ARTICLE |
Correspondence to: Heide S. Cross, Dept. of General and Experimental Pathology, Waehringer Guertel 1820, A-1090 Vienna, Austria. E-mail: heide.cross@akh-wien.ac.at
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Summary |
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We identified the parathyroid type Ca2+-sensing receptor (CaR) in normal human colon mucosa and in cancerous lesions at the mRNA and protein level. Polymerase chain reaction produced an amplification product from reverse-transcribed large intestinal RNA which corresponded in size and length to a 537-bp sequence from exon 7 of the CaR gene. With a specific antiserum against its extracellular domain, the CaR could be detected by immunostaining in normal human colon mucosa in cells preferentially located at the crypt base. The CaR protein was also expressed in tumors of the large bowel in all 20 patients examined. However, the great majority of CaR-positive cells in the adenocarcinomas inspected were confined to more differentiated areas exhibiting glandulartubular structures. Poorly or undifferentiated regions were either devoid of specific immunoreactivity or contained only isolated CaR-positive cells. In the normal mucosa and in glandulartubular structures of cancerous lesions, the CaR was exclusively expressed in chromogranin A-positive enteroendocrine cells and in only a small fraction of PCNA-positive cells. (J Histochem Cytochem 48:595601, 2000)
Key Words: calcium-sensing receptor, growth control, colorectal cancer, chromogranin A, endocrine cells, PCNA, differentiation, immunohistochemistry, reverse transcription, PCR
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Introduction |
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ALTHOUGH there is suggestive evidence from epidemiological studies that the incidence of human colorectal carcinoma is inversely related to calcium consumption (see, e.g.,
Caco-2 cells, although originally derived from a human colon adenocarcinoma, are still able to spontaneously differentiate in culture in an enterocyte-like fashion (
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Materials and Methods |
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Immunohistochemical Procedures
Archival blocks of formalin-fixed, paraffin-embedded tissue from 20 patients who had undergone surgery for colon carcinoma were investigated in this study. All tumors were of the adenocarcinoma type and were graded according to WHO criteria (
For gross inspection of tumor morphology, 5-µm sections were deparaffinized and stained with hematoxylin/eosin. Samples from the center and the periphery of a tumor as well as from adjacent histologically normal mucosa were chosen for subsequent immunostaining. In each case, sections from three to five blocks were used. For comparison, normal mucosa was obtained from three diverticulitis patients undergoing stoma re-operation.
For specific immunostaining, the following mouse monoclonal antibodies were used: an anti-CaR antibody directed against a synthetic peptide corresponding to amino acids 214235 in the extracellular domain (generously provided by Drs. Allan Spiegel and Paul Goldsmith, NIDDK, NIH, Bethesda, MD, through NPS Pharmaceuticals, Salt Lake City, Utah; dilution 1:300); anti-chromogranin A (Biomeda, Foster City, CA; dilution 1:100); and anti-PCNA (DAKO, Carpinteria, CA; dilution 1:10).
For immunostaining of the CaR, an indirect horseradish peroxidase-labeled antibody technique was used. Five µm serial paraffin sections on poly- L-lysine-coated slides were incubated for 20 min at 60C, deparaffinized, and rehydrated. After washing three times in PBS, pH 7.2, endogenous peroxidase activity was blocked by incubation in H2O 2 in methanol at room temperature (RT) for 10 min. Sections were stained according to the Histostain-Plus kit instructions (Zymed Laboratories; San Francisco, CA) with 3,3'-diaminobenzidine (DAB; Zymed) as substrate. Slides were counterstained with hematoxylin and then mounted with Histomount (Zymed).
Negative controls for CaR were carried out by incubation of the sections with the anti-CaR antibody preabsorbed with 6 µg/ml of the immunogenic peptide.
For double immunostaining, an indirect alkaline phosphatase-labeled antibody technique was used. After washing three times in PBS, pH 7.2 (the last washing also contained 0.1% Tween-20), endogenous alkaline phosphatase activity was blocked by incubation in 0.2 N HCl at RT for 8 min. Sections were incubated in 2% normal horse serum in PBS for 30 min at RT to reduce unspecific staining and then with the primary antibody overnight at 4C in a moist chamber. Then, biotinylated anti-mouse IgG and Vectastain ABC-AP kit (both from Vector; Burlingame, CA) were used according to the manufacturer's recommendations.
Specifically bound alkaline phosphatase was visualized by incubation with either 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT; Vector) or with the Fast Red substrate system (DAKO). The development of the color reaction was followed by light microscopy. After visualization of one antigen, the sections were intensively washed three times in PBS for 10 min on a rocker platform, with the last washing also containing 0.1% Tween-20. Then the second antigen was detected using the same protocol, except that the procedure for blocking endogenous alkaline phosphatase activity was omitted. Controls for specificity of immunostaining for chromogranin A or PCNA, respectively, were performed by substituting the primary antibody with PBS or preimmune mouse serum, and were consistently negative.
