[CANCER RESEARCH54, 3305—3314, June 15, 1994)
Expression of Carcinoembryonic
Antigen and Nonspecific Cross-Reacting
50-kDa
Antigen in Human Normal and Cancerous Colon Mucosa: Comparative Ultrastructural Study with Monoclonal Antibodies' Vladimir Baranov,2 Moorix Mo-Wai Yeung, and Sten Hammarström3 Department of Immunology, 115478,Russia[V.B.J
University of Umed, S-901 85 Umeâ,Sweden [M. M-W. Y., S. H.J, and Laboratory
ABSTRACT The precise localization of carcinoembryonic antigen (CEA) and non specific cross-reacdng 50-kDa antigen (NCA SO)in normal colon mucosa and colon adenocarcinoma was investigated by using an indirect immu noperoxidase electron antibodies. In normal
microscopic technique with specific monoclonal adult colon both antigens were localized to ml
crovesicles and filaments of the “fUzzy coat―on the apical surface of the epithelial between
cells. In addition, adjoining
microvilli.
NCA 50 was found Mature
columnar
in the narrow
spaces
cells at the free luminal
surface coatained most ofthe antigen positive material. CEA and NCA 50 were also detected
as intracellular
components
of goblet cells.
In multilayered tumor glands, the cell surface expression of the anti gem was dependent on the position of the tumor cell in the gland. The neoplastic cells showed either a predominant apical labeling or a positive staining of almost the entire cell surface. Some of the neoplastic cells contained CEA in so-called “intracellular luminL― In contrast to normal
colon epitheial cells most tumor cells synthesized NCA SOactively. In normal colonic mucosa, unlike in cancerous tissue, CEA and NCA 50 appear to be released via vesicles formed from the microvillous
membrane
of mature columnar cells. These results are consistent with the hypothesis that CEA and NCA play a role in the nonspecific defense against micro organisms in the large intestine.
INTRODUCTION
CEA4(1) is widely used as a tumormarker,particularlyfor cancers of the gastrointestinal tract. It is a heavily glycosylated protein with a molecular mass of 180—200kDa. In spite of its clinical importance relatively little is known about its biological function in normal colonic epithelium and in colorectal cancer (see Refs. 2—4).CEA, nonspecific cross-reacting antigen with molecular mass of 50—90kDa (NCA 50/90) and BGP have been shown to function as intercellular adhesion molecules in vitro (5—8),suggesting that these molecules play a role in the development and organization of normal tissues and probably also in the process leading to the development of a malignant tumor (9). However, CEA and NCA have also been shown to act as receptors for type 1 fimbriated Escherichia coli, Salmonella, etc. (10—12), andmouseBGP hasbeenshownto functionasa receptorfor mouse hepatitis virus (13), suggesting that these molecules may be involved in the recognition of microorganisms in the gut (10). Molecular cloning has revealed that the CEA gene belongs to the immunoglobulin gene superfamily (14) and that it is the prototype for a family of related genes, the CEA/PSG gene family, (reviewed in Received 3/3/94; accepted 4/19/94. 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
of Immunochemistry,
Cancer Research
Center, Moscow
Refs. 3 and 4). To date, 29 genes have been identified (15—17).The genes can be divided into three different subgroups: the first group containing the genes coding for CEA and the classical CEA-related antigens, NCA 50/90 (18—22),NCA 95 (also called CGM6) (23—25) and BGP (26, 27). This group contains 12 genes and pseudogenes. These glycoproteins have a complex tissue distribution and are ex pressed on the outer membrane of cells. CPA, NCA 50/90, and CGM6 are linked to the membrane via phosphatidylinositol (25, 28, 29), while BGP, CGM1, and CGM7 are transmembrane glycoproteins (3, 4). The genes of the second subgroup encode the pregnancy-specific glycoproteins (n 11) (30, 31), which are abundantly expressed in placenta (30, 31). Recently, a third group of genes (n = 6) intermin gled among the pregnancy-specific glycoprotein genes on chromo some 19q13.2 was discovered through gene mapping studies (15—17). The tissues in which these genes are expressed remain unknown. To study the function of CEA and closely related NCA 50/90 in normal colon physiology and in colon cancer we have determined their precise tissue localization by using specific mAbs in an immu noelectron microscopic technique with high resolution power. Im munoelectron microscopic studies on the tissue localization of CEA have previously been performed with the use of polyclonal antibodies (32—35).However, the complexity of the gene family and the high degree of immunological cross-reactivity between individual proteins prompted us to reexamine this question by using highly specific anti-CEA mAbs (36, 37). NCA 50 has not been studied at the immunoelectron microscopic level previously. Our study shows that both CEA and NCA 50 are specifically localized to microvesicles and filaments making up the so-called fuzzy coat of normal
colon epithelial
cells, and to intracellular
corn
ponents of goblet cells. In colon tumors, CEA and NCA 50 show either a predominantely apical localization or a localization on the entire cell surface, depending on the position of the tumor cell in the gland.
MATERIALS AND METHODS Tissue. Tissues fromtwo patientswith colon cancerwere studied.Of these two adenocarcinomas,
one was located in the cecum and the other in the
sigmoid. Samples of normal and cancerous tissues were obtained from the same tissue specimens.
Morphologically
normal
colon wall was taken at least
8—10cm away from the tumor margin. Cancer tissue was taken from non necrotic zones within the tumor mass. Antibodies. The following murine mAbs were utilized: two CEA specific mAb, Bu-103 and 11-7, a NCA 50-specific mAb, Mox-34, a mAb specific for
intestinal alkaline phosphatase, IAP4, and a mAb against human thyrotropic hormone. The specificity, affinity, and epitope group reactivity of the anti 18 U.S.C. Section 1734 solely to indicate this fact. CEA mAb: Bu-103 (IgG1; epitope group Gold 5; K,@, 8 X 10 ‘° M;a gift from I This work was supported by a grant from the Swedish Cancer Society. Dr. Patrick Seguin, Oris Industrie, Bagnols sur Ceze, France) and 11-7(IgG1; 2 Recipient of ajoint Fellowship from the Royal Swedish Academy of Sciences and the epitope group Gold 1; K,@, 2 X 108 M;produced in this laboratory) have been Russian Academy of Sciences. described in detail (36, 37). MAb Mox-34 (IgG2b) was produced in the 3 To whom requests for reprints should be addressed, at Department of Immunology, University of UmeA,5-901 85 UmeA,Sweden. laboratory by immunization with purified NCA isolated from liver metastases 4Theabbreviations used are:CEA, carcinoembryonic antigen; NCA, nonspecific of a patient with colon cancer. The antigen used for immunization was purified cross-reacting antigen; BGP, biliary glycoprotein; mAb, monoclonal antibody; PBS, phosphate-buffered saline; DAB, 3,3'-diaminobenzidine tetrahydrochloride; K,@,dissoci ation constant.
by a conventional procedure (38) combined with affinity chromatography the use of a CEA-specific
3305
mAb to remove
residual
with
CEA and CEAIOW (39).
FUZ2Y COAT WCAUZATION OF CEA AND NCA IN HUMAN COLON
b
a
12
180 —
__..NCA-50 C
kDa
2
E LO
Fig. 1. a, reactivity of mAb Mox-34 with CEA related antigens as determined by enzyme-linked
immunosorbent assay. Serial dilutions of mAb Mox-34 were incubated with antigen coated to tin crotiter plates (1 @.tglml) followed by alkaline phos phatase-labeled rabbit anti-mouse immunoglobu lin. b, reactivity of mAb Mox-34 with NCA 50
tO 0
62
CEA
0
NCA-90 BGP (bile, serum)
from colorectal carcinoma. Sodium dodecyl sin fate-polyacrylamide gel electrophoresis analysis of ‘@‘I-labeled components extracted from colorectal carcinoma by phosphatidylinositol phospholipase
C and then immunoprecipitated by mAb Mox-34 and anti-CEA mAb 11-7. Material was bound to
Sepharose 4B coated with (Lane 1) Mox-34 and
0
(Lane 2) 11-7 and eluted with 2% sodium dodecyl sulfate plus 0.1 M dithiothreitol. Samples were run
32
DilutionofmAb
under reducing conditions on a linear acrylamide gel (5.6—15%)in a discontinuous buffer system.
