Detection and characterization of sucrase-isomaltase in adult human colon and in colonic polyps

GASTROENTEROLOGY

1990;98:1467-1477

Detection and Characterization of Sucrase-Isomaltase in Adult Human Colon and in Colonic Polyps JEAN-FRANCOIS LEMUEL

BEAULIEU,

HERRERA,

MILTON

and ANDREA

M. WEISER,

QUARONI

Division of Gastroenterology, Hepatology and Nutrition, Buffalo General Hospital, Buffalo, New York; Surgical Developmental Oncology, Roswell Park Memorial Institute, Buffalo, New York; and Section of Physiology, Cornell University, Ithaca, New York

A panel of monoclonal antibodies specific for sucraseisomaltase, but differing in their ability to stain the proliferative crypt cells in human jejunum, was used to investigate expression of this enzyme in adult human colon and colonic tumors. Immunofluorescence staining on cryostat sections demonstrated the presence of sucrase-isomaltase in the apical region of normal colonic crypt cells but not on surface epithelium. Colonic sucrase-isomaltase was purified by immu.noprecipitation with selected monoclonal antibodies and identified predominantly as high-mannose and complex glycosylated single-chain precursors endowed with relatively low levels of enzyme activities. Most polyps examined (10/16) were also found to express significant amounts of sucraseisomaltase. In contrast, only 3 of 45 adenocarcinomas were positive by immunofluorescence staining; no correlation was found between enzyme expression and tumor classification either by “Dukes” stage or degree of histological differentiation. These results demonstrate that colonic crypt cells and some benign tumor cells synthesize and express at their cell surface a form of sucrase-isomaltase immunologically distinct from that present in the brush borders of small intestinal villose cells.

S

ucrase-isomaltase (SI) is one of the best-characterized marker enzymes of the absorptive villose cells in adult small intestine. Its structure, biosynthesis, and intracellular processing have been investigated in great detail both in animal model systems and in humans [for recent reviews see Semenza (1,211. Several !physiological factors are known to regulate its expression by intestinal cells in vivo and in tissue culture. In rodents, SI protein and the corresponding messenger RNA are absent during fetal and early

postnatal life and become detectable in the small intestine only at the time of weaning (3-6). In contrast, in humans SI is expressed very early during fetal development, by 10 wk of gestation (7). At this stage, it is found exclusively in the form of the mature complex precursor (220 kilodaltons] and is expressed in the entire intestinal tract, including the colon (8-10). Later, by about 30 wk of gestation, extracellular cleavage of the precursor into the sucrase (S) and isomaltase (I) subunits is observed as a result of the maturation of pancreatic functions. In the colon, as the transient villi typical of the fetal stage gradually disappear, SI enzyme activities begin to decrease, reaching very low or negligible levels at term [see Semenza et al. (11) and M6nard (12) for recent reviews]. It is generally assumed that in adults only the differentiated absorptive villose cells in the small intestine express this digestive enzyme. However, recent findings by several groups have questioned this conclusion. Sucrase-isomaltase expression has been demonstrated in some primary colon tumors from patients (13) and in colonic tumor cell lines such as HT-29 and Caco-2 when allowed to differentiate in culture (14-17) or after inoculation in nude mice (18). In the HT-29 cells, synthesis and rapid intracellular degradation of SI have also been demonstrated in “undifferentiated” cells, which lack detectable enzymes at their cell surface (191. The presence of an enzymatically inactive SI on proliferative small intes-

Abbreviations used in this paper: cP, complex glycosylated sucrase-isomaltase precursor; hmP, high-mannose sucrase-isomaltase precursor; I, isomaltase; P, palatinase; PAGE, polyacrylamide gel electrophoresis; S, sucrase; SDS, sodium dodecyl sulfate: SI, sucrase-isomaltase. 0 1990 by the American Gastroenterological Association 0016-5085/90/$3.00

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tinal crypt cells in vivo has been suggested in both humans (20) and rabbits (21). We have recently demonstrated (22) that, in human jejunum, it represents a form of the enzyme conformationally distinct from that expressed by differentiated villose cells. Taken together, the above observations suggest that SI may also be synthesized at significant rates in cells and tissues that have very low or undetectable sucrase or palatinase. In the present study we have demonstrated the presence of a form of SI endowed with low levels of enzyme activity in normal adult colonic crypts and in most polyps examined. It was observed in only a small fraction of colonic adenocarcinomas, suggesting that in most cases SI expression is repressed with malignant transformation of colonic epithelial cells.

