. For the co-localization of S l O O A 2 and S100A6, FITC-conjugated donkey anti-goat IgGs and TRITC-conjugated donkey anti-rabbit IgGs (purchased from Accurate Chemical, New York, NY; diluted 1:200) were used. Control incubations were done with pre-immune serum.

lmmunohistochemistty Multi-tissue slides containing a composite of mounted

normal and cancerogenic human tissue sections embedded in paraffin (Biomeda, Foster City, CA) were incubated for 15 min with normal rabbit serum, then for 2 hr at RT with the affinity-purified IgGs against SlOOAl (60 pg/ml), S100A4 (180 pg/ml) or SlOOA6 (13 pg/ml) in RPMI 1640 medium (GIBCO, Gaithersburg, MD) with 4% powdered milk. After washing in 150 mM NaCI, 50 mM Tris, pH 7.6, the tissue sections were incubated for 1 hr with rabbit anti-goat/alkaline phosphatase (1:200, Cappel) in RPMI 1640 medium. For the immunolocalization of S100A2, the human tissue sections were incubated with normal goat scrum for 15 min, then with rabbit antiserum against SlOOA2, diluted 1:200, for 1 hr. After 30-min incubation steps with mouse anti-rabbit (1:25) and rabbit anti-mouse IgGs (1:20), anti-phosphatase IgGs and alkaline phospatase (APAAP, 150) were added. Enzyme activity was visualized with naphthol-AS-BI-phosphate and new fuchsin as sub- strates.

Control incubations were done with pre-immune serum (S100A2) or with affinity-purified antibodies pre-absorbed with SlOO proteins (S100A1, S100A4, and S100A6) as dc- scribed above.

RESULTS Expression, prcrification, and biochemical properties of recombinant human SIOOAI, SlOOA2, SIOOA4, SlOOA6, and SlOOB

Recombinant SlOO proteins were produced in large amounts to investigate their biochemical properties and for the production of specific polyclonal antibodies to study their cellular distribution in normal tissues and different tumors. Usually, SlOO protcins can occur as monomers and/or homo/het- erodimers in vitro and in vivo. To investigate whether the recombinant S 100 proteins were also able to form tetramers and higher polymers, we subjected them to taurine SDS- PAGE under reducing and non-reducing conditions. SlOOB migrated as a monomer (apparcnt molecular weight of 6 kDa) in thc reducing (Fig. la , lane 2) and non-reducing (Fig. 16,

Ca"-BINDING PROTEINS IN TUMORS 327

lane 2) gels. SlOOAl and SlOOA6 (Fig. la and b , lane 1 and 3) migrated as monomers in both gels and as dimers (bands at approx. 22 rcsp. 18 kDa) in the non-reducing gel. SlOOA4 migrated as a monomer in the presence of DTT (Fig. la , lane 4). However, in the absence of DTT (Fig. lb, lane 4) a protein smear was observed at approx. 20 kDa (dimer) and a faint band at molecular weight 40 kDa (tetramer). SlOOA2 migrated as a monomer under reducing conditions (Fig. la, lane 5) but formed higher polymers under non-reducing conditions (Fig. 16, lane 5). We conclude that SlOOB migrates on SDS-PAGE as a monomer, whereas SlOOAl, SlOOA2, SlOOA4 and SlOOA6 are able to form dimers, trimers, tetramers, or higher polymers under non-reducing conditions.

The apparent molecular weights of all SlOO protein monomers estimated from SDS-PAGE are always too low when compared with the molecular masses calculated from the sequence data. To verify the correct molecular weight and correct synthesis of the SlOO proteins in bacteria, we determined their exact masses by electrospray ionization mass spectrometry (ESI-MS): 2 values were obtained for recombinant SlOOB, 10,714 Da and 10,742 Da. The first mass of 10,714 Da corresponded exactly to the calculated value. The second mass of 10,742 Da (calculated mass +28) corresponded to the protein with a formylated N-terminal methionine. The masses for SlOOAl were 10,548 Da (calculated: 10,547 Da) and 10,575 Da (calculated mass +28) for the formylated protein. The N-terminal methioninc is usually removed in bacterial systems (-131 mass units), and minor amounts of these forms were also detected (measured values: 10,583 Da for SlOOB and 10,417 Da for SlOOAI). The mass for SlOOA2 was 11,116 Da (calculated: 11,118 Da). The molecular masses for SlOOA4 and SlOOA6 determined by ESI-MS have been reported previously (Pedrocchi et al., 1 9 9 4 ~ ) . These data confirmed that all SlOO proteins used in this study arc expressed in bacteria as full-length proteins.

