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Original Studies |
Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, CH-3010 Berne, Switzerland
Address all correspondence and requests for reprints to: J. C. Reubi, M.D., Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, Murtenstrasse 31, CH-3010 Berne, Switzerland.
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Abstract |
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Introduction |
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There is evidence for abnormal gastrin expression in certain types of gastrointestinal tumors, in particular neuroendocrine tumors such as islet cell carcinomas (gastrinomas) and, to a lesser extent, in colorectal cancers (2, 4, 5, 6, 7, 8), as well as in several tumor cell lines (5, 9). CCK is much less frequently expressed in primary human tumors (2, 10) and tumor cell lines (11, 12). The receptors for CCK and gastrin, named CCK-A and CCK-B receptors (13), which mediate the actions of CCK (CCK-A and CCK-B) or gastrin (CCK-B), have also been shown to be expressed in several types of human tumors (8, 10, 14, 15, 16, 17, 18, 19) and cell lines (20, 21). This has led to the concept of an autocrine feedback stimulation of tumor growth by gastrin and CCK in those tumors and tumor cell lines expressing both the peptides and their receptors (8, 10, 22, 23).
Most of the gastrin and CCK research in the last few years has focussed on colorectal cancers, whereas many other tumor types have been largely neglected in this respect. Primary human neuronal, neuroectodermal, renal, and muscular tumors, for instance, have been only poorly investigated for gastrin and CCK peptide and receptor expression, although some preliminary data on cell lines of pediatric tumors have suggested that at least CCK is expressed by some of them (24, 25, 26), in particular Ewing sarcoma and neuroepithelioma cell lines.
The aim of the present study was therefore to evaluate the gene expression of CCK and gastrin by in situ hybridization of their messenger ribonucleic acids (mRNAs) and the presence of their respective CCK-A and CCK-B receptors by in vitro receptor autoradiography in a series of human neuronal, renal, and myogenic stem cell tumors. The use of morphological methods (in situ hybridization and receptor autoradiography) was considered essential due to the complex tissue composition of the tumor samples.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Aliquots of surgically resected tumors or of biopsies submitted for diagnostic histopathology were frozen immediately after surgical resection and stored at -70 C. The specimens originated from several different clinical institutions, and some have been previously used for other purposes. The following tumors were investigated: 11 medulloblastomas, 11 Ewing sarcomas, 5 central primitive neuroectodermal tumors (PNET), 10 neuroblastomas, 4 Wilms’ tumors, 5 rhabdomyosarcomas, and 10 leiomyosarcomas. Moreover, human brain (cerebellum and cortex), pituitary, duodenum, and stomach samples and human gastrinomas were used for control purposes as tissues expressing CCK and/or gastrin mRNA.
In situ hybridization histochemistry
Consecutive cryostat sections of human normal and tumoral
tissues were used for gastrin and CCK mRNA detection by in
situ hybridization. The protocol followed was essentially that
described in detail previously (27). Oligonucleotide probes
complementary to the nucleotides coding for amino acids 51–70 (28) or
75–82 (29) of the human gastrin gene and for amino acids 1–11 or
80–95 (30) of the human CCK gene were synthesized and purified on a
20% polyacrylamide-8 mol/L urea sequencing gel (Microsynth, Balgach,
Switzerland). They were labeled at the 3'-end using
[
-32P]deoxy-ATP (>3000 Ci/mmol; Amersham, Aylesbury,
UK) and terminal deoxynucleotidyltransferase (Boehringer Mannheim,
Mannheim, Germany) to specific activities of 0.9–2.0 x
104 Ci/mmol.
The absorbance was measured in the autoradiograms over the tissue area with a computer-assisted image-processing system, as described previously (31). A tissue was considered positive for the respective mRNA when the absorbance measured in a normally hybridized section was at least twice that in a parallel section in which hybridization was blocked with a 20-fold excess of the corresponding probe. Tissue sections were hybridized with an oligonucleotide complementary to bp 45–92 of the human ß-actin mRNA (32) to confirm and normalize the presence of mRNA in the tissues analyzed; only those tumors with a high abundance of ß-actin mRNA were included in the study.
