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Immunohistochemical Localization of CCK₁ Cholecystokinin Receptors in Normal and Neoplastic Human Tissues

Departments of Pharmacology and Toxicology (St.S.), Pathology (C.R.), Neuropathology (C.M.), Obstetrics and Gynecology (So.S.), Otto-von-Guericke University, 39120 Magdeburg, Germany

Address all correspondence and requests for reprints to: Stefan Schulz, Department of Pharmacology and Toxicology, Leipziger Strasse 44, 39120 Magdeburg, Germany. E-mail: Stefan.Schulz{at}Medizin.Uni-Magdeburg.de.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Background: The biological effects of cholecystokinin (CCK) are mediated by two distinct G protein-coupled receptors, CCK₁ and CCK₂. Although it is well established that CCK receptors are widely distributed throughout the normal gastrointestinal tract, little is known about their cellular and subcellular localization in human normal and neoplastic tissues.

Methods: We developed and characterized a novel antipeptide antibody to the carboxyl-terminal region of the human CCK₁ receptor. Specificity of the antiserum was demonstrated by 1) detection of a broad band migrating at a relative molecular mass of 85,000–95,000 in Western blots of membranes from CCK₁-expressing tumors and CCK₁-transfected cells, 2) cell surface staining of CCK₁-transfected cells, 3) translocation of CCK₁ receptor immunostaining after agonist exposure, and 4) abolition of tissue immunostaining by preadsorption of the antibody with its immunizing peptide. The distribution of CCK₁ receptors was investigated in 74 human tumors and their tissues of origin.

Results: The presence of CCK₁ receptors was rarely detected in human tumors except for carcinoids, insulinomas, pituitary adenomas, and meningiomas. CCK₁ receptors were clearly located at the plasma membrane and uniformly present on nearly all tumor cells. In the gastrointestinal tract, CCK₁ receptor immunoreactivity was highly abundant in chief cells of the gastric mucosa, in myenteric ganglion cells, and in myenteric nerve fibers.

Conclusion: This is the first localization of CCK₁ receptors in human formalin-fixed, paraffin-embedded tissues at the cellular level. The overexpression of CCK₁ receptors in a subset of human neuroendocrine tumors may provide a molecular basis for efficient targeting of these tumors with radiolabeled CCK analogs.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
CHOLECYSTOKININ (CCK) AND gastrin are regulatory peptide hormones with multiple functions in the gastrointestinal tract and the brain (1). In the gastrointestinal tract, CCK and gastrin act as regulators of gastric acid secretion, gastric emptying, and gall bladder motility (1). In addition, they act as physiological growth factors in most parts of the gastrointestinal tract (2, 3, 4). CCK and gastrin have also been shown to stimulate growth of several neoplasms such as colonic, gastric, and brain tumors (5, 6, 7). The biological effects of CCK are mediated by two distinct G protein-coupled receptors, CCK₁ (formerly CCK-A) and CCK₂ (formerly CCK-B), which can be distinguished pharmacologically by their low affinity (CCK₁) vs. high affinity (CCK₂) for gastrin (8, 9). The CCK₁ receptor is highly selective for sulfated CCK molecules, whereas the CCK₂ receptor has similar high affinities for sulfated and nonsulfated peptides containing the carboxyl-terminal domain of both gastrin and CCK (8, 9).

CCK₁ and CCK₂ have been identified in several normal tissues. CCK₂ receptors are present predominantly in the gut mucosa, in the endocrine pancreas, and in the brain; CCK₁ receptors are present in the gall bladder, in gastric smooth muscle cells and mucosa, in myenteric neurons, and in the brain (10, 11, 12, 13, 14). In addition, important species differences have been noted, e.g. human pancreatic acinar cells do not express significant levels of CCK receptors in contrast to rat pancreatic acinar cells (15). The presence of CCK receptors in human tumors has also been reported (16, 17). It has been established for a long time that small-cell lung cancers often express CCK₂ receptors, whereas non-small-cell lung cancers do not (18, 19). However, contradictory findings have been reported regarding the presence of CCK receptors in carcinomas of colon and stomach. Whereas some studies have reported the presence of CCK receptor mRNAs (20, 21), other investigations have failed to detect high-affinity CCK binding sites in most of these tumors (16). The same may be true for exocrine pancreatic carcinomas; although CCK₁ and CCK₂ receptor mRNAs were identified in most tumors (22, 23), CCK binding sites were difficult to detect on the tumor cells (24).

