Novel isoglobo-neolacto-series hybrid glycolipid detected by a monoclonal antibody is a rat colon tumor-associated antigen

Biochimica et Biophysica Acta, 1002 (1989) 267-272 Elsevier

267

BBA 53075

Novel isoglobo-neolacto-series hybrid glyeolipid detected by a rnonoclonal antibody is a rat colon tumor-associated antigen * Jan Thurin 1,2, Thomas Brodin 3 Bethany Bechtel 1, Per-}~ke Jovall 2,4, Hasse Karlsson 2 Nicklas $trSmberg 2, Susann Teneberg 2, Hans Olov SjSgren 3 and Karl-Anders Karlsson 2

9

t The Wistar Institute, Philadelphia, PA (U.S.A.) and 2 Department of Medical Biochemistry, UniversitFof GiJteborg, G6teborg~ The Department of Tumor lmmunolog),, Wallenberg Laboratory. University of Lund, Lund and 4 The Departmet~t of Biochemistry. University of GiJteborgand Chalmers Institute of Technology. G~teborg (Sweden) (Received 21 November 198[~)

Key words: Glycolipid; Monoclonal antibody; Adenocarcinoma; Colon tumor; (Rat)

lsogiobotetransyleeramide (GalNAc(flt-3)Gal(al-3)Gal(fil-4)Gle(fit-l)Cer), the major glycolipid species in dhnethylhydrazine-lnduced rat tumors of colorectal origin, was not detected in epithelial cells of normal colon but was present in the non-epithelial stroma and could be extracted from each of nine tumors studied. Monoclonal antibodies produced against isogiobetetraosylceramide detected this and another novel rat tumor tissue-associated glycolipid not present in epithelial cells nor in non-epitbelial stroma of normal rat colon (Brodin, T., Thur|n, J., Str6mberg, N., Karlsson, K.-A. and Sj6gren, H.O. (1985) Eur. J. |mmunol. 16, 951-956). This novel giycoiipid was present in 8 / 9 of the studied tumors and was also present it, two in vitro cell clones. These were originally obtained from a W 4 9 / T 4 colon tumor isograft. The novel giycolipid was characterized by mass spectrometry, IH-NMR, and methylafion analysis as a hybrid between the isogiobo, and neolacto-series, with the structure GalNAe(fll-3)Gal(al-3)Gal(fll-4)G|cNA(/~l-3)Ga| (fll-4)Gle(fll-l)Cer.

Introduction

Glycolipids are well-known tumor-associated markers in human and animal systems [1-3]. However, detailed studies to assess the potential usefulness of carbohydrate antigens in tumor diagnosis and therapy mediated by monoclonal antibodies would benefit from the availability of suitable animal models, lit the present study, we have focused on rat large intestine, in which glycolipid expression has been well-characterized [4-6], together with a defined rat colon tumor model [7]. In the tissue of dimethylhydrazine-induced tumors of colorectal origin, glycolipids of the isoglobo-series are found. These include the previously described isoglobotetraosylceramide (for structures, see Table I), and based on this an iso-Forssman glycolipid [6], present in the stroma * Nomenclature follows the IUPAC-IUB Commission on Biochemical Nomenclature (1978) Biocliem. J. 171, 21-35 and the Svennerholm nomenclature for gangliosides (1963) J. Neurochem. 10, 613-623. Correspondence: K.-A. Karlsson, Department of Medical Biochemistry, University of GtReborg, P.O. Box 33C~31, S-400 33 GiSteborg, Sweden.

but not in the epithelium of normal rat colon. We describe here the isolation and characterization of a novel hexaglycosylceramide present in these tumors but not in any of the normal colon compartments. Methods Animals. Wistar Furth (W/F) and Brown Norway (BN) rats were kept as inbred strains by strict brother to sister mating. They were fed on a standard pellet diet and water ad libidum. Tumors. Rat colon adenocarcinomas were induced by dimethylhydrazine (DMH) in W / F or BN rats. They were grown in vivo by serial passage in the syngeneic strain [7]. Cells. A tissue-cultured cell-line, DMH-W49, was estabhshed from in vivo-passaged tumor tissue and was cloned and subcloned by limiting dilution. These clones grew as solid tumors when inoculated intra-pefftoneally to syngeneic animals. Monoclonai antibodies. The monoclonai antibody 14.2 was produced by immunization of mice with the pufffled novel hexaglycosylceramide by a modification of the liposome-immunization technique described [8]. This