In enzyme-linked immunosorbent assay, mAb Mox-34 reacts with the
30 mm to prevent
low-molecular-weight form (NCA 50), but not with the high molecular-weight form (NCA 90), of NCA 50/90 or with CEA, BGP, or
room temperature with mAb solution (50 p@g/ml)containing 0.05% sapo nm, followed by 3 washings with PBS for 1 h. (e) Incubation overnight at 4°Cwith sheep anti-mouse Ig F(ab')2 fragments conjugated with horse radish peroxidase (Amersham, Buckinghamshire, England) diluted 1:100 in PBS containing 0.05% saponin. (J) Three washings in PBS for 1 h, and detection of peroxidase activity with DAB (Sigma); the sections were first incubated with 0.05% DAB in 0.05 MTris-HC1 buffer, pH 7.6, for 20 min and then incubated in the same DAB solution containing 0.003% fresh hydrogen peroxide for 20 mm. (g) Successive washings by distilled water, followed by postfixation in 1.33% 0504 for 1 h. (h) Dehydration in graded acetone and flat embedding in Epon-Araldite mixture (Fluka Chemie, Buchs, Switzerland). The ultrathin sections of the normal colonic mucosa were cut longitudinally through the crypts and the free luminal surface to enable exact localization of the columnar cells. The sections of the crypts and the free luminal surface were usually studied separately. The ultrathin
BGPIOW
(Fig.
la)
Moreover,
mAb
Mox-34
immunoprecipitates
NCA
50,@
but not NCA 90 or CEA, from supernatants of phosphatidylinositol-specific phospholipase
C-treated
homogenates
of colon
carcinoma
(Fig.
ib)
or
normal submandibular salivary gland (data not shown). The 32-kDa band immunoprecipitated by Mox-34 from supernatants of colon cancer and nor mal submandibular salivary gland most probably represents unglycosylated NCA 50/90. The mAb against intestinal alkaline phosphatase (IAP4, 1gM;Ref. 41), was kindly provided by Dr. Torgny Stigbrand, University of UmeA.The mAb to thyrotropic hormone was of the IgGi subclass. Immunoelectron Microscopy. Immediately after surgical removal, tissue
specimens were washed in a cold Hanks' balanced salt solution for 15 mm. Small 1-mm cubes of the specimens were fixed by immersion in a mixture of 4% paraformaldehyde
(Merck,
Darmstadt,
Germany)
plus 0.01% glutaralde
hyde (Sigma, St. Louis, MO) in 0.1 M sodium cacodylate buffer, pH 7.3, containing 0.05% saponin (Merck) for 4 h on ice. The fixed tissue blocks were then washed in the same buffer containing
3.5% sucrose and 0.05% saponin for
nonspecific
sections were examined
antibody
without
binding.
any additional
(d) Incubation
for 4 h at
staining by using a Zeiss
EM 109 electron microscope. Control reactions included: (a) incubation with mAb to human thyro tropic hormone
or intestinal
alkaline phosphatase
instead of anti-CEA
or
16 h at 4°C,frozen,and sectionedon a cryostatmicrotome.Fifteen-sm anti-NCA 50 mAb; (b) incubation with peroxidase-labeled sheep anti cryostat sections were cut at —16°C.In the case of the normal colonic mucosa, mouse Ig F(ab')2 without previous treatment with mAb; (c) incubation with the sections were cut longitudinally. The free-floating cryosections were then DAB solutions only. transferred to conical centrifuge tubes containing 0.02 MPBS, 0.05% saponin, and processed according to the indirect immunoperoxidase method. (a) Inca bation with 5 mM periodic acid (Sigma) for 10 min, washing in PBS for 15
min, and incubation with 3 ms@i sodium borohydride (Sigma) for 20 mm in order to block endogenous peroxidase activity (42). (b) Washing in several
RESULTS
laboratories(range, 40—65and 85—110kDa). Our values are on the high side ofthe range,
Normal adult colonic epithelium can be divided into two regions: (a) the epithelium of the free luminal surface containing fully differ entiated, mature columnar cells and goblet cells, as well as the aging or dying cells in the process of being shed from the luminal surface; (b) the epithelium of the crypts which consists of undifferentiated cells and differentiating columnar, goblet, and enteroendocrine cells.
i.e., °-60and
A well-defined zone at the crypt mouth separates the “proliferating
baths of PBS for 30 mm, then incubation with 0.1 M lysine monohydro chloride (Sigma) in PBS for 15 mm to block free aldehyde groups. (c)
Incubation with 0.3% bovine serum albumin and 0.05% saponin in PBS for 5 The
recorded
molecular
weights
for
the
110 kDa, respectively (40).
two
forms
of
NCA
50/90
vary
between
3306
FUZZY COAT LOCALIZATION OF CEA AND NCA IN HUMAN COLON
compartment―of the crypt from the “mature, functional compartment― of the free luminal surface. CEA in Normal Colonic Mucosa. Both anti-CEA mAb 11-7 and Bu-103 gave the same staining pattern but Bu-103 exhibited a higher staining intensity. The reaction product revealed a conspicuous, thick coat situated along the outer face of the microvillar apical surface of the mature columnar cells lining the free luminal surface (Fig. 2a). At higher magnification, this coat was mainly seen over and around the tops of the microvilli masking their fine structure. The stained coat contrasted
strongly with the virtually unstained core and plasma membrane of the lower parts of the microvilli (Fig. 2.b). The thickness of the coat was
as a rule about 0.3 p.m but sometimes more than 0.5
[email protected] CEA-positive material of the coat consisted of fine matted filaments showing wide variation in length, and extending out from the surface of the tops of the microvilli (Fig. 2b). Since the microvillus plasma membrane was also stained for CEA at the sites where it makes contact with the filaments, it appears as if the majority of the filaments are intimately associated with the membrane. Numerous vesicles limited by a thick CEA-containing membrane were detected between
a
MV
@l
e ,@.
b
C C
MV
Fig. 2. Immunoelectron microscopy ofCEA in normal human colon. a, low power micrograph ofthe apical pole ofmature columns cells lining the free luminal surface. The reaction
product stains an additional surface coat (arrows) coated on microvilhi(MV). X4,000. b, high power micrograph of the apical part of a mature columnar cell. The CEA-positive coat over the tops of microvilhi(MV) consists of fine matted filaments (arrows) and small vesicles (V) limited by a thick CEA-containing membrane (arrowheads). X40,000. c, a few of
theCEA-coatedvesicles(V)arepresentbetweenandovermicrovilhi(MV).Oneof themicrovillishowsa distinctiveblebbingor vesiculation(arrow).X90,000.d, thestrikingfeature of the fuzzy coat of a mature columnar cell is the presence of the numerous CEA-coated vesicles (V) and lamellar structures (arrow). X41,000. e, a mature goblet cell at the free luminal surface. Electron-dense deposits are located on membrane-like material in the mucus mass (arrows) and on the internal limiting membrane (arrowheads). The fuzzy coat of columnar cells adjacent to the goblet cell is positively stained. X6,000. Inset, details of the stained membrane-like material and internal limiting membrane. X24,000. f, a discharged goblet cell shows the CEA-positive staining of the microvillous limiting membrane (arrows) around a large apical cavity (C) open into the colon lumen. X12,000. 3307
FUZZY COAT LOCALIZATION OF CPA AND NCA IN HUMAN COLON
ied somewhat, and predominance of one or the other components described above could be seen on the apical surface of individual columnar cell (Fig. 2€!).No CEA was found along the basolateral plasma membrane of the mature columnar cells and the crypt cells, or inside these cells. Nondischarging mature goblet cells at the free luminal surface had rare and short microvilli which were covered by a thin layer of CPA-positive filaments. Usually, mucous granules were not stained
the microvilli and in the filarnentous coat (Fig. 2b). The thickness of the limiting membrane of the vesicles can be explained by the pres ence of short CEA-positive filaments attached to the membrane. The CEA-coated vesicles, often arranged as clusters or rows, were formed by budding off from the microvillus membrane (Fig. 2c). In addition, the surface coat comprised positively labeled granulations, membrane fragments, and spherical bodies showing a larnellar, myelin-like, structure. The composition of the CEA-positive material usually var
4
a
d
b
e
MV
f
C
Fig. 3. Immunoelectron microscopy of CEA (a, b, c) and NCA 50/90 (d, e, f) in normal human colon. a, cells of the lower part of a colonic crypt. The reaction product is present on the apical surface covered by numerous short microvilli (arrows). X9,000. b, high power appearance of the apical surface of a columnar cell at the base of a colonic crypt.