Despite the large number of studies describing various CCK effects on gastrointestinal functions via the activation of CCK₁ receptors, the cellular sites of CCK₁ receptors in human normal and neoplastic tissues still need to be fully elucidated (1). The expression of CCK₁ receptors has previously been detected using binding autoradiography or RT-PCR. However, the anatomic resolution of autoradiographic receptor binding is limited, and RT-PCR does not discriminate between receptor transcripts originating from individual target cells. Further morphological examination of the cellular and subcellular sites of CCK₁ expression in human tissues has been hampered by the lack of specific antibodies for immunohistochemical detection of human CCK₁ receptor proteins.

In the present study, we have generated and characterized antibodies directed to the carboxyl-terminal tail of the CCK₁ receptor. We have also developed an immunohistochemical protocol that allows efficient detection of this receptor in formalin-fixed, paraffin-embedded human tissues. The generation of this novel antibody enabled us to determine the cellular distribution of CCK₁ receptor proteins in a variety of human tumors and their tissues of origin.

	Patients and Methods

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Patients, tumors, and tissue preparation

Seventy-four tumor specimens were retrieved from the archives of the Departments of Pathology and Neuropathology. All tissue specimens had been fixed in formalin and embedded in paraffin. The following specimens were investigated: colorectal adenocarcinoma (n = 5), ductal pancreatic adenocarcinoma (n = 5), breast carcinoma (n = 5), ovarian carcinoma (n = 10), prostate cancer (n = 4), thyroid carcinoma (n = 6), carcinoid (n = 15), pancreatic insulinoma (n = 8), normal pituitary (n = 4), pituitary adenoma (n = 4), pheochromocytoma (n = 4), glioblastoma (n = 4), and meningioma (n = 4). Several of the neuroendocrine tumors contained adjacent normal tissue that was also analyzed. In addition, several tumor specimens were obtained immediately after surgical resection, frozen in liquid N₂, and stored at –70 C until Western blot analysis. The following tumors were investigated: colorectal carcinoma (n = 6), breast carcinoma (n = 8), ovarian carcinoma (n = 8), thyroid carcinoma (n = 4), carcinoid (n = 4), and glioblastoma (n = 8).

Generation and purification of antipeptide antibodies

Polyclonal antisera were generated against the carboxyl-terminal tails of the CCK receptor subtypes CCK₁ and CCK₂. The identity of the peptides is given in Table 1. Peptides were synthesized, purified, and coupled to keyhole limpet hemocyanin as described (25, 26). The conjugates were mixed 1:1 with Freund’s adjuvant and injected into groups of three rabbits each; 9028–9030 for CCK₁ and 9031–9033 for CCK₂ antisera production. Animals were injected at 4-wk intervals, and serum was obtained 2 wk after immunizations beginning with the second injection. The specificity of the antisera as well as possible cross-reactivity with other CCK receptor subtypes was initially tested using immuno-dot blot analysis as described (25). For subsequent analysis, antibodies were affinity purified against their immunizing peptides using the Sulfo-Link coupling gel according to the instructions of the manufacturer (Pierce, Rockford, IL).

