Human α2,3-Sialyltransferase (ST3Gal II) Is a Stage-specific Embryonic Antigen-4 Synthase

Monosialosyl globopentaosylceramide (MSGb5), originally described as stage-specific embryonic antigen-4, is expressed in testicular germ cell tumors and in aggressive cases of human renal cell carcinoma (RCC). Clarification of the molecular mechanisms regulating synthesis of MSGb5 is very important to understand testicular carcinogenesis and the malignant progression of human RCC. For this purpose, we have investigated α2,3-sialyltransferase involved in the synthesis of MSGb5. We used the method of expression cloning combined with polymerase chain reaction targeted to sialylmotif to isolate a cDNA clone from RCC cell line ACHN library. The cloned cDNA was found to be identical to the previously cloned ST3Gal II in sequence. A soluble recombinant form of the protein in COS-1 cells showed an enzyme activity of α2,3-sialyltransferase toward globopentaosylceramide (Gb5) in addition to asialo-GM1 and GM1a. Transient transfection of COS-7 and ACHN cells with this cDNA induced an increase of MSGb5, whereas stable transfection of antisense ST3Gal II cDNA suppressed expression of MSGb5 in ACHN cells. The ST3Gal II mRNA level was increased in 7 of 8 RCC cell lines and in all six RCC tissues surgically obtained, although it was not necessarily consistent with the MSGb5 level in RCC cell lines. This study indicates that ST3Gal II is a MSGb5 (stage-specific embryonic antigen-4) synthase and that its increased expression level is closely related to renal carcinogenesis. Monosialosyl globopentaosylceramide (MSGb5), originally described as stage-specific embryonic antigen-4, is expressed in testicular germ cell tumors and in aggressive cases of human renal cell carcinoma (RCC). Clarification of the molecular mechanisms regulating synthesis of MSGb5 is very important to understand testicular carcinogenesis and the malignant progression of human RCC. For this purpose, we have investigated α2,3-sialyltransferase involved in the synthesis of MSGb5. We used the method of expression cloning combined with polymerase chain reaction targeted to sialylmotif to isolate a cDNA clone from RCC cell line ACHN library. The cloned cDNA was found to be identical to the previously cloned ST3Gal II in sequence. A soluble recombinant form of the protein in COS-1 cells showed an enzyme activity of α2,3-sialyltransferase toward globopentaosylceramide (Gb5) in addition to asialo-GM1 and GM1a. Transient transfection of COS-7 and ACHN cells with this cDNA induced an increase of MSGb5, whereas stable transfection of antisense ST3Gal II cDNA suppressed expression of MSGb5 in ACHN cells. The ST3Gal II mRNA level was increased in 7 of 8 RCC cell lines and in all six RCC tissues surgically obtained, although it was not necessarily consistent with the MSGb5 level in RCC cell lines. This study indicates that ST3Gal II is a MSGb5 (stage-specific embryonic antigen-4) synthase and that its increased expression level is closely related to renal carcinogenesis. Gangliosides are glycosphingolipids containing sialic acids, which are classified as ganglio-, lacto-, and globo-series, depending on the core structure. Gangliosides undergo qualitative and quantitative changes during development, differentiation, and carcinogenesis (1Hakomori S. Cancer Res. 1996; 56: 5309-5318PubMed Google Scholar). In relation to malignant progression, they play roles in tumorigenicity (2Deng W. Li R. Ladisch S. J. Natl. Cancer Inst. 2000; 92: 912-917Crossref PubMed Scopus (87) Google Scholar), in the inhibition of T cell function (3McKallip R. Li R. Ladisch S. J. Immunol. 1999; 163: 3718-3726PubMed Google Scholar, 4Uzzo R.G. Rayman P. Kolenko V. Clark P.E. Cathcart M.K. Bloom T. Novick A.C. Bukowski R.M. Hamilton T. Finke J.H. J. Clin. Invest. 