ARTICLE |
Correspondence to: Agnes Kittel, Inst. of Experimental Medicine, Hungarian Academy of Sciences, PO Box 67, 1450 Budapest, Hungary. E-mail: kittel@koki.hu
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Summary |
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Elevated levels of extracellular ATP have been observed in many tumors. We have localized NTPDase1/CD39, one of the principal extracellular nucleotide-hydrolyzing enzymes, in normal and cancerous human pancreas. NTPDase/E-ATPDase activity was demonstrated with an enzyme histochemical technique on cryosections of human pancreas. Acinar and duct epithelial cells were devoid of E-ATPDase activity in both normal and transformed tissue. Endothelial cells and smooth muscle around blood vessels and larger ducts showed strong activity. Nerves, connective tissue, and the ß-cells of the islets were also stained. In cancerous tissue this activity was diminished in the smooth muscle around the ducts and was absent from newly formed connective tissue. Immunostaining for CD39 supported these results but revealed the presence of inactive CD39 in the duct epithelial cells. We hypothesize that the significantly diminished activity of NTPDase1 in the tissues surrounding the ducts may be associated with the processes that lead to tumor formation in human pancreas. (J Histochem Cytochem 50:549555, 2002)
Key Words: NTPDase1/CD39, extracellular nucleotides, ATP, purinoceptors, pancreas, tumor, islets
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Introduction |
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The metabolism of extracellular nucleotides plays an important role in nucleotide signaling mediated by ATP- and ADP-selective P2 receptors. ATP is degraded via ADP and AMP to adenosine and phosphate. Several surface-located enzymes may be involved in this hydrolysis chain, e.g., ecto-alkaline phosphatase, ecto-alkaline phosphodiesterase, ecto-apyrase or NTPDase1 (referred to as E-ATPDase), ecto-ATPase, and ecto-5'-nucleotidase (
Few data are available regarding extracellular ATP and NTPDase activity in the pancreas. In pig pancreas, two isoforms of NTPDase1 have been characterized, purified, and identified (
According to the authors, the E-ATPDase is strategically positioned to modulate purine-mediated actions such as fluid and electrolyte secretion, smooth muscle contraction, and blood flow (
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Materials and Methods |
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All reagents were purchased from Sigma (St Louis, MO) unless otherwise specified. Affinity-purified rabbit anti-CFTR (cystic fibrosis transmembrane conductance regulator) was prepared in the lab of A. Nairn (Rockefeller University; New York, NY). LP2K, a mouse monoclonal anti-cytokeratin 19, was obtained from Dr. E. B. Lane (University of Dundee; Dundee, Scotland).
Preparation of Cryosections
Human pancreatic tissue blocks were obtained at surgery (IMIM; University of Barcelona). A certified clinical pathologist at the location characterized each sample. Tissue blocks were frozen in isopentane cooled by liquid nitrogen. Three- to 5-µm cryosections were cut and, after drying, fixed with acetone (-20C) for 10 min. Each slide contained both normal and tumor sections. Staining procedures were carried out in a Shandon immunostainer (Shandon Scientific; Cheshire, UK).
Enzyme Histochemistry
A lead precipitation enzyme histochemical technique was used for demonstration of NTPDase (E-ATPDase) activity (,ß-methylene-ADP (5'-nucleotidase inhibitor), and KCl (5 mM) in Tris-maleate buffer (70 mM, pH 7.4) for 30 min at room temperature (RT). Incubation was followed by three rinses in Tris-maleate buffer. The precipitate was converted to PbS with 1% (NH4)2S (1-min incubation).
Immunohistochemistry
After the enzyme histochemical reaction, immunostaining was performed to identify the main cell types in the human pancreatic cryosection. -LP2K (1:4 dilution) was used to identify epithelial cells, amylase antibody (in undiluted solution) identified the acinar cells, or a chromogranin antibody (Biomeda; Foster City, CA; 3:1 dilution) was applied to distinguish the islet B-cells.
After blocking endogenous phosphatase activity (3 mM levamisole, 0.01% NaN3 in PBS, 10 min) and washing, nonspecific binding sites were blocked with 5% dried milk and 0.04% Tween-20 in PBS for 20 min. The primary antibody was diluted in 1% BSAPBS and the incubation time was overnight at 4C. After washing with PBS, the secondary antibody (alkaline phosphatase-conjugated IgG, diluted in 1% BSAPBS) was applied for 60 min at RT. After washing and preincubation with Tris (0.1 M, pH 8.2), staining was visualized with Fast Red (0.1 M, pH 8.2, 5 min). Sections were counterstained in 20% Papanicolau in PBS and, after washing with tapwater, mounted in Aquatex. Images were obtained with an Olympus BP 50 digital camera (Olympus Optical (Europa); Hamburg, Germany).
Immunostaining was also performed with -CD39 monoclonal antibody (Ancell; Bayport, MN) on the human pancreatic cryosections. Fixation was carried out as above. After rinsing with PBS, the nonspecific binding sites were blocked with 5% BSA in PBS for 30 min and the sections were incubated overnight with CD39 antibody in 1:400 dilution at 4C. The ABC method was used and Vector VIP as chromogen (Vector Laboratories; Burlingame, CA) was applied according to the manufacturer's instructions. After washing with distilled water, sections were mounted in Aquatex and imaged on a Nikon Eclipse E 600 microscope with a Spot digital camera (Nikon; Kumagaya City, Japan) using a x20 objective.
