Journal ofNeuroimmunology, 36 (1992) 41-55 ©1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00

41

JNI 02103

Tenascin expression in human glioma cell lines and normal tissues Joseph B. Ventimiglia

a

Carol J. Wikstrand a, Lawrence E. Ostrowski a,., Mario A. Bourdon Virginia A. Lightner c,d and Darell D. Bigner a,e

b,

Departments of a Pathology, ¢ Medicine and a Cell Biology, and e Preuss Laboratory for Brain Tumor Research, Duke University Medical Center, Durham, NC 27710, USA, and b California Institute of Biological Research, La Jolla, CA 92037, USA

(Received 1 April 1991) (Revised, received 13 August 1991) (Accepted 13 August 1991)

Key words: Tenascin; Extracellular matrix; Glioma; Isoforms; Alternative mRNA splicing; Immunotherapy

Summary Tenascin expression was evaluated in 21 human glioma cell lines and in normal adult tissue extracts by Western and Northern blotting. The cell lines differed in their relative expression of tenascin in the cell-associated and supernatant compartments. Glioma cell line tenascin production was not uniformly stimulated by changes in fetal bovine serum concentration in the growth media. In most glioma cell lines and normal tissue extracts, reducing Western blots and Northern blots revealed two tenascin species, respectively: a major 340 kDa polypeptide and a 9 kb RNA transcript accompanied by a less intense 250 kDa polypeptide and 7 kDa RNA species. In U-87 MG and in normal adult kidney extracts, however, the 250 kDa band and 7 kb transcript were more prominent. Quantitation of tenascin in the glioma lines revealed variable levels that were significantly higher than those in the tissue extracts.

Introduction Tenascin is a large extracellular matrix glycoprotein identified in numerous species and in the stroma and mesenchyme of tissues at various stages of differentiation (for reviews see Erickson

Correspondence to: Darell D. Bigner, M.D., Ph.D., Department of Pathology, Duke University Medical Center, Box 3156, Durham, NC 27710, USA. * Present address: N.I.E.H.S., P.O. Box 12233, Laboratory of Pulmonary Pathobiology, Mail Drop D201, Research Triangle Park, NC 27709, USA.

and Lightner, 1988; Erickson and Bourdon, 1989). The role(s) of tenascin are controversial, but putative functions and activities include chondroitin sulfate proteoglycan binding (Hoffman et al., 1988), fibronectin binding (Chiquet-Ehrismann et al., 1988; Hoffman et al., 1988), DNA and heparin binding (Marton et al., 1989), hemagglutination and growth factor-like activity (ChiquetEhrismann et al., 1986), cell attachment (Spring et al., 1989), and integrin binding (Bourdon and Ruoslahti, 1989). Electron microscopic examination of tenascin reveals a spider-like six-armed, disulfide-linked polymer with a characteristic terminal knob at the end of each arm, a thickened

42 distal portion of each arm, a thinner proximal arm, two T-shaped junctions (each joining three arms), and a central globular core joining the two trimers at their T-junction (Erickson and Iglesias, 1984; Erickson and Taylor, 1987). Expression of tenascin in several human tumors (Bourdon et al., 1983; Stamp, 1989; Howeedy et al., 1990; Lightner et al., 1990) has suggested its potential for diagnosis and therapy. Bourdon et al. (1983) described intense staining in unfixed tissue sections of 14 of 16 (88%) gliomas, 1 of 3 (33%) neuroblastomas, 1 of 7 (14%) melanomas, and 2 of 6 (33%) sarcomas. The high frequency of tenascin-positive gliomas and the absence of tenascin in normal brain detectable by 81C6 monoclonal antibody (MAb) immunohistochemistry have made immunotherapy of gliomas possible in xenografts (Lee et al., 1988) and in patients (Zalutsky et al., 1989). An obstacle to improved efficacy of therapy, however, has been the presence of tenascin in other normal human tissues. Bourdon et al. (1983) found tenascin in liver, spleen, kidney, and cultured fibroblasts by immunohistochemistry of unfixed frozen tissue sections or acetone-fixed cultured cells with MAb 81C6, and Lightner et al. (1989, 1990) detected tenascin in normal human skin. If malignant tissues express tenascin species with epitopes or isoforms that are absent or insignificant in normal tissues, directing a-tenascin MAbs against these epitopes or isoforms would offer a straightforward molecular approach for the e n h a n c e m e n t of tenascin-directed immunotherapy of tumors. Gene sequence data for the chicken protein (Jones et al., 1989) and for human tenascin (Nies et al., 1991) have demonstrated multiple isoforms for tenascin that differ in the number of fibronectin type III domains present. Existence of multiple tenascin isoforms and the precedent of tumor/fetal-specific variants of fibronectin (Matsuura and Hakamori, 1985; Matsuura et al., 1988; Loridon-Rosa et al., 1990) suggest that tumor-associated isoform expression of tenascin is a possibility. We report here the analysis of tenascin isoform expression in 21 established glioma cell lines and compare this with expression in normal human tissues. We found that glioma cell lines and normal tissue extracts expressed two tenascin species by reduc-

ing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Northern blotting but differed widely in their relative levels of tenascin production.

Materials and methods

Cell lines U-87 M G and U-373 M G were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and assayed at passages 130 and 181, respectively. All other lines were established or maintained in this laboratory (Mark et al., 1977; Bigner et al., 1987, 1988). All cell lines used in this study were negative for Mycoplasma contamination as determined by the method of Kurtzberg and Hershfield (1985). Cell lines were grown in Richter's zinc option medium (ZO, Gibco, Grand Island, NY, USA) (Richter et al., 1972) supplemented with 10 mM Hepes, 0.22% NaHCO 3, and 1, 10, or 20% heat-inactivated fetal bovine serum (FBS, Gibco). Material for immunoblotting was prepared from two parallel dishes of confluent cells, after replenishment of the medium with Z O + 1% FBS or Z O + 10% FBS on days 0 and 2, and harvested on day 4. Conditioned medium was centrifuged at 300 × g at room temperature for 10 min. Cell pellets were prepared following incubation with 0.2% E D T A for 10 min at room temperature. The resultant suspension was centrifuged as noted above, the supernatant discarded, and the cell pellet resuspended in 2 ml of 115 mM phosphate buffer (26 mM N a H 2 P O 4 d H 2 0 + 88 mM N a 2 H P O 4, pH = 7.4). Preparations were frozen at - 2 0 ° C or - 135 °C until used for Western blotting and at - 1 3 5 °C prior to Northern analysis. Cell pellets for R N A harvest were prepared from confluent 150 cm 2 culture flasks of cells grown in Z O + 10% FBS and were at the same passage level as the cells harvested for immunoblotting. Tissues Tissues were collected from autopsies begun no more than 3 h post-mortem from adult patients with no clinical, gross, or histological evi-

