This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Meichenin, M. Articles by Le Pendu, J. Search for Related Content PubMed PubMed Citation Articles by Meichenin, M. Articles by Le Pendu, J.

Tk, a New Colon Tumor-associated Antigen Resulting from Altered O-Glycosylation¹

INSERM U419, Institut de Biologie, 44093 Nantes, France [M. M., J. R., J. L. P.]; Shemyakin Institute for Bioorganic Chemistry, 117871 Moscow, Russia [O. G., N. B.]; Zelinsky Institute of Organic Chemistry, 117334 Moscow, Russia [N. N., A. S.]; Department of Pathology, University Hospital, 44093 Nantes, France [E. C., M. F. H.]; INSERM U482, Saint Antoine Hospital, 75571 Paris, France [J. B.]; and Blood Transfusion Centre, ML8 5ES Carluke, Scotland [R. H. F.]

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Erythrocyte polyagglutination antigens T and Tn are truncated O-glycan chains that are also carcinoma-associated antigens. We investigated whether Tk polyagglutination antigen could similarly be a carcinoma-associated marker and a target of immunotherapy. Monoclonal antibody LM389 was raised against Tk erythrocytes and tested by immunohistochemistry. LM389 strongly reacted with 48% human colorectal carcinomas. Labeling of normal tissues was visible on epithelial cells, mainly digestive, but was confined at a supranuclear level. Expression of the antigen on cloned human carcinoma cells correlated with sialosyl-Tn expression. O-Sialoglycoprotein endopeptidase treatment revealed that on carcinomas and cell lines, the epitope was present on O-glycans. Antibody specificity was determined using synthetic carbohydrates. Direct binding and inhibition studies indicated that LM389 best ligands were terminated by two branched N-acetylglucosamine units. Screening of murine cellular cell lines with LM389 allowed development of an experimental model with Tk-positive and -negative cells in syngeneic BDIX rats. Vaccination of rats with Tk erythrocytes provided a protection against growth of rat Tk-positive, but not of Tk-negative, tumor cells in association with the development of antibodies. Taken together, the results indicate that Tk polyagglutination antigen is a new colorectal carcinoma-associated antigen, absent from the normal cell surface, resulting from alteration of O-glycans biosynthesis and with potential as a target of immunotherapy.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Aberrant glycosylation is a well-known characteristic of cancer cells, and many tumor-associated antigens defined by mAbs³ raised against cancer cells are cell surface carbohydrate epitopes (1 , 2) . Regarding digestive cancers, most epitopes recognized are related to blood group antigens. Indeed, overexpression of ABH, Lewis b, and Lewis y antigens occurs in distal colonic tumors. Similarly, expression of sialyl Lewis a and sialyl Lewis X is strongly increased (3, 4, 5, 6, 7, 8, 9, 10, 11) . Colonic neoplasia is also characterized by the appearance of T, Tn, and STn antigens (12, 13, 14, 15) . These last antigens represent short O-glycan chains of mucin-type glycoproteins. Tn is formed by the addition of an GalNAc residue to serines or threonines of the polypeptide backbone, thus corresponding to the initial step of O-glycan synthesis. Further addition of a galactose in ß1,3 produces the T antigen. These antigens are normally cryptic because they undergo further extension. Their cell surface expression on cancer cells results from a lack of extension of O-glycan chains. The molecular mechanisms responsible for this defect in O-glycan biosynthesis are still unknown. The STn antigen is obtained by addition of a sialic acid residue in the 2,6 position on the Tn epitope. It is normally present in the colonic epithelium in an O-acetylated form. In cancer tissues, loss of the O-acetyl group from the sialic acid generates a tumor-associated STn epitope (15) . In addition, increased expression provides clustered epitopes that can be specifically recognized by mAbs (16) . The presence of H/Le^y/Le^b, sialyl Lewis X, and STn has been associated with poor prognosis in colon cancer patients, suggesting that their presence can be of functional relevance in the biology of carcinoma cells (10 , 17 , 18) . Most interestingly, because of their limited expression on normal tissues, T, Tn, and STn epitopes can provide targets for cancer immunotherapy. Indeed, patients immunized against such synthetic epitopes conjugated to a carrier protein could raise specific antibody responses, and a correlation between antibody titer and good clinical course has been reported (19) .

Natural antibodies of low titers against T and Tn antigens are present in the serum of all individuals. Some infectious agents can release glycosidases able to degrade erythrocyte glycans, revealing the cryptic T and Tn epitopes. Because of the presence of the natural antibodies, such erythrocytes are agglutinated by the sera from all individuals, defining these antigens as polyagglutination antigens (20) . There exists another polyagglutination antigen, called Tk, observed after infectious episodes. Although cases of Tk polyagglutination are rare, it is known that it concerns a cryptic epitope unmasked from the erythrocyte surface after glycan degradation by bacterial endo-ß-galactosidases (21 , 22) . Because altered glycan biosynthesis occurring in carcinomas leads to surface expression of normally cryptic epitopes such as T and Tn, we reasoned that Tk epitopes could similarly be unmasked on carcinoma cells. To test this hypothesis, a mAb was raised against endo-ß-galactosidase-treated red cells. The carbohydrate specificity of this anti-Tk mAb was defined, and it could be shown that Tk epitopes can strongly be expressed at the surface of a significant proportion of human colorectal carcinomas but not of normal cells. In addition, using a rat model of colon carcinoma, we present results indicating that immunization against human endo-ß-galactosidase-treated red cells can protect from the growth of rat Tk-positive tumor cells.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Development of Monoclonal Anti-Tk.
Washed, packed human group O red cells were incubated with 10 volumes of papain (BDH Chemicals, Poole, England) at 1 g/l in 0.1 M phosphate buffer (pH 5.4) for 15 min at 37°C. After four washes, 100 µl of these cells were then incubated with 100 µl of 0.1 M phosphate buffer (pH 6.0) and 5 µl of endo-ß-galactosidase from Bacteroides fragilis (Calbiochem, La Jolla, CA) for 3 h at 37°C and washed an additional four times. BALB/c mice were given i.v. injections of these cells (50 µl of packed cells suspended in 0.15 M NaCl to a total volume of 150 µl) on days 0, 2, 5, 8, 10, and 12, and splenocytes were fused to the murine myeloma line Sp2/0-Ag-14 on day 15, as described previously (23) .