1999; 104: 769-776Crossref PubMed Scopus (107) Google Scholar), and as adhesion receptors followed by activation of signal transducers leading to enhanced motility and invasiveness (5Hakomori S. Handa K. FEBS Lett. 2002; 531: 88-92Crossref PubMed Scopus (118) Google Scholar). Monosialosyl globopentaosylceramide (MSGb5), 1The abbreviations used are: MSGb5, monosialosyl globopentaosylceramide; SSEA-4, stage-specific embryonic antigen-4; Gb5, globopentaosylceramide; RCC, renal cell carcinoma; mAb, monoclonal antibody; FACS, Fluorescence-activated cell sorter analysis; PBS, phosphate-buffered saline; ST, sialyltransferase; TLC, thin layer chromatography. also known as stage-specific embryonic antigen-4 (SSEA-4) (6Shevinsky L.H. Knowles B.B. Damajanov I. Solter D. Cell. 1982; 30: 697-705Abstract Full Text PDF PubMed Scopus (300) Google Scholar, 7Kannagi R. Cochran N.A. Ishigami F. Hakomori S. Andrews P.W. Knowles B.B. Solter D. EMBO J. 1983; 2: 2355-2361Crossref PubMed Scopus (440) Google Scholar), is expressed maximally in murine four-cell stage embryo (8Fenderson B.A. Eddy E.M. Hakomori S. Bioessays. 1990; 12: 173-179Crossref PubMed Scopus (183) Google Scholar), in testicular germ cell tumors (9Wenk J. Andrews P.W. Casper J. Hata J. Pera M.F. Von Keitz A. Damajanov I. Fenderson B.A. Int. J. Cancer. 1994; 58: 108-115Crossref PubMed Scopus (45) Google Scholar, 10Olie R.A. Fenderson B.A. Daley K. Oosterhuis J.W. Murphy J. Looijienga L.H.J. Br. J. Cancer. 1996; 74: 133-140Crossref PubMed Scopus (16) Google Scholar) and in aggressive cases of human renal cell carcinomas (RCC) (11Saito S. Orikasa S. Ohyama C. Satoh M. Fukushi Y. Int. J. Cancer. 1991; 49: 329-334Crossref PubMed Scopus (55) Google Scholar, 12Saito S. Orikasa S. Satoh M. Ohyama C. Ito A. Takahashi T. Jpn. J. Cancer Res. 1997; 88: 562-569Google Scholar). Recent work on MSGb5 showed that monoclonal antibody (mAb) RM1 specific to MSGb5 stimulated invasive properties of human breast carcinoma cell line MCF-7 (13Steelant W.F. Kawakami Y. Ito A. Handa K. Bruyneel E.A. Mareel M. Hakomori S. FEBS Lett. 2002; 531: 93-98Crossref PubMed Scopus (47) Google Scholar). On the other hand MSGb5 (SSEA-4) is also important as a cell surface marker for embryonic stem cells (14Thompson J.A. Kalishman J. Golos T.G. Durning M. Harris C.P. Becker R.A. Hearn J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7844-7848Crossref PubMed Scopus (932) Google Scholar, 15Thompson J.A. Itskovitz-Eldor J. Shapiro S.S. Waknitz M.A. Swiergiel J.J. Marshal V.S. Jones J.M. Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12479) Google Scholar). Therefore, cloning of α2,3-sialyltransferase determining synthesis of MSGb5 may contribute to understanding not only of testicular carcinogenesis and malignant progression of RCC and breast carcinoma but also of mouse embryogenesis and stem cell research. To clone the cDNA of α2,3-sialyltransferase responsible for synthesis of MSGb5, which is the first ganglioside in the globo-series ganglioside pathway (Fig. 1), we utilized a transient expression cloning system in COS-7 cells according to the procedure by Seed and Aruffo (16Seed B. Aruffo A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3365-3369Crossref PubMed Scopus (789) Google Scholar). By means of this system combined with polymerase chain reaction targeted to sialylmotif, cDNA coding the gene that determines the expression of MSGb5 was isolated with mAb RM1, from a cDNA library of human RCC cell line ACHN. The cloned cDNA was found to be identical to ST3Gal II (17Kim Y.-J. Kim K.-S. Kim S.-H. Kim C.-H. Ko J.H. Choe I.-S. Tsuji S. Lee Y.-C. Biochem. Biophys. Res. Commum. 