Mucins are the major macromolecules of the mucus produced by epithelial or glandular cells. They are highly heterogeneous membrane-bound or secreted oligomeric or non-oligomeric molecules characterized by high molecular masses ranging from 200 to many thousands of kDa(1, 2, 3, 4) . These proteins are heavily O-glycosylated through serine and/or threonine residues, which account for 30-40% of the total amino acid composition of their protein backbone. The diversity of the carbohydrate chains has been shown to contribute largely to the heterogeneity observed in mucins. However, molecular cloning of cDNAs encoding mucins has revealed another level of heterogeneity and a higher degree of complexity. Up to now at least eight human mucin genes have been identified, namely MUC1 (5, 6) , MUC2(7, 8) , MUC3(9) , MUC4(10) , MUC5B-MUC5AC(11) , MUC6(12) , and MUC7(13) . They are characterized by variable numbers of tandemly repeated sequences, the exact number of repetitions differing from one individual to another(14) . The growing number of identified genes, the genetic polymorphism due to the variable number of tandem repeats, along with the unpredictable number of glycoforms of these proteins explain their marked heterogeneity and polymorphism.

Mucins have long been known to play an important role in the physiopathology of a number of diseases(1, 3) . However, the recent finding of aberrant expression of mucin gene products in cancers has stimulated a rapidly growing interest in them. Because of their unique features, cancer mucin antigens are ideally suited as tumor markers for cancer diagnosis and prognosis, and also as immunostimulants potentially useful in the design of cancer vaccines. Numerous tumor-associated antigens defined by mAbs ()were found to be expressed on mucins(15, 16, 17, 18, 19, 20, 21, 22, 23, 24) and many of them are already clinically useful(25, 26) . Although mucins are natural components of normal epithelial tissues, their abnormal expression in cancers may be due either to aberrant regulation of mucin gene expression or aberrant glycosylation of the gene products. The heterogeneity of mucin gene expression has been studied in several cancer types(27, 28, 29, 30, 31) . Mucin genes may be up- or down-regulated in cancers originating from tissues where they are constitutively expressed, or they may be ectopically expressed in cancers derived from tissues that do not normally express them. Aberrant glycosylation, on the other hand, is often responsible for the appearance of cancer determinants identified by mAbs(32, 33, 34, 35, 36, 37) . The abnormal glycosylation process in cancer cells leads in most instances to a shortening of the glycan chains, exposing new carbohydrate epitopes and enhancing the accessibility to some protein epitopes. To date, most mAbs reactive with tumor-associated mucins identify protein epitopes of MUC1 product or carbohydrate epitopes found on multiple mucins.

We have identified a tumor-associated antigen of human superficial bladder tumors using a series of mAbs of which mAb M344 was the prototype used in several clinical studies(38, 39, 40) . The antigen is expressed in 70% of papillary bladder tumors, the most common form of bladder cancer(41, 42) , but not in normal bladder urothelial cells or other normal adult or fetal tissues(38) . The M344 antigen has also been identified on a small subset of adenocarcinomas of various origins (43) . The measure of this antigen provides a basis for promising diagnostic and prognostic tests for the management of bladder cancer (40, 44, 45) . Biochemical studies have shown that mAb M344 reacts with a sialylated carbohydrate epitope expressed on a very high molecular mass protein. ()Several other characteristics of the antigen such as the molecular mass variability between individuals, the decrease of the apparent molecular mass upon thiol group reduction, the association of the antigen with cytoplasmic vacuoles and its secretion by tumor cells all indicate that the M344 antigen belongs to the mucin family. ()()We named this antigen MAUB for mucin antigen of the urinary bladder. It is not known at this time whether MAUB is a new mucin or a product of a known mucin gene.

Little is known on the expression of mucin genes in normal bladder and bladder cancer. However, reports based on immunohistology studies with mAbs have suggested the expression of at least the MUC1 and MUC2 mucins. MUC1 epitopes were found expressed in the superficial layer of normal urothelium and at higher frequency in the most aggressive forms of bladder cancer(46, 47, 48, 49) . On the other hand, MUC2 protein epitopes were not expressed in normal bladder but were expressed in approximately 40% of bladder cancers of all types in the only study reported(50) .

The objective of the present study was to establish the relationship between MAUB and the other known MUC gene products. We took advantage of the characteristics of MAUB expression in the human bladder cancer line MGH-U3 established from a low grade papillary bladder tumor. In this cellular system, MAUB is expressed at very high levels when MGH-U3 cells are grown as nude mice xenografts but is not expressed on the cells grown as monolayer in vitro. We report here the coexpression of at least three mucins in this bladder cancer line and that MAUB is distinct from those mucins already identified.