Culture supernatants were screened with papain-modified (Tk-negative) and papain/endo-ß-galactosidase-modified (Tk-positive) red cells, resulting in the isolation and subsequent cloning of one possible anti-Tk (LM389/870.35). This mAb was identified as a murine IgM using a dipstick isotyping kit (Life Technologies, Inc., Paisley, Scotland) and in tests with in vivo modified T, Tk, Th, Tn, Cad, Sd^a, and acquired B cells, the mAb reacted only with the Tk cells. Additionally, using red cells modified in vitro by B. fragilis supernatant, endo-ß-galactosidase, and neuraminidase, only the first two of these, the Tk-activated cells, were agglutinated (24) .

Tissue Samples.
Normal tissues were obtained either from surgical specimens or from kidney donors 5 min after death. These last human samples were obtained before the law 88-1138 (December 20, 1988) concerning resection of human tissues after death for scientific investigations. They were fixed in 95% ethanol for 48 h and paraffin embedded. Normal rat tissues were also fixed in 95% ethanol. Primary colorectal carcinoma tissues were obtained from 56 patients undergoing surgical resection. In 8 of these cases, lymph node or hepatic metastases were also obtained. The uninvolved mucosa adjoining the tumor was collected in 10 cases. This mucosa is referred to as transitional mucosa. Of the carcinomas, 34 originated from the left colon, 6 from the right colon, 8 from the rectum, and 8 were of nonspecified location. Their Tumor-Node-Metastasis classification was obtained for 49 cases. Tumor samples were fixed either in formalin or in 95% ethanol and embedded in paraffin.

Immunohistochemistry.
Sections (5 µm) were rehydrated in graded ethanol and washed in PBS. They were then incubated in methanol/H₂O₂, 0.3% for 20 min to block endogenous peroxidase, and washed 5 min in PBS. The tumor or normal tissue sections were then covered with PBS:3% BSA for 20 min at room temperature in a humidified atmosphere. After washing in PBS, sections were incubated with the primary antibody diluted in PBS:1% BSA at 4°C overnight. Sections were then rinsed twice with PBS and incubated with biotinylated secondary antibody (Vector Labs, Burlingame, CA) for 1 h at room temperature. After washing in PBS, sections were covered with peroxidase-conjugated avidin (Vector) for 45 min and washed with PBS, and reactions were revealed with 3-amino-9-ethylcarbazol. Counterstaining was performed with 1% Harris hematoxylin. To release O-glycans immediately after rehydration, sections were incubated in the presence of O-sgp (Cedarlane, Hornley, Ontario, Canada) and diluted in RPMI 1640 at a concentration of 120 µg/ml for 4 h at 37°C. Control sections were incubated similarly in the absence of the enzyme. Sections were then washed three times in PBS, incubated in PBS:3% BSA, and stained with the anti-Tk as described above. Within each tumor section, the percentage of positive cells was semiquantitatively estimated by two independent observers at low power field (x10). Tumors were scored as negative below 5% positive cells, as weakly positive below 25% positive cells, as moderately positive below 75% positive cells, and as strongly positive from 75 to 100% positive cells.

Cell Culture and Selection of Tk-positive Cells.
The SW707 human colon carcinoma cell line was obtained from the American Type Culture Collection (Rockville, MD). LSC and LSB are two clones derived from the human colon adenocarcinoma cell line LS174T. These two clones have been selected for their positive and negative expression of Tn and STn antigens, respectively (25) . These cells were kindly provided by Dr. S. H. Itzkowitz (Mount Sinai School of Medicine, New York, NY). The PROb cells, obtained from Dr. F. Martin (Faculty of Medicine, Dijon, France), is a clone derived from a dimethylhydrazine-induced rat cell line (DHDK12). The clone originates from a cell line selected for its high resistance to trypsin detachment and was chosen for its high tumorigenicity in syngeneic BDIX rats (26) . A15A5 cells, selected from a chemically induced BDIX rat glioma, were obtained from Dr. G. J. Pilkington (Institute of Psychiatry, De Gespigny Park, London, United Kingdom). Cells were cultured in DMEM, except PROb, which were cultured in RPMI 1640. Media were supplemented with 10% FCS, 2 mmol/L L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. (Life Technologies, Inc., Cergy-Pontoise, France). They were subcultured at confluency after dispersal with 0.025% trypsin in 0.02% EDTA. Cells were routinely checked for Mycoplasma contamination by Hoechst 33258 (Sigma Chemical Co., St. Louis, MO) labeling.

