Ž .Brain Research 755 1997 175–179

Short communication

a 2,3-Sialyltransferase mRNA and a 2,3-linked glycoprotein sialylation areincreased in malignant gliomas

Hirotaka Yamamoto a, Tasuku Saito a, Yoichi Kaneko a, Donna Kersey a, Voon Wee Yong b,Eric G. Bremer a, Edward Mkrdichian a, Leonard Cerullo a, Jan Leestma a, Joseph R. Moskal a,)

a The Chicago Institute of Neurosurgery and Neuroresearch, 2515 N. Clark St., Suite 800, Chicago, IL 60614, USAb Department of Neurology and Neurosurgery, Neuroimmunology Unit, Montreal Neurological Institute, 3801 UniÕersity, Montreal, Que. H3A 2B4,

Canada

Accepted 18 February 1997

Abstract

Ž . Ž .CMP-NeuAc: Galb1,3 4 GlcNAc a 2,3-sialyltransferase a 2,3-ST mRNA was expressed in human glioma specimens, human fetalastrocytes, and a panel of brain tumor cell lines. Maackia amurensis agglutinin staining revealed the presence of a 2,3-linked sialic acidson glioma cell surfaces and extracellular matrices whereas normal human adult astrocytes were negative. Increased expression ofa 2,3-linked glycoprotein sialylation may play a role in glial tumorigenesis.

Keywords: Sialyltransferase; Brain tumor; Glioma; Glycoprotein; Sialic acid

Alterations in the expression of terminal sialic acidresidues on glycoconjugates are common phenomena in

w xoncogenic transformation 8,9,14,17,20,23,25 . Increasedcell-surface sialylation has been implicated in invasivityw x w x5 , tumor cell-mediated platelet aggregation 1 , resistance

w xto T-cell mediated cell death 27 , adhesion to endothelialw xcells and extracellular matrices 6 , and metastatic potential

w x15 .There are at least 10 distinct enzymes that transfer sialic

acid to the termini of the oligosaccharide moieties ofglycosphingolipids and glycoproteins. These sialyltrans-ferases comprise a structurally related family of moleculesthat display substrate specificity, tissue specificity, and are

w xall developmentally regulated 11 .While a number of investigators have used cell lines

derived from vertebrate brain tumors to study the expres-wsion and regulation of various glycosyltransferases 2–

x4,13,16,22 , studies using primary human brain tumorw xmaterial have been very limited. Shen et al. 21 reported

that serum sialyltransferase, using desialylated fetuin as theacceptor, did not significantly differ from controls in glioma

w xpatients and Gornati et al. 7 found that the sialyltrans-

) Ž .Corresponding author. Fax: q1 773 935-2132.

ferase involved in the biosynthesis of GD3 from GM3ganglioside was altered in meningiomas. We have exam-ined the expression of the CMP-NeuAc: Galb1,4GlcNAc

w x Ž .a 2,6-sialyltransferase EC 2.4.99.1 a 2,6-ST in a varietyw xof human brain tumors 9,28 . a 2,6-ST is one of the two

characterized sialyltransferases that transfers sialic acid toN-linked glycoprotein oligosaccharides. While this enzymehas been suggested to play an important role in the onco-

w xgenic transformation of colon mucosa 19 and be anw xindicator of metastatic and invasive potential 2,12 , no

a 2,6-ST expression was found in gliomas or metastases tothe brain.

As part of a continuing effort to elucidate the molecularmechanisms that underlie the changes in cell-surface sialy-lation that accompany brain tumorigenesis, we report here

Ž .on the expression of CMP-NeuAc: Galb1,3 4 GlcNAcŽ . w xa 2,3-sialyltransferase a 2,3-ST mRNA 26 and the ex-

pression of cell-surface a 2,3-linked sialic acid expressionin primary brain tumor specimens, established brain tumorcell lines, and normal brain tissue. This study was under-taken because this a 2,3-ST, along with the a 2,6-STdescribed above, are the two enzymes responsible foreffectively all terminal sialylation of N-linked glycoprotein

w xoligosaccharides 6,23 .ŽFor the Northern analyses shown in Fig. 1 upper

.panel , a panel of surgical specimens was used that con-

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 97 00241-2

( )H. Yamamoto et al.rBrain Research 755 1997 175–179176

Ž . Ž .Fig. 1. The expression of a 2,3-ST in glioma specimens upper panel and brain metastases lower panel . 30 mg of total RNA per lane were used forNorthern analysis. Upper panel – lane 1: normal human brain, lanes 2–14: clinical glioma specimens, lane 15: U-373MG human glioma cell line. Lowerpanel – lane 1: normal human brain, lanes 2–10: clinical specimens of brain metastases, lane 11: U-373MG human glioma cell line. Human a 2,3-ST