EXPERIMENTAL PROCEDURES

Cell Culture Conditions and Tissues

Protein Extractions

Antibodies

For some immunoassays, M344 mAb and a goat anti-mouse (GAM) polyclonal antibody (Bio/Can Scientific, Missaussauga, Ontario, Canada) were labeled with I according to the IODOGEN method (60) . For double labeling immunofluorescence assay, mAb M344 was labeled with biotin as follows. Briefly, 200 µl of biotinamidocaproate N-hydroxysuccinimide ester (Sigma) dissolved in N-dimethylformamide at a concentration of 2 mg/ml was added dropwise to 10 ml of a 1 mg/ml solution of purified M344 mAb in 0.2 M bicarbonate buffer, pH 8.8, containing 0.15 M NaCl. After 15 min of agitation at room temperature, the solution was dialyzed against 0.1 M sodium phosphate buffer, pH 7.4.

Western Blot Analyses

Immunoassays

For enzyme-linked immunosorbent assay (ELISA), each well of MaxiSorb immunoplates (Nunc, Life Technologies, Inc.) was coated with 5 µg of solubilized proteins, blocked, and then incubated with first antibody as described for RIA. After several washes in TBS plates were incubated at room temperature for 1 h with alkaline phosphatase-conjugated GAM (Bio/Can Scientific) diluted at the manufacturer's recommended dilution in 1% BSA-TBS. Plates were washed and then incubated for 30 min in presence of p-nitrophenyl phosphate (Sigma) at a concentration of 0.5 mg/ml in 1 M diethanolamine, 0.5 mM MgCl, pH 9.8. After stopping the reaction by adding an equal volume of 0.1 N NaOH, the plates were read at 405 nm on an automatic plate reader.

For double determinant immunoradiometric assays, two different techniques were used. The first was a sandwich assay in which each well of polyvinyl chloride microwell plates was coated with 1 µg of GAM polyclonal antibody as described above. After blocking the unreacted binding sites with 5% BSA in TBS, calculated amounts of antibody solutions were added in order to capture an equivalent amount of antibodies (50 ng) in each well. This incubation was performed at 4 °C overnight. Plates were washed extensively in TBS, and then 5 µg of the appropriate antigen was added to each well. The capture of the antigen was allowed to proceed at 37 °C for 3 h. Plates were washed several times in TBS, and then available GAM paratopes were blocked with a solution of 0.01% purified normal mouse immunoglobulins (Bio/Can Scientific) in TBS for 5 h at 37 °C in a humid chamber. After this blocking step, 2 10 cpm of I-labeled M344 mAb was added to each well and the plates were incubated for 1 h at room temperature. After extensive washing with TBS, each well was counted. The second technique was a slightly modified version of that described by Würzner et al.(63) . It is similar to the first except that the captured antigens were detected using I-labeled immune complexes instead of using radiolabeled mAbs. Immune complexes were obtained by incubating optimal amounts of mAbs in microtubes with I-labeled GAM polyclonal antibody (2 10 cpm/50 µl) for 30 min at 37 °C. Normal mouse immunoglobulins were then added to a final concentration of 0.002% and incubated for an additional 30 min. The immune complexes were added to captured antigen in wells and the plates incubated for 1 h at 37 °C. After several washes in TBS, each well was counted.

Confocal Fluorescence Microscopy

Indirect Immunofluorescence

Double Labeling

Specimens were analyzed with a Bio-Rad MRC-600 confocal imaging system mounted on a Nikon Diaphot-TDM. A 60 objective lens with a 1.5 numerical aperture was used. Confocal settings were as follows: 0.3-milliwatt laser power, 1.5 zoom, 1 s/scan kalman filter, and six frames/image. The photomultiplier gain was set at maximum, and the confocal aperture was adjusted to obtain maximum resolution.

Oligodeoxynucleotides (Oligos) and cDNA Probe

Oligos were end-labeled using [-P]ATP and T4 polynucleotide kinase (Life Technologies, Inc.) according to the manufacturer's protocol. A 0.8-kilobase pair partial MUC2 cDNA insert from plasmid SMUC41, kindly provided by Young S. Kim (University of California, San Francisco), was labeled by random priming using [-P]dCTP and Klenow enzyme (Life Technologies, Inc.) (65) . Specific activities of labeled probes ranged from 10 to 10 cpm/µg.

Northern Blot Analyses

For analyses with oligo probes, membranes were prehybridized at either 42 °C (MUC1, MUC3, MUC5B, MUC5C, and MUC6) or 37 °C (MUC4) for 2-4 h in a solution containing 6 SSC (1 SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 5 Denhardt's reagent (1 Denhardt's reagent is 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA), 0.05% sodium pyrophosphate, 100 µg/ml denaturated salmon sperm DNA, and 100 µg/ml yeast tRNA. The probe was then added (1-4 10 cpm/ml), and hybridization was carried out overnight under the same conditions. Membranes were washed at room temperature for 20 min in 3 SSC, 0.05% sodium pyrophosphate, then washed twice for 2 min in the same solution, at 50 °C for MUC4, at 55 °C for MUC3, MUC5B, MUC5C, and MUC6, or at 60 °C for MUC1, before autoradiography.