Plasmids encoding CCK₁ or CCK₂ were kindly provided by Dr. C. Seva (Toulouse, France). Human embryonic kidney 293 (HEK-293) cells were stably transfected with either CCK₁ or CCK₂. Cells were grown on coverslips overnight and either not exposed or exposed to 1 µM sulfated CCK octapeptide (sCCK-8) (Bachem, Weil am Rhein, Germany). Cells were then fixed and incubated with 1 µg/ml anti-CCK₁ (9030) or anti-CCK₂ (9032) antibodies followed by cyanin 3.18-conjugated secondary antibodies (Amersham, Braunschweig, Germany). Specimens were mounted and examined using a Leica TCS-NT laser scanning confocal microscope as described (27).

Western blot analysis

Membranes were prepared from stably transfected HEK-293 as well as native tumor specimens. Cells and tissues were lysed in homogenization buffer (5 mM EDTA, 3 mM EGTA, 250 mM sucrose, 10 mM Tris-HCl, pH 7.6, containing 1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 10 µg/ml leupeptin, and 2 µg/ml aprotinin), and membranes were pelleted at 20,000 x g for 30 min at 4 C. Membranes were then dissolved in lysis buffer (150 mM NaCl, 5 mM EDTA, 3 mM EGTA, 20 mM HEPES (pH 7.4) containing 4 mg/ml dodecyl-ß-maltoside and proteinase inhibitors as above) and incubated with 150 µl wheat germ lectin agarose beads (Amersham) for 90 min at 4 C. Beads were washed five times in lysis buffer, and adsorbed glycoproteins were eluted with SDS-sample buffer for 20 min at 60 C. Samples were then subjected to 8% SDS-PAGE and immunoblotted onto nitrocellulose. Blots were incubated with 1 µg/ml anti-CCK₁ (9030) or anti-CCK₂ (9032) antibodies followed by peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection (Amersham). For adsorption controls, antisera were preincubated with 10 µg/ml of their cognate peptides for 2 h at room temperature.

Immunohistochemistry

Seven-micrometer paraffin sections were cut and floated onto positively charged slides and immunohistochemically stained as described (26, 28). Briefly, sections were dewaxed, microwaved in 10 mM citric acid (pH 6.0) for 20 min at 600 W, and subsequently incubated with 2 µg/ml anti-CCK₁ (9030) antibodies overnight at 4 C. Staining of primary antibody was detected using biotinylated goat antirabbit IgG followed by an incubation with avidin-biotinylated peroxidase solution (Vector, Burlingame, CA). Tissue was then rinsed and stained with 3,3'-diaminobenzidine-glucose oxidase for 15 min. Cell nuclei were lightly counterstained with hematoxylin. For immunohistochemical controls, the primary antibody was omitted, replaced by preimmune sera, or adsorbed with several concentrations ranging from 1–10 µg/ml of homologous or heterologous peptides for 2 h at room temperature. A tumor known to stain positively was included in each batch of staining as a positive control.

Assessment of staining patterns

All slides were evaluated by the same investigator. The presence or absence of staining and the depth of color were noted as well as the number of cells showing a positive reaction and whether or not the staining was localized to the plasma membrane. Tumors were categorized as positive only when they exhibited a moderate to strong plasma membrane and/or cytoplasmic staining in the majority of tumor cells that were easily visible with a low-power objective.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Characterization of CCK₁ receptor antibodies