Materials and Methods Preparation

of Human Sucrase-Isomaltase

Monoclonal

Antibodies

Monoclonal antibodies specific for human SI (HSIseries antibodies) were prepared by the hybridoma technique as previously described (23-25). Human SI was purified from Triton X-100-solubilized jejunal brush-border membranes by affinity chromatography using antibody CaCo3/73, previously shown to be specific for the SI precursor produced by Caco-2 cells (171, coupled to cyanogen bromide activated Sepharose 4B beads (Pharmacia Fine Chemicals, Piscataway, N. J.). After extensive washing, the beads with bound antibody-antigen complexes were resussaline (PBS) mixed pended in 150 ~1 phosphate-buffered with 150 ~1 of complete (primary immunization) or incomplete (booster injections) Freund’s adjuvant (Difco Laboratory, Detroit, Mich.) and injected intraperitoneally into BALB/c mice (Charles River Breeding Laboratory, Wilmington, Mass.]. Each mouse received approximately 20-50 pg SI protein per injection. Three days after the last injection, spleen cells were obtained from the mouse exhibiting the highest serum titer and fused with NSI myeloma cells as previously described (25). Hybridomas were selected with hypoxanthine, aminopterin, thymidine-containing medium [ZS]and screened for antibody production by immunofluorescence staining of frozen sections of human jejunum. Cultures of interest were cloned twice by dilution plating in the presence of mitomycin C-treated 3T3 cells. Double-cloned hybridomas were used for antibody characterization and large-scale antibody production in ascites form.

Characterization

of Monoclonal

Antibodies

Hybridoma-conditioned media were used for determination of immunoglobulin subtype, which was performed with a Mouse Immunoglobulin Subtype Identification Kit (Boehringer Mannheim Biochemical, Indianapolis, Ind.] following the protocol suggested by the manufacturer. Monoclonal antibodies of immunoglobulin G class were purified from culture media and ascitic fluids by affinity chromatogra-

phy on a Protein A-CL Sepharose previously described (25).

4B

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column

Determination of the Antigen Specificity Human Sucrase-lsomaltase Antibodies

(27)

as

of

Affinity-purified monoclonal antibodies were bound to cyanogen bromide-activated Sepharose 4B and tested for their ability to bind Triton X-100-solubilized human jejunal brush-border enzymes (sucrase, maltase, lactase, aminopeptidase N, dipetidylpeptidase IV, alkaline phosphatase] as described previously (23,241. Proteins of brush-border membranes purified from human jejunum and from Caco-2 cells were labeled by reductive alkylation with [‘“Cl formaldehyde (New England Nuclear, Boston, Mass.] and sodium cyanoborohydride (17,24,25), solubilized with Triton X-100, and incubated with monoclonal antibodies bound to Sepharose 4B beads. The specifically bound antigens were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE] under reducing conditions and detected by fluorography [see below].

lmmunofluorescence

Staining

Segments of human jejunum, colon, and colonic polyps or tumors were rinsed with 0.155 M NaCI, cut into small fragments (0.5-l cm long], embedded in optimum cutting temperature (OCT) compound, and quickly frozen in liquid nitrogen. Sections 4-6 Iurn thick were spread on glass slides and allowed to dry at room temperature for at least 1 h, fixed with formaldehyde, and stained by the doubleantibody fluorescence technique as described previously (25). Late-confluent Caco-2 cells were rinsed 3 times with PBS then fixed and stained while still attached to the dishes (28). Monoclonal antibodies were used in the form of straight conditioned culture media or ascites fluids diluted 1:100-l: 1000 in PBS. Negative controls used in all experiments included fresh hybridoma culture medium, nonimmune mouse serum, and monoclonal antibodies to rat brushborder enzymes (24) known not to react with the corresponding human antigens.

Caco-2 Cell Culture The human colonic tumor cell line Caco-2 [15] was obtained from the American Type Culture Collection (Rockville, Md.) and grown in lOO-mm plastic petri dishes [Falcon, Becton, Dickinson Labware, Oxnard, Calif.) at 37°C in an atmosphere of 95% air and 5% CO, in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (M.A. Bioproducts; Walkersville, Md.), 50 U/ml penicillin, 50 Kg/ml streptomycin, and 4 mM glutamine. The cultures were refed twice weekly with 10 ml of fresh medium and subcultured serially when approximately 80% confluent. All experiments were performed with cells 11-15 days after confluence.

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SLJCRASE-ISOMALTASE IN THE HUMAN COLON 1469

Membrane

Purification

The luminal (brush-border) membrane of intestinal epithelial cells was purified from human jejunum and from Caco-2 cells by the method of Kessler et al. (29). Caco-2 cells were washed 3 times with PBS before they were suspended in buff#er by scraping with a rubber policeman and homogenized. A crude total cell membrane fraction was obtained from adult jejunum, adult colon, colonic polyps and tumors, 1%wk-old fetal intestine, and Caco-2 cells as follows. Tissue specimens and pelleted cultured cells were suspended in 2 mM Tris and 50 mM mannitol and homogenized in a Waring blender [Fisher Sci., Rochester, N.Y.) (tissue fragments] or a glass-Teflon Potter-Elvehjem homogenizer (Caco-2 cells]. A mixture of proteases inhibitors (1 mM phenylmethylsulfonyl fluoride, 50 wg/ml leupeptin, 50 pg/ml antipain, 0.1 mg/ml aprotinin) was added to all buffer and solutions used for homogenization and membrane purification. Homogenates were centrifuged at 2700 g for 10 min, and the supernatants obtainled were spun at 207,000 xga” for 30 min. The resulting pellets were designated “crude membrane fractions.” Protein concentrations were determined by the method of Lowry et al. (30). Brush-border membrane fractions were monitored for purification (31) by measuring the increase in specific activity of sucrase, which was 12-20 times higher than in the corresponding homogenates.