Ž .cDNA was cloned by using the reverse-transcriptase polymerase chain reaction RT-PCR and poly Aq RNA from U-373 MG cells based on thew x X X Ž . Xsequence reported previously 11 . A sense primer, 3 -CTGGACTCTAAACTGCCTGC-5 bp 196–215 and an antisense primer, 5 -CCCAGAGACTTGT-

X Ž . Ž .TGGC-3 bp 524-508 were used. A 329 bp PCR product was subcloned into pT7 Blue T vector Novagen, Madison, WI and the sequence of the insertŽ .was confirmed by the dideoxy termination method Sequenase, United State Biochemical, Cleveland, OH . The cDNA coding for human a 2,3-ST cDNA

Ž .was gel purified following Xba I and Bam HI digestion of the vector and used as the template. All glioma specimens expressed a 2,3-ST mRNA A and 7Ž . Ž .out of 9 metastases expressed a 2,3-ST mRNA B . Ethidium bromide staining of total RNA panel B .

sisted of 13 gliomas: 1 astrocytoma grade II, 1 high-gradeoligodendroglioma, 1 mixed glioma, three cases of astrocy-toma grade III and seven cases of astrocytoma grade IV,

Ž w x.i.e. glioblastoma, WHO Brain Tumor Classification, 10 .Although the expression appeared variable, it is clear that12 of 13 gliomas, as well as normal brain, expresseda 2,3-ST mRNA. The only negative specimen was the

Žgrade II astrocytoma in Fig. 1, lane 2. In Fig. 1 lower.panel , a panel of surgical specimens was used that con-

sisted of nine metastases to the brain: four adenocarcino-

Fig. 2. Expression of a 2,3-ST in human brain tumor cell lines and fetalastrocytes. All established human neural cell lines were maintained using

ŽDulbecco’s modified Eagle’s medium DMEM, containing 4.5 grl glu-. Žcose supplemented with 10% heat-inactivated fetal bovine serum Whit-

.taker BioProducts, Walkersville, MD . Fetal astrocytes were preparedw xaccording to a method described previously 29 . 20 mg of total RNA per

lane were used for Northern analysis. Upper panel, A: lanes 1–5: humanglioma cell lines, SNB-19, SW1088, U-118MG, U-373MG, and U-87MG,respectively. Lanes 6–8: human neuroblastoma cell lines, SKN-MC,LAN-5, and IMR 32, respectively. All brain tumor cell lines expressed

Ž .a 2,3-ST mRNA. Ethidium bromide staining of total RNA B . Lowerpanel, A: lane 1: human neuroblastoma IMR 32; lane 2: human neurob-lastoma LAN-5; lane 3: cultured human fetal astrocytes; lane 4: humanglioma U-373MG; lane 5: human glioma U-118MG. Ethidium bromide

Ž .staining of total RNA lower panel, B .

mas of lung origin, three adenocarcinomas of unknownorigin, one papillary clear cell tumor of renal origin andone large cell neoplasm of unknown origin. Seven of these

( )H. Yamamoto et al.rBrain Research 755 1997 175–179 177

nine samples expressed a 2,3-ST mRNA. Both negativeŽ .samples Fig. 1, lower panel, lanes 2 and 7 were adeno-

carcinomas of unknown origin. The expression of a 2,3-STmRNA was also detected in all human glioma and neurob-lastoma cell lines examined, and was particularly high in

w x Žcultured human fetal astrocytes 29 Fig. 2, upper panel.and Fig. 2, lower panel, respectively .

Based on these data, there appeared to be little differ-ence in the level of a 2,3-ST mRNA expression betweenglioma specimens and control normal brain tissue. In orderto identify the cells expressing glycoproteins bearinga 2,3-linked sialic acids, Maackia amurensis agglutininŽ . w xMAA lectin staining was performed 24 . Fig. 3 showsthat, while normal adult astrocytes and neurons were notstained with MAA, robust staining of glioblastoma tissue

Žwas observed. For example, a glioblastoma specimen thespecimen used in lane 4 of the Northern analysis shown in

.Fig. 1, upper panel displayed heavy cell-surface stainingŽ .of pleomorphic tumor cells Fig. 3A as well as at the

Ž .invasion front proximal to the surrounding tissue Fig. 3C .