CEA-positive material is visible as short spike-like filaments covering the entire apical plasma membrane (arrows). Note the positive staining of the plasma membrane (arrowheads). In the crypt lumen is present filamentous and granular material (M) stained by the reaction product. X42,000. c, micrograph of differentiating cells at the upper part of a colonic crypt. The CEA-positive material of the fuzzy coat consists of mailed filaments and vesicles (arrows) and is seen at the tops of microvilli (MV). X11,000. d, low power micrograph of a mature columnar cell at the free luminal surface. The NCA-positive material forms a thick coat (arrows) on the apical cell surface. X6,000. e, a higher magnification of the apical surface of a mature columnar cell. The NCA-positive filamentous or granular material is seen between the sides and over the tops of microvilli (arrows). A few of the NCA-coated vesicles (V) are also visible. Note the positive staining of the entire plasma membrane (arrowheads).
X41,000. f, micrograph shows the apical pole of colomc crypt cells NCA-positive
surrounds microvilli and forms a compact superficial layer (arrows). The reaction product also stains the crypt contents (CC). X11,000. 3308
material
r@zz@COATLOCALIZATION OFCEAANDNCAINHUMAN COLON even though they were located very close to the apical cell surface. The predominant location of the NCA-positive material in the inter However, in goblet cells in the process of discharging mucus the microvillus spaces smoothed away the sharp increase in thickness of reaction product was seen at the internal microvillous membrane the stained layer at the transition from the crypt to the free luminal surrounding a large aggregate of mucus droplets, as well as inside the surface that was seen in case of CEA. mucus mass as numerous insertions of flocculent or membrane-like The crypt content also contained NCA-positive material. It ap material (Fig. 2e). The reaction product was also found on streamers peared as large aggregates of tiny granules and fine short filaments. of flocculent or membranous material within the mucus discharged These aggregates usually included numerous small vesicles with a into the colonic lumen. Some goblet cells which appeared to have positively labeled membrane surrounding them. exhausted their mucous contents showed the reaction product on the Some goblet cells expelling mucus and facing the free lurninal microvillous limiting membrane of a large cavity bordering on a surface at the upper part of the crypt showed positively labeled “shell― of cytoplasm (Fig. 2f). granular and membranous material inside the mucus. In the crypts, the apical surface of the epithelial cells were clearly CEA in Colon Tumors. Both colon tumors were classified mor stained by the reaction product; however, the staining pattern de phologically as moderately differentiated adenocarcinornas. In the pended on the level of the crypt (Fig. 3a). The undifferentiated cells same tumor, well-differentiated areas with glandular structures coex lining the crypt base and the differentiating cells of the lower one isted with areas that showed a tendency to solidify. third and middle part of the crypt had sparse, short, and wide mi At the electron microscopic level, the neoplastic glands were usu crovilli with a tendency to variable orientation. Occasional branched ally multi-layered. A majority of the neoplastic cells facing intratu and fused microvihi were also seen. In these cells, in contrast to the moral lumens had conserved close cell-cell contacts which were mature columnar cells, the whole apical plasma membrane both characterized by tight junctions and desmosomes. These cells had a covering the microvihi and forming areas between the roots of the thin irregular layer of CEA-positive material on the luminal surface. adjacent microvilli, was stained by the reaction product (Fig. 3b). A The material consisted of granular and filamentous deposits which delicate supramembranous layer which consisted of a branching net were attached to the plasma membrane. The latter covered poorly work of numerous short or spike-like filaments radiating out from the developed microvilli and was positively stained for CPA (Fig. ‘Ia). apical plasma membrane as well as fine granular material and vesicles Furthermore, in these cells positive staining was also detected at their were also visualized by the reaction product (Fig. 3b). abluminal surfaces, but was interrupted in areas of cell-cell contacts The CEA-positive surface layer became thicker on the differenti with adjacent cells. The reaction product appeared to be located not ating cells in the upper one-third of the crypt. This change in thickness only on the plasma membrane but also just beyond it. was associated with a gradual accumulation of longer CEA-positive In the inner areas of tumor glands the neoplastic cells were attached filaments and an increase in the number of the CEA-coated vesicles by simple apposition of contacting cell surfaces and, sometimes, by (Fig. 3c). desmosomes. On such “free― cells the noncontacted surfaces were A sudden additional increase in thickness of the CEA-positive labeled by the reaction product. The intercellular space between such surface layer as well as a change in its ultrastructure took place at the cells occasionally contained granular material that was labeled by the transition from the crypt to the free luminal surface (the crypt mouth reaction product. In contrast, the areas of cell-cell contact showed area) where the layer turned into a prominent CEA-positive coat weak or no staining. Individual neoplastic cells displayed an intense covering the microvilli tops (see above). In the crypt lumen, an abundant ifiamentous or granular material as well as clusters of the staining reaction on almost the entire cell surface (Fig. 4a). So-called “intracellular lumina―(43) were observed in some neo characteristic CEA-coated vesicles were revealed by the reaction plastic cells situated on the inner areas of tumor glands. Interestingly, product. NCA 50 in Normal Colonic Mucosa. The NCA-positive material the microvilous limiting membrane of these structures was labeled by the reaction product (Fig. 4b). formed a thick coat on the apical microvillar surface of the cells lining The reaction product also stained the luminal contents of tumor the free luminal surface. Thus, at low magnification the anti-NCA glands. It appeared as a complex medley of granular or filamentous staining looked similar to that seen with anti-CEA (Fig. 3d). How material and vesicles. ever, at higher magnification several differences were seen. (a) The NCA-positive material was more compact and granular and consisted NCA 50 in Colon Tumors. Contrary to the normal colonic mu of supramembraneous aggregates of very short filaments and parti cosa, NCA was seen in the contents and probably on the limiting des. (b) This material was present not only at the top of the microvilli membrane of numerous cytoplasmic vesicles and small vacuoles in but also completely filled the narrow spaces between adjoining mi the majority of neoplastic cells (Fig. 4c). Some of the NCA-contain crovilli (Fig. 3e). (c) The reaction product could be easily seen on the ing vesicles and vacuoles were observed in the region of the Golgi entire apical plasma membrane, including its invaginations between complex and/or underneath the plasma membrane, although at times the bases of the microvilli. The labeling intensity of the plasma they were fused with the cell surface (Fig. 4d). When the Golgi membrane was higher than that of the supramembranous NCA-posi complex was in the plane of the section it was usually stained for tive material. (d) The contents of sparse cytoplasmic vesicles located NCA 50 (Fig. 4e). near the apical plasma membrane of the mature columnar cells was In individual tumor cells the reaction product stained a few rounded occasionally labeled by the reaction product, however, the staining granules which looked like the mucus granules in goblet cells. These intensity was very low. granules were occasionally seen in the process of being discharged to The NCA-positive material on the apical cell surface also corn the adjacent intercellular space (Fig. 4f). prised different numbers of vesicles. Some vesicles showed distinctly On the cell surface the labeling intensity of NCA 50 was weaker positive staining of the limiting membrane. than that of CEA, and varied from region to region. However, the In the crypts, the NCA-positive material was more uniformly dis majority of the examined neoplastic cells showed the reaction product tributed on the external surface of the apical plasma membrane than along almost the entire cell surface. The positive staining was more was CEA. The reaction product stained a thin compact layer of fine intense on the apical surface of the cells lining intratumoral lumens granular or filamentous masses which were located along the labeled and appeared as thin electron-dense granulations covering the mi apical plasma membrane and “embedded― the microvilli (Fig. 3!). crovillous plasma membrane. 3309
@
@;@—
FUZZY COAT LOCALIZATION OF CEA AND NCA IN HUMAN COLON
To detect CEA and NCA 50 we used selected mAb with proven specificity for the two antigens. The reason for using both anti-CEA mAb 11-7 and Bu-103 was that they recognize physically separated
Control Reactions. No cell surface or intracellular staining was seen in sections of normal colonic mucosa or cancerous tissue treated with mAb to intestinal alkaline phosphatase or human thyrotropic hor mone or treated with the peroxidase-labeled specffic F(ab')2 fragments of anti-mouse immunoglobulin or with DAB solution only (Fig. 5).