43 dence of disease in the harvested tissue. Tissues were kept at - 1 3 5 ° C until RNA or tenascin extraction. Tenascin extraction was performed according to the method of Erickson and Taylor (1987). Briefly, tissue was mixed with 5 ml of extraction buffer (200 mM 3-[cyclohexylamino]1-propane-sulfonic acid (CAPS)+ 0.15 M NaC1, pH l l ) / g of tissue, homogenized in a Sorvall homogenizer on ice, mixed for 1 h at 4 ° C, titrated to pH 7.3 with 1 M NaH2PO4, supplemented with phenylmethylsulfonyl fluoride to 1 mM, and centrifuged at 30,000 × g at 4 ° C for 4 h. After the addition of NaN 2 to a final concentration of 0.06%, the supernatant was withdrawn and stored at - 20 ° C or - 135 ° C until further analysis.

Tenascin purification Tenascin was initially purified from U-251 MG-C3 supernatant by immunoaffinity chromatography using the murine a-tenascin MAb 81C6 (Bourdon et al., 1983). Culture supernatant was passed over an 81C6-Sepharose 4B affinity column at room temperature, the column was washed with 10 mM Tris plus 500 mM NaC1 (pH 8.0), and the tenascin was eluted with 100 mM CAPS in 500 mM NaC1 (pH 11.0) into tubes containing 30 mg of glycine per ml of eluate to neutralize the pH to approximately 8.3. Tenascin used for polyclonal antibody preparation was subjected to an additional glycerol gradient-sedimentation purification step (Erickson and Taylor, 1987).

Production of polyclonal antisera Polyclonal antiserum to tenascin was prepared against affinity purified tenascin. 5/xg of tenascin in Freund's complete adjuvant was injected s.c. into rabbits; nine subsequent monthly i.v. boosts of 5 /zg were administered, with high titers (1 : 50,000 against purified human tenascin) noted after the second boost. Antiserum from a bleed drawn 11 days after the second boost was used for all studies. No reactivity of this antiserum to ZO + 10% FBS or to purified human fibronectin was noted on immunoblots (data not shown).

Protein assay Total protein was determined with the Bio-Rad protein assay (Bio-Rad, Rockville Center, NY,

USA) according to the manufacturer's instructions using bovine serum albumin (BSA, fraction V, Boehringer-Mannheim Biochemicals, Indianapolis, IN, USA) or human plasma fibronectin (Sigma, St. Louis, MO, USA) as the standard.

SDS-PAGE Proteins were resolved by discontinuous SDSPAGE under reducing conditions using the BioRad Mini-Protean II system. A 3.75% stacking gel in 0.125 M Tris-HCl (pH 6.8) was layered over a 5% resolving gel of 30:0.8 acrylamide/bisacrylamide in 0.375 M Tris-HC1 (pH 8.8). Samples were dissolved 1 : 1 (v/v) in reducing sample buffer (4% SDS, 10%/3-mercaptoethanol, 22.5% glycerol, and 0.001% bromophenol blue in 0.125 M Tris-HC1, pH 6.8) and boiled in a 95 °C water bath for 4 min. For nonreduced fibronectin, /3mercaptoethanol was omitted. The gels were run in reservoir buffer (0.025 M Tris-HCI, 0.192 M glycine, pH 8.3) at 200 V for approximately 1 h. High molecular weight standards were obtained from Sigma (myosin, 205 kDa; human plasma fibronectin, 500 kDa nonreduced and 250 kDa reduced); and from Gibco (Engelbreth-HolmSwarm laminin, 400, 215 and 205 kDa). 5-10/~g of each molecular weight standard were run per lane.

Electrophoretic transfer and blotting Proteins were transferred to nitrocellulose (Trans-Blot transfer medium, Bio-Rad) in 20 mM Tris-HC1, 192 mM glycine, 0.1% SDS, 20% methanol (pH 8.2) in an LKB 2117 Multiphor II semi-dry apparatus (Pharmacia LKB Biotechnology, Piscataway, NJ, USA) at 0.8 m A / c m 2 for 2 h at room temperature. Lanes with molecular weight standards were separated after transfer and stained with amido black (Root and Reisler, 1989). Blots were blocked in 1% BSA in phosphate buffer (pH 7.4) at room temperature for 3-14 h. Duplicate sheets were incubated with rabbit polyclonal a-tenascin antiserum or normal rabbit serum (Gibco) for 2 h at room temperature at identical dilutions (1 : 1750 to 1 : 8000) in blotting buffer (1% BSA, 0.05% Tween 20 in phosphate buffer, pH 7.4). After blotting buffer washes, sheets were incubated with 1 tzCi/ml of 125I goat

44 antirabbit IgG (New England Nuclear, Boston, MA, USA) in blotting buffer for 1 h at room temperature, washed, arranged in X-ray film cassettes, and exposed to Kodak X-Omat AR film at - 1 3 5 ° C for 9-72 h before film development. Alternatively, sheets were stained by a fl-galactosidase staining system. After incubation with the primary reagent, sheets were incubated with 1:667 biotinylated goat antirabbit IgG (BRL, Gaithersburg, MD, USA) and 1:500 streptavidin /3-galactosidase (BRL) and developed with a solution containing 0.42 mg/ml Bluo-gal (BRL), 1 mM MgCI2, 150 mM NaCI, 3 mM potassium ferricyanide, and 3 mM potassium ferrocyanide for approximately 50 min.