ŽIn another glioblastoma specimen lane 10 in Fig. 1, lower.panel , the matrices of clusters of undifferentiated small

cells were stained with MAA, while proliferating endothe-lial cells derived from glomeruloid neovascularization were

Ž .not stained Fig. 3B ; large vascular lumina in glioblas-Ž .tomas were rarely stained data not shown . The predomi-

nant MAA-positive cells found in normal adult cerebralcortex and white matter were vascular endothelial cells,suggesting that a 2,3-ST activity may play an importantrole in neovascularization. It should be noted that, becauseof the inherent limitations in the sensitivity of the detectionmethod used in these studies, we cannot entirely rule outthe possibility that normal adult astrocytes do expressa 2,3-linked sialo-glycoproteins at very low levels. Torecapitulate, under the conditions employed in these stud-

Žies, no expression of a 2,3-linked sialic acids as demon-.strated by MAA lectin histochemistry could be detected in

adult human astrocytes but robust staining of fetal astro-cytes, normal adult brain vascular endothelial cells, andprimary human glioma specimens were observed. With

Ž . Ž .Fig. 3. Increased Maackia amurensis agglutinin lectin MAA staining in gliomas. The sections 6 mm thick were dewaxed, hydrated and soaked inŽ . ŽTris-buffered saline TBS, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 for 1–18 hours at 378C, then incubated in 0.5% blocking reagent Boehringer

. ŽMannheim, Indianapolis, IN in TBS for 45 min. After rinsing twice with TBS and once with Buffer 1 TBS with 1 mM MgCl , 1 mM MnCl , 1 mM2 2. Ž .CaCl , pH 7.5 for 10 min each, digoxigenin-labeled MAA Boehringer Mannheim 10 mgrml in Buffer 1 was overlaid for 1 h. After washing with TBS2

Ž . Ž .3=10 min , the sections were incubated with anti-digoxigenin Fab-conjugated with alkaline phosphatase Boehringer Mannheim at concentration ofŽ .either 0.75 or 1.5 Urml TBS for 1 h. After washing three times with TBS, BCIPrNBT solution Sigma, St. Louis, MO was overlaid as chromogen for

Ž .3–40 min. The sections were rinsed with deionized water and lightly counterstained with nuclear fast red. Surfaces of glioblastoma cells A , extracellularŽ . Ž . Ž .matrices between glioblastoma cells B and glioblastoma parenchyma C were heavily stained, while vasculatures within the tumors B, C remained

Ž .negative. Positive MAA staining was observed in capillaries of normal cerebral cortex, but not in neurons or glial cells D . Barss50 mm.

( )H. Yamamoto et al.rBrain Research 755 1997 175–179178

respect to a 2,3-ST mRNA expression, it too was observedin human fetal astrocytes, established glioma cell lines,and primary human glioma specimens. Northern analysisfor a 2,3-ST mRNA was performed on whole brain andwas found to be positive. However, lectin histochemicalanalysis with MAA revealed that only vascular endothelialcells were positively stained. Thus we have concluded thata 2,3-ST mRNA expression in normal adult brain is ex-pressed in vascular endothelial cells and at best, expressedat very low levels by normal adult glia.

The differential MAA lectin staining of glioma cellsurfaces but not normal adult glia and the heavy MAAstaining of glioma-associated extracellular matrices sug-gests the presence of glioma-associated glycoproteins bear-ing a 2,3-linked sialic acids. Since a 2,3-ST mRNA ex-pression was detected in normal human fetal astrocytes,perhaps, like other cell-surface glycoconjugates, they may

w xbe onco-fetal antigens under developmental regulation 11 .Since gliomas synthesize various extracellular matrixglycoproteins such as fibronectin, collagens, vitronectin

w xand tenascin 18,30 , a 2,3-linked sialic acids may bepresent on one or more of these proteins. To date, neitherthe identification of glioma-associated glycoproteins carry-ing a 2,3-linked sialic acids nor their function has beenexamined.