Specificity of the antisera was monitored using Western blot analysis. When membrane preparations from stable transfected HEK-293 cells were electrophoretically separated and blotted onto nitrocellulose, the antiserum (9030) detected a broad band migrating at a relative molecular mass (M_r) of 85,000–95,000 only in CCK₁- but not in CCK₂-expressing cells (Fig. 1A). When the same samples were incubated with antiserum (9032) that was generated against the carboxyl-terminal region of the CCK₂ receptor, a broad band migrating at M_r 80,000–90,000 and a second band migrating at M_r 170,000 was detected only in CCK₂- but not in CCK₁-expressing cells (Fig. 1B). Given that the band migrating at M_r 80,000–90,000 represents the receptor monomer, and that the second band exhibits exactly twice the size of the receptor monomer, this band most likely represents the previously reported dimeric form of the CCK₂ receptor (29). These results indicate that both CCK₁- and CCK₂-expressing cells contain high levels of receptor protein and that the anti-CCK₁ antibody 9030 selectively detected its cognate receptor and did not cross-react with the CCK₂ receptor. Antisera were further characterized using immunofluorescent staining of transfected cells. When HEK-293 cells stably transfected with CCK₁ were stained with anti-CCK₁ antibody (9030), prominent immunofluorescence localized at the level of the plasma membrane was detected (Fig. 2A). In contrast, no immunofluorescence was detected when CCK₂-expressing cells were stained with anti-CCK₁ antibody (9030) (not shown). When stably transfected HEK-293 cells were stained with anti-CCK₂ antibody (9032), prominent immunofluorescence localized at the level of the plasma membrane was detected only in CCK₁- but not in CCK₂-expressing cells (Fig. 2C). Again these results indicate that both CCK₁ and CCK₂ antibodies selectively detected their cognate receptor and did not cross-react.

View larger version (21K): [in this window] [in a new window]   FIG. 1. Western blot analysis of the specificity of anti-CCK1 and anti-CCK2 antibodies. Membrane preparations from HEK-293 stably transfected to express either CCK1 or CCK2 were separated on 8% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were then incubated with affinity-purified anti-CCK1 (9030) (A) or anti-CCK2 (9032) (B) antibodies at a concentration of 1 µg/ml. Blots were developed using enhanced chemiluminescence. Two additional experiments gave similar results. Migration of protein molecular weight markers (Mr x 10–3) is shown to the left of the gels.

View larger version (56K): [in this window] [in a new window]   FIG. 2. Characterization of anti-CCK1 and anti-CCK2 antibodies by immunofluorescent staining of transfected cells. A and B, HEK-293 cells stably transfected to express CCK1 were either not exposed (A) or exposed to 1 µM sCCK-8 for 30 min (B) and subsequently fixed and immunofluorescently stained with anti-CCK1 (9030) antibody. C and D, HEK-293 cells stably transfected to express CCK2 were either not exposed (C) or exposed to 1 µM sCCK-8 for 30 min (D) and subsequently fixed and immunofluorescently stained with anti-CCK2 (9032) antibody. Note that in untreated cells, prominent immunofluorescence was localized at the level of the plasma membrane and that exposure to sulfated CCK induced a rapid translocation of both CCK1 and CCK2 receptors from the plasma membrane into the cytosol. Representative results from one of three independent experiments are shown. Scale bar, 20 µm.

View larger version (46K): [in this window] [in a new window]   FIG. 3. Western blot analysis of the specificity of anti-CCK11 antibodies in human tumors. A, Membrane preparations from a human carcinoid were separated on an 8% SDS-polyacrylamide gel, blotted onto nitrocellulose, and incubated with 1 µg/ml anti-CCK1 antibody (9030) in the absence (–) or presence (+) of 10 µg/ml peptide antigen. B, Membrane preparations from a human glioblastoma were separated on an 8% SDS-polyacrylamide gel, blotted onto nitrocellulose, and incubated with 1 µg/ml anti-CCK1 antibody (9030) in the absence (–) or presence (+) of 10 µg/ml peptide antigen. Blots were developed using enhanced chemiluminescence. Representative results from one of four independent experiments are shown. Migration of protein molecular weight markers (Mr x 10–3) is shown to the left of the gels.