cDNA clones A human tenascin cDNA clone designated Tn8 was isolated from a human fibroblast cDNA library in 7gtll. Rabbit polyclonal antibody to human tenascin was used to screen 7gtll library plaque lifts for expression of tenascin. Secondary antibody horseradish peroxidase-conjugated goat antirabbit IgG (Bio-Rad) was used at a dilution of 1 : 2000. Immunoreactive clones were detected using 3,3'-diaminobenzidine substrate. The Tn8 cDNA was subsequently subcloned into the EcoRI site of plasmid Bluescript KS (Stratagene, La Jolla, CA, USA). The Tn8 cDNA is 1440 bp in length and contains coding sequences for the three carboxyl-terminal fibronectin type III repeats and the majority of the carboxy terminal globular domain of tenascin. The preparation of the 1.9 kb cDNA clone for /3-actin from the squamous cell carcinoma-producing cell line PDVc5 v has been previously described (Ostrowski et al., 1989). Northern analysis Total RNA was isolated from stored cell pellets and tissues by the method of Krieg et al. (1983) and enriched for mRNA by one cycle of oligo(dT) affinity chromatography (BRL, Pharmacia). Size fractionation was performed on a gel containing 1.0% agarose and 0.66 M formaldehyde according to established procedures (Davis et al., 1986). The gel was soaked briefly in alkali before being transferred to Genescreen by positive pressure (Possi-Blot apparatus, Stratagene)

and cross-linked with 1200/~J of ultraviolet light (Stratalinker, Stratagene). Prehybridization and hybridization were carried out according to the manufacturer's instructions (Genescreen, Dupont). An RNA ladder (BRL) was used to estimate transcript size. Probes for/3-actin (Ostrowski et al., 1989) and tenascin (a 1.44 kb cDNA fragment isolated from a human fibroblast line kindly provided by Dr. Mario Bourdon, California Institute of Technology) were labeled with [32p]dCTP (New England Nuclear) by random hexamer priming with the Oligolabelling Kit (Pharmacia). Filters were then developed in X-ray cassettes with a Lightning Plus intensifying screen (Kodak) and exposed to Kodak X-Omat AR film at -135 °C prior to development.

Densitometry The /3-galactosidase-developed sheets, photographic positives of silver-stained gels, and autoradiographically developed immunoblots were scanned using a Xerox Datacopy 730 GS gray scale flatbed scanner connected to a Macintosh SE/30 running Xerox's MacImage software and Image 1.27 software. For routine quantitation of tenascin, total tenascin-reactive staining was determined for each of the samples and calibrated against the same staining for immunoaffinitypurified tenascin standards on /3-galactosidase visualized blots. This quantitation system was capable of detecting > 50 ng of tenascin (2.8 ~g tenascin/ml original sample).

Results

Quantitative comparison of tenascin production in glioma cell lines maintained in 1% and 10% FBSsupplemented media To define optimal conditions for measuring intrinsic tenascin production, we examined the response of the glioma lines to 1 or 10% FBSsupplemented media. Fig. 1 summarizes the ratio of soluble tenascin levels (expressed as p.g supernatant tenascin/mg cell pellet protein) in 10% FBS maintenance conditions to the soluble tenascin levels in cells switched to 1% FBS for these 17 of the 21 cell lines that secreted tenascin detectable by quantitative immunoblotting in both

45 a

1 and 10% FBS-supplemented media. Tenascin expression was less in 10% FBS-supplemented medium than in 1% FBS in 13/17 (76%) of these confluent glioma cell lines.

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Glioma cell lines produce two major individual tenascin arm species Tenascin expression in 21 established human glioma cell lines was examined by 5% SDS-PAGE under reducing conditions in order to resolve different tenascin arm species. From 19 glioma cell line conditioned supernatants, four major qualitative categories emerged (Fig. 2): two isoforms detectable, with the large isoform predominating; two isoforms detectable, with the smaller isoform predominating; only the large isoform detectable; and no tenascin species detectable. Calculated molecular weights of the large and small isoforms based on standards run in Fig. 2 were 340 and 250 kDa, respectively. These glioma lines demonstrated little or no qualitative variations in tenascin expression from 1 to 10% FBS, essentially adhering to the patterns described below at both FBS concentrations.

m

Fig. 2. Polyclonal Western blot of individual tenascin arm species from glioma supernatants. A 30/zl aliquot of conditioned medium from 1% FBS grown cells was collected and run in 5% SDS-PAGE in the presence of/3-mercaptoethanol as detailed in Materials and methods, stained with polyclonal antiserum to tenascin and radioactive secondary antibody, and exposed to X-ray film for about 24 h. The positions of nonreduced fibronectin (500 kDa), reduced fibronectin (250 kDa), and myosin (200 kDa) are also indicated. Lanes: a = A172; b = D-54 MG; c = D-65 MG; d = U-118 MG; e = U-251 MG-C3; f = U-373 MG; g = U-105 MG; h = U-343 MG; i = U-410 MG; j = U-373 MGatcc; k = U-87 MGatcc; 1= D-423 MF; m = D-392 MG; n = D-336 MG; o = D-270 MG; p = D263 MG; q = D-247 MG; r = D-245 MG; s = U-138 MG.

Supernatant tenascin expression in 16/21 glioma lines fell into the first category; the large isoform appeared as a sharper, more distinct band than the small isoform, which was highly variable in intensity. Stronger intensities of the small arm

C e l l Line

Fig. 1. Ratio of secreted tenascin expression between confluent cells maintained in ]0% FBS in those switched to 1% FBS. Supernatants from the 17 glioma cell lines that produced detectable amounts of tenascin in both 1 and ]0% FBS-supplemented media were blotted and quantified for total tenascin expression as detailed in Materials and methods. The ratio of the ]0% to 1% FBS media-corrected tenascin content (/~g tenascin in supernatant/mg total cell protein) is shown. Values greater than ] indicate higher tenascin levels in the 10% FBS media, and values less than 1 indicate higher levels in l % media. Note that this ratio is not reflective of absolute tenascin content in either 1 or 10% FBS-containing media.