As further biochemical characterization, the isoeleetric points (IEPs) of the recombinant SlOO proteins were determined under various conditions. The native SlOO proteins migrated to IEPs of 4.3 for SlWA1, 4.7 and 7.1 for SlOOA2,4.9

t

t

t

t

f-

FIGURE 1 - Electrophoresis of purified SlOO proteins under reducing and non-reducing conditions. SDS-PAGE was performed in a taurine buffer system in the presence (a) and absence (b) of D7Twith Slo()AI (lane l), SlOOB (lane 2), SlOOA6 (lane 3), S100A4 (lane 4), and S100A2 (lane 5 ) , 5 J L ~ of each recombinant protein were applied. Proteins were visualized by Coomassic blue. Arrows indicate monomer up to pentamer.

for SlOOA4, 5.3 for SlOOA6, and 4.4 and 6.8 for SlOOB. The native proteins had lower isoelectric points than the reduced and denatured SlW proteins. This could be explained by exposing acidic residues to thc surface of the proteins upon formation of the quaternary structure. So far it cannot be explained why native SlOOB and SlOOA2 formed 2 protein bands with different isoelectric points. The calculated points from the sequence were comparable to the isoeleetric points measured with proteins under reducing and denaturing conditions. We conclude that native SlOO proteins are strongly acidic proteins.

Production and characterization of specijic antiseru ruised uguinst recornbinant human ,7100 proteins

In order to study the expression pattern and localization of SlOO proteins in normal and caneerogenic human cell lines and tissues, we raised polyclonal antisera against SlWA1, SlOOA4 and SlOOA6 in goat, and S100A2 in rabbit. Figure 2 shows a Western-blot analysis using recombinant SlOO proteins. SlOO antibodies rccognized only the corresponding antigens and did not cross-react with the other human SlOO proteins including SlOOA8 (MRP-8) and SlWA9 (MRP-14). In addition, there was no cross-reactivity against other Ca2+-binding proteins, such as calmodulin, troponin-C, parvalbumin and calbindin D-28K (data not shown). The dimeric forms of SlOOA2, SlOOA4 and SlWA6, barely visible in the Ponceau-stained blot, gave also strong reactions on Western-blot analysis (data not shown). This analysis therefore demonstrates that antibodies against SlOOAl, SlOOA2, S100A4 and SlOOA6 are specific and recognize the polymeric forms as well.

In comparison, commercially available antibodies to SlOO proteins, routinely used in many hospitals for the immunohistochemical classification of various tumors of children and adults, were also tested for their specificity on Western blots (Fig. 2). A polyclonal antibody against SlOO from Immunoteeh was highly reactive with human recombinant SlOOB. Only a faint reaction was visible with SlOOAl at the high antibody concentration of 1:lOO. The polyclonal antibody from Da- kopatts reacted strongly with human SIOOB, but also cross- reacted with human SlOOAl and, less strongly, with SlOOA6. The polyclonal antibody from Sigma rcacted strongly with human SlOOB and SlOOAl; however, it was also reactive against SlOOA2, SlOOA3, SlOOA4 and SlOOA6. We conclude that the antibody available from Immunotech against SlW protein recognizcs SlOOR most specifically.

The specificity of our SlOO antibodies was further tested against a whole-cell protein extract from thc breast-cancer cell line MDA-MB-231 (Fig. 3), known to express several SlOO proteins (Pedrocchi et al., 39946). The antibodies against SlOOA6 (lane 3) and SlOOA2 (lane 5 ) recognized only the corresponding monomeric proteins. The antibody against SlOOA4 (lane 4) also produced additional faint reactions, probably with polymeric forms of native SlOOA4. The antibody from Immunoteeh cross-reacted with unknown proteins of approx. 20 and 23 kDa. This analysis demonstrates that our SlOO antibodies arc specific for the different SlOO proteins and should be suitable for immunohistochemical studies.