For selection of Tk-positive cells, 1 x 10⁶ SW707 cells were suspended in PBS containing 3% BSA and supernatant from hybridoma LM389 diluted 1/4. Cells were incubated for 1 h at 4°C and washed with PBS containing 0.5% BSA and 5 mmol/L EDTA. Twenty µl of antimouse IgG coupled to MACS magnetic microbeads (Miltenyi Biotech, Bergish Gladbach, Germany) were added to the cells suspended in 500 µl of the same buffer and incubated for 20 min at 4°C under gentle agitation. Cells bound to beads were collected by a magnet using a MiniMACS column (Miltenyi Biotech). The collected cells were placed in culture. Once sufficient growth was attained, cells were submitted to a second round of selection using the same method. The resulting cellular population was then cloned by limiting dilution in 96-well plates. Cells originating from wells where only single colonies could be observed were expanded, and their expression of Tk antigen was assessed by flow cytometry. A few positive and negative clones were kept for further study.

Cytofluorimetric Analysis.
Anti-Tn and anti-STn IE3 and TKH2, respectively, were kind gifts from Dr. H. Clausen (Faculty of Dentistry, Copenhagen, Denmark). Viable cells (2 x 10⁵/well) were incubated with primary mAbs as a cell supernatants, diluted 1:10 in PBS containing 0.1% gelatin for 30 min at 4°C. After three washes with the same buffer, a 30-min incubation with the secondary antimouse FITC-labeled antibody (Sigma) was performed. After washings, fluorescence analysis was performed on a fluorescence-activated cell sorter FACScan (Becton Dickinson, San Jose, CA).

Western Blot Analysis.
For Western blotting, total proteins were solubilized as cells reached confluency by a 30-min incubation at 4°C in PBS (pH 7.4) containing 1 mmol/l EDTA, 10 mmol/l NaF, and 0.1% sulfobetaine-14. The preparations were centrifuged at 13,000 x g for 15 min. Protein concentration of supernatants was measured using bicinchoninic acid. Twenty-five µg of proteins were subjected to electrophoresis in 5–15% SDS-polyacrylamide gels in the presence of 5% ß-mercaptoethanol. Separated proteins were electrophoretically transferred to nitrocellulose filters in 25 mmol/l Tris, 192 mmol/l glycine, and 20% methanol at 200 mA for 1 h. The efficiency of transfer was monitored by Ponceau S staining of the nitrocellulose filter. After transfer, membrane strips were incubated with O-sgp at a concentration of 120 µg/ml in PBS/1% Triton X-100 for 6 h at 37°C. A control was done by incubating the membrane in the same conditions without the enzyme. Afterward, blots were incubated for 1 h in 3% defatted milk in PBS. The anti-Tk and anti-actin (Boehringer Mannheim, Mannheim, Germany) antibodies were then incubated (v/v) overnight at 4°C in PBS containing 1% defatted milk. After three washes in PBS/Tween 0.05%, a 2-h incubation was performed with an antimouse peroxidase-labeled antibody diluted in PBS containing 3% BSA. After washing, the bound antibodies were revealed by chemiluminescence using the ECL kit from Amersham (Little Chalfont, United Kingdom).

Inhibitions of Glycosylation.
To inhibit the maturation of N-glycans, cells were cultured for 48 h in the presence of DMJ from Boehringer Mannheim and diluted in culture medium at a concentration of 1 mmol/L. O-Glycans from the cell surface were also cleaved by the O-sgp enzyme (Cedarlane). For this, confluent cells in six-well plates were incubated for 4 h with the enzyme, diluted in fetal calf serum-free medium at a concentration of 120 µg/ml. Cells were then tested by flow cytometry with the anti-Tk mAb or with the lectin L-PHA as a control for efficiency of the DMJ treatment.

Determination of mAb LM389 Specificity.
For adsorption on SYNSORB^r, synthetic oligosaccharides coupled to a silica solid support (SYNSORB^r) were obtained from Chembiomed Ltd. and from Dr. R. U. Lemieux (Edmonton, Alberta, Canada). One hundred µl of mAb LM389 as a cell supernatant diluted 1:2 in PBS containing 0.1% gelatin were incubated on 10 µg of wet SYNSORB and incubated for 1 h at room temperature under gentle agitation. After centrifugation for 3 min at 3000 x g, the supernatant was recovered and tested by flow cytometry on PROb cells. Binding of the antibody was quantified by the decrease in mean fluorescence intensity relative to that given by the supernatant incubated on the same solid support lacking a coupled oligosaccharide. The following immobilized oligosaccharides were used: Galß1–4GlcNAcß1–6(Galß1–4GlcNAcß1–3)Galß-R, Galß1–4GlcNAcß1–3Galß1–4Glcß-R, and GlcNAcß1–3Galß1–4Glcß-R, where R is the linking arm and the silica solid support.