The different oligonucleotide probes designed for the same mRNA, when used independently as hybridization probes in consecutive sections of tissues, showed the same in situ hybridization patterns, with similar exposure times. Among the two gastrin probes, however, the one designed by Larsson and Hougaard (29) was the more sensitive. The thermal stability of the hybrids was close to the theoretical melting temperature. In all types of tissues tested, disappearance of the hybridization signal was observed when consecutive sections were hybridized with the radiolabeled probe in the presence of a 20-fold excess of the corresponding unlabeled probe, proving the specificity of the signal obtained. This last control was particularly important when considering the heterogeneity of the tissue samples investigated.
Northern analysis
Total RNA was isolated from different human tumors (gastrinoma and Ewing sarcoma) as described by Chomczynski and Sacchi (33), and polyadenylated [poly(A)+] RNA was purified by chromatography through oligo(deoxythymidine)-cellulose. Human brain poly(A)+ RNA was obtained from Clontech (Palo Alto, CA), and RNA mol wt markers were purchased from Boehringer Mannheim. RNA was denatured with glyoxal (34), separated by electrophoresis on 1% agarose gels, and blotted onto nylon membranes (Hybond N, Amersham). Blots were hybridized with the labeled 24-mer (gastrin) and 33-mer (CCK) oligoprobes for 18 h at 37 and 42 C, respectively, in a buffer containing 600 mmol/L NaCl, 80 mmol/L Tris-HCl (pH 7.5), 4 mmol/L ethylenediamine tetraacetate, 0.1% sodium pyrophosphate, 0.2% SDS, 5 x Denhardt’s solution, 500 µg/mL yeast transfer RNA, and 50% formamide. Filters were washed twice for 5 min at room temperature and twice for 15 min at 42 C in 2 x sodium chloride-sodium citrate buffer (SSC; 1 x SSC = 150 mmol/L NaCl-15 mmol/L sodium citrate, pH 7.0)-0.1% SDS. Filters were apposed to an x-ray film and exposed for the appropriate period of time.
Receptor autoradiography
Receptor autoradiography was performed on 10- and 20-µm thick cryostat (1720, Leitz, Rockleigh, NJ) sections of the tissue samples, mounted on microscope slides, and then stored at -20 C for at least 3 days to improve adhesion of the tissue to the slide, as described previously (31). Each tissue underwent receptor autoradiographic processing with 125I-labeled D-Tyr-Gly-Asp-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-Phe-amide ([125I]CCK), a radioligand identifying both CCK-A and -B receptors, as described previously (17, 35). The sections were preincubated in 50 mmol/L Tris-HCl, 130 mmol/L NaCl, 4.7 mmol/L KCl, 5 mmol/L MgCl, 1 mmol/L ethylene glycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 0.5% BSA, pH 7.4 (preincubation solution), for 30 min at 25 C. The slides were then incubated in a solution containing the same medium as the preincubation solution but without BSA, and the following compounds were added: 0.025% bacitracin, 1 mmol/L dithiothreitol, 2 µg/mL chymostatin, 4 µg/mL leupeptin (pH 6.5), and the radioligand, 45 pmol/L [125I]CCK (2000 Ci/mmol; Anawa, Wangen, Switzerland). The slides were incubated at room temperature with the radioligand for 150 min. On completion of the incubation, the slides were washed six times for 15 min each time in ice-cold preincubation solution, pH 7.4. The slides were rinsed twice in ice-cold distilled water for 5 s each time. They were then dried under a stream of cold air at 4 C, apposed to 3H-Hyperfilm (Amersham, Little Chalfont, U.K.), and exposed for 1–7 days in x-ray cassettes.