For the analysis of MUC2, the membrane was prehybridized at 42 °C for 2 h in a solution consisting of 50% formamide, 5 SSC, 5 Denhardt's reagent, 0.1% SDS, and 100 µg/ml denatured salmon sperm DNA. The cDNA probe was added (1-4 10 cpm/ml), and incubation was continued overnight. The membrane was washed at room temperature for 20 min in a 1 SSC, 0.1% SDS solution, and then three times at 68 °C for 20 min in the same solution and subjected to autoradiography.

Inhibition of Protein Expression with Oligos

RESULTS

Expression of Mucin Gene mRNA in MGH-U3 Bladder Cancer Cells

Figure 1: Northern blot analysis of the levels of seven mucin mRNAs in the MGH-U3 cellular system. Total RNA (10 µg/lane) from M-MGH-U3 cells (lane 1) and T-MGH-U3 cells (lane 2) have been tested for mucin gene expression with MUC1, MUC3, MUC4, MUC5AC, MUC5B, and MUC6 oligo probes or with a MUC2 cDNA probe. Controls (lanes C) were total RNA from MCF-7 cells (MUC1), CAPAN-1 cells (MUC4), nude mouse xenografts of LS180 cells (MUC2), and from normal stomach mucosa (MUC3, MUC5AC, MUC5B, and MUC6).

Expression of Tumor-associated Mucin Antigens

Western Blot Analysis

Figure 2: Western blot analysis of the expression of some carcinoma-associated mucins in the MGH-U3 cellular system. M-MGH-U3 (lane 1) and T-MGH-U3 proteins (lane 2) (50 µg/lane) were analyzed with mAbs DF3, HMFG-2, B72.3, M344, and LDQ10 for band pattern comparison. The arrow represents the interface between the stacking and running gels (I detection).

Double Determinant Analysis

Confocal Immunofluoresence Microscopy

Figure 3: Confocal immunofluorescence micrographs of T-MGH-U3 cells double-labeled with M344 and LDQ10 mAbs. A, micrograph showing cells where LDQ10 reactivity (green fluorescence) is diffusely distributed in the cytoplasm and where M344 reactivity (red fluorescence) is found in vacuolar structures. B, micrograph showing the diversity of the expression patterns observed. Scale bars represent 10 µm.

Inhibition of Mucin Gene Expression with Antisense Oligos

Figure 4: Inhibition of the MUC2 gene expression with MUC2 antisense oligos. T-MGH-U3 cells were treated with 25 and 50 µg/ml MUC2 antisense oligos (2as). Control cells were treated with identical amounts of MUC1 sense (1s) and MUC1 antisense oligos (1as). After 72 h of treatment, proteins from the treated cells were extracted and analyzed by ELISA with M344 and LDQ10 mAbs. The asterisk (*) indicates experiments showing statistical difference by Kruskal-Wallis test. , 0 µg/ml; &cjs2106;, 25 µg/ml; , 50 µg/ml.

Tumor Sample Analysis

Figure 5: Analysis of the expression of MAUB and MUC2 mucins in a human superficial bladder tumor specimen. A, proteins from T-MGH-U3 cells (lane 1) and from a superficial bladder tumor sample (lane 2) were analyzed by Western blot with M344 and LDQ10 mAbs. The arrow represents the interface between the running and the stacking gels (ECL detection). B, Northern blot analysis of the expression of the MUC2 gene in T-MGH-U3 cells (lane 1) and in the same human superficial bladder tumor sample (lane 2) expressing MAUB but not MUC2 mucin as assessed by Western blot analysis.

DISCUSSION

Superficial papillary tumors are the most common form of bladder cancer, representing more than 70% of cases at initial diagnosis. These tumors can be effectively treated by endoscopic excision, but more than 60% of patients will experience multiple tumor recurrences and thus require careful monitoring(41, 42) . Intravesical immunotherapy with bacillus Calmette-Guerin is currently one of the most effective methods to prevent bladder tumor recurrence and bladder cancer is certainly the best example of success of nonspecific immunotherapy in the treatment of cancer(69, 70) . MAUB defined by mAb M344 has several characteristics of a promising tumor marker for the management of bladder cancer. The expression of MAUB in 70% of superficial tumors and its complete lack of expression in normal cells provided a basis for the design of an effective non-invasive diagnostic test on exfoliated cells of urine(44) . Other studies also indicated that primary superficial bladder tumors expressing MAUB had a significantly higher rate of tumor recurrence(71) . This observation may find an explanation in the fact that MAUB is expressed at high frequency in the normal appearing urothelium of patients with a MAUB-positive tumor, thus suggesting that MAUB expression is occurring early in the process of bladder tumorigenesis(45) . The identification of MAUB as a new mucin antigen associated with bladder cancer may have important implications for the treatment of superficial bladder tumors, since cancer mucins appear to have immunomodulatory properties and thus are good candidates for the design of specific cancer vaccines(72, 73) .