Binding to Polyacrylamide-based Neoglycoconjugates.
Neoglycoconjugate probes were prepared by conjugating synthetic oligosaccharides to PAA. Three types of spacers between the oligosaccharides and the polyacrylamide were used: Sp¹, -O(CH₂)₃; Sp², -O(CH₂)₂; and Sp³ , -O(HOCH₂CHOH)-(CHOH)₂CHNHAcCH_2. The methods for synthesis of oligosaccharides, of poly(4-nitrophenylacrylate), and for preparation of oligosaccharide polyacrylamide conjugates have been reported (27) . All of the probes contained equal amounts of oligosaccharide (20 mol%). The neoglycoconjugates used in the present study are listed in Table 4 . Reactivity of mAb LM389 toward these probes was tested by ELISA. Probes were coated onto 96-well plates (Nunc, Roskilde, Denmark) at 10 µg/ml in sodium carbonate buffer 0.05 mol/l (pH 9.6) by a 1-h incubation at 37°C, followed by an overnight incubation at 4°C. Plates were then incubated for 1 h at 37°C in PBS containing 1% BSA. Between each step, plates were washed three times with PBS:0.1% Tween 20. mAb LM389, as a culture supernatant, was added in 2-fold serial dilutions from 1:10 in PBS:1% BSA and incubated for 1 h at 37°C. Its binding was then revealed by addition of peroxidase-labeled antimouse (Vector) diluted to 1:3000 in PBS:1% BSA for 1 h at 37°C. After washings, plates were incubated with orthophenylene-diamine (Sigma) at 0.4 mg/ml in sodium citrate buffer, 10 mmol/l, pH 6.0. Reactions were stopped by addition of 50 µl of H₂SO₄ at 30% and read at 405 nm. Inhibitions of binding were also performed by ELISA. In this case, plates were coated with the probe GlcNAcß1–4GlcNAc-Sp³ -PAA, as described above. Then, after a blocking step, inhibitory substances, either as PAA neoglycoconjugates or as monomeric oligosaccharides (listed in Table 4 ) were added in a volume of 50 µl in 2-fold serial dilutions starting from 1 mmol/l. Fifty µl of LM389 cell supernatant, diluted 1:5 in PBS:1%BSA, were then added in each well to obtain a 1:10 final dilution. Incubation was performed for 1 h at 37°C. Antibody binding was then revealed as described above.

Detection of anti-Tk Antibodies in the Sera of Immunized Rats.
Blood samples from immunized rats were taken at an eye sinus site before immunization (preimmune serum) and after immunization (immune serum). Sera were obtained and kept frozen at -80°C, before being assayed for the presence of anti-Tk antibodies by ELISA. Ninety-six-well plates (Nunc) were coated with the structure GlcNAcß1–4GlcNAc-Sp³ -PAA at 10 µg/ml in 0.05 mol/l sodium carbonate buffer (pH 9.6) overnight at 4°C. Plates were then incubated with 3% BSA containing PBS. Between all steps, plates were washed three times with PBS containing 0.1% Tween 20. Serum samples were then added, diluted 1:50 in PBS containing 0.3% BSA, and incubated for 1.5 h at 37°C. After being washed, plates were incubated with anti-rat IgG alkaline phosphatase conjugate (Sigma) for 1 h at 37°C. Finally, reactions were developed by incubation with p-nitrophenylphosphate substrate (Sigma) diluted at 1 mg/ml in 0.05 mol/l sodium carbonate buffer and read at 405 nm.

Statistical Analysis.
In the survival experiment, the significance of the observed differences between survival rates was determined by the log-rank test.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Expression of the Epitope Recognized by mAb LM389 in Normal Tissues and Colorectal Carcinomas.
Antibody LM389 was selected for its ability to selectively agglutinate erythrocytes with a Tk phenotype. On the basis of its serological reactivity, it can be considered as a specific anti-Tk reagent because it reacts exclusively with endo-ß-galactosidase-treated erythrocytes and in vivo-modified Tk red cells (24) . To know if the epitope recognized could be present on other cell types, the antibody was tested by immunohistochemistry on normal tissues. The results are summarized in Table 1 . Reactivity was mainly visible on epithelial cells of the digestive tract. Thus, the esophagus epithelium, the stomach, the small and large intestines, as well as the exocrine pancreas were stained by the anti-Tk reagent. In addition, epithelial cells of the trachea and to a lesser extent of the urinary bladder were also stained. However, in all of these tissues, the staining was intracellular, mostly limited to a supranuclear area typical of the Golgi region. The strongest staining was observed in the colon, as depicted on Fig. 1A . None of the other tissues tested showed reactivity. The binding of the anti-Tk mAb was also tested in normal rat tissues. Strikingly, the staining of tissues from this animal species paralleled that of human tissues and was similarly restricted to a supranuclear location, suggesting that the carbohydrate epitope recognized could correspond to a core structure masked at the cell surface by further elongation of the glycan chain. This observation prompted us to look for the presence of the Tk epitope in adenomas and colorectal carcinomas. As shown in Table 2 , it appeared that none of the 16 adenomas tested reacted with mAb LM389. However, strong reactivity could be detected in the case of carcinomas. It was no longer restricted to the Golgi area because the strongest staining was observed on cell membranes and secretory material (Fig. 1, B and C) . Of 56 tumors, 13 showed a uniform strong staining of the carcinoma cells. The remaining tumors were separated into three groups: those with large areas, estimated between 25 and 75% of the cancer cells, presenting a strong staining; those where the positive areas were restricted to 5–25% of the cancer cells; and finally, those that were either completely devoid of positive cells or that contained only some rare such cells. Altogether, the positive cases represented 71% of the total. Yet, the two first groups with large positive areas represented 48% of the cases. No association could be observed with either the location of the primary tumor or the Tumor-Node-Metastasis staging. In transitional mucosa, staining was similar to that in normal mucosa, which is restricted to the supranuclear area of epithelial cells.