0005-2760/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

268

antibody binds to isoglobo-series glycolipids, in immunohistochemistry to rat colon adenocarcinomas and antigens expressed on the surface of cultured cells (Brodin et al.. unpublished data). Glycolipid purification. The frozen tumor tissue was first lyophilized and then extracted in a Soxhlet apparatus. Further fractionafion by ion exchange chromatography and silicic acid chromatography [9-11] yielded about 150 mg of total non-acid glycolipids, which were acetylated and separated on a silicic acid column, 1 × 50 cm (latrobeads 6RS-8060, latron Laboratories, Tokyo) by eluting with 300 fractions of 1 ml each, using a linear gradient of methanol in chloroform (0~10~). Aliquots of these fractions were deacetylated and tested for reactivity with the monoclonal antibody in the chromatogram binding assay. Positive fractions were pooled, de-acetylated and separated on the same type of column with a linear gradient of chloroform/methanol/water (60 : 35 : 8-50 : 40 : 10, v/v) as native glycolipids. Analysis of these fractions by thin-layer chromatography and chromatogram binding assay revealed that the slower migrating ceramide band of the novel glycolipid (as detected by the monoclonal antibody) was free of visible contamination. These positive fractions were then pooled to yield 1 mg of the glycolipid for further characterization. The glycolipids from the two cell lines were prepared essentially as described [12] from 100/~1 of packed cells each. Thin.layer chromatography (TLC). Silica Gel 60 HPTLC plates (Merck, Darmstadt, F.R.G.) were de. veloped with chloroform/methanol/water (60: 35 : 8, v/v) and detection was made with the aniuddehyde reagent [13]. Chromatofram binding assay. The assay [14] was performed as described [15]. Glycolipids separated on

A

thin-layer plates were overlayed with monoclonal antibody 14.2, as ascites diluted 1/100, and bound immunoglobulin was detected with t25I-labeled goat anti-mouse F(abr)2-specific serum (2000 cpm//tl). The autoradiogram ~'as developed overnight at - 70 ° C with an intensifying screen. Mass spectrometry. 10 ~tg sample was analyzed on a ZAB-HF mass spectrometer (VG Analytical, Wythenshire, U.K.) with electron energy of 70 eV, trap current of 200/~A and acceleration voltage 8 kV. The sample was distilled off between 260 and 300 °C and spectra were recorded on conventional paper as well as on a computer as 21 scans over 10 rain, using the direct inlet 'in beam' technique [16,17]. M/z values indicate nominal masses. Nuclear magnetic reasonance (NMR). 500/tg of the permethylated derivative of the novel glycolipid were analyzed in CaHCI3 at 312 K on a 270 MHz Bruker spectrometer using 10000 semis as described [18]. 200 /tg of the native novel glycolipid and 5 mg of isoglobo. tetraosylceramide reference were analyzed in 2H20 / water 98:2 (v/v) at 338 K on a 500 MHz Bruker spectrometer using 3 000 scans as described [19]. Methylation analysis. The permethylated glycolipid derivatives were degraded and acetylated as described [20]. Gas chrom~ography wi~s done on a Carlo Erba instrument (Carlo Erba Stru~lentasione, Milano, Italy) on a fused silice ~olumn 0.22 mw x 20 m [21]. l~eselts isoglobotetraosylceramide was found to be the major 81ycolipid presen~ in dimethylhydrazine-induced rat colon tumous ~22]. This glycolipid could be extracted from all 12 (representing 9 individual tumors) tumor

B

Fig. 1, (A) Total neutral glycolipid fractions of 12 different dimethylhydrazine induced rat colon tumors (representing nine individual tumors as three tumors we~ tested at two different numbers of in vivo passage) separated on HPTLC. (B) Same fractions analyzed by the chromatogram a ~ y with antibody 14.2. An amount of 20 pg/tumor was appfied per lane, corresponding to an average of 0.65~ of the dry tumor we/gh~ which were 3.1+ 1.2 mg/g. Upper arrow indicates isoglobotetrao~iceramide and lower arrow indicates novel isogloboneolacto hybrid hexaglycosylceramide.