1996; 228: 324-327Crossref PubMed Scopus (72) Google Scholar) in sequence. Analyses of the newly expressed gangliosides and the sialyltransferase activity in transfected cells indicate that ST3Gal II is a SSEA-4 (MSGb5) synthase. We also found that the ST3Gal II mRNA level was indeed increased in most RCC cell lines and renal tumor tissues examined. Materials—Asialo-GM1, GM1, GM2, and GD1a were purchased from Sigma. Globopentaosylceramide (Gb5), monosialosyl globopentaosylceramide (MSGb5), and GM3 were prepared from pooled renal tumor tissues as described previously (18Saito S. Levery S.B. Salyan M.E.K. Goldberg R.I. Hakomori S. J. Biol. Chem. 1994; 269: 5644-5652Abstract Full Text PDF PubMed Google Scholar). Cells and Monoclonal Antibodies—The human RCC cell line ACHN was purchased from Dainihonseiyaku Co. (Osaka, Japan). Human RCC cell lines SMKT-R2, -R3, and -R4 were kindly provided by Dr. N. Miyao (Sapporo Medical College) (19Miyao N. Tsukamoto T. Kumamoto Y. Urol. Res. 1989; 17: 317-324Crossref PubMed Scopus (49) Google Scholar, 20Ohtani N. Tsukamoto T. Kumamoto Y. Miyao N. Jpn. J. Urol. 1991; 82: 613-619Crossref PubMed Scopus (22) Google Scholar), and TOS-1, -2, and -3 and 3LN were established in our laboratory (21Satoh M. Moosavi Nejad F. Nakano O. Ito A. Kawamura S. Ohyama C. Saito S. Orikasa S. Tohoku J. Exp. Med. 1999; 189: 95-105Crossref PubMed Scopus (15) Google Scholar). COS-7 and COS-1 cells were obtained from Human Science Research Resources Bank (Osaka, Japan), and CV-1 cells were from Riken Cell Bank (Wako, Japan). Mouse IgM was purchased from Sigma. The mouse IgM anti-SSEA3 mAb (MC631) was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa. The mouse IgM anti-MSGb5 mAb RM1 (18Saito S. Levery S.B. Salyan M.E.K. Goldberg R.I. Hakomori S. J. Biol. Chem. 1994; 269: 5644-5652Abstract Full Text PDF PubMed Google Scholar) was purified by ionic exchange chromatography (Takara Shuzo, Kyoto, Japan). Goat anti-mouse IgM was purchased from Zymed Laboratories, Inc. (San Francisco, CA). Construction of cDNA Library—Double-stranded cDNAs were synthesized with oligo(dT) primers using poly(A+) RNA prepared from human ACHN cells. After addition of BstXI adapters (Invitrogen), the cDNA was size-fractionated (>0.4 kb) and ligated into expression vector pME18S, which was kindly provided by Dr. Maruyama (Tokyo Medical and Dental College). The library contained 2 × 106 independent clones in Escherichia coli strain DH10B (Invitrogen). Isolation of a Human α2,3-Sialyltransferase cDNA Clone—TLC immunostaining and fluorescence-activated cell sorter (FACS) analysis showed that COS-7 cells express Gb5 and the precursor of MSGb5 but not MSGb5 (Fig. 2). Eight samples of 7 × 106 COS-7 cells were transfected with 30 μg of plasmid DNA each in HEPES-buffered saline (20 mm HEPES, pH 7.05, 137 mm NaCl, 5 mm KCl, 0.7 mm Na2HPO4, 6 mm glucose) by electroporation using a Gene Pulser II (Bio-Rad) at 220 V and 975 microfarad. Three days after transfection, the exponentially growing cells were harvested, blocked with 5% fetal calf serum in PBS for 1 h, and incubated with anti-MSGb5 mAb RM1 (10 μg/ml) for 1 h. The cells were washed twice with PBS and incubated in a Petri dish (Falcon 1007) coated with anti-mouse IgM goat IgG (Zymed Laboratories Inc.). Plasmid DNA was rescued from adherent cells by Hirt extraction (22Hirt B. J. Mol. Biol. 1967; 26: 365-369Crossref PubMed Scopus (3352) Google Scholar) and used to transform E. coli DH10B (Invitrogen) by electroporation. Adherent cells were observed under the inverted microscope to increase from 100–200/dish after the first round of transfection and panning and about 500–1000 after the second round to ∼1 × 104 after the fifth round. After seven rounds of transfection and panning, we tried to isolate a cDNA clone that directs synthesis of MSGb5 by sibling selection but failed to identify positive transfectants. We then employed PCR with degenerate primers to detect a sialylmotif (23Datta A.K. Paulson J.C. J. Biol. Chem. 1995; 270: 1497-1500Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) that might be concentrated as the round of transfection and panning advanced. The primers were sense 5′-TGCCGSCGCTGYGTGGTBGTGGGG and the antisense 5′-GGTGGTYYKGSWSCCAACATC (S = C, G; Y = C, T; B = C, G, T; K = G, T; W = A, T). PCR was with by of for 30 for 1 and for The PCR was concentrated as the of transfection and panning advanced. The concentrated PCR was isolated and cloned to and the samples were of the samples of PCR were identical to the sialylmotif of ST3Gal II in sequence. we isolated a cDNA of identical to ST3Gal II in using the PCR as a PCR of the α2,3-Sialyltransferase the ST3Gal II have (17Kim Y.