Immunohistochemical localization of SI 00 proteins in normal and cancerogenic human cell lines and tissues

SlOO proteins are known to be individually regulatcd in human breast-cancer cell lines. While there is a clear correlation between expression of SlOOA4 and enhanced invasiveness in rodents, this issue remains to be clarified in humans. To investigate whethcr specific antibodies recognizing individual SlOO proteins would be useful in the immunohistoehemieal classification of tumors, we initially used them in an immuno-

328 ILG E T A L

1 2 3 4 5 6 anti S l OOAl

anti S1 OOAZ

anti S1 OOA4

anti S100A6

anti S I 00 (Dakopatt!

anti s100 (Immuno- tech)

anti SIOO (Sigma)

loading control

k 1 c . u ~ 2 - Specificity of polyclonal antisera tested by Western- blot analysis. The human recombinant proteins SlOOAI (lane I), SlOOB (lane 2), S100A6 (lane 3), SlOOA4 (lane 4), S100A2 (lane 5). and S100A3 (lane 6) (2 kg each) were blotted onto nitrocellulose membrane and stained with Ponceau S (loading control). Western blots were performed with antibodies against SlOOAl (1:5,000), SIOOA2 (1:5,000), S100A4 (1:2,000), and S100A6 (1: S,000), and with commercially available polyclonal antibodies against bovine S1OO proteins from Dakopatts (1:2,SOO), Immuno- tech (1: IOO), and Sigma (1:SOO).

fluorescent staining of the metastatic breast-cancer cell line MDA-MB-231 in comparison with the non-tumorigenic breast epithelial cell line HBL-100 (Fig. 4a, b ) . S100A6 was detected in hoth cell lines. In contrast, S100A4 and S100A2 were seen to be regulated in an oppositc manner to each other: S100A4 was highly cxpressed in all MDA-MB-231 cells but not in HBL-I00 cells. In contrast, strong staining with SIOOA2-specific antibodies was found in a subset of HBL-l(H) cells, but only faint staining in a few MDA-Ml3-231 cells was seen. Interestingly, we also observed different intracellular localizations of individual SlO0 proteins. S 100A2 was localized predominantly in the cell nucleus, whereas S100A6 was concentrated around the perinuclear membrane, decreasing toward the cell periphery

k1cum 3 - Western blot of protein extract of the breast cancer cell line MDA-MB-231. Approx. SO pg of total protein were run on SDS-PAGE. Lane 1, protein extract visualized by Coomassie blue. Lane 2, incubation with anti-Sl00Al sera (1:1200); lane 3, with anti-Sl00A6 sera (1:800); lane 4, with anti-SlOOA4 sera (1:4OO); lane 5, with anti-Sl00A2 sera (1:200); and lane 6, with thc antibody from Dakopatts against bovine SlOO. +, incubation with serum; -, incubation with preimmune serum as a control.

(Fig. 4c). The functional significance of these observations is not yet clear. We conclude that human SlOO proteins, despite their strong sequence similarity, are differentially expressed both in different cell lines and with respect to their intraccllular localization.

To analyzc the expression of SlOO proteins in normal and cancerogenic tissues, a panel of human tissues was immuno- stained. Table I lists the expression patterns of SlOO proteins in different tumors and corresponding normal tissues. In comparison, the staining pattern with the antibody availablc from Immunotech against SlOO protein, routinely used as a tumor marker, is also listed. SlOOAl and S100A2 could not be found in tumor tissues. SlOOAl immunoreactivity was detected only in epithclial cells of the thyroid follicles. S100A2 staining was found in lung, some cells of the epithelial layer in the kidney, and in some cells of the prostate and skin. S100A4 and S100A6, rcstricted to a specific subclass of cells in normal tissue, were much more highly expressed in tumor tissues (Table I). S100A6 immunoreactivity was detected in most tumor cells, especially in breast, colon and thyroid carcinoma, as well as in malignant fibrohistiocytic (MFH) sarcoma. In contrast, S100A4 immunostaining was mainly restricted to MFH sarcoma, breast carcinoma, and a few as yet unidcntificd cells in colon carcinoma (Fig. 5). Control incubations with prc-immune serum (S100A2) or prc-absorbed, affinity-purificd antibodies (S100A1, S100A4, S100A6) showed only weak hackground staining or were complctcly negative in all tissues examined. As an example, pre-absorption of S100A6 antibody with S100A6 antigen is shown in Figure 5. We conclude that our antibodies against S100A1, SlOOA2, SlOOA4 and S100A6 work well on formalin-fixed arid paraffin-embedded tissue sections and can now be used for more detailed localization studies and a better immunohistochemical classification of human tumors.