269 TABLE I Specificity of monoclonalantibody 14.2 with structuresand trivial names of g;ycolipids used in this work The nomenclature is according to IUPAC-IUBCommission of BiochemicalNomenclature, Biochem.3. (1978) 171, 21--35.

Gal(al-4)Gal(fll-4)Glc(fll-1)Cer Gal(al-3)Gal(/31-4)Glc(/~l-1)Cer GalNAc(/~l-3)Gal(al-4)Gal(/31-4)Glc(fll-1)Cer GalNAc(fll-3)Gal(al-3)Gal(fll-4)Glc(/]l-1)Cer Gal(/]l-3)GlcNAc(fll-3)Gal(fll-4)Gic(fll-1)Cer Gad(/31=4)GIcNAc(/]1-3)Gal(fl1-4)G1¢(~81- 1)Cer Gal(fll-3)GalNAc(/31-4)Gal(/31-4)Glc(/~l-1)Cer GalNAc(~l-3)Gal(al-3)Gal(/~l-4)GlcNAc(fll-3)Gal(fll-4)Glc(/]l_l)Cer Gal(al-3)(Fuc(al-2))Gal(/]l-4)(Fuc(al-3))GlcNAc(/31-3)Gal(/~l-4)Gal(ASl-1)Cer Gal(a1-3XFuc(al-2))Gal(/31-3)(Fuc(a1-4))GlcNAc(/]l-3)Gal(/~l-4)Gal(/~1-1)Cer Gal(el- 3XFuc(al-2))Gal(fll- 3)GlcNAc(fll- 3)Gal(~l-4)Glc( ~l-1)Cer Gal(al-3)(Fuc(al-2))Gal(fll-4)Gic(fll-1)Cer Fuc(al-2)Gal(/31=3)GlcNAc(/~l-3)Gal(/~l-4)GIc(fll-1)Cer Fuc(ed-2)Gal(fll-4)GlcNA¢(/~l=3)Gal(fl1-4)Glc(//l-1)Cer Gal(/31-3)(Fuc(al=4))GlcNAc(/~l=3)Ga|(/~l=4)Gic(~l=l)Ccr Gal(//1-4)(Fuc(al=3))GlcNAc(fll-3)Gal(fll-4)GIc(//1-1)Cer Fuc(at-2)Gal(~l-3)(Fuc(al-4))GlcNA~(/31-3)Gal(~81-4)Glc(181-1)Cer Fuc(a1-2)Ga~(~l-4)(Fuc(a1-3))GlcNAc(ill- 3)Gal(~l =4)GIc(fll- 1)Cer

tissues tested, as demonstrated in Fig. 1A, but not from normal rat colon epithelial cells [5]. Several monoclonal antibodies to this glycolipid have been generated [8]. The specificity of ,~..ze of these antibodies, designated 14.2, is described in Table I. It was used to demonstrate the presence of two antigens in nine different colon tumors, as shown in Fig. lB. The isoglobotetraosylceramide is present in approximately the same amount in all tumors, while the novel hexaglycosylceramide is detectable in variable amounts in all but one of the tumors. Both antigens were also detected in glycolipid extracts from two cloned cell lines established from the syngeneic rat tumcx W49/T4, as shown in Fig. 2. The novel glycolipid was not detected in normal colonic tissues, neither epithelial nor non-epithelial [5].

1

2

3

Fig. 2. Autoradiogram of chromatogram binding assay with antibody 14.2 on non-acid glycolipid fractions from (1) tumor W49/T4, (2) cultured cells of clone 1, and (3) cultured cells of clone 2. Approx. 20 jag applied per lane of each fraction. Only the six-sugarantigen is seen under these conditions(comparerelative reactivitiesin Table I).