The study of MAUB and other mucin antigens on the MGH-U3 cell line was very informative. The MGH-U3 cell line is derived from a grade I non-invasive papillary bladder tumor(74) . It is tumorigenic in nude mice, and the xenografts obtained reproduce the histopathologic appearance of the original tumor. There are, however, important differences between MGH-U3 cells grown as nude mouse tumors or as monolayer in vitro. Ultrastructural studies showed that T-MGH-U3 cells contain electron-lucid cytoplasmic vacuoles typical of mucin secretion, which are not found in M-MGH-U3 cells cultured in vitro. The expression of MAUB followed closely the pattern of appearance of these vacuoles, and immunogold electron microscopy studies clearly demonstrated reactivity of mAb M344 with these vacuoles. In the present study, the expression of MUC2 mucin detected by mAb LDQ10 was only observed in T-MGH-U3 cells and was located to cytoplasmic granules as assessed by confocal immunofluorescence microscopy. MUC2 mRNA was also present in T-MGH-U3 but not in M-MGH-U3 cells. A similar finding was reported with the pancreatic cancer cell line SW1990, which did not express MUC2 mRNA while the cells from tumor xenografts showed intense expression(75, 76) . Of the seven mucin genes tested, MUC1 was the only other gene expressed in MGH-U3 cells. The presence of MUC1 mRNA in both M-MGH-U3 and T-MGH-U3 cells, the differences in band patterns observed in Western blots between MUC1 and MAUB, and the results of immunocaptures convincingly ruled out the possibility that MAUB is related to MUC1. To determine whether MAUB is a new mucin or is a glycoform of MUC2 mucin required more detailed analysis.

Several carcinoma-associated antigens were found to result from early sialylation of shorter carbohydrate chains in cancers compared to normal cells(32, 36, 77) . The results of the present study indicate that such changes in glycosyltransferase activity may also be influenced by the three-dimensional conformation of the cancer cells as shown previously for mAb B72.3 reactivity(78) . In addition to revealing MUC1 bands in T-MGH-U3, mAb B72.3 also showed in Western blots a band comigrating with the high molecular mass one revealed by mAbs M344 and LDQ10. Immunocapture experiments using T-MGH-U3 protein extracts showed that B72.3 reactivity was limited to the antigen captured by mAb M344 and not by mAb LDQ10 to MUC2 mucin. Even though the M344 epitope is a sialylated carbohydrate, the absence of reactivity of mAb M344 with the MUC1 bands and also the lack of reactivity with bovine submaxillary mucin, which is rich in sialyl-Tn antigen, rule out that mAb M344 reacts with the epitope recognized by mAb B72.3. The immunocapture experiments also showed no reactivity of mAb M344 with MUC2 mucin captured by mAb LDQ10, and conversely no LDQ10 reactivity with the antigen captured by mAb M344, suggesting that MAUB and MUC2 are two distinct molecules.

That MAUB and MUC2 mucins are distinct is also suggested by their very different patterns of expression in normal and cancerous human tissues. Several mAbs reactive with the core protein of MUC2 gene product have been described(58, 79, 80, 81) . In at least two studies of human tissues, expression of MUC2 mucin measured by immunohistochemistry was found to correlate with expression of MUC2 mRNA as measured by in situ hybridization and by semiquantitative analysis of gene expression by reverse transcription followed by polymerase chain reaction(82, 83) . All studies showed restricted expression of MUC2 gene product to normal epithelium of stomach and small and large bowel. The LDQ10 mAb that was used in the present study reacts with deglycosylated colon cancer mucin and with a synthetic peptide encompassing the MUC2 tandem repeat sequence. LDQ10 showed strong reactivity with goblet cells in the gastrointestinal tract and with a majority of colorectal, stomach, pancreatic, and breast cancers(58) . By contrast, in two different studies, mAb M344 was unreactive with any normal adult or fetal tissue tested and was reactive with only few colon and breast carcinomas(38, 43) . MAUB is not expressed in normal urothelium but is expressed in 70% of superficial (stages pTa and pT1) bladder tumors and in less than 15% of muscle invasive cancers (stage T2+)(38) . The expression of MUC2 in urothelial cancers was only studied in depth by one group using mAb 4F1 also reactive with MUC2 core protein. MUC2 was not expressed in normal urothelium, but in contrast with MAUB it was expressed in 40% of muscle-invasive cancers and in only 40% of superficial pTa and pT1 bladder tumors(50) . Since immunodetection of MUC2 mucin was found to correlate well with MUC2 mRNA expression, the lack of expression of MUC2 in tumors positive with mAb M344 strongly suggests that MAUB is distinct from MUC2. This conclusion is further supported by the lack of colocalization of MUC2 and MAUB mucins in confocal microscopy studies of T-MGH-U3 cells co-expressing both antigens.