-J. Kim K.-S. Kim S.-H. Kim C.-H. Ko J.H. Choe I.-S. Tsuji S. Lee Y.-C. Biochem. Biophys. Res. Commum. 1996; 228: 324-327Crossref PubMed Scopus (72) Google Scholar, Paulson J.C. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Paulson J.C. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). To which sialyltransferase was most during the of transfection and panning, we the level of the sialyltransferase cDNA ST3Gal and ST3Gal by PCR in the from the adherent cells of transfection and The primers were sense and antisense for ST3Gal sense and antisense for ST3Gal and sense and antisense for ST3Gal PCR was as described for ST3Gal and ST3Gal and by 30 for ST3Gal DNA cDNA was with a DNA using a with and Construction of the into the cDNA the enzyme in RCC cell the cDNA was from with and into vector (Invitrogen), and the sense and the antisense cDNA ligated into were Construction of a of the form of which from the of the was prepared by PCR as described previously using containing an and a of the with (17Kim Y.-J. Kim K.-S. Kim S.-H. Kim C.-H. Ko J.H. Choe I.-S. Tsuji S. Lee Y.-C. Biochem. Biophys. Res. Commum. 1996; 228: 324-327Crossref PubMed Scopus (72) Google Scholar). The PCR was into of the vector containing the protein A kindly provided by Dr. J. C. Paulson Research to expression plasmid of a of the and the expression plasmid was transfected into COS-1 cells on a dish using transfection After h, the cell from was combined and 5 of the was concentrated by using a The of the was with of followed by for at The were by and washed with The were then in of and used for sialyltransferase A of the after with the was also and concentrated as described The the concentrated after with the and the after with the were for sialyltransferase activity using as The reaction contained mm mm pH 2 mm mm of the concentrated of the of the and mm in of After at for 2 h, the reaction was by of The was isolated by and to chromatography on a thin layer chromatography with a system of and containing The on a was with a Japan). with the cDNA into sense antisense cDNA was transfected into ACHN cells to the sense cDNA MSGb5 level and the antisense cDNA MSGb5 For transfection, transfection was used as by the were under RNA and RNA was prepared from RCC cell and renal tumor tissues by the method P. N. Biochem. 1987; PubMed Scopus Google Scholar). 5 μg of RNA from cell line μg of that from surgically obtained was on and then to The form of ST3Gal II was used as a The was with cDNA at in a containing mm Na2HPO4, mm 1 mm and DNA and then washed in containing at The was with a We obtained the from the to the transfected with the were by in PBS, incubated with antibody for on washed twice with serum in PBS, and then incubated with goat to mouse IgG and IgM for on After the cells in PBS were to Isolation and of Gangliosides from of gangliosides and immunostaining were as described (18Saito S. Levery S.B. Salyan M.E.K. Goldberg R.I. Hakomori S. J. Biol. Chem. 1994; 269: 5644-5652Abstract Full Text PDF PubMed Google Scholar). of a MSGb5 (SSEA-4) the expression cloning of the cells that express but and a specific for the are COS-7 cells were by mAb but not by RM1 as in Therefore, we used COS-7 cells as a for the expression cloning of MSGb5 synthase and mAb RM1 as a specific for the We a cDNA MSGb5 synthesis with of PCR using degenerate primers targeted to The PCR was concentrated as the of transfection and panning (Fig. the PCR as a a cDNA of identical to ST3Gal II in was the