Sections were counterstained with hematoxylin or, in some cases, with methyl green (Vector) as indicated, and then mounted with Histomount (Zymed) or Glycergel (DAKO).
The percentage of epithelial cells co-expressing the CaR and chromogranin A or PCNA was assessed by counting at least 250 epithelial cells at a magnification x200 on each section of normal colon mucosa (from three non-cancer, i.e., diverticulitis, patients), and of cancerous colon mucosa and of normal mucosa adjacent to the cancerous lesion from the same patient. Sections from the tumor center and, if necessary, also from the tumor periphery were inspected for the presence of representative differentiated areas exhibiting glandulartubular structures. Results are presented as means ± SD from four tumors, of which two were of low grade (Stage pT2) and two of high grade (Stage pT3 and pT4) (cf. Table 1).
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Semiquantitative Reverse Transcription-PCR
For analysis of CaR mRNA in relation to cytokeratin-8 mRNA expression by reverse transcription-PCR, snap-frozen surgical specimens of tumor tissue and adjacent normal mucosa from 16 patients were used. Tumor staging was done according to the TNM system: pT2, n = 4; pT 3, n = 6; pT4, n = 6. Five samples of normal mucosa from diverticulitis patients undergoing stoma re-operation were included in the analysis.
Total RNA was extracted with the TRIzol reagent (Life Technology; Palo Alto, CA) according to the instructions of the manufacturer. RNA was quantitated by absorbance at 260 and 280 nm. Two µg was used for synthesis of sin-gle-stranded cDNA (SUPERSCRIPT II kit; Life Technol-ogy). PCR was performed at a final concentration of 1 x DyNAzyme buffer, 0.25 mM dNTP, 0.25 µM of forward and reverse primers, and 1 U DyNAzyme II DNA polymerase (Finnzymes OY; Riihitontuntie, Finland). Conditions were 33 cycles at 94C for 15 sec, 61C for 30 sec, 72C for 1 min, with a final extension at 72C for 10 min.
Cytokeratin 8, a common marker for intestinal epithelial cells, was used as an internal control for the RT-PCR reaction. Primers were designed to span an intron to ensure that the products were not the result of amplification of contaminating genomic DNA segments. To avoid differences among tubes, RT-PCR was performed in the same tube for amplification of the CaR mRNA and cytokeratin 8.
The amount of RNA and the number of amplification cycles was optimized to ensure that PCR products were in the exponential phase of amplification. Aliquots of the PCR products were resolved by electrophoresis on a 2% agarose gel. Validation of the PCR products was done by multiple digestion with restriction enzymes and size evaluation of the fragments obtained thereby. For quantitation of transcripts, photographs of ethidium bromide stained gels were analyzed by densitometry.
Primers used for CaR were 5'-CAGCAAGAGCAACAGCGAAG-3', sense; 5'-GAAACCTCTCTGCATTCTCC-3', antisense; for cytokeratin 8: 5'-AGTGGGCAGCAGCAACTTTCG-3', sense; 5'-TTCAGCTTCTCCTGGCCCAGAG-3', antisense.
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Results |
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CaR mRNA Expression in Normal and Malignant Mucosa
For analysis of CaR mRNA expression in human colon adenocarcinomas, in adjacent mucosa from the same cancer patients, and in normal mucosa from non-cancer patients, semiquantitative RT-PCR was used. Primers were selected to allow amplification of a 537-bp fragment within exon 7 of the CaR gene and of a 360-bp of the cytokeratin 8 gene. The latter is uniformly expressed in normal and malignant colon epithelial cells and can therefore be used as appropriate reference to exclude any contribution from stromal and other non-epithelial cells. Fig 1A shows that after 33 cycles PCR products appeared as single bands which, with respect to size and restriction fragment length, indicated the presence of mRNA specific for CaR and CK 8. Densitometric analysis ( Fig 1B) revealed no significant difference in the CaR/CK 8 ratio among normal mucosa, adjacent mucosa, and cancerous tissue. It should be noted that CaR-specific mRNA fragments could be amplified from any of the 16 cancerous tissue samples inspected, which were derived from four tumors of stage pT2, six of pT3 , and six of pT4. There was no correlation between tumor stages and CaR/CK 8 mRNA expression (not shown).