Besides secreting their own extracellular matrix compo-nents, malignant gliomas are very invasive. Many studieshave shown a correlation between increased terminal sialy-lation of cell-surface glycoproteins and both the metastatic

w xand invasive potential of a variety of tumors 5,14,15,23 .In agreement with these reports, a 2,3-ST was also foundin most of the metastases to the brain. It has also beenreported that terminal sialylation of glycoproteins found inhuman chronic myelogenous leukemia K562 cells in-

w xcreases their resistance to T-cell-mediated cell lysis 27 .Since malignant gliomas are resistant to T-cell mediatedlysis, increased terminal sialylation may be important intheir ability to escape immune surveillance.

In conclusion, based on the experiments presented herew xand previously 28 it appears that it is the a 2,3-ST as

opposed to the other terminal N-linked glycoprotein sialyl-transferase, a 2,6-ST, that is the principal enzyme involvedin the increased terminal sialylation of N-linked oligo-saccharides found on glycoproteins in malignant braintumors.

Acknowledgements

The authors would like to gratefully acknowledge Mr.Kevin Cramer for his assistance in obtaining fresh tumorspecimens. Work was supported in part by grants from the

Ž .Illinois division of The American Cancer Society H.Y. ,Ž .The Buchanan Foundation J.M. , The Brach Foundation

Ž . Ž .J.M. , and The Falk Foundation J.M. .

References

w x1 E. Bastida, L. Almirall, G.A. Jamieson, A. Ordinas, Cell surfacesialylation of two human cell lines and its correlation with their

Ž .platelet-activating activity, Cancer Res. 47 1987 1767–1770.w x2 R.S. Bresalier, R.W. Rockwell, R. Dahiya, Q.-Y. Duh, Y.S. Kim,

Cell surface sialoprotein alterations in metastatic murine colon can-cer cell lines selected in an animal model for colon cancer metasta-

Ž .sis, Cancer Res. 50 1990 1299–1307.w x3 P. Broquet, H. Baubichon-Cortay, P. George, M.J. Peschard, Effect

of desipramine on a glycoprotein sialyltransferase activity in C6Ž .cultured glioma cells, J. Neurochem. 54 1990 388–394.

w x4 P. Broquet, P. George, J. Geoffroy, P. Reboul, P. Louisot, Study ofO-glycan sialylation in C6 cultured glioma cells: evidence forpost-translational regulation of a beta-galactoside alpha 2,3 sialyl-tranferase activity by N-glycosylation, Biochem. Biophys. Res. Com-

Ž .mun. 178 1991 1437–1443.w x5 J.G. Collard, J.F. Schijven, A. Bikker, G. LaRifiere, J.G.M. Bolscher,

E. Roos, Cell surface sialic acid and the invasive and metastaticŽ .potential of T-cell hybridomas, Cancer Res. 46 1987 3521–3527.

w x6 J. Dennis, C. Waller, R. Timpl, V. Schirrmacher, Surface sialic acidreduces attachment of metastatic tumor cells to collagen type IV and

Ž .fibronectin, Nature 300 1982 274–276.w x7 R. Gornati, S. Basu, G. Montorfano, B. Berra, Glycosyltransferase

activites in human meningiomas. Preliminary results, CancerŽ .Biochem. Biophys. 15 1995 1–10.

w x8 W.J. Grimes, Sialic acid transferases and sialic acid levels in normalŽ .and transformed cells, Biochemistry 12 1973 990–996.

w x9 Y. Kaneko, H. Yamamoto, D. Kersey, K. Colley, J. Leestma, J.Moskal, Expression of Galb1,4GlcNAc a 2,6 sialyltransferase anda 2,6-linked sialoglycoconjugates in human brain tumors, Acta Neu-

Ž .ropath. 91 1996 284–292.w x10 J.J. Kepes, Review of the WHO’s proposed new classification of

brain tumors. Proceedings of the XIth International Congress ofNeuropathology, Kyoto, September 2–8, 1990. Japanese Society ofNeuropathology, Kyoto, Japan.

w x11 H. Kitagawa, J.C. Paulson, Differential expression of five sialyl-Ž .transferase genes in human tissues, J. Biol. Chem. 269 1994

17872–17878.w x12 N. Le Marer, D. Stehelin, High alpha-2,6-sialylation of N-acetyllac-

tosamine sequences in ras-transformed rat fibroblasts correlates withŽ .high invasive potential, Glycobiology 5 1995 219–226.

w x13 J.R. Moskal, D.A. Gardner, S. Basu, Changes in glycolipid glycosyl-transferases and glutamate decarboxylase and their relationship todifferentiation in neuroblastoma cells, Biochem. Biophys. Res. Com-

Ž .mun. 61 1974 751–758.w x14 G.L. Nicholson, Cancer metastasis-organ colonization and the cell

surface properties of malignant cells, Biochem. Biophys. Acta 695Ž .1982 113–176.

w x15 A. Passaniti, G.W. Hart, Cell surface sialylation and tumor metasta-Ž .sis, J. Biol. Chem. 263 1988 7591–7603.

w x16 P. Reboul, P. Broquet, P. George, P. Louisot, Effect of retinoic acidon two glycosyltransferase activites in C6 cultured glioma cells, Int.