epitopes
DISCUSSION The aim of this study was to determine the precise tissue and cellular localization at the ultrastructural level of CEA and NCA 50 in normal human colon and colon cancer.
on CEA, thus increasing
the probability
of detecting
all CEA
in the tissue. Gold 1 epitopes (mAb 11-7) are located in the COOH terminal part of CEA, while Gold 5 epitopes (mAb Bu-103) are located in the NH2-terminal domain (44). As it turned out the two mAb gave identical staining patterns. CEA and NCA 50 in Normal Adult Colon. CEA was found: (a) in the external coat spreading over the microvillous apical surface of epithelial cells facing both the free lurninal surface and the crypts; (b)
4
@
•‘2'-@M
“j?@, • r .
‘l
(•
C .@
4k
.
•
•1
d
‘•I
•@1@h,•
a
I
.@
4.
Fig. 4. lmmunoelectron microscopy of CEA (a, b) and NCA 50/90 (c, d, e, f) in colon cancer. a, cells facing intratumoral lumen have a thin layer of the CEA-positive material on their luminal surface (arrows). Neoplastic cells of the inner portion of tumor gland show CEA on almost the entire cell surface (arrowheads). X9,000. Inset, details of the CEA
staining on the luminal surface. X15,000. b, micrograph shows the CEA staining on the microvillous limiting membrane of the “intracellular lumen― (arrows). X12,000. c, within neoplastic cells NCA is present in small or medium-sized vesicles and vacuoles (arrows). The positive staining can also be seen on the cell surface (arrowheads). X5,000. d, a higher magnification micrograph shows two NCA-containing vesicles fused with the cell surface (arrows). CV, cytoplasmic vacuole. X20,000. e, the reaction product is present in the Golgi
complex (GC) and vesicles of its region (arrows). X25,000. f, the NCA staining shows three rounded cytoplasmic granules (CG). X15,000. 3310
FUZZY COAT LOCALIZATION OF CEA AND NCA IN HUMAN COLON
free luminal surface as compared with the fuzzy coat of crypt cells. The undifferentiated and differentiating cells in the lower one-half of the crypt show only a thin layer of the short CEA-positive filaments and sparse CEA-coated vesicles located on the entire stained apical membrane. When the differentiating cells reach the upper one-half of the crypt, the CEA-positive layer becomes thicker because of a gradual accumulation of the CEA-containing filaments and vesicles. A sudden increase in thickness of the CEA-positive layer is observed at the transition from the crypt to the free luminal surface. The mature columnar cells facing the free luminal surface have the most abundant surface CEA-positive layer consisting of numerous long filaments and vesicles. Here these structures have a tendency to be concentrated at the tops of the microvilli. MV A comparison of these findings with former electron microscopic descriptions of the fuzzy coat in normal colonic mucosa (46, 51) shows that the observed differences directly reflect differences in ultrastructure of the fuzzy coat in various compartments of the colonic mucosa. This once more suggests that CEA is a major constituent of the fuzzy coat and that CEA and other glycoproteins of the fuzzy coat appear to share a common biosynthetic pathway. Our observations also indicate that the cell surface expression of Fig. 5. Immunoelectron microscopy of alkaline phosphatase in normal human colon. Thecontrolsectionshowsnostainingof areaof thefuzzycoat.MV,microvilli.X40,000. CEA varies in one and the same columnar cell during its differenti ation and migration along the crypt to the free luminal surface. Maximum CEA expression is attained only when the epithelial cell in mature goblet cells discharging mucous granules and in some of the has migrated up to the level of the free luminal surface, that is to the nondiseharging goblet cells showing coalescence of the granules; and functional compartment of colonic mucosa. Thus, surface expression (c) in the abundant luminal contents of the crypts. of CEA is controlled by the degree of cytodifferentiation and in some With respect to the first site, our results with anti-CEA mAb agree way by the position of the cell in the normal colonic mucosa. Differ with and extend those obtained in previous immunoelectron micro entiation-dependent surface expression of CEA in normal colonic scopic studies with polyclonal antibodies demonstrating the presence mucosa and colon tumors has already been noted by other investiga of CEA in the glycocalyx (32—35)or, more precisely, in the fuzzy coat tors (32—34,53—55). of the normal colon epithelium. Why do the mature columnar cells express much more CEA within The term “glycocalyx― denotes a carbohydrate-rich extraneous cell their fuzzy coat than do the immature cells? It is generally accepted surface coat. In the gastrointestinal mucosa only the glycocalyx of the that surface expression of a protein is determined by the balance apical cell surface is distinctly detected (45, 46). This special apical between its rate of synthesis and rate of release by shedding. If this is domain is generally known as the fuzzy coat (45). Each intestinal cell so, the observed surface overexpression of CEA on mature epithelial produces its own fuzzy coat consisting of carbohydrate chains and cells could be explained by an increased synthesis of CEA together extracellular domains of membrane-bound glycoproteins and glyco with increased release rate. This explanation is the most plausible as lipids as well as macromolecules that are secreted by cells and then previous in situ hybridization studies have shown that in the normal adsorbed onto the cell surface. The carbohydrate chains of the colonic mucosa the highest levels of CEA mRNA are expressed in the fuzzy coat appear to be important for maintenance of the filamen differentiated epithelial and goblet cells at the free luminal surface, tous conformation of the extracellular domain of the membrane with low levels in the middle and lower thirds of the crypts (56, 57). bounded glycoproteins (47), for protease resistance (48), as well as Furthermore, these topographical differences in CEA mRNA were for cell-cell interactions including the recognition and elimination paralleled by similar differences in the pattern of CEA protein ex of microorganisms (49, 50). pression as demonstrated by immunohistochemistry on serial sections The fuzzy coat is usually invisible in conventional electron micro (56, 57). Unfortunately, at present little is known about release of scopic specimens and special cytochemical staining is required to CEA in normal colonic mucosa. Several lines of indirect data can reveal it. However, our anti-CEA mAb clearly develop the fuzzy coat nonetheless be considered. First, although normal colon mucosa and both on the mature columnar cells at the free luminal surface and in cancerous tissue were reported to show very similar levels of CEA the crypt cells. The CEA-positive material within the fuzzy coat transcripts (58, 59), only small amounts of CEA are detected in consists of fine filaments linked to and radiating out from the apical normal colonic mucosa (59—61). This strongly suggests that in nor mal colonic mucosa CEA is rapidly released into the colonic lumen plasma membrane and small CEA-coated vesicles formed by budding and excreted via feces. Second, as first demonstrated by Matsuoka off from the microvillus membrane. Previous cytochemical investiga tions of the fuzzy coat ultrastructure in normal colonic mucosa have et aL (62), about 50—70mg of CEA (or related antigens) are recovered in normal feces evacuated during 1 day. This suggests once again that demonstrated that the presence of filaments and vesicles is the most normal colonic mucosa very actively releases CEA into the lumen. distinctive feature of the coat (46, 51, 52). Thus, at least two major Third, Fantini et al. (63, 64) have reported that the levels of CEA structural elements of the fuzzy coat in normal colonic epithelium, released in the medium rose in a time-dependent manner when a colon filaments, and vesicles, contain CEA and it consequently is a major cancer cell line, HT-29-D4, was induced to differentiate. Finally, our integral glycoprotein of the fuzzy coat (the glycocalyx). Our results demonstrate that in normal colonic mucosa CEA is results show that the accumulation of CEA within the fuzzy coat of the mature columnar cells is structurally related to the presence of present along the entire length of the fuzzy coat. However, a closer numerous CEA-coated vesicles formed by blebbing of the microvillus analysis shows that there are obvious differences in thickness and fine membrane and subsequent pinching off. This vesiculation of the structure of the CEA-positive material in the fuzzy coat of cells at the 3311