Complex influences may come into play in the detection of various mucin epitopes by mAbs. It was thus important to obtain further evidence at the molecular level to substantiate the hypothesis raised by immunohistochemistry studies suggesting that MAUB and MUC2 are distinct mucins. One indication is provided by the results of the inhibition of MUC1 and MUC2 gene expression using antisense oligos. While the antisense MUC1 oligos had no effect on MUC2 nor MAUB expression, the antisense MUC2 oligos resulted in specific inhibition of MUC2 mucin expression and increased expression of MAUB. Further evidence came from the identification of a superficial bladder tumor, which strongly expressed MAUB and had no expression of MUC2 peptide and mRNA. Thus MAUB is a mucin distinct from those identified so far, although it shares several common features with MUC2 mucin. Indeed, both are typical secreted mucins and their expression in cancer cell lines appear to be influenced by the spatial configuration of tumor cells. The complete characterization of MAUB and its accurate tissue distribution will await the cloning of cDNA encoding its core protein. It is, however, possible to already conclude that cells from MGH-U3 tumor xenografts express three distinct mucins. Coexpression of distinct mucins is a normal feature of mucus secreting epithelium such as stomach and colon. A recent study reported that increased heterogeneity of mucin gene expression in gastric adenocarcinomas was associated with advanced cancer stage(31) . The finding of multiple mucin expression in a well differentiated bladder tumor originating from a typically non-mucous-secreting epithelium suggests that ectopic expression of mucin gene products may be an early feature of urothelial tumorigenesis.

FOOTNOTES

*

This investigation was supported by National Cancer Institute of Canada Grant 3515 with funds from the Canadian Cancer Society. This work was presented at the XXIIIth Meeting of the International Society for Oncodevelopmental Biology and Medecine, Montréal, September 10-13, 1995.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom reprint requests should be addressed. Tel.: 418-691-5281; Fax: 418-691-5562; yfradet{at}crhdq.ulaval.ca.

()

The abbreviations used are: mAb, monoclonal antibody; MAUB, mucin antigen of the urinary bladder; M-MGH-U3, MGH-U3 cells cultured as monolayer; T-MGH-U3, MGH-U3 cells grown as nude mouse tumors; RIA, radioimmunoassay; ELISA, enzyme-linked immunosorbent assay; GAM, goat anti-mouse; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ECL, enhanced chemiluminescence; oligo, oligonucleotide.

()

A. Bergeron, H. LaRue, and Y. Fradet, manuscript in preparation.

()

Y. Fradet, R. Pankov, A. Bergeron, and C. Parent-Vaugeois, manuscript in preparation.

()