46

band generally correlated with higher overall expression in the 15 lines (data for D-37 MG not shown) presented in Fig. 2 (cf. lanes d, e, f, q, and r to lanes m and o). One cell line, U-87 MGatcc (lane k), expressed the smaller 250 kDa arm at a greater intensity than the large 340 kDa arm. Two lines, U-105 MG (lane g) and D-259 MG (not shown), fell into the third category and showed only the large isoform, but also gave very weak overall signals. D-65 MG and U-343 MG (lanes c and h, respectively) produced no detectable tenascin. Further analysis of D-65 MG, however, using /3-galactosidase visualized blots with high titers of primary antibody (1 : 2000) and long development times, revealed a very faint large isoform (data not shown). Scanning densitometry of a photographic positive of the blot in Fig. 2 confirmed the conclusions reached by visual inspection regarding the presence and predominance of isoforms in these lines. Analysis of cell-associated tenascin (Fig. 3) revealed individual arm species detectable in cell suspensions of the glioma lines that were essentially identical in electrophoretic mobility to those found in the supernatants, with variable amounts of the 340 kDa species and the 250 kDa species detected. Two specific observations were noted, however. First, a faint small isoform band was visible in the cell-associated compartment of U105 MG (lane g), the supernatant of which only revealed the large isoform. Second, the small isoform predominance in U-87 MG~tcc cells (lane

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Fig. 3. Polyclonal Western blot of individual tenascin arm species from glioma cell samples were prepared from 10% FBS grown cells resolved by reducing 5% SDS-PAGE, transferred to nitrocellulose and blotted with polyclonal antiserum to tenascin as previously described. Molecular weight markers of 250 and 200 kDa are as in Fig. 2. Total protein loaded: lane k, 4.4 ~g; r, 1.5 /xg; q, t, p, o, j, u, n, 6.3 /xg; 1, m, g, 15.5 ,ag. Film exposed for 48 h. Lanes correspond to same cell lines as in Fig. 2 except t = D-259 MG and u = D-37 MG.

k) was more striking than that in supernatants (Fig. 2, lane k), further emphasizing the uniqueness of this line. The isoform expression and predominance characteristics of these cell lines, along with their final assignment to one of the categories described above, are summarized in Table 1. Detection of small monomeric arm species is dose and antibody dependent In purified tenascin dose curves, dose-dependent appearance of the small arm was seen clearly with silver staining (Fig. 4A), with polyclonal

TABLE 1 I N D I V I D U A L T E N A S C I N A R M E X P R E S S I O N C H A R A C T E R I S T I C S O F E S T A B L I S H E D H U M A N G L I O M A C E L L LINES Data from polyclonal immunoblots of conditioned supernatants and cell suspensions are summarized above. Expression of one, both, or neither of the two individual tenascin arm species is indicated, as is the predominant (more intense band) of the 250 kDa or 340 kDa arm variants. The categories in the right-hand column are described in the text. The four cell lines characterized from data not presented in Figs. 2 and 3 are shown in parentheses. Cell line

Isoforms expressed

Arm size predominance (kDa)

Category

(D-37 MG), A142, D-54 MG, U-118 MG, U-251 MG-C3, U-373 MG, (U-105 MG), U-410 MG, U-138 MG, D-245 MG, D-247 MG, D-263 MG, D-270 MG, D-336 MG, D-392 MG, D-423 MG, U-373 MGatcc U-87 MGatcc (D-65 MG), (D-259 MG) U-343 M G

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Fig. 4. Dose and antibody dependence of detection of small tenascin arm isoform. Indicated nanogram a m o u n t s of immunoaffinity-purified tenascin from 1% FBS grown U-251 MG-C3 conditioned media were subjected to reducing 5% S D S - P A G E and silver stained ( A ) or transferred to nitrocellulose and visualized with /~-galactosidase staining of murine monoclonal 81C6 (B) or polyclonal (C) immunoblots. Polyclonal antiserum was used at a 1:8000 dilution, and 81C6 was used at 5 ~ g / m l . Graph of total tenascin reactive intensity quantified by densitometry for autoradiograpbically developed 81C6 or polyclonal immunoblots (D, page 48). Comparison of tenascin a m o u n t s at which an equivalent staining intensity was noted in the linear portion of both curves reveals an approximately 9-fold difference in sensitivity (951 ng vs. 110 ng).

immunoblotting using /3-galactosidase visualization (Fig. 4C), and with autoradiographic development of immunoblots using either murine antitenascin MAb 81C6 or polyclonal antiserum

(data not shown). In the least sensitive case, immunoblotting using MAb 81C6- and fl-galactosidase-based visualization did not reveal the small arm isoform, even at the highest tenascin amount loaded (Fig. 4B). These data indicate that the apparent absence of a small isoform band in a cell line may simply be due to a lack of sufficient sensitivity to detect this species at the given total tenascin concentration of the preparation. The overall sensitivity of polyclonal antiserum was superior to that of MAb 81C6 (compare 4B and 4C). Increasing the 81C6 concentration (5 /zg/ml to 20 /~g/ml) did not improve sensitivity of the monoclonal immunoblots (data not shown). Densitometric analysis of total tenascin intensity versus tenascin load per lane for monoclonal and polyclonal immunoblots was visualized autoradiographically (Fig. 4D). This analysis revealed an approximately 9-fold lower sensitivity of 81C6 (at 5 /xg/ml) relative to polyclonal antiserum (at 1 : 2000 dilution). The minimum tenascin concentrations detectable in our system for 81C6 and polyclonal antiserum are approximately 16.7 and 1.7/zg/ml, respectively. Glioma cell lines synthesize two major tenascin mRNAs To investigate the hypothesis that alternative RNA splicing generates multiple tenascin isoforms (Jones et al., 1988, 1989; Gulcher et al., 1989), we investigated the tenascin RNA produced in glioma cell lines using a probe capable of hybridizing with all published human tenascin sequences. The data presented in Fig. 5 show the expression of tenascin m R N A in a panel of 17 selected glioma lines. Human tenascin probe Tn8 hybridized to two major messages in these glioma 'lines. In total RNA blots (panel A), all 17 glioma lines demonstrated variable intensities of a large, approximately 9 kb transcript. In only one cell line, U-87 MGatcc, was a smaller 7 kb transcript detected (panel A, lane a). Using purified m R N A (panel C), we were able to demonstrate the presence of the smaller 7 kb transcript in two large arm-predominant cell lines (lane c, U-251 MG-C3; and lane k, U-118 MG). D-65 MG (lane 1) served as the negative control cell line for these blots, as