FUZZYCOATLOCALIZA11ON OF CPA ANDNCA IN HUMANCOLON
microvilli is a normal process and is the most distinctive feature of the fuzzy coat in human colonic epithelium (46). Villin the microvillus core protein appears to be directly involved in the process of vesic ulation. In the presence of @2+,villin induces depolymerization and a rapid shortening of the actin filaments in the core (65, 66). The shortening of the microvillus core results in a rapid vesiculation of the plasma membrane along the entire length of the microvillus (67). Precise mechanisms for regulation of the depolymerizing villin activ ity are unclear, however, it seems that vesiculation of microvilli is a common response to conditions which affect the intestine microvillus membrane. It has been observed in vivo as a response to fasting, thermal injury, and treatment with antibodies and lectins (66, 68—70). According to Matsudaira and Burgess (71) the vesicles released from the microvilli perhaps serve as a rapid mechanism for the removal of membrane-active agents from gut luminal surface. Taken together, the data suggest that it is the mature columnar cells in normal colon mucosa that release CEA and that this occurs via CEA-coated vesicles. This agrees with the findings of Matsuoka et aL (62) who showed that almost all CEA in feces exists in a membrane bound form. However, we do not agree with their assumption that the release of CEA is normally associated with natural extrusion of moribund or dead mature epithelial cells from the free luminal sur face. In humans it has been estimated that 20 to 50 million cells are lost each minute from the intestine (46). However, it is well known that prior to extrusion the mature intestinal epithelial cells undergo degenerative changes. These dying cells, first of all, lose their fuzzy coat (together with CEA) since exfoliated cells virtually lack the fuzzy coat (45, 46). It is obvious that the efficiency of the postulated mechanism of CEA release depends not only on the presence of the microvilli but also on their mature organization in the colonic epithelial cells. This implies that the crypt cells and the neoplastic cells which have rare, immature, and badly developed microvilli may release only a low amount of CEA. Our results showing a low number and a gradual accumulation of CEA-coated vesicles in the fuzzy coat of the crypt cells during their differentiation support this. Intracellular localization of CEA was observed in the mature goblet cells. However, the intensity of the positive staining in these cells was variable. This might be due to the discharge of the mucus before and during the preparation of the specimens. In the positive cells, CPA was seen within mucus as well as on the internal microvillous mem branes limiting mucus granules. These findings are in agreement with previous reports (33—35)and suggest that goblet cells synthesize CPA and secrete it into the lumen together with the mucus. It is tempting to speculate that this non-membrane-bound CPA correspond to the small fraction of naturally solubiized CPA that was detected in normal feces by Matsuoka et aL (62). We did not find intracellular CPA in the normal colonic columnar cells, although in some previous studies CPA was found in protein synthetic organelles of these cells (33—35).This discrepancy is prob ably due to our using mAb against conformation-dependent protein epitopes, while earlier investigators used polyclonal antibodies. A conformation-dependent protein epitope in a highly glycosylated pro tein like CPA probably has a different conformation when the protein is not glycosylated, leading to unreactively or only weak reactivity with a mAb against the native glycoprotein. False-negative results due to poor antibody penetration is excluded in our experiments since we were able to localize intracellular CPA and NCA in tumor tissue by using the same immunocytochemical procedure. Our results also agree with the presence of the CPA-positive material in the colonic content (32, 54, 72). This abundant material contains granular masses, filaments, vesicles, lamellar dense bodies which apparently represent
structures derived from the colonic cells, as well as products of their degradation. NCA 50 was present within the fuzzy coat of the mature columnar cells and of the crypt cells. However, in the fuzzy coat of the mature columnar cells NCA 50, in contrast to CEA, was present not only at the tops of the microvihi but also in the spaces between the microvilli. Moreover, the NCA-positive material of the fuzzy coat was more compact and granular than that of CPA. The parallel distinct staining of the entire apical membrane, furthermore, suggests that NCA 50 is anchored to the plasma membrane. In addition, NCA 50 appears to be present in the limiting membrane of the same vesicles which contain CPA. On this basis, it seems likely that NCA 50 is released by the same vesicle-mediated mechanism as was proposed for CPA. Earlier light microscopic data have demonstrated that NCA is present in normal colonic mucosa (23, 32) and that NCA and CPA transcripts are coexpressed in normal colonic mucosa (58, 73). Intracellular localization of NCA 50 was definitely detected only in mature goblet cells. In the mature columnar cells NCA 50 appeared also to be associated with cytoplasmic vesicles. However, they were difficult to delimit and identify. The finding that CPA and NCA 50 appear to be released as membrane vesicles from the microvilli of preferentially mature colon epithelial cells is of considerable interest in light of a possible function of these molecules in normal colon physiology. Leusch et aL (10, 11) and Sauter et aL (12) have recently reported that CPA and NCA 50 bind to E. coli and Salmonella bacteria expressing type 1 fimbriae. This raises the possibility that these molecules are involved in the regulation of host colonization by fixation of symbiotic microorgan isms and that they play an essential role in the host defense mecha nism preventing the binding of pathogenic bacteria by rapid shedding of CPA and NCA 50 from the colonic mucosa. In this context, the constant and rapid release of the CEA-coated vesicles from the CPA-containing microvillus membrane may represent a mechanism not only for regulating the steady state level of the normal microbial flora in human colon but also for “catching― and rapidly eliminating of pathogenic bacteria and, perhaps viruses (13). CPA and NCA 50 in Colon Cancer. Our studies show that there are variations in the surface distribution of CPA between different cell layers of tumor glands. The neoplastic cells facing intratumoral lu mens, which are morphologically polarized and have intercellular junctions, mainly express CPA on the apical microvillous membrane. In contrast, the cells located deep inside tumor glands, which are morphologically unpolarized and have few or no intercellular junc tions, exhibit CPA on almost the whole surface of the neoplastic cells. Thus, one of the basic characteristics of malignant colonic cells as compared to their normal counterpart, is the loss of polarity in the cell surface localization of CPA. These results confirm and extend previ ous electron microscopic studies on the surface expression of CPA in cancerous colonic cells (32, 33, 35, 55, 74). The presence of CEA over the entire cell surfaces of the tumor cells suggests that the tumor cells have lost the ability to selectively transport newly synthesized pro teins to the different surfaces of cell. In our studies this conclusion is supported by the appearance of a new storage organelle in some tumor cells, the so-called intracellular lumen (43) or vacuolar apical com partment (75), in which apically directed proteins may accumulate. Our studies show that the microvillous luminal membrane of these lumens and their contents do contain CPA. It is believed that the presence of the intracellular lumens is connected with dysfunction of the Golgi complex (76). The dysfunction so frequently observed in cancer cells leads to the formation of cytoplasmic cavity bordered by a new internal “apical― membrane. As a result, the epithelial cell reconstructs its polarity and revives its ability to continue the transport of apically targeted proteins, which is the next step toward differen