View larger version (133K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Reactivity of mAb LM 389 with paraffin-embedded tissue sections using the avidin-peroxidase technique. Sections are counterstained with hematoxylin. A, normal human colon epithelial cells showing strong perinuclear reactivity of mAb LM389. B, colonic adenocarcinoma with strong staining of cancer cells and intraluminal material. C and D, O-glycoprotein endopeptidase treatment of colonic adenocarcinoma serial sections, control without enzyme (C), enzyme-treated section (D). Bar, 100 µm (A, C, and D) and 50 µm (B).

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Cytofluorimetric analysis of Tk antigen expression on human and rat cells using mAb LM389. Shown are examples of clones derived from the SW707 human colon carcinoma cell line, clones C8 and D5; clones LSC and LSB derived from the LS174T human colon carcinoma cell line; clone PROb derived from the DHDK12 rat colon carcinoma cell line and rat A15A5 glioma cell line. The log of fluorescence intensities in arbitrary units is plotted against cell number. Fluorescence intensities from cells incubated in the presence of mAb LM389 (black lines) are superimposed on fluorescence intensities from control cells incubated with the FITC antimouse immunoglobulin only (gray lines).

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Cytofluorimetric analysis of cell surface glycosylation using FITC-labeled lectins and mAbs. Clonal cells were derived from SW707 after selection using mAb LM389 and magnetic beads followed by limiting dilution. Shown are examples of two clones labeled by mAb LM389 (C6 and F2) and of two unlabeled clones (B2 and G6). Mean intensities of fluorescence obtained with each reagent are given in arbitrary units. Control corresponds to the mean fluorescence intensity of cells incubated in the presence of the FITC antimouse immunoglobulin only.

View larger version (66K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Western blot analysis of total cell extracts using mAb LM389. Extracts were from clones LSC and LSB derived from the LS174T human colon carcinoma cell line and clone PROb derived from the DHDK12 rat colon carcinoma cell line and rat A15A5 glioma cell line. Molecular weight markers (in thousands; kDa) are indicated.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Determination of the type of glycan-carrying Tk epitopes on LSC human colon carcinoma cells. A, cells were treated or not (Control) with the O-sgp, and their reactivities with mAb LM389 were tested by flow cytometry. B, after SDS-PAGE and blotting on nitrocellulose, cell extracts were treated or not (Control) with O-sgp and simultaneously immunostained with mAb LM389 and an anti-actin mAb. The major band stained by LM389 is visible at about Mr 150,000, and the Mr 42,000 band corresponds to actin. C, cells were treated with the N-glycosylation inhibitor DMJ, and their reactivities with mAb LM389 and the lectin L-PHA, which detects the ß1–6 branch of N-glycans, were tested by flow cytometry. The log of fluorescence intensities in arbitrary units is plotted against cell number. Fluorescence intensities from cells incubated in the presence of mAb LM389 and L-PHA (black lines) are superimposed on fluorescence intensities from control cells incubated with the FITC antimouse immunoglobulin only (gray lines).

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Adsorption of mAb LM389 on oligosaccharides coupled to silica beads (SYNSORBr). mAb LM389 was incubated on beads substituted with three different oligosaccharides, the structures of which are indicated, and substituted with uncoupled beads for use as a nonspecific adsorption control. Reactivity of the supernatants was then tested by flow cytometry on PROb rat cells. The percentage of adsorption was calculated from the ratio of mean fluorescence from supernatants incubated on oligosaccharide-substituted beads to that from the nonspecific adsorption control. Values represent means of four independent experiments.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. Inhibition of the binding between mAb LM389 and GlcNAcß1–4GlcNAcß1-Sp3-PAA by two oligosaccharides determined by ELISA. Percentage inhibitions were calculated from absorbance values obtained in absence of inhibitor. Values represent means of triplicates. Sp3, -O(HOCH2CHOH)-(CHOH)2CHNHAcCH2.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Fig. 8. Immunization against Tk-positive human erythrocytes. Groups of five to six rats were immunized by i.v. injections of native erythrocytes (A, E, and G), papain-treated erythrocytes (B), endo-ß-galactosidase-treated erythrocytes (C), papain and endo-ß-galactosidase-treated erythrocytes (D, F, and H). After the last injection, rats received s.c. 1 x 106 Tk-positive PROb (A, B, C, D, G, and H) or Tk-negative A15A5 cells (E and F). Tumor growth was monitored, each line representing growth in a single animal (A–F), and serum antibody reactivities toward GlcNAcß1–4GlcNAcß1-Sp3-PAA were detected by ELISA (G and H). Preimmune () and immune () individual rat sera were tested at a 1:50 dilution.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 9. Survival of rats immunized against Tk-positive human erythrocytes. Groups of five rats were immunized by i.p. injections of native erythrocytes () or of papain and endo-ß-galactosidase-treated erythrocytes (). After the last injection, rats received 1 x 106 Tk-positive PROb cells () or Tk-negative A15A5 cells (----).