The autoradiograms were quantified using a computer-assisted image-processing system, as described previously (31). Tissue standards for iodinated compounds (Amersham) were used for this purpose. A tissue was defined as receptor positive when the absorbance measured over a tissue area in the total binding section was at least twice the absorbance of the nonspecific binding section.
Tumors were considered as expressing CCK-A receptors when the [125I]CCK analog was fully displaced by 50 nmol/L sulfated CCK-8 but was not displaced by 50 nmol/L gastrin. Conversely, tumors were considered as expressing CCK-B receptors when the [125I]CCK ligand was fully displaced by nanomolar concentrations of sulfated CCK-8 and gastrin (17).
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Results |
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Northern blot analyses were performed with oligonucleotide probes
identical to those used for the in situ hybridization
described below. The stringencies for the Northern and in
situ hybridizations were approximately equal. As shown in Fig. 1
, poly(A)+ RNA extracted
from various human tissues and hybridized with 32P-labeled
CCK and gastrin probes, as described in Materials and
Methods, revealed single discrete bands of 0.8 and 0.7 kb,
respectively. The normal human brain expressed CCK mRNA, the human
gastrinoma expressed gastrin mRNA, and the Ewing sarcoma expressed both
CCK and gastrin mRNAs. The mol wt found are in agreement with those
reported previously (5, 24).
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The specificity of the probes was controlled by localizing the
corresponding target mRNAs in normal human tissue specimens known to
express gastrin or CCK (2). As shown in Fig. 2, the gastrin probes labeled glandular
structures arranged in small nests and located in the middle third of
the antral mucosa, as shown previously by others to represent
gastrin-producing cells (28, 29). The human pituitary gland also
expressed gastrin mRNA; a higher level of expression was observed in
the adenohypophysis than in the neurohypophysis (data not shown), in
agreement with the preprogastrin products levels found in the
corresponding porcine lobes (36). Finally, human cerebellar cells
expressed gastrin mRNA (data not shown), as reported previously at the
peptide level for the porcine cerebellum (37).
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where its laminar distribution, i.e. the superficial layers
(I–III) and especially the deep layers (V–VI) exhibiting higher
expression of CCK mRNA than the middle layers, was the same as that
previously described (30). The CCK probes also labeled the duodenal
mucosa (not shown), where CCK-producing cells are known to be localized
(38). The normal fundic mucosa, as a negative control (10), was not labeled by the gastrin or CCK probes used in this study (data not shown). Moreover, gastrinomas, which contained elevated amounts of gastrin mRNA, gave negative results with the CCK probes; conversely, the brain cortex, which was highly positive for CCK mRNA, was negative with the gastrin probes.
Gastrin mRNA in tumors
As shown in Table 1, the
overwhelming majority of the tested tumors expressed gastrin mRNA. This
has been the case for Ewing sarcomas, central PNETs, and
medulloblastomas. It was also found in the majority of neuroblastomas
(8 of 10) as well as in all Wilms’ tumors and rhabdo- and
leiomyosarcomas. Figure 3 shows examples
of gastrin mRNA in a medulloblastoma, a central PNET, a neuroblastoma,
and a rhabdomyosarcoma. Figure 4 shows gastrin mRNA
in an Ewing sarcoma.
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The expression of CCK mRNA is much less frequent (Table 1) and is
mainly restricted to the Ewing sarcomas and half of the
leiomyosarcomas. In several Ewing sarcomas, a very high abundance of
CCK mRNA was found. This abundance is illustrated in the Northern blot
of Fig. 1 and can be compared to the much lower levels of gastrin mRNA
in the same tumor. Figure 4 shows a CCK mRNA containing Ewing sarcoma,
and Fig. 5 illustrates CCK mRNA in a
leiomyosarcoma, both using in situ hybridization
methods.