Ž .J. Biochem. 22 1990 889–893.w x17 J. Roth, Cellular sialoglycoproteins: a histochemical perspective,

Ž .Histochem. J. 25 1993 687–710.w x18 J.T. Rutka, G. Apodaca, R. Stern, M. Rosenblum, The extracellular

matrix of the central and peripheral nervous system: structure andŽ .function, J. Neurosurg. 69 1988 155–170.

w x19 T. Sata, J. Roth, C. Zuber, B. Stamm, P.U. Heitz, Expression ofa 2,6-linked sialic acid residues in neoplastic but not in normal

Ž .human colonic mucosa, Am. J. Pathol. 139 1991 1435–1448.w x20 V. Schirrmacher, P. Altevogt, M. Fogel, J. Dennis, C.A. Waller, D.

Barz, R. Schwartz et al., Importance of cell surface carbohydrates incancer cell adhesion, invasion, and metastasis, InÕasion MetastasisŽ .2 1982 313–360.

( )H. Yamamoto et al.rBrain Research 755 1997 175–179 179

w x21 A.L. Shen, M.D. Chou, C.W. Chi, L.S. Lee, Alterations in serumsialyltransferase activities in patients with brain tumors, Surg. Neu-

Ž .rol. 22 1984 509–514.w x22 L.I. Stoykova, M.C. Glick, Purification of an alpha-2,8-

sialyltranferase, a potential initiating enzyme for the biosynthesis ofpolysialic acid in human neuroblastoma cells, Biochem. Biophys.

Ž .Res. Commun. 217 1995 777–783.w x23 A. Varki, Biological roles of oligosaccharides: all of theories are

Ž .correct, Glycobiology 3 1993 97–130.w x24 W.-C. Wang, R.D. Cummings, The immobilized leukoagglutinin

from the seeds of Maackia amurensis binds with high affinity tocomplex-type Asn-linked oligosaccharides containing terminal sialicacid-linked a-2,3 to penultimate galactose residues, J. Biol. Chem.

Ž .263 1988 4576–4585.w x25 L. Warren, J.B. Fuhrer, C.A. Buck, Surface glycoproteins of normal

and transformed cells: a difference determined by sialic acid and agrowth-dependent sialyltransferase, Proc. Natl. Acad. Sci. USA 69Ž .1972 1838–1842.

w x26 D.X. Wen, B.D. Livingston, K.F. Medzihradszky, S. Kelm, A.L.Ž .Burlingame, J.C. Paulson, Primary structure of Galb1,3 4 GlcNAc

a 2,3-sialyltransferase determined by mass spectrometry sequenceŽ .analysis and molecular cloning, J. Biol. Chem. 267 1992 21011–

21019.w x27 J.A. Workmeister, H.F. Pross, J.C. Roder, Modulation of K562 cells

with sodium butyrate. Association of impaired NK susceptibilitywith sialic acid and analysis of other parameters, Int. J. Cancer 32Ž .1983 71–78.

w x28 H. Yamamoto, Y. Kaneko, D. VanderMeulen, D. Kersey, E.Mkrdichian, L. Cerullo, J. Leestma, J.R. Moskal, The expression ofCMP-NeuAc:Galb1,4GlcNAc a 2,6 sialyltransferase and glyco-proteins bearing a 2,6-linked sialic acids in human brain tumors,

Ž .Glycoconjugate J. 12 1995 848–856.w x29 W.V. Yong, T. Tejada-Berges, C.G. Goodyer, J.P. Antel, F.P. Yong,

Differential proliferative responses of human and mouse astrocytesŽ .to gamma-interferon, Glia 6 1992 269–280.

w x30 D. Zagzag, D.R. Friedlander, D.C. Miller, J. Dosik, J. Cangiarella,M. Kostianovsky, H. Cohen, M. Grumet, M.A. Greco, Tenascinexpression in astrocytomas correlates with angiogenesis, Cancer

Ž .Res. 55 1995 907–914.