48

its tenascin expression was low enough that the presence of both the 7 and 9 kb tenascin transcripts could be demonstrated only with very long exposure times (data not shown). Note that several lanes in the total RNA Northern blot (panel A) show a band migrating above the 9 kb tenascin message that is not present in the poly(A) + enriched blot (panel C). This band may represent tenascin pre-mRNA. The same blots shown in panels A and C were stripped and reprobed with the /3-actin probe (panels B and D, respectively). All lanes showed a single, intact /3-actin band, demonstrating the integrity of the RNA used for these blots.

tive intensities between the cell pellet and supernatant compartments under these loading conditions was observed, with some lines demonstrating greater cell-associated intensity (U-87 MGatcc and D-392 MG), approximately equal intensities (U-138 MG), no detectable signal in either compartment (D-65 MG), or greater supernatant intensity (U-251 MG-C3 and D-247 MG). These variations in partitioning of tenascin occurred in confluent cell lines and in both 1 and 10% FBSs.upplemented media, making differences in growth phase or growth factors unlikely explanations for this phenomenon. A m o u n t of tenascin secreted by glioma lines varies over a 5- to lO-fold range Table 2 shows the quantitation of total tenascin concentration in the supernatants of all 21 glioma cell lines. Corrected tenascin content (supernatant tenascin concentration/total cellular pro-

Glioma cell lines vary in their relative compartmentalization of tenascin Individual tenascin-arm species of supernatant and cell pellet samples were analyzed by polyclonal immunoblotting (Fig. 6). A range of rela-

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Fig. 5. Northern blot analysis of glioma cell lines. 10 /xg of total cellular R N A ( A , B) or 5 /~g of purified poly(A) ÷ m R N A (C, D) were fractionated on a 1% agarose gel electrophoresis in the presence of 0.66 M formaldehyde and transferred to Genescreen as detailed in Materials and methods. The blot was probed with the tenascin-specific probe, Tn8 ( A , C), then stripped and rehybridized to the /3-actin specific probe (B, D). Panels A and B are the composite of two separate experiments, with lanes a - c coming from one Northern blot and lanes d - j coming from another. O n e of the common samples rerun between experiments is shown in lanes b and j. Lanes: a = U-87 MGatcc; b = U-373 MGatcc; c = U-251 MG-C3; d = U-373 MG; e = U-105 MG; f = U-410 MG; g = U-138 MG; h = D-247 MG; i = D-423 MG; j = U-373 MGatcc; k = U-118 MG; 1 = D-65 MG.

moderately high producers of tenascin when synthesis was corrected for cellular protein. Human brain and kidney extracts show two major species on both Western and Northern blotting that correlate with those seen in glioma lines Tenascin expression in selected normal tissues was examined by Western and Northern analysis (Fig. 7). The polyclonal immunoblot of resolved individual arms from two different samples of human kidney (panel A, lanes b, c) revealed two isoforms comigrating with those from glioma lines (lanes a, d). Of note was the strong predominance of the small 250 kDa isoform, similar to that seen in U-87 MGatcc (lane d). Analysis of tenascin expression in a sample of normal human cerebral cortex also revealed 250 and 340 kDa arm isoforms, but with the large isoform predominance typical of most of the glioma lines (lane e). Two additional human temporal lobe extracts revealed

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250 k D tein) is also shown. With the exception of U-343 MG, the one cell line with undetectable tenascin levels in these supernatants, supernatant tenascin concentration ranged from a low value of 3 Izg/ml to a high value of 17.8 Ixg/ml. Total biosynthetic activity (as judged by cellular protein concentration) and production of secreted tenascin are largely independent of one another (Spearman's rank correlation coefficient, r s = 0.37, P = 0.10). For example, the third highest producer of tenascin by concentration, D-336 MG, was overwhelmingly superior to the other lines in corrected tenascin production; and two of the apparently weaker producers of secreted tenascin, D-423 MG and D-37 MG, were actually

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SC

Fig. 6. Compartmentalization of tenascin between secreted and cell-associated forms. 35 ~zl of a 1:1 aliquot of the indicated samples in reducing sample buffer were resolved by reducing 5% SDS-PAGE, transferred to nitrocellulose, blotted with 1:2000 diluted of rabbit polyclonal o~-tenascin, and 'visualized by the/3-galactosidase system as noted in Materials and methods. For each cell line, the aliquot loaded corresponds to 0.175% of the 10 ml of total supernatant and 0.875% of the 2 ml of total cell suspension harvested. For the cell lines U-251 MG-C3, U-87 MGatcc, D-392 MG, and D-247 MG, samples were obtained from cells maintained in media with 10% FBS; for D-65 M G and U-138 MG, cells were maintained in 1% FBS supplemented media prior to harvesting. S = supernatants; C = cells. Total protein loaded in the cell suspension compartments was as follows: U-87 MGatcc, 5.25 g g ; D-65 MG, 31.5 /~g; U-251 MG-C3, 21 /.Lg; D-392 MG, 14 ~g; U-138 MG, 17.5 # g ; and D-247 MG, 22.75/xg.