H. LaRue, C. Parent-Vaugeois, A. Bergeron, S. Champetier, and Y. Fradet, manuscript in preparation.

ACKNOWLEDGEMENTS

REFERENCES

1. Strous, G. J., and Dekker, J. (1992) Crit. Rev. Biochem. Mol. Biol. 27, 57-92 [Medline] [Order article via Infotrieve]
2. Devine, P. L., and Mckenzie, I. F. C. (1992) Bioessays 14, 619-625 [CrossRef][Medline] [Order article via Infotrieve]
3. Rose, M. C. (1992) Am. J. Physiol. 263, L413-L429
4. Verma, M., and Davidson, E. A. (1994) Glycoconjugate J. 11, 172-179 [CrossRef][Medline] [Order article via Infotrieve]
5. Gendler, S. J., Burchell, J. M., Duhig, T., Lamport, D., White, R., Parker, M., and Taylor-Papadimitriou, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6060-6064 [Abstract/Free Full Text]
6. Gendler, S. J., Lancaster, C. A., Taylor-Papadimitriou, J., Duhig, T., Peat, N., Burchell, J., Pemberton, L., Lalani, E. N., and Wilson, D. (1990) J. Biol. Chem. 265, 15286-15293 [Abstract/Free Full Text]
7. Gum, J. R., Byrd, J. C., Hicks, J. W., Toribara, N. W., Lamport, D. T., and Kim, Y. S. (1989) J. Biol. Chem. 264, 6480-6487 [Abstract/Free Full Text]
8. Gum, J. R., Hicks, J. W., Toribara, N. W., Rothe, E. M., Lagace, R. E., and Kim, Y. S. (1992) J. Biol. Chem. 267, 21375-21383 [Abstract/Free Full Text]
9. Gum, J. R., Hicks, J. W., Swallow, D. M., Lagace, R. L., Byrd, J. C., Lamport, D. T., Siddiki, B., and Kim, Y. S. (1990) Biochem. Biophys. Res. Commun. 171, 407-415 [CrossRef][Medline] [Order article via Infotrieve]
10. Porchet, N., Nguyen, V. C., Dufosse, J., Audie, J. P., Guyonnet-Duperat, V., Gross, M. S., Denis, C., Degand, P., Bernheim, A., and Aubert, J. P. (1991) Biochem. Biophys. Res. Commun. 175, 414-422 [CrossRef][Medline] [Order article via Infotrieve]
11. Crepin, M., Porchet, N., Aubert, J. P., and Degand, P. (1990) Biorheology 27, 471-484 [Medline] [Order article via Infotrieve]
12. Toribara, N. W., Roberton, A. M., Ho, S. B., Kuo, W. L., Gum, E., Hicks, J. W., Gum, J. R., Byrd, J. C., Siddiki, B., and Kim, Y. S. (1993) J. Biol. Chem. 268, 5879-5885 [Abstract/Free Full Text]
13. Bobek, L. A., Tsai, H., Biesbrock, A. R., and Levine, M. J. (1993) J. Biol. Chem. 268, 20563-20569 [Abstract/Free Full Text]
14. Gendler, S. J., and Spicer, A. P. (1995) Annu. Rev. Physiol. 57, 607-634 [CrossRef][Medline] [Order article via Infotrieve]
15. Johnson, V. G., Schlom, J., Paterson, A. J., Bennett, J., Magnani, J. L., and Colcher, D. (1986) Cancer Res. 46, 850-857 [Abstract/Free Full Text]
16. Hilkens, J., Buijs, F., Hilgers, J., Hageman, P., Calafat, J., Sonnenberg, A., and Van der Valk, M. (1984) Int. J. Cancer 34, 197-206 [Medline] [Order article via Infotrieve]
17. Arklie, J., Taylor-Papadimitriou, J., Bodmer, W., Egan, M., and Millis, R. (1981) Int. J. Cancer 28, 23-29 [Medline] [Order article via Infotrieve]
18. Lan, M., Finn, O. J., Fernsten, P. D., and Metzgar, R. S. (1985) Cancer Res. 45, 305-310 [Abstract/Free Full Text]
19. Davis, H. M., Zurawski, V. R., Bast, R. C., and Klug, T. L. (1986) Cancer Res. 46, 6143-6148 [Abstract/Free Full Text]
20. Miotti, S., Aguanno, S., Canevari, S., Diotti, A., Orlandi, R., Sonnino, S., and Colnaghi, M. I. (1985) Cancer Res. 45, 826-832 [Abstract/Free Full Text]
21. Prat, M., Medico, E., Rossino, P., Garrino, C., and Comoglio, P. M. (1989) Cancer Res. 49, 1415-1421 [Abstract/Free Full Text]
22. Prat, M., Medico, E., Garrino, C., and Comoglio, P. M. (1989) Int. J. Cancer 44, 67-74 [Medline] [Order article via Infotrieve]
23. Abe, M., and Kufe, D. (1989) Cancer Res. 49, 2834-2839 [Abstract/Free Full Text]
24. Samuel, J., Noujaim, A. A., Maclean, G. D., Suresh, M. R., and Longenecker, B. M. (1990) Cancer Res. 50, 4801-4808 [Abstract/Free Full Text]
25. Jacobs, E. L., and Haskell, C. M. (1991) Curr. Probl. Cancer 15, 300-350
26. Lamerz, R. (1993) In Vivo 7, 607-613 [Abstract/Free Full Text]
27. Ogata, S., Uehara, H., Chen, A., and Itzkowitz, S. H. (1992) Cancer Res. 52, 5971-5978 [Abstract/Free Full Text]