3312
FUZZY COAT LOCALIZATION OF CEA AND NCA IN HUMAN COLON
tiation (43). Neither the presence of the intracellular lumens nor alteration in the polarity of proteins are specific for neoplastic trans formation of epithelial cell (43, 77). As was the case for normal colonic mucosa, we failed to detect CPA in protein-synthesizing organelles of the neoplastic cells, as has been observed by others (33, 35, 74). In contrast to normal colonic mucosa, the intracellular localization of NCA in tumor cells is constant and dominant. The cells show NCA in the Golgi complex and in numerous cytoplasmic vesicles and vacuoles. The NCA-containing vesicles and vacuoles are found near the plasma membrane or fused with the cell surface. In a majority of the neoplastic cells NCA is also detected on almost the whole plasma membrane. These findings suggest that the neoplastic cells actively synthesize NCA and transport it to the cell surface. This agrees with previous data suggesting that the levels of NCA mRNA are much higher in colonic carcinomas compared to very low levels in normal colonic mucosa (58, 73, 78). The results are also consistent with the finding that NCA mRNA, in comparison to CPA mRNA, is over expressed in colon carcinoma (78). Taken together, these data support the conclusions of Chi et aL (78), suggesting that NCA may be a more useful marker of the malignant cell phenotype in colon carcinoma than CEA, and that the NCA gene is more suitable for studies of genetic alterations in colon carcinoma than the CEA gene. It must, however, be recognized that the NCA 50/90 gene is active in a number of normal tissues and cell types, including lung, spleen, and granulocytes (3, 4), and high levels of NCA (79) are found in normal serum.
ACKNOWLEDGMENTS The authors wish to thank Professor G. I. Abelev and Dr. Marie-Louise HammarstrOm
for critical reading of the manuscript,
and R. SjOberg for
assistance in use of the electron microscope.
12. Sauter, S., Rutherfurd, S., Wagener, C., Shively, J., and Hefta, S. Binding of non-specific cross-reacting antigen, a granulocyte membrane glycoprotein, to Esch erichia coli expressing type 1 fimbriae. Infect. Immunol., 59: 2485—2493,1991.
13. Williams, R., Jiang, G. S., and Holmes, K. Receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins. Proc. NatI. Acad. Sri. USA, 88: 5533-5536, 1991. 14. Paxton,
R., Mooser,
0.,
Paude,
H., Lee, T., and Shively,
3. Sequence
analysis
of
carcinoembryonic antigen: identification of glycosylation sites and homology with the immunoglobulin supergene family. Proc. Nail. Acad. Sci. USA, 84: 920—924,1987. 15. Olsen, A., Teglund, S., Nelson, D., Gordon, L, Copeland, A., Georgesco,
A.,
Carrano, A., and HammarstrOm, S. Gene organization of the pregnancy-specific glycoprotein region on human chromosome 19: assembly and analysis of a 700 kb cosmid contig spanning the region. Genomics, in press, 1994. 16. Teglund,
S., Olsen,
A., Khan,
W. N., Frangsmyr,
L,
and HammarstrOm,
S. The
pregnancy-specific glycoprotein (PSG) gene cluster on human chromosome 19: identification of six new genes forming a third subgroup within the carcinoembryonic
antigen (CEA) family and structure and organization of the eleven PSG genes. Genomics, in press, 1994. 17. Khan, W., Frangsmyr, L, Teglund, S., Israelsson, A., Bremer, K., and HammarstrOm, S. Identification of three new genes and estimation of the size of the carcinoembry onic antigen family. Genomics, 14: 384—390, 1992. 18. von Kleist, S., Chavanel, G., and Burtin, P. Identification of an antigen from normal human tissue that crossreacts with the carcinoembryonic antigen. Proc. Natl. Acad. Sci. USA, 69: 2492—2494, 1972. 19. Mach, i-P., and Pusztaszeri, 0. Carcinoembryonic antigen (CEA): demonstration of a partial identity between CEA and a normal glycoprotein. Immunochemistry, 9: 1031—1034, 1972.
20. Neumaier, M., Zimmerman, W., Shively, L, Honoda, Y., Riggs, A. D., and Shively, J. Characterization of a cDNA clone for the nonspecific cross-reacting antigen (NCA) and a comparison of NCA with carcinoembryonic antigen (CEA). J. Biol. Chem., 263: 3202—3206,1988.
21. Grunert, F., Kolbinger, F., Schwarz, K., Schwaibold, H., and von Kleist, S. Protein analysis of NCA-5Oshows identity to NCA cDNA deduced sequences and indicates posttranslational 1988.
modifications. Biochem. Biophys. Res. Commun., 153: 1105—1115,
22. Hefta, S., Paxton, R., and Shively, .1.Sequence and glycosylation site identity of two distinct glycoforms of nonspecific cross-reacting antigen as demonstrated by se quence analysis and fast atom bombardment mass spectrometry. 3. Biol. (1cm., 265: 8618—8626,1990. 23. Buchegger, F., Schreyer, M., Carrel, S., and Mach, I. P. Monoclonal antibodies identify a CEA crossreacting antigen of 95 kD (NCA-95) distinct in antigenicity and tissue distribution from the previously described NCA of 55 kD. Int. J. Cancer, 33:
643—649,1984. 24. Kuroki, M., Matsuo, Y., Kinugasa, T., and Matsuoka, Y. Three different NCA species, CGM6/CD67, NCA 95 and NCA 90 are comprised in the 90 to 110 kDa band of granulocyte NCA detectable upon SDS-polyacrylamide
gel electrophoresis.
Biochem. Biophys. Rca. Commun., 182: 501—506,1992. 25. Berling, B., Kolbinger, F., Grunert, F., Thompson, J., Brombacher, F., Buchegger, F., von Kleist, S., and Zimmermann, W. Cloning of carcinoembryonic antigen family
REFERENCES 1. Gold, P., and Freedman, P. Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques. J. Exp.
member expressed in leukocytes of chronic myeloid leukemia patients and bone marrow. Cancer Res., 50: 6534—6539,1990. Med., 121: 439—452, 1965. 26. Svenberg, T., HammarstrOm,S., and Hedin, A. Purification and properties of biliary 2. Thomas, P., Toth, C. A., Saini, K., Jessup, J. M., and Steele, 0. The structure, glycoprotein I (BGP I). Immunochemical relationship to carcinoembryonic antigen. metabolism and function of carcinoembryonic antigen gene family. Biochim. Bin Mol. Immunol., 16: 245—252,1979. phys. Acts, 1032: 177—189,1990. 27. Barnett, T., Kretschmer, A., Austen, D., Goebel, S., Hart, 3., Elting, J., and Kamarck, 3. Thompson, J., Grunert, F., and Zimmerman, W. Carcinoembryonic antigen gene M. Carcinoembryonic antigen: altemative splicing accounts for multiple mRNAs that family: molecular biology and clinical perspectives. I. Gin. Lab. Anal., 5: 344—366, code for novel members of the carcinoembryonic antigen family. I. Cell. Biol., 108: 1991. 4. HammarstrOm, S., Khan, W. N., Teglund, S., Hammarstrflm, M-L, Ramos, T.,
267—276,1989. 28. Hefta, S., Hefta, L., Lee, T., Paxton, R., and Shively, J. Carcinoembryonic
Baranov, V., Yeung, M. M-W., and FrAngsmyr,L The carcinoembryonic antigen family. In: M. H. V. Regenmortel (ed.), Structure of Antigens, Vol. 2, pp. 341—376. ben Raton, FL: CRC Press, 1993. 5. Benchimol, S., Fuks, A., Jothy, S., Beauchemin, N., Shirota, K., and Stanners, C.
Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule. Cell, 57: 327—334,1989. 6. Oikawa, S., Inuzuka, C., Kuroki, M., Matsuoka, Y., Kosaki, G., and Nakazato, H. Cell
adhesion activity of non-specific cross-reacting antigen (NCA) and carcinoembryonic antigen (CEA) expressed on CHO cells surface: homophilic and heterophilic adhe sion. Biochem. Biophys. Res. Common., 164: 39-45,
1989.
7. Rojas, M., Fuks, A., and Stanners, C. P. Biiary glycoprotein a member of the immunoglobulin supergene family, functions in vitro as a Ca2@-dependent intercel lular adhesion molecule. Cell Growth & Differ., 1: 527—533,1990. 8. Oikawa, S., Kumki, M., Matsuoka, Y., Kosaki, G., and Nakazato, H. Homotypic and heterotypic Ca@ @-independent cell adhesion activities of biliary glycoprotein, a
antigen is
anchored to membranes by covalent attachment to a glycosylphosphatidylinositol moiety: identification of the ethanolamine linkage site. Proc. Natl. Acad. Sci. USA,
85: 4648—4652, 1988. 29.
Kolbinger,
F., Schwartz,
K., Brombacher,
F., von Kleist,
S., and Grunert, F. Expres
sion of an NCA cDNA in NIH/3T3 cells yields a 110 kD glycoprotein, which is anchored into the membrane via glycosylphosphatidylinositol. Biochem. Biophys. Res. Commun., 161: 1126—1134,1989. 30. Tatarinov, Y. S., and Masyukevich, V. N. Immunochemical identification of a new beta-globulin in blood serum ofpregnant women. Bull. Eksp. Biol. Med. (USSR), 69: 66—78, 1970. 31. Watanabe, S., and Chou, I. Y. Isolation and characterization ofcomplementary DNAS encoding human pregnancy-specific @1-g1ycoprotein.i. Biol. Chem., 263: 2049—
memberof carcinoembryonic antigenfamily,expressedon CHOcell surface.Bio
2056, 1988. 32. Huitric, E., Laumonier, R., Burtin, P., von Kleist, S., and Chavanel, G. An optical and ultrastructural study of the localization of carcinoembryonic antigen (CEA) in normal and cancerous human rectocolonic mucosa. Lab. Invest., 34: 97—107,1976.
chem. Biophys. Res. Commun., 186: 881-887, 1992.
33.
9. Neumaier,M., Paululat,S., Chan,A., Mattheas,P., andWagener,C. Biliaryglyco protein, a potential human cell adhesion molecule, is down-regulated in colorectal carcinomas. Proc. NatI. Acad. Sci. USA, 90: 10744-10748,
non-specific crossreacting antigen (NCA). FEBS LetL, 261: 405-409, 1990.
11. Leusch,H. 0., Drzeniek,Z., Markos-Pusztai,Z., and Wagener,C. Bindingof coil and Salmonella
strains to members of carcinoembryonic
D., Nakane,
P., and Brown,
W. Ultrastructural
localization
of carcinoembry
onic antigen in normal intestine and colon cancer. Abnormal distribution of CEA on
the surfaces of colon cancer cells. Cancer (Phila.), 49: 2077—2090,1982. 34. Ahnen, D., Kinoshita, K., Nakane, P., and Bmwn, %V.Differential expression of
1993.
10. Leusch,H.0., Hefta,S.,Drzeniek,Z.,Hummel,K.,Markos-Puztai, Z.,andWagener, C. Escherkhiacoli of humanoriginbindsto carcinoembryonic antigen(CEA)and Escherichia
Ahnen,
carcinoembryonic antigen and secretory component during colonic epithelial cell differentiation and in colonic carcinomas. Gastroenterology, 93: 1330—1338,1987. 35. Shirota, K., Minassian, H., and Jothy, S. Protein G-gold immunoelectron microscopy of colon carcinoma: the effect of tumor differentiation on carcinoembryonic antigen immunostaining.
antigen
family: differential binding inhibition by aromatic-glycosides of mannose. Infect. Immun., 59: 2051—2057,1991. 3313
Exp. Mol. Pathol., 49: 305—315,1988.
36. Hammarström,S., Shively, J., Paxton, R., Beatty, B., Larsson, A.,Ghosh, R., Börmer,
0., Bucheger,F., Mach,J. P., Burtin,P., Seguin,P., Darbouret,B., Degorce,F.,
Fuzzy COATWCALIZA1'IONOF CEAAND NCA IN HUMANCOLON Sertour, I., Jolu, I., Fuks, A., Kaithoff, H., Schmiegel, W., Arndt, R., Kloppel, G., von Kleist, S., Grunert, F., Schwarz, K., Matsuoka, Y., Kuroki, M., Wagener, C., Weber,
1., Yachi, A., Imai, K., Hishikawa, N., and Tsujisaki, M. Antigenic sites in carcino embryonic antigen. Cancer Res., 49: 4852—4858,1989. 37. Nap, M., Hammarstrom,
M-L, Bormer, 0., Hammarstrom,
nonmalignant human tissues. Cancer Res., 48: 3153—3157,1988. 59. Kuroki, M., Arakawa, F., Yamamoto, H., Shimura, H., Ikehara, Y., and Matsuoka, Y. Active production and membrane anchoring of carcinoembryonic antigen observed in normal colon mucosa. Cancer Lett, 43: 151—157, 1988. 60. Fritsche, R., and Mach, I. P. Isolation and characterization
S., Wagener, C., Handt,
S., Schreyer,M., Mach,i-P., Buchegger,F., von Kleist,S., Grunert,F., Seguin,P., Fuks, A., Holm, R., and Lamerz@R. Specificity and affinity of monoclonal antibodies against carcinoembryonic antigen. Cancer Res., 52: 2329—2339, 1992.
38. Hammarström,S., Engvall, E., and Feld, S. Quantitation of CEA by enzyme linked immunoadsorbent
of carcinoembryonic
antigen (CEA) extracted from normal human colon mucosa. Immunochemistry, 14: 119—127, 1977.
61. Quetmeler, A., Moller, P., Schwartz, V., Abel, U., and Schlag, P. Carcinoembryonic antigen, CA 19-9 and CA 125 in normal and carcinomatous human colorectal tissue. Cancer (Phila.), 60: 2261—2266, 1987.
assay (ELISA) and specificity studies. In: K. GriffIths, K. Neville,
and C. Pierrepoint(eds.), Tumor Markers, pp. 252—258. Cardiff: Alpha Omega, 1978. 39. Hedin, A., HammarstrOm,S., Svenberg, T., and Sundblad, G. Low molecular weight CEA. Isolation and characterization. Scand. J. Immunol., 8(Suppl. 8): 423—428,1978. 40. Hammarström, S., Svenberg, T., Hedin, A., and Sundblad, G. Antigens related to carcinoembryonic antigen. Scand. J. Immunol., 7(Suppl. 6): 33—46,1978.