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Immunohistochemistry of Normal Colon Mucosa
In sections of histologically normal mucosal tissue adjacent to a colon carcinoma, a monoclonal antibody specific for the amino acid sequence 214235 in the extracellular domain of the CaR produced positive immunoreactivity exclusively in cells interspersed in the crypt epithelium, where they appeared to be located preferentially in or next to the crypt base (Fig 2A). No specific staining was detectable when the anti-CaR antibody had been preincubated with the immunogenic peptide (Fig 2B).
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On perpendicular sections counterstained with methyl green (not shown), it became obvious that cells which stained positive with the anti-CaR antibody differed in appearance from most other cells because they had round nuclei and abundant cytoplasm. In contrast, CaR-negative epithelial crypt cells exhibited more elongated nuclei and rather scanty cytoplasm. Such morphological criteria suggested that CaR-positive cells might be endocrine cells. However, because of their position at the crypt base (cf.
To address this question, sections were double-stained with an antibody against PCNA, a marker of proliferative cells, and with the anti-CaR antibody. Fig 2D shows that PCNA-positive cells, which can be easily recognized by intense nuclear staining, are almost uniformly distributed within epithelial cells at the crypt base, whereas CaR immunoreactivity is confined to cells at the very base of the crypt (Fig 2D). From a quantitative analysis of expression of PCNA and CaR, it became clear that of all PCNA-positive cells only a minute fraction co-express the CaR (Table 1). Conversely, almost one half of CaR-positive cells were devoid of PCNA ( Table 1).
When sections of normal colon mucosa adjacent to the tumor were stained for CaR and chromogranin A (Fig 2C), CaR positivity was found exclusively in cells that also stained positively for the enteroendocrine cell marker chromogranin A (Table 1). However, a similarly large number of chromogranin A-positive cells showed no specific staining for the CaR (Table 1, see also Fig 2C).
Identical results were obtained when normal colon mucosa from three non-cancer patients was analyzed for co-expression of CaR with chromogranin A or PCNA (not shown).
It should be noted that, apart from the mucosal epithelium, the CaR could be detected by specific immunostaining in the neurons of the myenteric plexus but not in connective tissue, smooth muscle, or vascular endothelial cells (not shown).
Immunohistochemistry of Colon Adenocarcinoma
Immunoreactivity for the CaR was also detected in all samples (20/20) of carcinomatous lesions inspected. However, the pattern of immunostaining in each tumor was rather heterogeneous because the great majority of CaR-positive cells were confined to more differentiated areas exhibiting glandulartubular structures, as illustrated in Fig 2E, whereas poorly or undifferentiated regions were either devoid of specific immunoreactivity (15/20 tumors) or contained only solitary CaR-positive cells (5/20 tumors) (not shown). Fig 2F shows that, in a pT2 carcinoma, distribution of CaR positive cells in glandular structures is similar to that seen in normal mucosa (cf. Fig 2A). In addition, the exclusive co-localization of the CaR to chromogranin A-positive cells, as noted in the normal adjacent mucosa (Table 1), is completely preserved in the more differentiated regions of colon carcinomas exhibiting glandulartubular structures (Table 1).
To check the proliferative activity of CaR-positive carcinoma cells, tissue sections were double-stained with an anti-CaR and an anti-PCNA antibody. In glandulartubular structures of cancerous lesions (see Fig 2G), the expression pattern of the CaR and PCNA was similar to that observed in colon mucosa outside the tumor border (Table 1). Whereas a large portion of tumor cells exhibited immunostaining of PCNA but not of the CaR, the majority of CaR-positive cells also stained positively for the proliferation marker.
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Discussion |
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The CaR, which is likely to transduce changes in [Ca++]o into specific cell functions in parathyroid gland, kidney, thyroid C-cells, and neuronal cells in various regions of the brain, is apparently also expressed by gastrointestinal epithelial cells. Apart from human colon adenocarcinoma-derived cell lines such as Caco-2 (
The notion that the CaR is present in enteroendocrine cells of human colon mucosa is substantiated by the observation of
It should be noted that another product of neuroendocrine cells, i.e., parathyroid hormone-related protein which, similar to the CaR, is rarely expressed in normal human colon mucosa, was detected by specific immunostaining in more than 90% of colon carcinomas (
A number of human colon adenocarcinoma-derived cell lines, e.g., Caco-2 and HT-29, display the CaR at the mRNA and protein level (
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Footnotes |
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1 Yuri Sheinin and Enikö Kállay should both be considered first authors.
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Acknowledgments |
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Supported by a grant from ASTA Medica GmbH (Vienna, Austria) and by grant no. 6152 from the Jubiläumsstiftung der Österreichischen Nationalbank (Vienna, Austria).
Received for publication July 15, 1999; accepted December 30, 1999.
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