Gel Electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed according to the method of Thomas and K.ornberg (32) on 10% acrylamide gels. Detection of labeled proteins by fluorography was as previously described (31).

fluent Caco-2 cells (17); this antibody is of the IgGl subtype and was previously shown to be specific for the isomaltase subunit of SI. It was found to recognize all forms of SI present in human small intestine, with the exception of free sucrase subunit, and to react with SI-related polypeptides on immunoblots (22). Other monoclonal antibodies used as negative or positive controls in the course of this work [both positive and negative controls were included in all the immunofluorescence staining experiments described here) were CaCo3/28 (specific for human keratin and staining the epithelial cells in all samples of human small intestine, colon, and tumors), BBC1/35, and BB4/33 (specific, respectively, for rat SI and rat aminopeptidase, and not reacting with the corresponding human enzymes]. Their preparation and characterization have been described elsewhere (17.24).

Tissue

Specimens

Samples of normal intestinal mucosa, colonic polyps, and adenocarcinomas were obtained by biopsy or from patients undergoing surgical resection. Colonic mucosa immediately adjacent to carcinomas was referred to as “transitional” mucosa. The specimens were obtained at the Buffalo General Hospital [Division of Gastroenterology, Hepatology and Nutrition] and at the Roswell Park Memorial Institute (Surgical Developmental Oncology] according to protocols approved by the corresponding human research review committees. Specimens to be analyzed by immunofluorescence staining were immediately embedded in OCT compound and frozen in liquid nitrogen. Larger specimens to be used for antigen immunoprecipitation and analysis were frozen unfixed on dry ice and stored at -70°C until they were used.

Results lmmunoblotting Proteins from SDS gels were electrophoretically transferred to nitrocellulose membranes (BioRad, Richmond, Calif.) according to the method of Towbin et al. (33). Following transfer, proteins and molecular weight markers were localized by staining with Ponceau red (34). Membrane.s were blocked in PBS containing 5% defatted dry milk [Blotto, see Johnson et al. (3511overnight and incubated with monoclonal antibodies (ascites fluids diluted 1:500 in DME containing 20% FCS) for 4 h at room temperature. After washing in PBS containing 0.05% Tween 20, the membranes were incubated with alkaline phosphataseconjugated goat anti-mouse IgG (H + L) antibody (Promega Biotec, Madison, Wis.) diluted I:7500 in PBS containing 0.2% bovine serum albumin [BSA), further washed in PBS-Tween, and incubated with freshly prepared NBT/ BCIP substrate for alkaline phosphatase detection (Protoblot System; Promega Biotec, Madison, Wis.]. Finally, the blots were rinsed with water, photographed, and air dried.

Other

Monoclonal

Antibodies

Used

Antibody CaCo3/73 was prepared from a mouse immunized with luminal membranes purified from con-

Antigen Specificity Sucrase-Isomaltase

of Human Monoclonal

Antibodies and Their Reactivity With Sucrase Isomaltase From Human lejunum and Caco-2 Cells

The monoclonal antibodies used in this study are listed in Table 1. They produced two markedly different patterns of immunofluorescent staining of the adult human j ejunal mucosa. Antibody HSI-5 was specific for the luminal membrane of the absorptive villus cells (Figure lB), while the others also stained, with different intensities, the crypt cells [Figure 1C). All antibodies stained the apical surface membrane of differentiated Caco-2 cells, which are typically present as islands or patches in postconfluent cultures (Figure IE and F]. In spite of the different staining patterns illustrated in Figure 1, all 5 HSI antibodies were found to share an absolute specificity for SI from both human jejunum and Caco-2 cells. This was demonstrated by measuring various brush-border-associated enzyme activities in the immunoprecipitates and by SDSPAGE electrophoretic analysis of antigens precipi-