28. Ho, S. B., Niehans, G. A., Lyftogt, C., Yan, P. S., Cherwitz, D. L., Gum, E. T., Dahiya, R., and Kim, Y. S. (1993) Cancer Res. 53, 641-651 [Abstract/Free Full Text]
29. Carrato, C., Balague, C., Debolos, C., Gonzalez, E., Gambus, G., Planas, J., Perini, J. M., Andreu, D., and Real, F. X. (1994) Gastroenterology 107, 160-172 [Medline] [Order article via Infotrieve]
30. Balague, C., Gambus, G., Carrato, C., Porchet, N., Aubert, J. P., Kim, Y. S., and Real, F. X. (1994) Gastroenterology 106, 1054-1061 [Medline] [Order article via Infotrieve]
31. Ho, S. B., Shekels, L. L., Toribara, N. W., Kim, Y. S., Lyftogt, C., Cherwitz, D. L., and Niehans, G. A. (1995) Cancer Res. 55, 2681-2690 [Abstract/Free Full Text]
32. Singhal, A., and Hakomori, S. (1990) Bioessays 12, 223-230 [CrossRef][Medline] [Order article via Infotrieve]
33. Hanski, C., Drechsler, K., Hanisch, F. G., Sheehan, J., Manske, M., Ogorek, D., Klussmann, E., Hanski, M. L., Blank, M., Xing, P. X., Mckenzie, I. F. C., Devine, P. L., and Riecken, E. O. (1993) Cancer Res. 53, 4082-4088 [Abstract/Free Full Text]
34. Itzkowitz, S. (1992) APMIS Suppl. 100, (Suppl. 27), 173-180
35. Kim, Y. S. (1992) J. Cell. Biochem. Suppl. 16G, 91-96
36. Hakomori, S. I. (1989) Adv. Cancer Res. 52, 257-331 [Medline] [Order article via Infotrieve]
37. Devine, P. L., Warren, J. A., Ward, B. G., Mckenzie, I. F. C., and Layton, G. T. (1990) J. Tumor Marker Oncol. 5, 11-26
38. Fradet, Y., Islam, N., Boucher, L., Parent-Vaugeois, C., and Tardif, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7227-7231 [Abstract/Free Full Text]
39. Fradet, Y. (1992) Semin. Urol. 10, 28-38 [Medline] [Order article via Infotrieve]
40. Fradet, Y., and Cordon-Cardo, C. (1993) Semin. Urol. 11, 145-153 [Medline] [Order article via Infotrieve]
41. Soloway, M. S., and Perito, P. E. (1992) J. Cell. Biochem. Suppl. 16I, 120-127
42. Itoku, K. A., and Stein, B. S. (1992) Hematol. Oncol. Clin. North Am. 6, 99-116 [Medline] [Order article via Infotrieve]
43. Cordon-Cardo, C., Wartinger, D. D., Melamed, M. R., Fair, W., and Fradet, Y. (1992) Am. J. Pathol. 140, 375-385 [Abstract]
44. Bonner, R. B., Hemstreet, G. P., Fradet, Y., Rao, J. Y., Min, K. W., and Hurst, R. E. (1993) Cancer 72, 2461-2469 [CrossRef][Medline] [Order article via Infotrieve]
45. Rao, J. Y., Hemstreet, G. P., Hurst, R. E., Bonner, R. B., Jones, P. L., Min, K. W., and Fradet, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8287-8291 [Abstract/Free Full Text]
46. Takahashi, M., Murase, T., Kinjo, T., Mitsuya, H., and Nagura, H. (1987) Urol. Int. 42, 170-175 [CrossRef][Medline] [Order article via Infotrieve]
47. Carbin, B. E., Collins, V. P., and Ekman, P. (1987) Urol. Res. 15, 191-194 [Medline] [Order article via Infotrieve]
48. Conn, I. G., Crocker, J., Emtage, L. A., and Wallace, D. M. A. (1988) J. Clin. Pathol. 41, 1191-1195 [Abstract/Free Full Text]
49. Anagnostaki, E., Skarlos, D., Tamvakis, N., Psaropoulou, P., Blana, E., Bamias, A., Legaki, S., Aravantinos, G., Deliveliotis, C., and Dimopoulos, K. (1990) Br. J. Cancer 62, 52-56
50. Walsh, M. D., Hohn, B. G., Thong, W., Devine, P. L., Gardiner, R. A., Samaratunga, M. L. T. H., and Mcguckin, M. A. (1994) Br. J. Urol. 73, 256-262 [Medline] [Order article via Infotrieve]
51. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
52. Fradet, Y., Larue, H., Parent-Vaugeois, C., Bergeron, A., Dufour, C., Boucher, L., and Bernier, L. (1990) Int. J. Cancer 46, 990-997 [Medline] [Order article via Infotrieve]
53. McKinney, M. M., and Parkinson, A. (1987) J. Immunol. Methods 96, 271-278 [CrossRef][Medline] [Order article via Infotrieve]
54. Burchell, J., Gendler, S., Taylor-Papadimitriou, J., Girling, A., Lewis, A., Millis, R., and Lamport, D. (1987) Cancer Res. 47, 5476-5482 [Abstract/Free Full Text]
55. Lan, M. S., Hollingsworth, M. A., and Metzgar, R. S. (1990) Cancer Res. 50, 2997-3001 [Abstract/Free Full Text]
56. Gold, D. V., and Mattes, M. J. (1988) Tumor Biol. 9, 137-144