41. Domar,U., Nilsson,B., Baranov,V., Gerdes,U., and Stigbrand,T. Expressionof intestinal phosphatase in human organs. Histochemistry, 98: 359—364,1992. 42. Isobe, Y., Chen, S., Nakane, P., and Brown, W. Studies on translocation of immu noglobulins across intestinal epithelium. I. Improvements in the pemxidase-labeled antibody method for application to study of human intestinal mucosa. Acts Histo
62. Matsuoka, Y., Matsuo, Y., Sugano, K., Ohkura, H., Kuroki, M., and Kuroki, M. Characterization of carcinoembryonic antigen-related antigens in normal adult feces. Jpn.J.CancerRes.,31: 514—519, 1990.
63. Fantini, J., Rognom, J. B., Culonson, J. H., Pommier, G., Marvaldi, i., and Tirard, A. Induction of polarized apical expression and vectoral release of carcinoembryonic antigen (CEA) during the process of differentiation of HT-29-D4 cells. J. Cell. Physiol., 141: 126—134,1989. 64. Fantini, J., Rognoni, J. B., Theveniau, M., Pommier, G., and Marvaldi, J. Impaired
carcinoembryonic antigenreleaseduringthe processof suramin-induced differenti
43. Remy, L. The intracellular lumen: origin, role and implications of a cytoplasmic neostructure. Biol. Cell (Paris), 56: 97—106,1986.
ation of human colonic adenocarcinoma cell clone HT-29-D4. i. Cell. Physiol., 143: 468—474,1990. 65. Weber, K., and Glenney, 1. Calcium-modulated multifunctional proteins regulating F-actin organization. Cold Spring Harbor Symp. Quant. Biol., 46: 541—552,1982.
44. Thou, H., Fuks, A., Alcaraz, G., Boiling, T. J., and Stanners, C. P. Homophilic
66. Mooseker, M., Bonder, E., Conzelman, K., Fishkind, D., Howe, C., and Keller, T.
chem., 10: 161—171,1977.
adhesion between Ig superfamily carcinoembryonic antigen molecules involves dou ble reciprocal bonds. i. Cell Biol., 122: 951—960,1993. 45. Ito, S. Structure and function of the glycocalyx. Fed. Proc., 28: 12—25, 1969.
Brush border cytoskeleton and integration of cellular functions. 1. Cell. Biol., 99(SuppL): 104—112,1984.
46. Wyburn, G. Intestine. In: P. Toner and K. Can (eds.), The Digestive System. An Ultrastructural Atlas and Review, pp. 55—127.London: Butterworths, 1971.
47. Rose, M., Voter, W., Sage, H., Brown, C., and Kaufman, B. Effects of deglycosyla tion on the architecture of ovine submaxillary mucin glycoprotein. 1. Biol. Chem., 259:3167—3172, 1984. 48. Variyam, E., and Hoskins, L. In vitro degradation of gastric mucin. Carbohydrate side chains protect polypeptide core from pancreatic proteases. Gastroenterology, 84:
67. Burgess, D., and Prom, B. A reevaluation of brush border motility: calcium inducers core filament solation and microvillar vesiculation. J. Cell. BioL, 94: 97—107, 1982. 68. Lorenzsonn,
V., and Olsen, W. Intestinal brush border membrane
degradation:
comparison of in vivo responses to antibody and lectins. J. Cell. Biol., 87: 95, 1980. 69. Misch, M., Giebel, P., and Faust, R. Intestinal microvilhi: response to feeding and fasting. Eur. J. Cell Biol., 21: 269—279,1980. 70. Ezzell, R., Carter, E., Yarmush,
M., and Tompkins,
R. Thermal injury-induced
changesin the rat intestinebrush bordercytoskeleton.Surgery(St. Louis),114:
591—597,1993. 533—537,1983. 49. Ofek, I., Beachey, E., and Sharon, N. Surface sugars on animal cells as determinants 71. Matsudaira, P., Burgess, D. Structure and function of the brush-border cytoskeleton. of recognition in bacterial adherence. Trends Biochem. Sci., 4: 159—160,1978. Cold Spring Harbor Symp. Quant. Biol., 46: 845—854,1982. 50. Ruoslahti, E. Proteoglycans in cell regulation. 1. Biol. Chem., 264: 13369—13372, 72. Freed, D., and Taylor, G. Carcinoembryonic antigen in faeces. Br. Med. J., 1:85-89, 1989. 1972. 51. Rifaat, M., iseri, 0., and Gottlieb, L An ultrastructural study of the “extraneous coat― 73. Sato, C., Miyaki, M., Oikawa, S., Nakazato, H., and Kosaki, G. Differential expres of human colonic mucosa. Gastroenterology, 48: 593—598,1965. sion of carcinoembryonic antigen and nonspecific crossreacting antigen genes in 52. Lorenzsonn, V., and Trier, J. The fme structure of human rectal mucosa. The humancolonadenocarcinomas and normalcolonmucosa.Jpn. J. CancerRes.,79: epithelial lining of the base of the crypt. Gastroenterology, 45: 88—94,1968. 433—437,1988. 53. O'Brien,
M., Zamcheck, N., Burke, B., Kirkham, S., Saravis, C., and Gottlieb, L
74. Wolf, B., Schwinde, A., Thompson, 1., and von Kleist, S. Ultrastructural localization
Immunocytochemical localization of carcinoembryonic antigen in benign and malig nant colorectal tissues. Am. 1. Gin. Pathol., 75: 283—290,1981. 54. Primus, J., Clark, C., and Goldenberg, D. Immunoperoxidase localization of carci
of carcinoembryonic antigen (CPA) on ultrathin cryosections of human tumours. Anticancer Res., 6: 91-96, 1986. 75. Vega-Salas, D., Salas, P., and Rodriguez-Boulan,
noembryonic antigen in normal human intestinal mucosa. 1. Nail. Cancer Inst., 67: 1031—1039, 1981. 55. Haynes, W., Shertock, K., Skinner, J., and Whitehead, R. The ultrastructural immu
nohistochemistry of oncofetal antigens in large bowel carcinomas. Virchows Arch. Abt. A Pathol. Mat., 405: 263—275,1985. 56. Abbasi, A., Chester, K., Macpherson, A., Boxer, G., Begent, R., and Malcolm, A. Localization of CEA messenger RNA by in situ hybridization in normal colonic mucosa and colorectal adenocarcinomas. 1. Pathol., 168: 405—41 1, 1992. 57. Jothy, S., Yuan, S-Y., and Shirota, K. Transcription of carcinoembryonic
antigen in
normal colon and colon carcinoma. in situ hybridization study and implication for a new in vivo functional model. Am. J. Pathol., 143: 250—257,1993. 58. Cournoyer, D., Beauchemin, N., Boucher, D., Benchimol, S., Fuks, A., and Stanners, C. Transcription of genes of carcinoembryonic antigen family in malignant and
E. Modulation of expression of an
apical membrane protein of Madin-Darby canine kidney cellular storage compart ment. J. Cell. Biol., 104: 1249—1259,1987. 76. Remy, L, and Marvaldi, J. Origin of intracellular lumina in HT 29 colonic adeno carcinoma cell line. An ultrastructural study. Virchows Arch. Abt. B. Zellpathol., 46: 297—305,1985. 77. Rodriguez-Boulan, E., and Nelson, J. Morphogenesis of the polarized epithelial cell phenotype. Science (Washington DC), 245: 718—725,1989. 78. Chi, K., Jessup, J. M., and Frazier, M. Predominant expression of mRNA coding for
nonspecific cross-reacting antigen in colorectal carcinomas. Tumor Biol., 12: 298— 308, 1991. 79. Reck, W., Daniel, S., Nagel, G., Him, M., von Kleist, S., and Grunert, F. Determi nation of CEA and different NCAS in aria of tumor patients using ELISAs. Tumor Biol., 13: 110, 1992.
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