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Table 1. Properties

Antibody

and Antigen Specificity of Human Sucrose-lsomaltase

Immunoglobulin subtype

HSI-3

IgGl

HSI-5 HSI-6 HSI-9 HSI-12

IgGl IgGl IgG2b IgGl

Immunofluorescence pattern” Jejunum

CaCo-2

Antibodies

Enzyme activityb

Figure 1C

Figure 1F

S,P

Figure Figure Figure Figure

Figure Figure Figure Figure

S.P S,P S,P S,P

1B lBd 1C 1C

1E 1E 1F 1F

Vol. 98. No. 6

Antigen band on SDS-PAGE” Jejunum

Caco-2 cells

225,148,134 225,148,134 225,148,134 225,148,134 225.148.134

212 212 212 212 212

“Immunofluorescence staining of frozen sections of adult jejunum and Caco-2 cells, fixed with 1% formaldehyde and stained by indirect immunofluorescence as described in Materials and Methods. The fluorescence patterns obtained with the different antibodies either are presented in Figure 1 or were found to be similar to the ones included in Figure 1. ‘Triton X-loo-solubilized membrane proteins, obtained from adult human jejunum or colon, were incubated with monoclonal antibodies bound to Sepharose 4B. Specifically bound antigens were tested for sucrase. palatinase. maltase, lactase, aminopeptidase, dipeptidylpeptidase, and alkaline phosphatase activities as described in Materials and Methods; only the indicated activities could be detected in the immunoprecipitates. “Apparent molecular masses of proteins subunits determined by SDS-PAGE under reducing conditions; these data were obtained from the gels included in Figure 2. dA weak staining of the apical portion of the crypt cells was observed with this antibody in human jejunum.

tated from [‘“Cl formaldehyde-labeled, Triton X-lOOsolubilized, brush-border membrane fractions from human jejunum and confluent Caco-2 cells. Only sucrase and palatinase [palatinose was used as a specific substrate for isomaltase; see Semanza (l)] activities were detected in the immunoprecipitates (Table 11. All five antibodies selectively precipitated, from jejunal brush borders, the single-chain complex glycosylated SI precursor (cP) (36) and the I and S subunits (Figure 2A). From Caco-2 luminal membranes (Figure 2B), the same antibodies precipitated only one protein corresponding to the single-chain precursor of SI, which has a slightly higher mobility or SDS-PAGE than the one produced by adult villose cells in vivo and is not split into S and I subunits by cultured cells (16,171. The five HSI antibodies were equally effective in precipitating, with a single incubation, >90% of the total sucrase and palatinase activities present in the solubilized brush-border membrane fractions tested; they produced antigen bands of nearly identical intensities on the fluorograms illustrated in Figure 2.

Detection and Characterization Sucrase-Isomaltase in Normal Mucosa

of Adult Colonic

In a separate study (22), we have demonstrated the presence of conformationally distinct forms of SI in crypt and villose cells of adult jejunum, accounting for marked differences observed in the ability of the various HSI antibodies to stain the proliferative crypt cells. The previously reported expression of this enzyme in large intestinal tumors (18) and cultured cell lines (14,15) prompted us to investigate the possibility that a previously undetected form of SI might also be present in normal adult colon. Indirect immunofluorescent staining with some of

our HSI antibodies demonstrated a specific reaction at the apical portion of the colonic epithelial cells located in the crypts (Figure 3). Only antibodies which had also been found to stain the crypt cells in human jejunum either moderately (HSI-6) or intensely (HSI3, HSI-9, and HSI-1.2; see Figure 1C) produced, respectively, a weak (HSI-6; see Figure 3B) and a strong (HSI-3, HSI-9, and HSI-12; Figure 3C) reaction in the colon. The entire colonic epithelium was negative with HSI-5 (Figure 3A), an antibody exclusively recognizing the form of SI expressed by differentiated jejunal villose cells (Figure lB]. All of the HSI antibodies failed to stain the surface epithelium of the colon significantly (see the upper right sections of Figure 3B and C]. The antigen detected by immunofluorescence staining in the colonic crypts by some of the HSI antibodies was purified and characterized by enzymatic and gel electrophoretic/immunoblotting analysis. Because previous studies (18) using similar techniques had failed to detect the presence of SI in normal human colon, a preliminary immunopurification step was used to purify the antigen from relatively large amounts of tissue and therefore improve the sensitivity of the analytical methods used. A crude total membrane fraction was obtained from specimens (0.5-4 g wet weight] of normal adult colon as described in the “Materials and Methods” section. Membrane proteins were solubilized with nonionic detergent (1% Triton X-100) and then incubated with the HSI-9 antibody covalently bound to Sepharose 4B beads. Both sucrase and palatinase activities were found to be associated with the washed beads, but at levels much lower (25-50-fold) that those present in comparable amounts of antigen purified from small intestinal specimens (the amount of antigen bound to the beads was estimated by SDS-PAGE analysis of eluted proteins,

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Figure 1. Immunofluorescence staining of adult human jejunum (A-C) and confluent Caco-2 cells (D-F) with HSI monoclonal antibodies. Frozen sections (&&pm thick] of jejunum were ilxed with 1% formaldehyde and stained by indirect immunofluorescence. Antibodies used were (A) negative control (unused culture medium), (S) HSI-5, and (C) HSI-9. Arrows point to the bottom of the crypts. A staining pattern identical to that shown in C was obtained with antibodies HSI-3 and HSI-12. Caco-2 cells, 29 days after reaching confluency, were fixed while still attached to their culture dishes, and stained with antibodies: (D)negative control (a monoclonal antibody not reacting with these cells); (0 HSI-5, and (F)HSI-9 (all figures, original magniflcatlon x 135; bar = 100 am).