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Similar to the Tn and sialyl Tn antigens, Tk antigen was not expressed on most cell lines tested, and only small subpopulations expressed the antigen in the few positive cell lines. It could explain why this antigen has not been described earlier as a tumor-associated antigen, because most mAbs defining tumor-associated antigens have been raised using cell lines as the immunogen. Surprisingly, the clones that uniformly expressed the Tk antigen also expressed STn. Because both antigens result from alterations of O-glycans biosynthesis, some of the underlying molecular defects responsible for their expression might be common. Two distinct molecular mechanisms can account for the expression of the STn antigen. Because it is present in an acetylated form in the normal colonic mucosa, a loss of acetyltransferase activity is sufficient to reveal the cancer-associated epitope (15 , 16) . This simple molecular defect cannot lead to expression of the Tk antigen because the latter is not sialylated, but it could occur early during carcinogenesis. At variance, in LSC cells, it has been established that more drastic defects of O-glycan biosynthesis were responsible for the synthesis of the short STn chains (34) . These cells, unlike LSB cells that do not present either STn or Tk epitopes, lack detectable core 1 galactosyltransferase and all N-acetylglycosaminyltransferases required to extend O-glycan chains. The presence of Tk epitopes on LSC O-glycan chains suggests that at least residual N-acetylglucosaminyltransferase activities are present in these cells. In absence of competition with core 1 galactosyltransferase, these residual enzymatic activities could be sufficient to synthesize the Tk antigen. Such major defects in O-glycan biosynthesis would only be found at later stages of the carcinogenesis process, explaining why the Tk reactivity was restricted to carcinomas.

Recent studies of cancer immunotherapy focused on cancer-associated peptide targets recognized by T cells (35 , 36) . However, the efficacy of passive immunotherapy using antibodies was documented recently for colorectal cancer patients (37) . Moreover, active immunization against carbohydrate antigens such as the T and sialyl Tn antigens yielded encouraging results in association with the development of antibody responses (19 , 38) . The absence of Tk antigen at the cell surface of normal tissues, together with the existence of natural antibodies, suggested that this antigen could be a new interesting target of colon cancer immunotherapy. To test this possibility, we searched for an appropriate animal model. Two cell lines from BDIX rats, PROb and A15A5, respectively, were found that express or are devoid of the antigen. The distribution of Tk epitopes in the rat normal tissues was similar to that in human normal tissues. This experimental model can thus be considered as a valuable tool to assay the usefulness of the Tk antigen as a target of immunotherapy and to set up immunization protocols. Preliminary experiments using endo-ß- galactosidase-treated erythrocytes as the immunogen, showed that growth of the Tk-positive tumor cells but not of Tk-negative tumor cells was retarded or completely abolished in immunized animals. This antitumor effect was related to the presence of antibodies cross-reactive with the antigen, indicating that indeed this new carcinoma-associated antigen could be an interesting target of immunotherapy. The antibody reactivities raised by the immunization were rather low. It should be noted that they were probably not tested against the optimal synthetic antigen. In addition, immunizations were performed in the absence of adjuvant. It can be expected that the use of adjuvants and of synthetic oligosaccharides such as GlcNAcß1–6(GlcNAcß1–3)Galß1–4Glc or GlcNAcß1–6(GlcNAcß1–3)GalNAc, coupled to a potent immunogenic carrier, should improve the immune response. The availability of the animal model will allow testing of such approaches. In addition, the availability of Tk-positive human cells, such as LCS- or the SW707-derived clones, will allow evaluation of the antibody responses against the cellular antigen and not only against synthetic oligosaccharides. These cell lines should also be useful models to define the molecular mechanisms responsible for the expression of the antigen at the cell surface and to define its potential biological role.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the Association pour la Recherche sur le Cancer, from La Ligue Départementale des Pays de la Loire, from the French Ministry for Education and Research (PECO Grant), and from Syntesome GmBH (Munich, Germany).

² To whom requests for reprints should be addressed, at INSERM U419, Institut de Biologie, 9 Quai Moncousu, 44093, Nantes, Cedex 1, France. Phone: 33-240-08-40-99; Fax: 33-240-08-40-82; E-mail: jlependu{at}nantes.inserm.fr

³ The abbreviations used are: mAb, monoclonal antibody; DMJ, deoxymannojirimycin; Fuc, fucose; Gal, galactose; Glc, glucose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; isoGln, isoglutamine; L-PHA, phytohemagglutinin isolectin L; MurNAc, N-acetylmuramic acid; O-sgp, O-sialoglycoprotein endopeptidase; PAA, polyacrylamide; Rha, rhamnose; Sp, spacer (linking arm); STn, sialosyl-Tn.