ST3Gal and ST3Gal have we which sialyltransferase cDNA was concentrated after each round of transfection and the PCR of ST3Gal II was of ST3Gal and ST3Gal were after the of transfection and panning (Fig. of MSGb5 the cloned cDNA directs MSGb5 the expression and were transfected into COS-7 and ACHN COS-7 cells were found to have COS-7 cells transfected by were with The with the was After the cells were harvested, incubated with RM1, and to COS-7 cells transfected with cDNA showed a increase of MSGb5 with transfected with vector (Fig. which was in the was not On the other the increase of MSGb5 was only for COS-7 cells and for ACHN cells with transfected with vector not To that the cDNA clone MSGb5 we a soluble recombinant form of the in 5 and the concentrated cell from COS-1 cells transfected with the plasmid showed the activity of to Gb5, and but was toward the other To that the sialyltransferase activity toward Gb5 was from the after with and the after with the were used for sialyltransferase activity was only for the to the but not in the after with the (Fig. In this of the activity in the was in the the to inhibition of the by the indicate that the by the cDNA MSGb5 The antisense cDNA ligated into was then transfected into ACHN cells to the antisense cDNA the MSGb5 analysis showed that expression of MSGb5 was suppressed in the stable with the antisense cDNA (Fig. To that the level of MSGb5 was analysis was to the (10 from the stable were to chromatography on the and by MSGb5 level from each was by with MSGb5, the of which was in The analysis were using and the were to Gb5 the level in all the transfectants. The of MSGb5 from the stable were and from of cell (Fig. the in MSGb5 synthesis by the antisense precursor (Gb5) was not observed with the antisense cDNA with vector (Fig. In an to increase MSGb5 level in ACHN stable transfection with the sense cDNA was of stable with the sense cDNA not of ST3Gal II in RCC Cell and the sialyltransferase is involved in renal we the of ST3Gal II mRNA with of MSGb5 in human RCC cell lines. A level of ST3Gal II mRNA was in all human RCC cell lines SMKT-R2, although of ST3Gal II mRNA were not necessarily consistent with of MSGb5 (Fig. the ST3Gal II mRNA level was in and renal tumor the level was in all six cases of RCC tissues (Fig. level of ST3Gal II mRNA in human RCC of ST3Gal II mRNA was in human RCC renal of and T is from the The study described the of a cDNA clone synthesis of MSGb5 which was found to be identical to ST3Gal COS-7 cells which express Gb5 but not MSGb5 to be an we failed to isolate a MSGb5 synthase gene by the expression cloning and sibling selection The may be to activity in COS-7 cells that may the of the the increase of cells cells adherent to the Petri dish was observed under the microscope during the expression cloning and we to isolate a positive clone by the adherent cells. For this we used the of the sialylmotif (23Datta A.K. Paulson J.C. J. Biol. Chem. 1995; 270: 1497-1500Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) with the that a sialyltransferase cDNA directs synthesis of MSGb5, of this cDNA be observed as the round degenerate we found that only a sialylmotif identical to that of ST3Gal II was concentrated but not the other of In of the clone MSGb5 we transfected the cDNA into COS-7 cells and ACHN cells. a increase of MSGb5 was observed in COS-7 cells transfected with the ST3Gal II but the addition of a in a increase of MSGb5. of a of the with the is to from We not at transfection of the sense cDNA in COS-7 cells to a of MSGb5 synthesis with a indicate that in addition to the specific antibody RM1, the sialylmotif was a to identify the clone with a increase of MSGb5 On the other the MSGb5 level was suppressed in the stable with the antisense cDNA in RCC cell line ACHN. of Gb5 was not observed in stable with that with A for this may be to to Gb5 under a level in ACHN cells. of the soluble recombinant form of ST3Gal II the sialyltransferase activity toward Gb5, and in the of the activity with indicates that it is from soluble recombinant form of ST3Gal II the of our study that ST3Gal II a of of sialic to of Gb5 in addition to that of asialo-GM1 and (17Kim Y.