followed by densitometric scanning of the Coomassie bluestained gels). The antigen purified from colonic specimens with HSI-9Sepharose 4B beads was further eluted by heating the beads at 100°C in a buffer containing 1% SDS and 50 mM dithiothreitol, separated by SDSPAGE, and electrophoretically transferred to nitrocellulose membranes. Sucrase-isomaltase-related polypeptifdes, including high-mannose and complex glycosylated forms of the single chain precursor and cleaved subunits (16,36) were detected by incubation of the blots with a mixture of HSI-9 and Caco3/73

antibodies, which were found to produce the strongest signals on immunoblots. These two antibodies were found to differ in their subunit specificity (HSI-9 is specific for sucrase, CaCo3/73 for isomaltase); thus when used in combination they recognized all molecular forms of SI on immunoblots. As demonstrated in Figure 4 (lane C), HIS-9 precipitated, from detergentsolubilized colonic cell membranes, the mature SI precursor (cP), the cotranslationally glycosylated form of the SI precursor (high-mannose form, hmP), and some I subunit. The identity of the colonic CP and hmP forms of SI was confirmed by endoglycosidase H

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1

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1

23456

Figure 2. Immunoprecipitation and SDS-PAGE analysis of antigens recognized by HSI monoclonal antibodies. [“Cl-labeled, Triton X-loo-solubilized brush-border (luminal) membrane proteins were incubated with HSI antibodies bound to Sepharose 4B beads. Specifically bound, labeled membrane proteins were analyzed by SDS-PAGE under reducing conditions (50 mM DTI’), and visualized by fluorography. A. Membrane proteins isolated from adult human jejunum. Lane 1 shows total [“Qlabeled brush-border membrane proteins. Lanes z-6 correspond to proteins specifically bound to antibodies HSI-3, -5, -6, -9 and -12, respectively. Arrows point to the position of the different SI forms: cP, I subunit, and S subunit. B. Membrane proteins and antigens from confluent Caco-2 cells. Lane 1 shows total [‘%I-labeled luminal membrane proteins. Lanes 2-6 correspond to proteins specifically bound to HSI-3, -5, -6, -9, and -12, respectively. Molecular weight markers indicated (in thousands) at the left margin were, respectively, myosin, 200 kilodaltons; phosphorylase B, 97.4 kilodaltons: and BSA, 69 kilodaltons.

digestion (36) of the immunoprecipitated proteins, which altered the mobility of the hmP band only (data not shown). For comparison, Figure 4 also shows the forms of SI immunoprecipitated, with the same HSI-9 antibody, from adult human jejunum [lane J] and confluent Caco-2 cells (lane Ca). Significant differences were observed among the forms of enzyme detected in these different tissues. In human jejunum, the cleaved S and I subunits were prevalent, together with a substantial amount of cP; the hmP could be barely detected. The SI from Caco-2 cells was present exclusively, or predominantly, as unsplit single-chain

precursor (cP). However, the hmPs detected in all of the above tissues and cells displayed identical mobilities on SDS-PAGE. This was expected because it represents the initial co-translationally glycosylated form of SI. The apparent molecular masses of the different forms of SI present in Figure 4 are summarized in Table 2. As previously reported (16,17), the CP produced by Caco-2 cells had a smaller apparent molecular weight than that found in all other samples, probably because of differences in composition and/ or number of the carbohydrate chains. Overall, the SI isolated from normal adult colon

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Figure 5.. Immunofluorescence staining of normal adult human colon (A-C), a polyp (D), and two different adenocarcinomas (E,FJwith HSI monoclonal antibodies. Frozen sections (&S-pm thick) of tissues were 6xed with 1% formaldehyde and stained by indirect immunofluorescence. Antibodies used were (A) HSI-5, (B) HSI-6, and (C-F) HSI-6; staining patterns identical to those shown in (C-F) were also observed with antibodies HSI-3 and HSI-12. SE, surface epithelium. (A,E, and F, original magni5cation x 135; B,C, and D, original magnification x 270; borrs = 100 pm).

exhibited

a distinct

electrophoretic

pattern,

excluding

that it could be derived from small intestinal enterocytes potentially present in the colonic lumen: the CP had a rslightly higher apparent molecular weight than that isolated from the jejunum, the CP and hmP were present in about equal amounts in colonic SI, and little or no S subunit could be detected in the colon.