Received 3/13/00. Accepted 8/ 3/00.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Hakomori S. I. Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipids metabolism. Cancer Res., 56: 5309-5318, 1996.[Abstract/Free Full Text]
2. Kim Y. J., Varki A. Perspective on the significance of altered glycosylation in cancer. Glycoconj. J., 14: 569-576, 1997.[Medline]
3. Yuan M., Itzkowitz S. H., Palekar A., Shamsuddin A. M., Phelps P. C., Trump B. F., Kim Y. S. Distribution of blood group antigens A, B, H, Lewis^a, and Lewis^b in human normal, fetal, and malignant colonic tissue. Cancer Res., 45: 4499-4511, 1991.[Abstract/Free Full Text]
4. Kim Y. S., Yuan M., Itzkowitz S. H., Sun Q. B., Kaizu T., Palekar A., Trump B. F., Hakomori S. Expression of Le^y and extended Le^y blood-group-related antigen in human malignant, premalignant, and nonmalignant colonic tissues. Cancer Res., 46: 5985-5692, 1986.[Abstract/Free Full Text]
5. Sakamoto J., Furukawa K., Cordon-Cardo C., Yin B. W. T., Rettig W. J., Oettgen H. F., Old L. J., Lloyd K. O. Expression of Lewis^a, Lewis^b, X and Y blood group antigens in human colonic tumors and normal tissue and in human tumor-derived cell lines. Cancer Res., 46: 1553-1561, 1986.[Abstract/Free Full Text]
6. Schoentag R., Primus J., Kuhns W. ABH and Lewis blood group expression in colorectal carcinoma. Cancer Res., 47: 1695-1700, 1987.[Abstract/Free Full Text]
7. Bara J., Mollicone R., Herrero-Zabaleta E., Gautier R., Daher N., Oriol R. Ectopic expression of the Y (Le^y) antigen defined by monoclonal antibody 12–4LE in distal colonic adenocarcinomas. Int. J. Cancer, 41: 683-689, 1988.[Medline]
8. Itzkowitz S. H., Yuan M. , Fukushi. Y., Lee,H.,Shi,Z.,Zurawski,J.,Hakomori,S.I.,andKim,Y.S.ImmunohistochemicalcomparisonofLe^a,monosialosylLe^a(Ca19-9)anddisialosylLe^aantigensinhumancolorectalandpancreatictissues.CancerRes.,48: 3834-3842, 1988.
9. Ruggiero F., Cooper H. S., Steplewski Z. Immunohistochemical study of colorectal adenomas with monoclonal antibodies against blood group antigens (sialosyl-Le^a, Le^a, Le^b, Le^x, Le^y, A, B and H). Lab. Investig., 59: 96-103, 1988.[Medline]
10. Nakamori S., Kameyama M., Imaoka S., Furukawa H., Ishikawa O., Saski Y., Kabuto T., Iwanaga T., Matsushita Y., Irimura T. Increased expression of sialyl Lewis X antigen correlates with poor survival in patients with colorectal carcinoma: clinicopathological and immunohistochemical study. Cancer Res., 53: 3632-3637, 1993.[Abstract/Free Full Text]
11. Nakayama T., Watanabe M., Katsumata T., Teramoto T., Kitajima M. Expression of sialyl Lewis^a as a new prognostic factor for patients with advanced colorectal carcinoma. Cancer (Phila.), 75: 2051-2056, 1995.[Medline]
12. Itzkowitz S. H., Yuan M., Montgomery C. K., Kjeldsen T., Takahashi H. K., Bigbee W. L., Kim Y. S. Expression of Tn, Sialosyl-Tn, and T antigens in human colon cancer. Cancer Res., 49: 197-204, 1989.[Abstract/Free Full Text]
13. Orntoft T. F., Morse N. P. O., Eriksen G., Jacobsen N. O., Poulsen H. S. Comparative immunoperoxidase demonstration of T antigens in human colorectal carcinomas and morphologically abnormal mucosa. Cancer Res., 45: 447-452, 1985.[Abstract/Free Full Text]
14. Itzkowitz S. H. Carbohydrate changes in colon carcinoma. Acta Pathol. Microbiol. Immunol. Scand., 100: 173-180, 1992.
15. Jass J. R., Allison L. J., Edgar S. G. Distribution of sialosyl-Tn and Tn antigens within normal and malignant colorectal epithelium. J. Pathol., 176: 143-149, 1995.[Medline]
16. Ogata S., Koganty R., Reddish M., Longenecker B. M., Chen A., Perez C., Itzkowitz S. H. Different modes of sialyl-Tn expression during malignant transformation of human colonic mucosa. Glycoconj. J., 15: 29-35, 1998.[Medline]
17. Naitoh H., Nakajima T., Nagamachi Y., Yazawa S. A clinicopathological evaluation of anti-fucosylated antigens antibody (YB-2) in colorectal carcinoma. Glycosyl. Dis., 1: 31-36, 1994.
18. Itzkowitz S. H., Bloom E. J., Kokal W. A., Modon G., Hakomori S. I., Kim Y. S. Sialosyl-Tn: a novel mucin antigen associated with prognosis in colorectal cancer patients. Cancer (Phila.), 66: 1960-1966, 1990.[Medline]
19. MacLean G. D., Reddish M. A., Koganly R. R., Longenecker B. M. Antibodies against mucin-associated sialyl-Tn epitopes correlate with survival of metastatic adenocarcinoma patients undergoing active specific immunotherapy with synthetic STn vaccine. J. Immunother., 19: 59-68, 1996.[Medline]
20. King M. J. Blood group antigens on human erythrocytes. Distribution, structure and possible functions. Biochim. Biophys. Acta, 1197: 15-44, 1994.[Medline]
21. Inglis G., Bird G. W., Mitchell A. A., Milne G. R., Wingham J. Effect of Bacteroides fragilis on the human erythrocyte membrane: pathogenesis of Tk polyagglutination. J. Clin. Pathol., 28: 964-968, 1975.[Abstract/Free Full Text]
22. Doinel C., Andreu G., Cartron J. P., Salmon C., Fukuda M. N. Tk polyagglutination produced in vitro by an endo-ß-galactosidase. Vox Sang., 38: 94-98, 1980.[Medline]
23. Inglis G., Fraser R. H., McTaggart S., Sheridan R., Forrest A. H., Mitchell R. Monoclonal antibodies to high-incidence Kell epitopes: characterization and application in automated screening of donor samples. Transf. Med., 4: 209-212, 1994.[Medline]
24. Inglis G., McTaggart S., Fraser R. H. Murine monoclonal antibodies specific for T, Tk and Tn cryptantigens. Transf. Med., 5S1: 28 1995.
25. Ogata S., Chen A., Itzkowitz S. H. Use of model cell lines to study the biosynthesis and biological role of cancer-associated sialosyl-Tn antigen. Cancer Res., 54: 4036-4044, 1994.[Abstract/Free Full Text]
26. Caignard A., Martin M. S., Michel M. F., Martin F. Interaction between two cellular subpopulations of a rat colonic carcinoma when inoculated to the syngenic host. Int. J. Cancer, 36: 273-279, 1985.[Medline]
27. Bovin N. Polyacrylamide-based glycoconjugates as tools in glycobiology. Glycoconj. J., 15: 431-446, 1998.[Medline]
28. Fenderson B. A., O’Brien D. A., Millette C. F., Eddy E. M. Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies. Dev. Biol., 103: 117-128, 1984.[Medline]
29. Symington F. W., Fenderson B. A., Hakomori S. Fine specificity of a monoclonal anti-testicular cell antibody for glycolipids with terminal N-acetyl-D-glucosamine structure. Mol. Immunol., 21: 877-882, 1984.[Medline]
30. Hanisch F. G., Koldovsky U., Borchard F. Monoclonal antibody 2B5 defines a truncated O-glycan, GlcNAcß1–3Galß1–4GlcNAcß1–6 (GalNAc), on mucins from deep gastric and duodenal glands as well as metaplasia and neoplasia of gastric differentiation. Cancer Res., 53: 4791-4796, 1993.[Abstract/Free Full Text]
31. Hounsell E. F., Davies M. J., Renouf D. V. O-Linked protein glycosylation structure and function. Glycoconj. J., 13: 19-26, 1996.[Medline]
32. Boland C. R., Deshmukh G. D. The carbohydrate composition of mucin in colonic cancer. Gastroenterology, 98: 1170-1177, 1990.[Medline]
33. Tabas I., Kornfeld S. The synthesis of complex-type oligosaccharides. III. Identification of an -D-mannosidase activity involved in a late stage of processing of complex-type oligosaccharides. J. Biol. Chem., 253: 7779-7786, 1978.[Free Full Text]
34. Brockhausen I., Yang J., Dickinson N., Ogata S., Itzkowitz S. H. Enzymatic basis for sialyl-Tn expression in human colon cancer cells. Glycoconj. J., 15: 595-603, 1998.[Medline]
35. Sogn J. A. Tumor immunology: the glass is half full. Immunity, 9: 757-763, 1998.[Medline]
36. Foon K. A., Yannelli J., Bhattacharya-Chatterjee M. Colorectal cancer as a model for immunotherapy. Clin. Cancer Res., 5: 225-236, 1999.[Free Full Text]
37. Riethmuller G., Holz E., Schlimok G., Schmiegel W., Raab R., Hoffken K., Gruber R., Funke I., Pichlmaier H., Hirche H., Buggisch P., Witte J., Pichlmayr R. Monoclonal antibody therapy for resected Dukes’ C colorectal cancer: seven-year outcome of a multicenter randomized trial. J. Clin. Oncol., 16: 1788-1794, 1998.[Abstract]
38. Springer G. F. Immunoreactive T and Tn epitopes in cancer diagnosis, prognosis, and immunotherapy. J. Mol. Med., 75: 594-602, 1997.[Medline]