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As seen in Table 1, only a minority of these embryonal and
mesenchymal tumors express CCK-A or CCK-B receptors, except for the
leiomyosarcomas and Wilms’ tumors. Most of these tumors had CCK
receptors present together with gastrin and/or CCK mRNA. The
leiomyosarcoma in Fig. 6 had gastrin and
CCK mRNAs as well as CCK receptors. The Wilms’ tumor in Fig. 7 had gastrin mRNA in the whole tumor
sample, but CCK-B receptors in a restricted region of the tumor
only.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We show that most Ewing sarcomas are characterized by the concomitant expression of gastrin and CCK mRNAs. That a single tumor area contains both gastrin and CCK mRNAs is documented not only with in situ hybridization, but also with Northern blots, in which the adequate bands for gastrin and CCK mRNAs are found in the same tumor. Based on previous studies in various other types of tumors by Rehfeld, it had been concluded that gastrin and CCK are usually not simultaneously produced by a given cell (2). Ewing sarcoma cells may therefore be an exception to this observation.
Several investigations, mainly by Rehfeld and others, have shown that gastrin mRNA can be processed to various forms of bioactive gastrins, including progastrin, glycin-extended gastrin, or amidated gastrin (2, 5, 6, 7, 9). Further studies with selective gastrin antibodies will be needed to evaluate which form of gastrin is predominantly expressed by these embryonal and mesenchymal tumors. The processing of CCK mRNA to CCK peptides appears complex as well (2, 25, 26, 41), and again, the predominant CCK form synthesized by these tumors needs to be assessed.
Recent data suggest that several of these forms of gastrin and CCK and several types of CCK receptors may be responsible for tumor growth stimulation. Whereas both amidated gastrin and CCK have growth-promoting properties, it was shown recently that immature forms of gastrin (i.e. glycine-extended gastrin) also had growth-stimulating effects (2, 9, 20, 22, 23, 42, 43, 44). These endogenous peptides may not only act through CCK-A and CCK-B receptors to mediate this growth stimulation, but perhaps also through CCK-C receptors (8, 45, 46) or through receptors selective for glycine-extended gastrin similar to the receptor described on the rat pancreatic carcinoma cell line AR4–2J (47). Here, the CCK-C receptors were not evaluated due to the low binding affinity of the CCK radioligand for this receptor subtype. It is further known that not only mature forms of gastrin, but also progastrin itself may be able to stimulate proliferation, as suggested by the data reported by Wang et al. (48) showing that an overexpression of unprocessed progastrin in transgenic mice increased colonic mucosal proliferation. However, the nature of the receptor responsible for this proliferative effect of progastrin is unknown. Whereas the present study indicates that selected human embryonal and mesenchymal tumors (leiomyosarcomas and Wilms’ tumors) have the molecular machinery (gastrin and CCK mRNAs, and CCK-A and CCK-B receptors) for a potential autocrine feedback growth regulation, future investigations will be necessary to elucidate its precise mechanism of action.
The presence of mRNA for the two growth factors, gastrin and CCK, in embryonal and mesenchymal tumors may be the basis for abnormal production of bioactive gastrins and CCK, which may, in turn, represent a potential hazard and risk for the cancer patient regardless of whether CCK receptors are present in the tumor itself. Indeed, these regulatory peptides may play a deleterious role in the growth regulation of normal CCK receptor-expressing tissues (44), such as gastric mucosa, as well as of CCK receptor-expressing tumors. In the future, it may be diagnostically worthwhile to measure gastrin and CCK levels in the blood circulation of such tumor-bearing patients. If increased peptide levels can be detected, the use of specific CCK receptor antagonists (49) may be indicated; such a therapy may, on the one hand, prevent gastrin/CCK-induced symptoms in healthy tissue; it may, on the other hand, also prevent a further growth of those tumors that express CCK receptors in addition to the peptide itself.
Received July 7, 1998.
Revised October 5, 1998.
Accepted October 12, 1998.
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