Expression

of Sucrase-Isomaltase

in Human

Colonic Tumors All specimens of normal colon and transitional mucosa examined were positive for SI expression using the HSI-3, HSI-9, or HSI-12 antibodies in indirect immunofluorescent staining of cryostat sections

GASTROENTEROLOGY Vol. 98. No. 6

1474 BEALJLIEU ET AL.

J

C

Ca

Table 2. Apparent Molecular Masses (kilodaltons) of Sucrase-Isomaltase Forms Isolated From Adult Jejunum, Adult Colon, Fetal Intestine, and Caco-2 Cells” Tissue Adult jejunum Adult colon Caco-2 cells

@-I

hmP=

IS-

Figure 4. Purification and analysis of SI from crude membrane fractions obtained from adult human jejunum 0, adult human colon (C), and late confluent Caco-2 cells (Ca). T&on X-lOOsolubilized membrane proteins were incubated with the HSI-9 antibody bound to Sepharose 4B beads. Specifically bound antigens were solubilized with sample solution at loo%, separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. Sucrase-isomaltase forms were detected on the nitrocellulose membrane by incubation with a mixture of HSI-9 and CaCo3/73 antibodies, followed by anti-mouse IgG conjugated to alkaline phosphatase, and developed with freshly prepared NBT/BCIP substrate for alkaline phosphatase detection (Protoblot System; Promega Biotec).

(Figure 3, Table 3). As described above, staining was in most cases confined to the region of the crypts, but also extended to the surface epithelium in some samples of mucosa adjacent to tumors. Three of the 12 specimens of normal rectum and cecum examined were negative. Sixty-two percent of the adenomas were found to express SI; the fluorescence was confined to the surface membrane of the epithelial cells [Figure 30) and varied markedly in intensity in different areas of each section. In contrast with previously published reports (13,181, only 3 of the 45 adenocarcinomas examined appeared to express SI (Table 3), and in all cases the fluorescence was confined to very small regions (composing tl% of the tumor cells] of the tumors. No correlation was found between degrees of invasiveness or histological differentiation of the tu-

CP

hmP

I

225

205 205 205

148 150 NE

232 212

S 134 (1381b NE

“Total membrane proteins, obtained from the indicated tissues, were solubilized with 1% Triton X-100 and incubated with the HSI-9 antibody bound to Sepharose 4B. Specifically, bound antigens were separated by SDS-PAGE under reducing conditions (50 mM DTT), electrophoretically transferred to nitrocellulose membrane, and stained for the detection of SI-immunoreactive proteins with HSI-9 and CaCo3/73 antibodies as described in legend to Figure 4. Molecular masses [in thousands) for SI forms were calculated from molecular weight marker proteins (NEN methylated [“Cl-labeled high-molecular weight market kit]. bFree S subunit was present in very small amounts in some colonic samples. NE, not expressed. mors and SI expression, although this information is of limited significance given the overall small number of positive samples observed. The above results were confirmed by immunoprecipitation and biochemical analysis of SI from selected samples of adenomas and adenocarcinomas. Crude total membrane fractions from each specimen were solubilized with Triton X-100 and incubated with HSI-9 antibody coupled to Sepharose 4B as described above for samples of normal colonic mucosa. The Table 3. Expression

of Sucrase-Isomaltase

in Human

Colon Specimens” No. of positive specimens/ samples tested Tissue Normal colon 46/46 9/12 Normal rectum and cecum Adenomas/polyps Colon B/12 Rectum Z/4 Adenocarcinomas Colon 4/50 o/11 Rectum Classification of tumors by Dukes stage Stage A l/3 Stage Bl l/l2 Stage B2 o/9 Stage Cl l/7 Stage C2 o/5 Stage D o/9 Classification of tumors by degree of histological differentiation Well differentiated l/12 Moderately well differentiated Z/23 Poorly differentiated O/8 ‘Tested as cryosections by immunofluorescence staining with antibodies HSI-3, HSI-9. and HSI-12 as described in the legend to Figure 3; intensity of fluorescence and fraction of stained epithelial cells varied markedly among positive specimens.

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c

12345

” ..

cP4

hmP-

Figure ti. Immunopurification and analysis of SI from crude membrane fractions obtained from adult human jejunum a adult normal human colon (C), two different polyps (I,& and three different adenocarcinomas (9-g). Antigens were immunopurified with antibody HSI-9 and analyzed by immunoblotting as described in the legend to Figure 4.

immunoprecipitated SI was separated by SDS-PAGE and analyzed by immunoblotting using a mixture of HSI-9 and CaCo3/73 antibodies. Representative results obtained are presented in Figure 5. The SI purified from the two adenomas (Figure 5, lanes 1 and 2). which had also been positive by immunofluorescence staining, had an electrophoretic pattern similar or identical to that of normal colonic SI (lane C of Figure 5). No SI could be detected in the three different adenocarcinoma specimens examined (Figure 5, lanes 3-5).