-J. Kim K.-S. Kim S.-H. Kim C.-H. Ko J.H. Choe I.-S. Tsuji S. Lee Y.-C. Biochem. Biophys. Res. Commum. 1996; 228: 324-327Crossref PubMed Scopus (72) Google Scholar), which that the and be by the of by ST3Gal II was S. O. O. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that ST3Gal II is a The that of ST3Gal II mRNA are not necessarily consistent with of MSGb5 in RCC cell lines may also that the enzyme the other The level of ST3Gal II mRNA was not necessarily consistent with that of MSGb5 in each RCC cell This may also that not only sialyltransferase but also the other involved in synthesis of globo-series Y. S. K. T. J. K. Y. T. M. K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, D. T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (49) Google Scholar) the level of MSGb5. all but of the RCC cell lines a level of ST3Gal II the of ST3Gal II mRNA were in all six cases of human RCC tissues which ST3Gal II with the that ST3Gal II mRNA level was very in (17Kim Y.-J. Kim K.-S. Kim S.-H. Kim C.-H. Ko J.H. Choe I.-S. Tsuji S. Lee Y.-C. Biochem. Biophys. Res. Commum. 1996; 228: 324-327Crossref PubMed Scopus (72) Google Scholar), an increased level of this sialyltransferase mRNA may be related to carcinogenesis of the although on cases are surgically obtained renal tissues not the of the from of renal to 1 to 2 in and in renal tissues surgically obtained might be We have to this into in the mRNA level in human We previously that the gangliosides are related to the of human RCC (11Saito S. Orikasa S. Ohyama C. Satoh M. Fukushi Y. Int. J. Cancer. 1991; 49: 329-334Crossref PubMed Scopus (55) Google Scholar, 12Saito S. Orikasa S. Satoh M. Ohyama C. Ito A. Takahashi T. Jpn. J. Cancer Res. 1997; 88: 562-569Google Scholar, S. Levery S.B. Salyan M.E.K. Goldberg R.I. Hakomori S. J. Biol. Chem. 1994; 269: 5644-5652Abstract Full Text PDF PubMed Google Scholar, M. Handa K. Saito S. S. Ito A. Miyao N. Orikasa S. Hakomori S. Cancer Res. 1996; 56: Google Scholar, A. Levery S.B. Saito S. Satoh M. Hakomori S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (55) Google Scholar). gangliosides MSGb5 are the of In this motility of ACHN cells transfected with antisense was observed with transfected with S. A. K. M. and Y. consistent with the by (13Steelant W.F. Kawakami Y. Ito A. Handa K. Bruyneel E.A. Mareel M. Hakomori S. FEBS Lett. 2002; 531: 93-98Crossref PubMed Scopus (47) Google Scholar). increased of ST3Gal II mRNA may be involved not only in carcinogenesis of but also in the malignant progression of human RCC. This may indicate that at the of as by increased level of ST3Gal II renal carcinoma cells may be for the malignant Studies on the of ST3Gal II mRNA in cases of RCC and on the roles of the enzyme on the of carcinogenesis as as the malignant of RCC. only a study on sialyltransferase in human RCC S. S. M. T. S. Ohyama C. R. Ito A. Satoh M. T. Paulson J.C. Y. T. 2002; Google Scholar). of ST3Gal mRNA was with the malignant progression of RCC, and the cases with level of ST3Gal mRNA showed a S. S. M. T. S. Ohyama C. R. Ito A. Satoh M. T. Paulson J.C. Y. T. 2002; Google Scholar). In this the expression level of ST3Gal II mRNA is in relation with that of ST3Gal mRNA in human RCC. expression of was observed in human tissues Paulson J.C. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), mechanisms of sialyltransferase expression were to be involved in carcinogenesis. of the mechanisms may be to understand the of the malignant The monoclonal antibody by D. Solter and B. B. Knowles was obtained from the Developmental Studies Hybridoma Bank under the of the of and by the University of of