This article has been cited by other articles:

				T. Satomaa, A. Heiskanen, I. Leonardsson, J. Angstrom, A. Olonen, M. Blomqvist, N. Salovuori, C. Haglund, S. Teneberg, J. Natunen, et al. Analysis of the Human Cancer Glycome Identifies a Novel Group of Tumor-Associated N-Acetylglucosamine Glycan Antigens Cancer Res., July 15, 2009; 69(14): 5811 - 5819. [Abstract] [Full Text] [PDF]

				W. Wang, B. Hause, W. J. Peumans, G. Smagghe, A. Mackie, R. Fraser, and E. J.M. Van Damme The Tn Antigen-Specific Lectin from Ground Ivy Is an Insecticidal Protein with an Unusual Physiology Plant Physiology, July 1, 2003; 132(3): 1322 - 1334. [Abstract] [Full Text] [PDF]

				M. M. Burdick, J. M. McCaffery, Y. S. Kim, B. S. Bochner, and K. Konstantopoulos Colon carcinoma cell glycolipids, integrins, and other glycoproteins mediate adhesion to HUVECs under flow Am J Physiol Cell Physiol, April 1, 2003; 284(4): C977 - C987. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Meichenin, M. Articles by Le Pendu, J. Search for Related Content PubMed PubMed Citation Articles by Meichenin, M. Articles by Le Pendu, J.