Discussion In this study, expression of SI has been demonstrated in adult colonic crypt cells. This finding has important implications with respect to both the program of differentiation of normal colonocytes and a posttranslational level of control of SI expression in intestinal epithelial cells. The form of the enzyme identified in the colon was biochemically and immunologically distinct from that predominantly expressed by absorptive villose cells in the small intestine.

IN THE HUMAN

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1475

First, only monoclonal antibodies that were also found to stain the proliferative crypt cells in the j ejunum reacted with the colonic form of SI. Antibody H SI-5 which is specific for an epitope exclusively associated with the fully enzymatically active SI expressed by the small intestinal absorptive villose cells (22), failed to stain any of the colonic specimens examined or to immunoprecipitate SI from colonic mucosa. This observation probably accounts for the discrepancy between the results obtained in our study and previous investigations that failed to detect SI in many samples of normal adult colon using a different panel of monoclonal antibodies (13,18). Second, the colonic form of SI was predominantly identified as uncleaved hmP and cP, present in about equal amounts, and had a low level of enzyme activities, like the hmP purified from the small intestine (37). This suggests that a major fraction of colonic SI may be present inside the cells (possibly in the endoplasmic reticulum or the Golgi complex], accounting for the diffuse fluorescence detected in the apical cytoplasm of colonic crypt cells [see Figure 3B and C). These findings could be explained by either a slow rate of transport among membrane compartments and/or to the cell surface or a high degree of intracellular storage and degradation, similar to that demonstrated in many cases of congenital SI deficiency (38,39). The difference in apparent molecular weight between the complex precursors purified from small intestine and colon [Figure 4, lanes J and C) can be ascribed to differences in structure or composition of their carbohydrate chains because the cotranslationally glycosylated hmPs had, in all cases, identical mobilities on gels. The presence of a small amount of free I subunit in the colon, with little or no free S, suggests that degradation of SI inserted into the surface membrane of the colonic crypt cells is mediated by extracellular proteases and, like in the small intestine, produces an excess of free I subunits (40). Conceivably, the free S subunit generated in this process may be more rapidly degraded in the colon than in the small intestine, explaining why it was not detected in many samples of normal colonic mucosa or polyps we have examined. Overall, these results suggest that it is very unlikely that a tissue-specific colonic SI gene exists, also in accordance with previous studies that have failed to identify more than 1 species of messenger RNA coding for SI in the intestines of different animals and humans (6,41-44). The lack of staining, with all monoclonal antibodies tested, of the surface epithelium in the normal colon suggests that the SI present in this region of the intestinal tract does not contribute significantly to the overall process of carbohydrate digestion. Rather, its expression in the colonic crypts may represent a transient phenomenon restricted to proliferative, mi-

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grating cells before they reach a fully differentiated state in the upper regions of the crypts (45,46). Alternatively, SI degradation may be much more rapid in the surface epithelial cells, producing a steady-state level of protein undetectable by immunofluorescence staining. This has been demonstrated to be the case for HT-29 cells cultured in the presence of glucose (19). In a separate study (221, we have demonstrated that a form of SI immunologically related to the one we have now detected in adult colon is synthesized and expressed at the apical cell surface by small intestinal crypt cells. A detailed investigation of its structure and transport from endoplasmic reticulum to Golgi to the cell surface indicated that processing of the cotranslationally glycosylated hmP and its conversion into a distinct conformational species are dependent on the state of differentiation of the enterocytes (22). Therefore, expression of SI in adult human intestine may be primarily regulated at the posttranslational level, in contrast with the transcriptional level of regulation demonstrated during prenatal and postnatal development of intestinal functions (6,441. A marked similarity in biological structure and functions between small and large intestine has been well documented at early stages of human fetal development (47-491, when both display a villose architecture and express the same brush-border enzymes, including SI (4,13,18]. This study suggests that such a similarity may extend to the proliferative crypts cells in the colon and small intestine after birth. Our investigation of a relatively large panel of colonic polyps and adenocarcinomas indicated that SI does not represent a reliable marker for tumor intestinal cells. Rather, its expression appeared to be repressed in the process of malignant transformation, being relatively high in many samples of benign polyps and very limited or absent in most adenocarcinomas examined. However, this does not imply a suppression of SI gene expression in tumor cells: as discussed above, other mechanisms, such as alterations in the intracellular transport of the enzyme or increased degradation, may be involved.

References 1. Semenza G. Intestinal oligo- and disaccharidases. In: Randle PJ, Steiner DF, Whelan WJ, eds. Carbohydrate metabolism and its disorders. London: Academic Press, 1981:691-706. Semenza G. Anchoring and biosynthesis of stalked brush border membrane proteins: glycosidases and peptidases of enterocytes and renal tubuli. Annu Rev Cell Biol1986;2:255-314. Doe11 RG, Rosen G. Kretchmer N. Immunochemical studies of intestinal disaccharidases during normal and precocious development. Proc Nat1 Acad Sci USA 1965;54:1268-1273. Koldovsky 0. Development of the functions of the small intestine in mammals and man. Basel: Karger, 1969. Henning SJ. Ontogeny of enzymes in the small intestine. Annu Rev Physiol 1985:47:231-245.