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Results |
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The enzyme histochemical reaction used in the present work demonstrates the active NTPDase1 enzyme. Whether ATP or ADP was used as a substrate, the distribution of enzyme activity indicated by the precipitate was similar. Ecto-ATPase/E-ATPDase activity was detected as a brown lead sulfide (PbS) deposit, which is generated at sites where phosphate is liberated from the extracellular ATP/ADP during hydrolysis. Immunohistochemical staining performed after enzyme histochemistry was used to identify the different cell types. Immunostaining was also done to localize the DC39/NTPDase1-positive cells. As controls for the enzyme histochemical reaction, the substrate ATP or ADP was omitted from the reaction mixture (Fig 1A). For the immunostaining, the incubation step with the first antibody was omitted (Fig 1I).
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As described in Materials and Methods, certified clinical pathologists identified the origin of the tissue blocks used. We studied only one type of pancreatic cancer, duct adenocarcinoma. The general appearance of this cancerous pancreas differs from the normal tissue in many details. We observed proliferation and disorganization of duct epithelial cells, an increased amount of connective tissue, and no staining in the acini when amylase immunostaining was applied (Fig 1D, inset).
Endothelial cells and smooth muscle cells around blood vessels showed the strongest E-ATPDase activity in cryosections of human pancreas (Fig 1B). Acini were unstained. When CD39/NTPDase1 immunostaining was applied, the result was the same for these types of cells (Fig 1C). Each presented staining was performed on normal tissue, but, as shown in Fig 1D, the E-ATPDase activity in the endothelial cells and smooth muscle cells was not altered in cancerous pancreas. The cryosection shown in Fig 1D was cut from a tissue block of tumor origin. The vessel endothelial and smooth muscle cells are covered with the brown PbS deposit because of the active ectoenzyme. Duct epithelial cells are negative for the enzyme reaction; only the connective tissue around the group of ducts shows some very weak staining. Although amylase staining was applied to identify the acini, they were unstained because of the cancerous transformation of the tissue. In normal tissues, when amylase immunostaining was applied (Fig 1D, inset) the acini were positive and showed the red color of the Fast Red. The brown color of the endothelial cells demonstrated E-ATPDase activity.
In normal pancreas, the smooth muscle cells around the larger ducts were also stained (Fig 1E) but the intensity of this staining decreased in the cancerous tissue and the number of ducts was increased because of proliferation (Fig 1E, inset). LP2K staining confirmed the identification of the duct cells (Fig 1E and inset). Although according to the result of enzyme histochemical staining the duct cells did not bear ATPDase enzyme activity, immunostaining showed strong immunoreactivity for CD39 (Fig 1F). Some E-ATPDase activity was also detected in nerves (Fig 1G). The presence of CD39/NTPDas1 was proved with immunostaining (Fig 1G, inset). We have also demonstrated for the first time the E-ATPDase activity of the ß-cells of the islets of Langerhans (Fig 1H). ß-Cells were identified with chromogranin staining (Fig 1H, upper inset). Immunostaining for CD39 confirmed that the E-ATPDase activity demonstrated by enzyme histochemistry belongs to NTPDase1 (Fig 1H, lower inset).
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Discussion |
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NTPDase1, one of the most intensively studied members of the ecto-NTPDase enzyme family, is considered to play an important role in purinergic signaling, in thromboregulation, and in cell-protective processes by converting ATP released by damaged cells to AMP. The source of extracellular ATP can also be nerve terminals and other types of cells. ATP can be released in response to endogenous ligands and is involved in a special mode of signal transmission, so-called cascade transmission (
Among the nucleotide receptors, P2 receptors are selective for ATP and ADP. In 1981, when the stimulatory effect of ATP on insulin secretion in isolated perfused rat pancreas was demonstrated, it was concluded that a purinoceptor of the P2-type is likely to be present on the ß-cells of the rat pancreas (-Cells possess adenosine receptors involved in the stimulation of glucagon secretion and ß-cells have P2 purinoceptors (for ATP and/or ADP) which increase insulin secretion (
The presence of E-ATPDase activity has previously been demonstrated on several cell types in the exocrine pancreas of rat and pig (
In the tumor samples examined in this study, the transformation was of duct adenocarcinoma origin. Given the overexpression of NTPDase1 observed previously in human melanoma cell lines (
Other cells in the human pancreas, such as endothelial cells, smooth muscle cells, nerves and, to a lesser extent, fibroblasts, showed both E-ATPDase activity and CD39 immunoreactivity. This finding is in agreement with previous experience in other types of tissue. Endothelial cells and smooth muscle cells were among the first cell types in which E-ATPase/E-ATPDase activity was demonstrated (
In conclusion, our findings suggest that NTPDase1 is not expressed in acinar cells of the human pancreas and that the duct cells express only an inactive form of the enzyme. This may indicate a different role of NTPDase1 in human pancreas compared with other species, and its differential distribution in the surrounding tissues may be associated with the processes that lead to tumor transformation.
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Acknowledgments |
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Supported by INCO program of the European Comission (ERB IC20-CT98-0202), Hungarian Committee for Technical Development (EU-98-D9-065), and Hungarian Medical Research Council 282/2000 grants, as well as Biomed contract BMH4-CT98-3805 and by grant PM97-0077 from the Direccion General de Enseñanza Superior e Investigación Científica (Spain) Grant.
We thank Drs F.X. Real and Anouchka Skoudy (IMIM; Barcelona, Spain) for their valuable help during the experimental work. We also thank Drs S.C. Robson, (Harvard BIDMC; Boston), J. Sevigny (Harvard BIDMC; Boston), and M.C. Steward (Manchester Univ., UK) for critical reading of the manuscript.
Received for publication April 23, 2001; accepted November 14, 2001.
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