The anti-CCK₁ antibodies were then subjected to immunohistochemical staining of a variety of human tissues. Initial experiments showed that all three anti-CCK₁ antisera yielded essentially identical immunohistochemical staining patterns although with different staining intensity. The anti-CCK₁ antibody (9030) produced the most prominent immunostaining and was therefore used throughout the study. Many neuroendocrine tumors contained adjacent noncancerous tissue, which enabled us to analyze the distribution of CCK₁ receptors in several parts of the normal gastrointestinal tract. Prominent localizations of the CCK₁ receptors in the stomach and small intestine are shown in Fig. 4, A–C. The highest densities of immunoreactive CCK₁ receptors were observed in the basal portion of the gastric mucosa (Fig. 4A). CCK₁ receptor immunoreactivity was predominantly confined to the plasma membrane of a subpopulation of cells in the gastric mucosa, which according to their size and appearance, most likely represent chief cells (Fig. 4A). CCK₁ receptor immunoreactivity was also abundant in many neurons of the myenteric plexus throughout the gastrointestinal tract (Fig. 4B). In addition, CCK₁ receptor immunoreactivity was also seen in many nerve fibers in the muscle layer (Fig. 4C). The anti-CCK₁ (9030) antibody was then subjected to immunohistochemical staining of 74 unselected human tumors. The prevalence of CCK₁ receptors in human tumors is summarized in Table 2. In general, CCK₁ receptors were rarely detected in human tumors except for neuroendocrine tumors and meningiomas. Immunoreactive CCK₁ receptors were frequently detected in carcinoids (53%), pancreatic insulinomas (50%), pheochromocytomas (50%), and meningiomas (75%) (Fig. 4, D–G). In addition, immunoreactive CCK₁ receptors were detected in a subset of cells in the normal pituitary as well as in 75% of GH-producing pituitary adenomas (Fig. 4, H and I). CCK₁ receptors were either rarely or not found in ductal invasive breast cancers, ductal pancreatic adenocarcinomas, or thyroid carcinomas. In addition, CCK₁ receptors were not detected in cancers of the colon, prostate, and ovary. A highly abundant expression of CCK₁ receptors was evident in glioblastoma samples; however, in each of these cases, immunoreactive CCK₁ receptors were clearly confined to contaminating nerve tissue and not to the tumor cells, suggesting that the prominent immunoreactive bands detected in Western blots of glioblastoma samples correspond to high levels of neuronal CCK₁ receptors. The anti-CCK₁ (9030) antibody yielded often prominent staining predominantly localized to the plasma membrane of the tumor cells (Fig. 4, E and F). In a subset of cases, immunoreactive CCK₁ receptors were observed in the cytosol as well as at the plasma membrane (Fig. 4, H and I). In contrast in a few cases, CCK₁ receptor immunoreactivity was mostly confined to cytoplasmic vesicles (Fig. 4, D and G). Immunostaining was completely abolished by preadsorption of the antibody with 10 µg/ml of its immunizing peptide (Fig. 4, F, inset, and I, inset).

View larger version (145K): [in this window] [in a new window]   FIG. 4. CCK1 immunohistochemical staining in human normal and neoplastic tissues. A–C, CCK1 immunohistochemical staining in gastric mucosa (A), myenteric neurons (B), and myenteric nerve fibers of the small intestine (C). D–F, CCK1 immunohistochemical staining in pancreatic insulinoma (D) and midgut carcinoid (E and F). G–I, CCK1 immunohistochemical staining in pheochromocytoma (G), normal pituitary (H), and GH-producing pituitary adenoma (I). Sections were dewaxed, microwaved in citric acid, and incubated with affinity-purified anti-CCK1 (9030) antibody at a concentration of 2 µg/ml. Sections were then sequentially treated with biotinylated antirabbit IgG and avidin-biotinylated peroxidase solution. Sections were then developed in diaminobenzidine-glucose oxidase and lightly counterstained with hematoxylin. Note that in the normal gastrointestinal tract, CCK1 receptor immunoreactivity was predominantly detected at the plasma membrane of chief cells in the gastric mucosa as well as in myenteric neurons and fibers. Whereas CCK1 receptor immunoreactivity was predominantly localized at the level of the plasma membrane in a midgut carcinoid, it was confined to cytoplasmic vesicles in a pancreatic insulinoma. For adsorption controls, primary antibody was incubated with 10 µg/ml of the peptide used for immunizations. Representative results from one of three independent experiments are shown. Insets in F and I, Peptide adsorption controls. Scale bars, 100 µm (A–D and G–I), 200 µm (E), and 100 µm (F).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