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6 Sebastio G, Hunziker W, Ballabio A, Auricchio S. Semenza G. On the primary site of control in the spontaneous development of small intestinal sucrase-isomaltase after birth. FEBS Lett 1986;208:460-464. 7. Grand RJ, Watkins JB, Torti FM. Development of the gastrointestinal tract. Gastroenterology 1976;70:790-810. 8. Skovbjerg H. High-molecular weight pro-sucrase-isomaltase in human fetal intestine. Pediatr Res 1982;16:948-949, 9. Triadou N, Zweibaum A. Maturation of sucrase-isomaltase complex in human fetal small and large intestine during gestation. Pediatr Res 1985;19:136-138, 10. Menard D, Pothier P. Differential distribution of digestive enzyme in isolated epithelial cells form developing human small intestine and colon. J Pediatr Gastroenterol Nutr 1987;6: 509-516. 11. Semenza G, Sebastio G, Hunziker W, Malo C, Menard D, Auricchio S. Biosynthesis of human small intestinal sucraseisomaltase. In: Lentze MJ, Sterchi EE, eds. Brush border membranes Stuttgart: Thieme Verlag, 1988:43-51. 12. Menard D. Growth promoting factors and the development of the human gut. In: Lebenthal E, ed. Human gastrointestinal development. New York: Raven, 1989:123-149. 13. Zweibaum A, Hauri HP, Sterchi E, Chantret I, Haffen K, Bamat J, Sordat B. Immunohistological evidence, obtained with monoclonal antibodies, of small intestinal brush border hydrolases in human colon cancer and foetal colons. Int J Cancer 1984;34:591598. 14. Pinto M, Appay MD, Simon-Assmann P, Chevalier G. Dracopoli N, Fogh J, Zweibaum A. Enterocytic differentiation of cultured human colon cancer cells by replacement of glucose by galactose in the medium. Biol Cell 1982;44:193-196. 15. Pinto M, Robine-Leon S, Appay MD, Kedinger M, Triadou N, Dussaulx E, Lacroix B, Simon-Assmann P, Haffen K, Fogh J, Zweibaum A. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol Cell 1983;47:323-330. 16 Hauri HP, Sterchi EE, Bienz D, Fransen JAM, Marxer A. Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells. J Cell Biol 1985;101:838-851, 17. Quaroni A. Crypt cell antigen expression in human tumor colonic cell lines. Analysis with a panel of monoclonal antibodies to CaCo-2 luminal membrane components. JNCI 1986;76:571585. 18. Zweibaum A, Triadou N, Kedinger M, Augeron C, Robine-Leon J. Pinto M, Rousset M, Haffen K. Sucrase-isomaltase: a marker of foetal and malignant epithelial cells of the human colon. Int J Cancer 1983;32:407-412. 19. Trugnan G, Rousset M, Chantret I, Barbat A, Zweibaum A. The posttranslational processing of sucrase-isomaltase in HT-29 cells is a function of their state of enterocytic differentiation. J Cell Biol 1987;104:1199-1205. 20. Silverblatt ER, Conklin K, Gray GM. Sucrase precursor in human jejunal crypts (abstr). J Clin Invest 1974;53:76a. 21. Dubs R, Gitzelmann R, Steinman B, Lindenmann J. Catalytically inactive sucrase antigen of rabbit small intestine: the enzyme precursor. Helv Paediatr Acta 1975;30:89-102. 22. Beaulieu JF, Nichols B, Quaroni A. Post-translational regulation of sucrase-isomaltase expression in intestinal crypt and villus cells. J Biol Chem 1989;264:20000-20011. 23 Hauri HP, Quaroni A, Isselbacher KJ. Monoclonal antibodies to sucrase-isomaltase: probes for the study of postnatal development and biogenesis of the intestinal microvillus membrane. Proc Nat1 Acad Sci USA 1980;77:6629-6633. 24. Quaroni A, Isselbacher KJ. Study of intestinal cell differentiation with monoclonal antibodies to intestinal cell surface components. Dev Biol 1985:111:267-279.

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Received August 31,1989. Accepted November 20,1989. Address requests for reprints to: Andrea Quaroni, Ph.D., Section of Physiology, 820 Veterinary Research Tower, Cornell University, Ithaca, New York 14853. This study was supported by grant no. DK-32656 from the National Institutes of Health, U.S. Public Health Service. Dr. Beaulieu was the recipient of a postdoctoral fellowship from the Fonds de la recherche en Sante’ du Quebec. Dr. Beaulieu’s present address is: Dep. anatomie et biologie cellulaire, Faculte’de Medicine, Universite’ de Sherbrooke, Sherbrooke, Quebec, Canada JlH 5N4.