(Received for publication, December 6, 1995; and in revised form, January 9, 1996)
From the
The M344 tumor-associated antigen, expressed in 70% of superficial bladder tumors, is a sialylated carbohydrate present on a high molecular mass thiol-reducible secreted mucin, which we named MAUB for mucin antigen of the urinary bladder. Herein we studied the relationship between MAUB and other known mucins in the MGH-U3 bladder cancer line where MAUB expression is modulated by culture conditions. Northern blots, immunoradiometric assays, and Western blots showed that only MUC1 and MUC2 are expressed in this MAUB-positive cell line. MUC1 differs from MAUB by its molecular mass and its non-oligomeric nature, while MUC2 has similar molecular mass and response to culture conditions. However, in double determinant immunoradiometric assays, MAUB and MUC2 did not cross-react. Moreover, confocal microscopy showed different subcellular localization of the two antigens. Treatment of MGH-U3 cells with MUC2 antisense oligodeoxynucleotides resulted in decreased expression of MUC2 and increased expression of MAUB, ruling out the possibility that monoclonal antibody M344 recognizes a different glycosylated form of MUC2. In addition, we identified a tumor specimen expressing MAUB but no MUC2 antigen or mRNA. Together, these results suggest that there is expression of at least three mucins in MGH-U3 cells and that MAUB is a cancer-associated mucin distinct from those identified so far.
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.
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.
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.
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.
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.
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.
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).
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).
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.
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.
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.
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.
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