Trivial name Gb3Cer iGbaCer Gb4Cer iGb4Cer Lc4Cer nLc4Cer Gg4Cer iGbnLc6Cer B-7-2 B-7-1

Reacti~~'.

+

+++

B.6.1 B.4 H-5-1

H-5-2 Le"-5 X-5 Leb.6 Y-6

Structural characterization of novel glycolipid The novel glycolipid was purified in the amount of 1 mg from 500 g (wet weight) of pooled W49/T4 tumors. Fig. 3 shows the mass spectrum of the permethylated glycolipid and a formula indicating the typical fragmentation of permethylated glycolipids [23]. The fragments at m / z 260 and the absence of fragments, at m / z 219 and 189, i.e., the masses for terminal hexose and deoxyhexose, respectively, indicated the presence of a terminal N-acetylhexosamine in the molecule. Evidence for a terminal tetrasaccharide consisting of HexN-HexHex-HexN is derived from peaks at m / z 913 and 881 ( 9 1 3 - 32). The latter fragment results from loss of methanol (i.e., 32) that accompanies cleavage internal to hexosamines. Peaks at m / z 1117 and 1321, together with a peak at m / z 1394 (1393 + 1), corresponding to a fragment containing the saccharide and part of the ceramide, indicated that the glycolipid was a hexaglycosylceramide with a saccharide sequence of HexNHex-Hex-HexN-Hex-tlex-Cer. Only one ceramide peak was fo~:,d, m / z 548, indicating that spingosine with 16:0 fatty acid was the only ceramide present in the sample (the procedures necessary to separate the closely migrating glycolipid species resulted in recovery of only the slower migrating ceramide, and long-chain fatty acid species may therefore also have been present in the tissue). Further sequence information was given by the intense ion at m / z 1201, which contained the ceramide plus the internal tdsaecharide. The total mass was therefore calculated as 1885 mass units (1321 + 16 + 548% consistent with the M - 1 peak at m / z 1884. The chemical shifts and coupling constants (when measurable) of the 1H-NMR spectrum obtained from

270

it~2)l~

, Lcoo

I

- ~

~--

,[

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HEXlI~c~ O •He~ ] 0

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-

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i

I1201

I

,768

~

100-][[

1

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"

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013

1253

xZ0

0

100

~..xl0

~17

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50

B93+1

ll~I~

1300

1500

UO0

1900

Fig. 3. Mass spectrum of the permethylated derivative of the novel hybrid glycofipid together with a simplified formula with typical fragmentation: for interpretation.

0.5 mg of the permetylated glycolipid in C3HC13 at 312 K are shown in Table II. The four/3-anomeric signals at 4.63, 4.34, 4.30 and 4.21 ppm were identical to those reported for neolactotetraosylceremide [18], susgesting a structure of HexN-Hex-Gal(/31-4)GlcNAc(/31-3) Oal(~l-4)GIc(~l-1)Cer. The ~-a.uomeric signal at 453 ppm was identical with the terminal GalNAc(/~l-3) resonance found in the spectrum of isoglobotetraosylceramide but not with that of 81obotetraosylceramide, which exists at 4,64 ppm [18]. An ~-anomcric signal at

5.13 ppm for 3Gal(al-3) should have been d¢:ected, especially since the signal for 3Gal(ed-4) at 4.95 ppm was absent, but this region was unfortunately obscured by a large signal from a solvent contaminant (ethanol, at 5.15 ppm). However, an a-anomeric signal at 4.91 ppm was observed when the native glycolipid was analyzed. This chemical shift is identical with that of the anomeric signal for a reference isoglobotetraosylceramide, which was obtained under the same conditions. Thus, this provides evidence for a 3Gal(al-3) at the

TABLE it NMlt

Chemical thifts in ppn~ relative tetramethyhilane, with coupling constants (Hz) in parentheses for the permethylated derivative of the novel glycolilgd and ~fereace 81ycolipkh obtained at 312 K in C2HC13. (a) and (0) stand for small (a-anomeric) and large (~-anomeric) coupling constants, respectively. GalNAc(•I-