1)ISCUSSION

Impairment of Ca2 ' homeostasis and an altered expression of Caz+-binding proteins (Heizmann and Braun, 1995) or

a

B

C

SIOOAl

Ca:'-BINDINC PROTEINS IN TUMOKS

SlUOA2 SlOOAJ

329

si 00.46

FIGURE 4 - Expression of different human SlOO proteins in the human cell lines MDA-MB-231 and HBL-100 with antisera a ainst different human SlOO proteins. (a) and (6) For double immunofluorescence, cells were fixed with formaldehyde, permeabilizef with methanol, and incubated with antisera against SlOOA1, S100A2, SlOOA4 and S100A6, and a second FITC-labeled antibody (top ofa and 6) . As controls, cells were stained with pre-immune serum (bottom of u and b). Calibration bar: 0.1 mm. (c) Co-localization of SlOOA2 and SlOOA6 in HBL-100 epithelial cells. Cells were incubated with rabbit anti-S100A2 sera and goat anti-SlOOA6 sera. Second antibodies were TRITC-conjugated donkey anti-rabbit lgGs and FITC-conjugated donkey anti-goat IgGs. Calibration bar: 0.01 mrn.

330 l l x j ETAL.

TABLE I -EXPRESSION OF SlOO PROTEISS IN NORMAL AND CANCEROGENIC TISSUES

al., 1994u), SlOOA4 is strongly expressed in breast tumors. Corroborating these reports, we found a high amount of S100A4 in breast adenocarcinoma tissues. In cell lines, S100A4 occurred only in the metastatic cell line MDA-MB-231 and could not be detected in the breast-cancer cell line MCF-7 or in the breast epithelial cell line HBL-100 (Fig. 4). To possibly correlate S100A4 expression with the metastatic potential of human tumor cells, further characterization of a large number of tumor samples is needed with respect to their altered invasiveness/motility and their chromosome lq21 rearrange- ments. Interestingly, we also observed high S100A4 expression in human ovary, colon and thyroid carcinoma and in sarcoma MFH. These tumors should be further analyzed. S100A6, restricted to a specific subclass of cells in normal tissue, is also highly expressed in nearly all tumor tissues and tumor cell lines analyzed. So far it is known to be present in melanoma (Weterman et al., 1992) and in breast-cancer cell lines (Pedroc- chi et al., 1994u), but has not been described in other tumors. We demonstrate here that S100A6 is highly expressed in breast, colon and thyroid tumors. Its functional importance for tumor development is not yet clear; however, it can be considered as a tunior marker in the future, since it is present in most tumors. One commercially available antibody (Immu- notech) that is highly specific for SlOOB stained only a subset of tumors, such as melanomas and prostate adenocarcinomas. In contrast to previously published data (Nagasaka et al., 1987), we could not detect SlOOB in thyroid or kidney adenocarcinoma. As shown in this report, some commercially available antibodies against bovine brain SlOO protein cross-react with other members of this large protein family. The use of such antibodies, especially those cross-reacting with S100A6, which is highly expressed in almost all tumors, therefore renders the interpretation of previously published results more difficult. Earlier experiments should be repeated using specific antibodies against Sl00 proteins. While, for most tumor types, a correlation between the expression of individual SlOO proteins and the degree of malignancy remains to be investigated, normal and tumorigenic cells can, in most cases, be distin- guished by analyzing the expression pattern of SlOO proteins.