50 TABLE 2 T]~NASCIN EXPRESSION IN GLIOMA CELL LINES GROWN IN 10% FBS Aliquots (30 /zl) of supernatant from all 21 glioma cell lines were resolved by reducing 5% SDS-PAGE, transferred to nitrocellulose, stained with 1:4000 dilution of rabbit polyclonal c~-tenascin, developed by the /3-galactosidase method, scanned, and quantified for total tenascin expression using immunoaffinity-purified U-251-C3 MG tenascin as the standard as detailed in Materials and methods. Cellular protein was determined as in Materials and methods. For corrected tenascin content, the tenascin measured for each cell line was multiplied by total supernatant volume (10 ml), then divided by the total cellular protein (cellular protein × cell suspension volume [2 ml]). Cell line

Cellular protein (mg/ml)

Supernatant tenascin (/xg/ml)

Corrected tenascin content (tzg/mg cell protein)

D-336 MG D-270 MG D-37 MG D-423 MG U-105 MG U-373 MGatcc D-247 MG U-373 MG U-87 MGatcc U-251 MG-C3 U-118 MG A142 U-410 MG D-392 MG D-259 MG D-263 MG D-54 MG U-138 MG D-245 MG D-65 MG U-343 MG

< 0.1 0.2 < 0.1 < 0.1 0.2 0.2 1.3 1.0 0.3 1.2 1.2 0.4 0.5 0.8 0.8 1.4 1.2 1.0 1.6 1.8 0.6

12.5 6.9 3.0 3.0 3.3 4.3 17.8 13.9 3.3 12.2 8.8 3.1 3.3 4.3 3.5 5.0 4.2 3.1 3.5 3.8 ND

>_625 158 _> 152 _> 152 93 86 69 68 57 50 38 35 34 28 22 18 18 16 11 10 ND

ND: not detected.

an identical pattern, but similar extracts of cerebellum revealed no detectable tenascin (data not shown). Northern

analysis of poly(A) ÷ RNA

from the

h u m a n k i d n e y s a m p l e s r e v e a l e d two splice variants of equivalent electrophoretic mobility to t h o s e p r e s e n t in U - 8 7 MGatcc, s u g g e s t i n g t h a t t h e m e c h a n i s m o f a l t e r n a t i v e R N A s p l i c i n g is a p o s s i ble explanation for the presence of the two

A.

a

b

c

d

e

3 4 0 k D --

25OkD~

a

B.

9kB

--

b

~

7kB -- i

Fig. 7. Analysis of normal human tissue extracts. (A) Western blotting. Aliquots of frozen human autopsy tissues were extracted as detailed in Materials and methods. 39 /xg of total protein for the kidney samples and 15 /zg for the cortex were resolved in reducing 5% SDS-PAGE, transferred to nitrocellulose, and blotted with 1:2000 rabbit polyclonal tr-tenascin. Glioma cell line reference lanes (a, d) were loaded with 30/zl of sample prepared from cells grown in 10% FBS. Lanes: a =U-251 MG-C3 supernatant; b = kidney, autopsy 1; c = kidney, autopsy 2; d = U-87 MGatcc cell suspension; e = cerebral cortex. (B) Northern analysis. Approximately 5/.tg of poly(A) ÷ enriched RNA prepared from the indicated tissues was resolved by agarose gel electrophoresis as previously noted and probed with the tenascin specific probe, Tn8. Exposure was time lengthened for lane a, which was not visible at the shorter exposure time shown for lane b. Lanes: a = kidney, autopsy 1; b = U-87 MGatcc.

51

tenascin isoforms seen in human kidney. Additional polyclonal immunoblots and tenascinspecific Northern analyses were performed on material from normal human spleen and liver extracts. Data from preliminary analyses of normal human spleen suggest the presence of the same two 340 and 250 kDa arm isoforms, with a large arm predominance (data not shown). Liver extracts were repeatedly negative for tenascin expression on Western and Northern blots, but a control immunoblot with an a-fibronectin MAb and a control Northern blot with the fl-actin probe also revealed no signal, thus indicating degradation. Human brain and kidney extracts contain similar amounts of tenascin Quantitation of tenascin in these human tissue extracts was carried out to provide a comparison to the values from glioma cells (Table 3). Four separate histologically normal samples of human brain (one cerebellum, one cortex, and two temporal lobes) and two samples of human kidney were subjected to quantitative Western blotting. For all of these samples, total protein was determined and actual tenascin expressed as the mean of multiple tenascin determinations (n) corrected for the total extract protein. Except for a lack of detectable tenascin in the human cerebellar extract, corrected tenascin content was in the range

of 2 - 5 / z g tenascin/mg extract protein, with values for cortical and temporal lobe being slightly higher than those in the kidney. Tenascin content is also expressed in terms of/zg tenascin/g whole tissue (calculated as described in the legend to Table 3), where the quantitative similarities between the cerebral cortex and kidney are even more obvious. Although tissue tenascin values were obtained from CAPS-extracted autopsy material, values for normal tissues are substantially below corrected tenascin content in the glioma supernatants ( 2 - 5 / z g soluble tenascin/mg cellular protein for tissue samples versus 10-625 /xg tenascin/mg glioma cell line extract protein, respectively).

Discussion

Biosynthesis of tenascin by chick embryo fibroblasts is stimulated when the cells are grown in 10% FBS-supplemented medium relative to their production in the presence of lower (0.3% or 1%) FBS concentrations (Pearson et al., 1988). There is evidence that this stimulation is due at least in part to TGF-/3 (Pearson et al., 1988; Chiquet-Ehrismann et al., 1989). In this study, a similar difference in FBS concentration did not uniformly stimulate tenascin production in human glioma cell lines. These diverse observations

TABLE 3 Q U A N T I T A T I O N O F T E N A S C I N E X P R E S S I O N IN H U M A N TISSUE E X T R A C T S Aliquots (17.5 tzl) of tissue extracts prepared as described in Materials and methods were diluted 1 : 1 with sample buffer and blotted by the /3-galactosidase system with immunoaffinity purified U-251 MG-C3 tenascin, run as the standard, and subjected to quantitative analysis as detailed in Materials and methods. The n u m b e r of independent quantitative determinations done for each sample is specified (n). The total protein in each of the extracts is noted, as is the corrected tenascin content in each sample _+ SD for the n separate determinations. Tenascin c o n t e n t / g of whole tissue is calculated by multiplying the ~ g t e n a s c i n / m g total protein x mg total p r o t e i n / m l extract solution x 5 ml of extract s o l u t i o n / g of whole tissue (Materials and methods). For the cerebellar extract, no detectable bands were present. Sample

n

Extract protein (mg/ml)

Tenascin c o n t e n t / m g total orotein (/~g)

Tenascin c o n t e n t / g whole tissue (~g)

Cerebellum, autopsy 1 Cortex, autopsy 1 Temporal lobe, autopsy 2 Temporal lobe, autopsy 3 Kidney, autopsy 4 Kidney, autopsy 5

2 7 2 2 4 4

1.32 1.31 2.44 2.21 2.02 3.01

ND 5+ 1 3 4 2 2

35 ± 6 37 41 26 34 ± 6

ND: not detected.