3)Gal(al-

3)Gal(pl-

4)GleNAc(~I-

3)Gal(~l-

4)GIc(~T-1)Cer

4.74 (,8)

5.13 (a)

4.34 (,8)

4.63 (,8)

4.30 (p)

4.21 (j~)

GaiNAc(Bi-

3)Gal(el-

3)Oal(a~l-

4)Glc(p1-1)Cer

4.74" (.8)

5.13 (a)

4,34 (B)

4.21 (.8)

Isoglobotetraosylceramide

G~(.81-

4)GleNAc(.BI -

3)Gal(,B1-

4)Glc(fll-1)Cer

4.34 (8.0)

4.63 (8,0)

4.30 (8.0)

4.19 (8.0)

OalNAc(~-

3~(,~-

4)o~(.m-

4)Ol~(~-l)Cer

4.64 (7,0)

4.95 (2,9)

4.32 (7.4)

4.21 (8,0)

Novel glycofipid

Neolactotetraosylceramide Globotetraosylceramide

Gal(,81.-

3)GIeNAc(,81-

3)Gal(,81-

4)Olc(~l-1)Cer

4.98 (8.0)

4.65 (8.1)

4.30 (7.4)

4.19 (8.0)

Chemical shifts obtained from isoslobotetraosylceramide,also prepared from rat colon adenocarcinoma.

Laetotetraosylceramide

271 penultimate binding position, and therefore favors a terminal structure of GalNAc(fll-3)Gal(od-3)Gal for the novel hexaglycosylceram/de. Methylation analysis confirmed this structure. Reference glycolipids were Gal(fll-3)GlcNAc(/~l-3)Gal(fll -4)Glc(/~l-l)Cer (prepared from human meconium, Ref. 24) and GalNAc(fll-3)Gal(/~I-4)GlcNAc (fll-3)Gal(fll-1)Cer (prepared from human blood group H erythrocytes, Ref. 25). Peaks for acetates of 3,4,6-trimethyl-GalNAcMe and 3,6-dimethyl-GlcNAcMe confirmed a GalNAcl and 4GIcNAcl, respectively A peak corresponding to 2,3,6-trimethyl-Glc and a 3-fold larger peak indicating 2,4,6-trimethyl-Gal, revealed a 3Gall at the penultimate position. Thus, the complete structure of the novel glycolipid isolated was concluded as GalNAc(/~l-3)Gal(al-3) Gal(fll-4)GlcNAc(/~l-3)Gal(fll-4)Glc(.~l-1)Cer, with sphingosine and nonhydroxy 16:0 fatty acid. Discussion

Isoglobotetraosylceramide, originally called cytolipin R, was one of the first non-neural glycoLpids to be described. This glycolipid was identified initially on the basis of its reactivity with antiserum raised in rabbit against a rat lymphosarcoma extract [26], which was preceded by studies demonstrating immunc~genicity of non-acid glycolipids by xenogeneic inmaunization [27]. The present study reveals the presence of isoglobotetraosylceramide and a novel hybrid glycol~pid in rat colorectal tumor tissue, with a carbohydrate terminal GalNAc(~l-3)Gal(al-3)Gal, both reactive with monoclonal antibodies generated in mice. These s~ructurally related glycolipids, normally absent in epithelial cells, were detected in 9/9 and 8 / 9 of the tumor tissues extracted, including tumors of a wide variety of in vivo passage times (4-60 passages). The presence of isoglobotetraosylceramide in normal rat small and large intestine is confined to the non-epithelial stroma [5]. Although isoglobotetraosylceramide could also be a compoaent of tumor tissue stroma, the detection of isoglobotetraosylceramide and the normal hybrid antigen in the tumor cell lines as demonstrated in Fig. 2 shows that they are authentic antigens expressed in the tumor cells and not degradation artifacts. Thus, the