The expression of Ca2+-binding proteins in rodents or other animals is quite often different from the expression in human tissues (Pedrocchi el al., 1993; Davies et al., 1995) and this must be taken into account in future clinical and functional studies. Becausc of their tissue-specific expression, quantification of SlOO proteins in blood and in cerebrospinal fluid has also been used as a measure of the extent and progression of tissue damage in myocardial hypertrophy or ischemia (Usui et al., 1990), chronic inflammation (Bhanvaj et al., 1992), and neuro- degenerative disorders (Heizmann and Braun, 1995). The availability of these new and specific antibodies against the human SlOO protein will improve and extend those studies.

In summary, SlOO proteins are potentially of great value in clinical applications, such as cancer diagnosis and therapy. We have now generated specific tools for their immunohistochemical localization and for functional studies capable of monitor- ing subtle pathological changes in normal and tumor tissues and in body fluids.

SlWAl S100A2 S100A4 SIWA6 S100B'

Ovary

Breast

Normal tissue Cancer cells






Normal tissue Canccr cells



All tissues

Skin

Colon

Thyroid

Lung

Kidney

Prostate

Pancreas

Controls

X

+, - -

++

X + X -

- ++

X ++ X ++, -

X -

X ++ X

++

- +, - -

+ +, - -

+ +

X

+, - X -

X - x, - +, -

X -

X +

+, positive staining; + +, strongly positive staining; -, negative staining; +, -, positive or negative staining, depending on tissue sample; X , subset of immunoreactive cells only.

'Antibody from Immunotech against bovine brain SlOO extract, recognizing predominantly S100B.

mutations of the corresponding genes (Schafer and Heizmann, 1996) have been associated with a number of human diseases. Members of the SlOO protein family arc of special interest because of their deregulated expression in neoplastic disorders, the clustered organization of SlOO genes on human chromosome lq21, a region frequently rearranged in tumors, and the widespread application of SlOO antibodies for tumor diagnosis by immunohistochemistry.

We have successfully isolated and partially characterized a number of human SlOO proteins. The biochemical and electro- phoretic properties of the individual SlOO proteins were quite different. On SDS-PAGE under non-reducing conditions (Fig. 1) SlOOAl migrated as a monomer, S100A6 mainly as a dimer, S100A4 as a protein smear, and SlOOA2 as monomer and dimer (double band). These observations arc important in view of the suggested functions for S l W proteins, since the dimeric form (in the case of SlOOB and S100A4) might be the extracellular active form, whereas the monomeric protein may have normal intracellular functions. Further experiments arc under way to identify the intra- and extracellular targets for individual SlOO proteins in order to obtain a better insight into their functions in tumor cells and to test them as targets for pharmacological interventions as a new approach for cancer therapy.

SlOO proteins are individually regulated in normal and tumor-cell lines and tissues. SlOOAl was absent from all tumors analyzed. As expected, S100A2 was down-regulated in the tumor-cell line MDA-MB-231 and the tumor tissues. The expression pattern of S100A4 is of particular interest: in normal tissues, S100A4 was present in smooth-muscle cells only. As previously reported (Davies et al., 1993; Pedrocchi et

ACKNOWLEDGEMENTS

We thank Mr. U. Redweik and Dr. J.A. Rhyner for technical help, Ms. M. Killcn for critical reading of the manuscript, Drs. P. Hunziker, P. Gehrig, and Th. Kuster for ESI-MS analyses, and Drs. G. Burg and M. Miintener for advice with the immunohistochcmistry. This work was supported by the Wilhelm Sander-Stiftung (Germany) and the Stipendienfonds der Basler Chemischen Industrie (Switzerland).

Ca?+-BINDING PROTEINS IN TUMORS

S100A4 S100A6 CONTROL

331

Breast Adeno Carcinoma

Sarcoma MFH

Thyroid Adeno Carcinoma

Colon Carcinoma

FIGURE 5 - Localization of SlOOA4 and S100A6 in human tumor tissues. Paraffin-embedded human tumor tissue sections were immunohistochemically stained with affinity-purified antibodies against human SlOO proteins. As a control, tissue sections were incubated with preabsorbed antibodies. Pre-absorption for S 100A6 is shown here. Calibration bar: 0.1 mm.

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Expression pattern of S100 calcium-binding proteins in human tumors