52 are attributable to several factors: differences in species, cell type, malignant versus nonmalignant phenotype, growth phase, and means of calculating protein induction. Perhaps most significant, however, is the possibility of differences in the endogenous synthesis of transforming growth factor-/3 (TGF-/3) or other growth factors between the glioma cell lines and the chick embryo fibroblasts. Detection of two major isoforms on reducing SDS-PAGE (Figs. 2 and 3, Table 1) in the majority of the human glioma cell lines analyzed agrees well with previously published data reporting analysis of glioma tenascin (Erickson and Lightner, 1988; Erickson and Bourdon, 1989; Taylor et al., 1989). Assignment of molecular weights of 320 kDa for the large form and 220-230 kDa for the small isoform was made on the basis of an appropriate set of molecular weight standards that migrate both below and above the tenascin species (see Lightner et al., 1989; Taylor et al., 1989). These values agree well with the estimates of 340 and 250 kDa for the large and small isoforms reported here. Previous reports from this laboratory (Bourdon et al., 1985) using the commercial high molecular weight standards underestimated the size of the large isoform. Gulcher et al. (1989) reported the appearance of two isoforms in the conditioned media of U-87 MG cultures. They assigned molecular weights of 240 kDa for the larger, more intense form and 180 kDa for the less intense, smaller form. Though it is difficult to be certain of the molecular weight of Gulcher's large isoform because of the absence of molecular weight standards above 200 kDa in their experiments, we propose that the intense 240 kDa band by Gulcher et al. (1989) corresponds to the intense 250 kDa band found in U-87 MG media in this study. As we found the larger 340 kDa isoform to be of such a weak intensity that it was nearly undetectable in some preparations of supernatant from U-87 MG, we believe this isoform went undetected in previously published work. We have not observed the 180 kDa species noted by Gulcher et al. (1989). No additional tenascin species were discovered in the analysis of the cell-associated compartment (Fig. 3) other than those already seen in the supernatants (Fig. 2). This suggests that no major

stable intracellular or cell-surface intermediates or metabolites of tenascin are present in glioma cells. Finally, the absence of both forms in the D-65 MG, D-259 MG, and U-343 MG lines may represent an actual absence of one or both isoforms or simply reflect an expression below the detection limits of our system. There is a single complete gene for tenascin in both chicken (Jones et al., 1988) and human (Gulcher et al., 1989, 1990) DNA. The presence of two major tenascin mRNAs in Northern blots (Figs. 5 and 6B) agrees with previously published results for human gliomas (Gulcher et al., 1989) and suggests that alternate mRNA splicing is the major mechanism for tenascin isoform generation. As with Western blots, cell lines that demonstrated only one of the mRNA transcripts (e.g., D-247 MG) may represent either actual expression deficits or undetectable signal levels. The fact that the D-65 MG cell line did not show a tenascin signal in the total RNA analysis but showed both transcripts on long exposure of poly(A) + Northern blots underscores the importance of assay sensitivity. Minimum estimated polypeptide molecular weights for the large- and small-arm isoforms, based on the estimated 9 and 7 kb sizes of the mRNA species, translation of the entire length of the mRNA, and an average amino acid molecular weight of 110 Da, are 330 kDa for the large-arm isoform and 257 kDa for the small. Glycosylation is reported to add only 10 kDa to the weight of both isoforms from U-251 MG-C3 tenascin (Taylor et al., 1989). The final estimate of 340 and 267 kDa for the large and small isoforms is in excellent agreement with the data from Western blots (Figs. 2 and 3) that dictate assignments of 340 and 250 kDa. Identification of two major arm variants in normal kidney and brain samples that comigrate with the arm species present in glioma cell lines (Fig. 7) suggests that no tumor-specific isoform patterns are present - - at least at the gross level of differing electrophoretic mobilities on SDSPAGE - - to differentiate gliomas from these normal tissues. Presence of the same 250 and 340 kDa isoform species in samples from different regions of cerebral cortex reveals no major regional differences in tenascin expression within

53 the cortex, but the absence of detectable tenascin in the sample of normal cerebellum is suggestive of potential variations within the central nervous system (CNS). As was the case with tenascin expression in glioma cell lines, Northern blotting data point to the alternative RNA splicing as the major factor in the generation of tenascin isoforms. The isoform predominance characteristics of normal brain and spleen match those of the majority of glioma cell lines in the preference for the large 340 kDa isoform. The small 250 kDa isoform predominance of normal kidney matches that seen in U-87 MG. Tenascin in human tissues was detected in the mesenchyme of spleen and kidney by immunohistochemistry with the a-tenascin MAb 81C6 (Bourdon et al., 1983), confirming the observations in this study. In contrast to this study, however, Bourdon et al. (1983) reported 81C6 binding to be present in normal liver. Expression of tenascin by human liver is also suggested by recent observations from paired-label localization studies using 81C6 and the murine IgG2b subclass control MAb 45.6, where 20-fold greater uptake of 81C6 relative to MAb 45.6 was noted in human liver and 3-fold greater specific localization was observed in livers of nonhuman primates (M. Zalutsky, unpublished data). Our inability to demonstrate tenascin in human liver extracts may be due to loss of protein and message by degradative proteases and RNases in the tissue or to poor extractability of tenascin from liver by our CAPS-based system. The potential for such degradation is not surprising in an organ with such high inherent metabolic activity. Accurate analysis of tenascin and tenascin mRNA expression will require improved methodology, such as analysis of freshly frozen surgical specimens, to reduce the impact of this degradation. The strong tenascin signal on polyclonal immunoblots of human cerebral cortical extracts reported here is consistent with several reports showing tenascin in normal brain in animal (Grumet et al., 1985; Crossin et al., 1989; Poltorak et al., 1989) and human (McComb and Miller, 1990) systems. Despite this evidence for the presence of tenascin in normal human brain, atenascin MAb 81C6 is unable to demonstrate significant specific binding to normal brain in