novel hybrid glycolipid is a tumor-associated molecule which is present neither in normal colon stroma nor in epithelial cells. Epithelial cells of the normal rat large intestine express both a blood group B-active tetraglycosylceramide (B-4) and a difucosyl type-2 chain heptaglycosylceramide (B-7-2) [4,5]. These and other blood group active fucolipids, are expressed to a varying degree in dimethylhydrazine-induced epithelial rat colon adenocareinomas passaged in vivo in syngeneic rats and in tissue cultured cell lines (data not shown). Table III gives the structures of the isoglobotetraosylceramide and the novel tumor-associated hybrid glycolipid as compared with the B-4 and B-7-2 glycolipids. The core structures of B-4 znd B-7-2 correspond to those of isoglobotetraosylceramide and the hybrid glycolipid, respectively. Furthermore, the relative amounts of these glycolipids in normal versus tumor tissue were the same, i.e., the B-4 and isoglobotetraosylceramide were both major components, each compound accounting for 30-40% ot the total neutral glycolipid content, whereas the B-7-2 and the novel hybrid glycolipid each comprised less than 570 of the total glyeolipid amount. A synthetic relationship, if it exists in these pairs, implies that when less fucose is added (in tumors with reduced expression of blood group fuco!ipids), possibly due to a decreased fucosyltransferase activity, then GalNAc(fll-3) is added efficiently (leading to the expression of isoglobotetraosylceramide and the novel hybrid glycolipid), implying a normal or enhanced galactosaminyltransferase activity. A similar synthesis of this linkage (Gal(al-3)Gal) and synthetic relationship have also been reported in rat granulomas [28], where it was detected either in a blood group B active hexaglycosylceramide or in isoglobotetraosylceramide. Tissue-cultured normal colon epithelial cells and tumor cells established from early passages of these tumors will be needed in further studies to confirm the existence of this relationship. This is necessary both to avoid contamination of stromal components (that could be expected to contain isoglobotetraosylceramide) and to be able to pinpoint the sequence of changes in glycolipid expression also in relation to other biochemical, morphological and biological aspects of the tumor cell phenotype.

TABLE111 Proposed changes of glycolipid s.ynthesis in dimethyihydrazine-induced rat tumors compared to normal colon

The tumorglycolipidsare suggestedto be a resultof decreasedfucosyltransferaseand increased N.acetylgalactosaminyltransferaseactivities. Gal(al-3XFuc(al-2))Gal(~l-4)(Fu~(al-3))GlcNAc(fll-3)Ga1(/~l-4)Glc(fl1-1)Cer GalNAc(/31-3)GaI(a1-3)Gal(fl1-4)GIcNAc(~1-3)Gal(/31-4)Glc(fll-I )Cer Gal(ed-3)(Fuc(al-2))Gal(fll-4)Glc(fl1-1)Cer

GalNAc(fll-3)Gal(al-3)Gal(fll-4)Glc(fll-1)Cer

B-7-2 iGbnL%-hybrid B-4 iGb4Cer

Colon Tumor Colon Tumor

272

Ad,m ~ t S a m a t s The skilled assistance of Weston Pimlott with mass spectrometry analyses is 8ready appreciated. We are also indebted to Marina Hoffmun for editorial help. This work was supported by 8rants from the Swedish Medical Reseasch Council (3967, 6498 and 6810) and National Institutes of Health (CA-10815 T-32CA09171).

I lqekomod, S.-L (1984) in Monodoaal Antibodies and Functional Cell Lines (llechtok If,.,ed.), pp. 67-100, Plenum Press, New Yo& 2 Petsi, 3'. (19~) Nature 314, $3-$7. 3 Thurin, L (1965) in C~wat topics in Micmbiololy and Immunolq y (Wilton, !, and Clmke, A,), Vol. 139, pp, 59-79, Springer4 ~