57. Leoni, L., Magnani, J. L., Miotti, S., Canevari, S., Pasquali, M., Sonnino, S., and Colnaghi, M. I. (1988) Hybridoma 7, 129-139 [Medline] [Order article via Infotrieve]
58. Gambus, G., Debolos, C., Andreu, D., Franci, C., Egea, G., and Real, F. X. (1993) Gastroenterology 104, 93-102 [Medline] [Order article via Infotrieve]
59. Beckett, M. L., Lipford, G. B., Haley, C. L., Schellhammer, P. F., and Wright, G. L. (1991) Cancer Res. 51, 1326-1333 [Abstract/Free Full Text]
60. Fraker, P. J., and Speck, J. C. (1978) Biochem. Biophys. Res. Commun. 80, 849-857 [CrossRef][Medline] [Order article via Infotrieve]
61. Laemmli, U. K. (1970) Nature 227, 680-685 [CrossRef][Medline] [Order article via Infotrieve]
62. Burnette, W. N. (1981) Anal. Biochem. 112, 195-203 [CrossRef][Medline] [Order article via Infotrieve]
63. Würzner, R., Oppermann, M., Zierz, R., Baumgarten, H., and Götze, O. (1990) J. Immunol. Methods 126, 231-237 [CrossRef][Medline] [Order article via Infotrieve]
64. Audie, J. P., Janin, A., Porchet, N., Copin, M. C., Gosselin, B., and Aubert, J. P. (1993) J. Histochem. Cytochem. 41, 1479-1485 [Abstract]
65. Hodgson, C. P., and Fisk, R. Z. (1987) Nucleic Acids Res. 15, 6295 [Free Full Text]
66. Ausubel, F. M., Brent, R., and Kingston, R. E. (1987) Current Protocols in Molecular Biology , John Wiley & Sons Inc., New York
67. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
68. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [Medline] [Order article via Infotrieve]
69. Herr, H. W., Schwalb, D. M., Zhang, Z. F., Sogani, P. C., Fair, W. R., Whitmore, W. F., and Oettgen, H. F. (1995) J Clin. Oncol. 13, 1404-1408 [Abstract]
70. Fradet, Y. (1995) Curr. Opin. Urol. 5, 267-271
71. Allard, P., Fradet, Y., Têtu, B., and Bernard, P. (1995) Clin. Cancer Res. 1, 1195-1202 [Abstract]
72. Taylor-Papadimitriou, J., Stewart, L., Burchell, J., and Beverly, P. (1993) Ann. N. Y. Acad. Sci. 690, 69-79 [Medline] [Order article via Infotrieve]
73. Apostolopoulos, V., and Mckenzie, I. F. C. (1994) Crit. Rev. Immunol. 14, 293-309 [Medline] [Order article via Infotrieve]
74. Lin, C. W., Lin, J. C., and Prout, G. R. (1985) Cancer Res. 45, 5070-5079 [Abstract/Free Full Text]
75. Yonezawa, S., Byrd, J. C., Dahiya, R., Ho, J. J., Gum, J. R., Griffiths, B., Swallow, D. M., and Kim, Y. S. (1991) Biochem. J. 276, 599-605
76. Byrd, J. C., Ho, J. J., Lamport, D. T., Ho, S. B., Siddiki, B., Huang, J., Yan, P. S., and Kim, Y. S. (1991) Cancer Res. 51, 1026-1033 [Abstract/Free Full Text]
77. Feizi, T., and Childs, R. A. (1987) Biochem. J. 245, 1-11 [Medline] [Order article via Infotrieve]
78. Hand, P. H., Colcher, D., Salomon, D., Ridge, J., Noguchi, P., and Schlom, J. (1985) Cancer Res. 45, 833-840 [Abstract/Free Full Text]
79. Devine, P. L., Mcguckin, M. A., Birrell, G. W., Whitehead, R. H., Sachdev, G. P., Shield, P., and Ward, B. G. (1993) Br. J. Cancer 67, 1182-1188 [Medline] [Order article via Infotrieve]
80. Xing, P. X., Prenzoska, J., Layton, G. T., Devine, P. L., and McKenzie, I. F. (1992) J. Natl. Cancer Inst. 84, 699-703 [Abstract/Free Full Text]
81. Durrant, L. G., Jacobs, E., and Price, M. R. (1994) Eur. J. Cancer 30A, 355-363 [CrossRef]
82. Chang, S. K., Dohrman, A. F., Basbaum, C. B., Ho, S. B., Tsuda, T., Toribara, N. W., Gum, J. R., and Kim, Y. S. (1994) Gastroenterology 107, 28-36 [Medline] [Order article via Infotrieve]
83. Blank, M., Klussmann, E., Kruger-Krasagakes, S., Schmitt-Graff, A., Stolte, M., Bornhoeft, G., Stein, H., Xing, P. X., Mckenzie, I. F. C., Verstijnen, C. P. H. J., Riecken, E. O., and Hanski, C. (1994) Int. J. Cancer 59, 301-306 [Medline] [Order article via Infotrieve]
84. Perey, L., Hayes, D. F., Maimonis, P., Abe, M., O'Hara, C., and Kufe, D. W. (1992) Cancer Res. 52, 2563-2568 [Abstract/Free Full Text]
85. Gambus, G., Jodas, G., Andreu, D., and Real, F. X. (1995) Int. J. Cancer 60, 146-148 [Medline] [Order article via Infotrieve]
86. Jerome, K. R., Bu, D. W., and Finn, O. J. (1992) Cancer Res. 52, 5985-5990 [Abstract/Free Full Text]

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