both immunohistochemical studies of normal human brain sections (Bourdon et al., 1983) and in in vivo paired-label localization studies in human brain tumor patients (Zalutsky et al., 1989). These differences in detectability of brain tenascin are likely due to both variations in antigen a n d / o r epitope availability and to sensitivity differences between 81C6 and polyclonal antisera. The blood-brain barrier and large diffusion distances between blood vessels and deep, watershed areas of cortex provide anatomic barriers to antibody access in vivo. The interaction of tenascin with other molecules at the histologic level both in vivo and in situ in tissue sections may well mask certain epitopes of tenascin, obliterating the binding of any MAbs that recognize these epitopes. The staining of the extensively homogenized, chemically extracted brain samples with polyclonal antiserum carried out in this study, however, is not likely to be limited by these factors. Additionally, reduced sensitivity of MAb 81C6 relative to polyclonal antiserum may account for the inability of 81C6 to react with available tenascin in vivo and in situ. Data showing that the sensitivity of 81C6 in these blots (> 16.7 /xg tenascin/ml sample; see Results for Fig. 4) is not sufficient to detect the tenascin levels in normal tissue extracts (all less than 8 /xg/ml), strongly suggest that antibody sensitivity may be important. Our quantitative data agree well with previously published values. U-251 MG-C3 is the only glioma cell line for which levels of secreted tenascin have been previously published, and our value of 12.2 /xg secreted tenascin/ml (Table 2) agrees well with the 5 - 2 0 / z g / m l found by Aukhil et al. (1990). Determination of tenascin content in normal cerebral cortical extracts of 34.7-41.1 /zg tenascin/g whole tissue is in good agreement with about 20/zg tenascin/g whole brain in embryonic brain reported by Lightner et al. (1990). Large differences in tenascin content between glioma cell line supernatants (median of 38 lzg tenascin/mg cell protein; Table 2) and normal brain and kidney (from 2 to 5 /xg/mg extract protein, Table 3), although not comparable directly, are highly suggestive of the possibility of similarly large differences between tenascin levels in normal and malignant tissues in situ.

54 In s u m m a r y , o u r studies into t h e p l e i o m o r p h i s m o f t e n a s c i n e x p r e s s i o n in h u m a n g l i o m a cell lines a n d a c o m p a r i s o n with that p r e s e n t in n o r m a l tissues have y i e l d e d i n f o r m a t i o n p o t e n tially useful to i m a g i n g a n d t h e r a p y o f m a l i g n a n t gliomas. W e have shown the p r e s e n c e of two m a j o r individual a r m a n d intact i s o f o r m variants for t e n a s c i n a n d two a l t e r n a t i v e l y spliced m R N A variants. A l t h o u g h the ratios of the large a n d small isoforms vary widely a m o n g t h e 21 g l i o m a lines a n a l y z e d in this study, we could find no e v i d e n c e of g l i o m a - s p e c i f i c isoform p a t t e r n s det e c t a b l e as d i f f e r e n c e s in e l e c t r o p h o r e t i c m o b i l i t y to distinguish g l i o m a s from n o r m a l b r a i n o r kidney. M a r k e d d i f f e r e n c e s in c o m p a r t m e n t a l i z a t i o n between cell-associated and secreted compartm e n t s a m o n g the g l i o m a lines m a y identify gliomas with s u p e r i o r t e n a s c i n clustering a n d c o n s e q u e n t l y i m p r o v e p o t e n t i a l for i m m u n o t h e r apy. T h e q u a n t i t a t i v e d i f f e r e n c e s in t e n a s c i n exp r e s s i o n o b s e r v e d b e t w e e n t h e g l i o m a cell lines a n d n o r m a l h u m a n tissue extracts a r e suggestive o f p o s s i b l e in vivo d i f f e r e n c e s in t e n a s c i n expression levels b e t w e e n n o r m a l tissues a n d m a l i g n a n t tumors. O n g o i n g w o r k in o u r l a b o r a t o r y is directly assessing t e n a s c i n e x p r e s s i o n in n o r m a l tissue sections, t u m o r xenografts, a n d surgical tum o r s p e c i m e n s with in situ hybridization. Differe n c e s b e t w e e n n o r m a l a n d m a l i g n a n t tissues in q u a n t i t a t i v e levels a l o n e m a y allow g o o d t h e r a p e u t i c efficacy of a p p r o p r i a t e a - t e n a s c i n M A b s , b u t w o u l d p e r m i t a m i n i m a l yet t h e r a p y - l i m i t i n g level of n o r m a l tissue b i n d i n g of a - t e n a s c i n M A b s . A l t h o u g h t h e i n t r o d u c t i o n of M A b s directly into t h e C N S via c o m p a r t m e n t a l t h e r a p y can h e l p c i r c u m v e n t this b i n d i n g of a - t e n a s c i n M A b s to low levels o f t e n a s c i n in e x t r a - n e u r a l tissues, m o r e d e t a i l e d b i o c h e m i c a l analyses of t e n a s c i n e x p r e s s i o n a r e n e c e s s a r y to u n c o v e r possible t u m o r - a s s o c i a t e d m o l e c u l a r p a t t e r n s t h a t can s u p p l e m e n t the q u a n t i t a t i v e d i f f e r e n c e s identified in this study, allowing f u r t h e r i m p r o v e m e n t o f t e n a s c i n - d i r e c t e d i m m u n o t h e r a p y o f tumors.

Acknowledgements This r e s e a r c h was s u p p o r t e d by N I H g r a n t s C A 11898, C A 32672, NS 20023, 7R29 C A 45586,

1R1 C A 52897, A R 38479, a n d T32 GM-07171-16. E d i t o r i a l assistance was r e n d e r e d by A n n T a m a riz.

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