O.C,, Kadsma, K.-A~ and Thrown,L (1980) Biochim. ~ephy~ Acta 520, 270-7Jt0. 5 Hmmmt, G,C,, lfJuqsm~ K.-A. and Thurln, J. (1984) Biochim. W~phyLAm 792, 281-292. 6 Fdk, P., H o ~ m m , J., ~Mn, P.-A, Lmo~, O., K=hson, K..A., p., SUemberS,N., Th.de~ J., erodin, 1". and S j ~ 14.O. (1956) _aL~h_l~Biophys.Acta 878, 29~.-299, 7~ 14,0. (1980) Cancer 45, 1229-.1233. 8 kodia, T., Thurin, $., Stt6mberll,N., Kadsson, K.-A. and SjOltren, a.o. (198.5)Fur.J. ~ le, 951-956. 9 aotma, E. and Htkemmi, S..i. (1982) J. BioL Chem. 257, 7698-.7703. 10 Bmimer, M.E, l~_~.m~_; G.C., Katisma, K..A. and Le~er, H. (1981) Exp. Cell. l ~ . 1~, 1-13. 11 ~ K.-A. ( 1 ~ ) Methods EmpTmoL138, 212-220. 12 HedyL M., Thufln, L, Balal~u~ O., BennicelU,J,L, Herlyn, D., Elder, D.A., ~ E., Ouerty, D, IV., Nowell, P., Clark, W.H., Jr, 8rid KoproMki. H. (19~) Cancer ~ 45, 5670--$676.

13 Stahl, E. (1962) in DUnnschichts-Chromatographie, SpringerVerla8, Berlin. 14 Magnani, J.L., BreclChaus, M., Smith, D.F. and Ginsburg, V. (1983) Methods Enzymol. 83, 235-240. 15 Hansson, G.C., Karlsson, K.-A., Larsson, G., McKibbin, J.M., Blaszczyk, M., Herlyn, M., Steplewski, Z. and Koprowski, H. (1983) L Biol. Chem. 258, 4091-4097. 16 Breimer,M.E., Hansson, G.C, Karlsson, K.-A. Leffler, H., Pimlott, W. and Samuel,on, B.E. (1981) FEBS Lett. 124, 289-303. 17 Burlinlpune, A.L., Dell, A. and Rusell, D.H. (1982) Anal. Chem. 54, R363-R409. 18 Falk, IL-E., Karlsson, K.-A. and Samuelsson, B.E. (1979) Arch. Biochem. Biophys. 192, 164-202. 19 BlaszczykoThurin,M., Thurin, J., Hindssanl, O., Karlsson, K.-A., Steplewski, Z. and Koprowski, H. (1987) J. Biol. Chem. 262, 372-379. 20 Stellner, K., Saito0 H. and Hakomori, S.-i. (1973) Aleh. Biochem. Biophys. 155, 464.-472. 21 Leffler, H., Hansson, G.C. and StrSmbers, N. (1986) J. Biol. Chem. 261, 1440-1444. 22 Hansson, G.C., Karlsson, K.-A., Larson, ¢3., StrSmber8, N., Teneber~ S,, Thurin, J., Brodin, 1". and Sjt~ren, H.O. (1983) in Glycomnjusates (Chester, A,M., Heine~rd, D., Lundbled, A. and Svensson, S., eds.), Proceedings of the 7th International Symposium on Glycoconjuipttes,pp. 854-855, Raluns, Land, Sweden. 23 K~rlsso~ K.-A., Pascher, !., Pimlott, W. and Samuelsson° B.E. (1974) Biomed. Mass Spectrum. 1, 49-56. 24 Karlumn, K.-A. and Larson, G. (1979) J. Biol. Chem. 254° 9311-9316. 2.5 ~nB~r~m, J., Karlsson, H., Karlsson, K.-A., Larson, G. and Nihon, K. (1986) Arch. Bier.hem.Biophys. 251, 440-449. 26 l~pport, M.M,, Schneider, H. and Graf, L. (19457) Biochim. Biophys. Acta 137, 409-.411. 27 Rapport, M.M. and Graf, L (1955) Cancer 8, 538-545. 28 Hanada, E., l.Janda° S., Konno, K. and Yamakawa, T. (1978) J. Biochem. (Tokyo) 83, 85-90.