CCK₁ receptor mRNA and binding sites have previously been identified in several normal tissues including gastric mucosa, gall bladder, and myenteric neurons (10, 12, 13). However, a precise identification of the cellular sites of CCK₁ receptor protein expression is not possible with in vitro receptor autoradiography because of the limited anatomic resolution of this technique (12). Antibodies have been generated against the rat CCK₁ receptor (30, 31, 32). Although these antibodies have been used to elucidate the localization of CCK₁ receptors in the rat stomach and pancreas, none of the antibodies available has been demonstrated to effectively stain human formalin-fixed, paraffin-embedded tissues (30, 31, 32). In addition, relevant species differences have been noted; although CCK₁ receptor binding sites were detected in the basal region of the human antrum, no CCK₁ receptors were detected in the canine antrum (10, 12). Thus, the exact cellular and subcellular localization of CCK₁ receptors in human tissues still needs to be fully elucidated. Using the novel anti-CCK₁ antibody (9030), we show that CCK₁ receptors in the basal portion of the human gastric mucosa are predominantly confined to the plasma membrane of chief cells, suggesting a role for CCK₁ in the regulation of gastric pepsinogen secretion. CCK₁ receptors also decorate numerous neuronal cell bodies and nerve fibers of the myenteric plexus in the gastrointestinal tract. These receptors are very likely involved in the CCK-mediated regulation of gastrointestinal motility. CCK₁ receptors were also observed in a subpopulation of cells in the normal pituitary. In addition, we show that CCK₁ receptors detected in human glioblastoma samples were predominantly confined to contaminating neuronal cell bodies and nerve fibers.

Our findings indicate that immunoreactive CCK₁ receptors are rarely detectable in human tumors. Overexpression of CCK₁ receptor protein in significant numbers of cases was found only in carcinoids, insulinomas, pituitary adenomas, pheochromocytomas, and meningiomas. In most tumors, immunoreactive CCK₁ receptors were predominantly localized to the plasma membrane of the tumor cells. In contrast, in a few cases, CCK₁ receptors were mostly confined to cytoplasmic vesicles. Given that some tumors express CCK (2), CCK₁ receptors contained in intracellular vesicles may represent a pool of internalized receptors. A potentially important clinical implication of the present results is the possible use of radiolabeled CCK₁ receptor ligands to visualize in vivo in patients CCK₁ receptor-expressing gastroenteropancreatic tumors and their metastases (33). Radiolabeled CCK analogs may also be useful for radiotherapy of a subset of CCK₁ receptor-expressing neuroendocrine tumors that do not respond to octreotide therapy. However, it should be noted that a high tracer uptake should be expected in the normal gastric mucosa after the diagnostic or therapeutic application of radiolabeled CCK analogs (33).

In conclusion, we have generated and extensively characterized anti-CCK₁ antibodies. Using these antibodies, we provide the first demonstration of CCK₁ receptors in human formalin-fixed, paraffin-embedded tissues. It is now possible to determine the exact cellular and subcellular sites of CCK₁ receptor protein expression in normal human tissues, which is needed for a better understanding of CCK actions in physiological target tissues. The rapid immunohistochemical CCK₁ receptor visualization may also be helpful to identify those tumors with a sufficient receptor overexpression for diagnostic or therapeutic intervention.

	Acknowledgments

 
We thank Beate Peter and Dana Mayer for skillful technical assistance and Dr. C. Seva for plasmids encoding CCK₁ and CCK₂.

	Footnotes

 
First Published Online August 16, 2005

Abbreviations: CCK, Cholecystokinin; CCK₁, CCK receptor 1; CCK₂, CCK receptor 2; HEK, human embryonic kidney; M_r, relative molecular mass; sCCK-8, sulfated CCK octapeptide.

Received January 26, 2005.

Accepted August 4, 2005.

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Top Abstract Introduction Patients and Methods Results Discussion References

 

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