Expression of transmembrane carbonic anhydrases IX and XII in ovarian tumours.

AIMS: Carbonic anhydrase (CA) isozymes IX and XII have been suggested to play a role in oncogenic processes. The aim of the present study was to investigate CA IX and XII expression in patients with ovarian tumours. METHODS AND RESULTS: A series of ovarian tumours was immunostained for CA IX and XII and the results were correlated with histopathological and clinical parameters. Most cases of borderline mucinous cystadenomas, mucinous cystadenocarcinomas and serous cystadenocarcinomas were moderately or strongly positive for CA IX. In malignant tumours, the staining was most prominent in hypoxic regions. Expression of CA XII was detected in all tumour categories, although the mean staining intensity was weaker than for CA IX in all groups except for clear cell carcinomas. CONCLUSIONS: The wide expression of CA IX and XII in ovarian tumours suggests that these isozymes could represent potential targets in ovarian cancer therapy. The expression pattern of CA IX suggests that it could also serve as a useful histopathological marker protein for hypoxia in malignant ovarian tumours.

Differential expression of cytoplasmic carbonic anhydrases, CA I and II, and membrane-associated isozymes, CA IX and XII, in normal mucosa of large intestine and in colorectal tumors.

This study compares the localization of carbonic anhydrase isozymes (CA) I and II and that of IX and XII in normal large intestine and in colorectal tumors. Immunohistochemical studies were performed on 69 colorectal lesions. While the normal mucosa of the large intestine showed high expression for CA I and II, the intensity of the immunostaining for both isozymes decreased in benign lesions and was very weak in malignant tumors. The reciprocal pattern of expression observed for these cytoplasmic isozymes and transmembrane CA IX and XII in intestinal tissue specimens supports the suggestion that CA IX and XII may be functionally involved in tumor progression to malignancy and/or in invasion. By contrast, while CA I and II are prominent in normal colorectal mucosa, where they play a role in regulation of pH homeostasis and water and ion transport, loss of expression of these cytoplasmic isozymes consistently accompanies progression to malignant transformation.

Expression of the membrane-associated carbonic anhydrase isozyme XII in the human kidney and renal tumors.

Carbonic anhydrase isozyme XII (CA XII) is a novel membrane-associated protein with a potential role in von Hippel-Lindau carcinogenesis. Although Northern blotting has revealed positive signal for CA XII in normal human kidney, this is the first study to demonstrate its cellular and subcellular localization along the human nephron and collecting duct. Immunohistochemistry with a polyclonal antibody (PAb) raised against truncated CA XII revealed distinct staining in the basolateral plasma membrane of the epithelial cells in the thick ascending limb of Henle and distal convoluted tubules, and in the principal cells of the collecting ducts. A weak basolateral signal was also detected in the epithelium of the proximal convoluted tubules. In addition to the normal kidney specimens, this immunohistochemical study included 31 renal tumors. CA XII showed moderate or strong plasma membrane-associated expression in most oncocytomas and clear-cell carcinomas. The segmental, cellular, and subcellular distribution of CA XII along the human nephron and collecting duct suggests that it may be one of the key enzymes involved in normal renal physiology, particularly in the regulation of water homeostasis. High expression of CA XII in some renal carcinomas may contribute to its role in von Hippel-Lindau carcinogenesis.

Expression of transmembrane carbonic anhydrase isoenzymes IX and XII in normal human pancreas and pancreatic tumours.

Carbonic anhydrase (CA) IX and XII are transmembrane isoenzymes which are expressed in several epithelia and overexpressed in some carcinomas. They have recently been linked to von Hippel-Lindau gene-mediated carcinogenesis in that both isoenzymes are downregulated by the product of the wild-type von Hippel-Lindau tumour suppressor gene. This paper describes the localisation of CA IX and XII in the normal human pancreas and pancreatic tumours. Both isoenzymes showed positive reaction in the basolateral plasma membrane of the normal acinar and ductal epithelia. The hyperplastic ductal epithelium in tumour specimens generally showed an increased staining for CA IX. Of 29 malignant tumours of exocrine pancreas, 10 showed moderate or strong immunoreaction for CA IX. The signal for CA XII remained weak in most malignant lesions. The present results show that both CA IX and XII are unevenly expressed in the ductal and acinar compartments of the human pancreas. The expression of these isoenzymes in a relatively low number of malignant tumour specimens suggests that they have a limited value in diagnostic evaluation of pancreatic carcinoma. However, the increased expression of CA IX in hyperplastic ductal epithelium may contribute to the pancreatic tumourigenesis.

Carbonic anhydrase inhibitor suppresses invasion of renal cancer cells in vitro.

Acidification of the extracellular milieu of malignant tumors is reported to increase the invasive behavior of cancer cells. In normal tissues, production of acid is catalyzed by carbonic anhydrases (CAs), some of which are known to be overexpressed in certain cancers. To investigate the functional role of CA activity in such cancer cells, we analyzed the effect of acetazolamide, a potent CA inhibitor, on the invasive capacity of four renal carcinoma cell lines (Caki-1, Caki-2, ACHN, and A-498). We found that 10 microM acetazolamide inhibited the relative invasion rate of these cell lines between 18-74%. The Caki-2 and ACHN cell lines displayed the highest responsiveness, and their responses clearly depended on the acetazolamide concentration in the culture medium. Immunocytochemical and Western blotting results identified the presence of CA isoenzyme II in the cytoplasm of all four cell lines and CA XII on the plasma membrane in three of four cell lines. Because acetazolamide alone reduced invasiveness of these cancer cells in vitro, we conclude that the CAs overexpressed in these renal cancer cells contribute to invasiveness, at least in vitro, and suggest that CA inhibitors may also reduce invasiveness in other tumors that overexpress one or more CAs.

Expression of a novel transmembrane carbonic anhydrase isozyme XII in normal human gut and colorectal tumors.

Carbonic anhydrase isozyme XII is a recently discovered member of the alpha-carbonic anhydrase gene family with a suggested role in von Hippel-Lindau gene-mediated carcinogenesis. Increased expression of its mRNA has been observed in renal and lung carcinomas. This paper presents the localization of CA XII in the normal human gut and in colorectal tumors. Immunohistochemistry performed using a polyclonal antibody raised against truncated CA XII revealed prominent polarized staining for CA XII in the basolateral plasma membrane of the enterocytes of the normal large intestine, the reaction being most intense in the surface epithelial cuff region. Most colorectal tumors displayed abnormal expression of CA XII; the most dramatic change was observed in the deep parts of the adenomatous mucosa, where the positive immunoreaction clearly increased along with the grade of dysplasia. Adenomas with severe dysplasia and carcinomas showed an equal, diffuse staining pattern. The results indicate region-specific regulation of CA XII expression along the cranial-caudal axis of the human gut, whereas its diffuse expression in the most malignant tumors seems to correlate with their biological behavior.

Salivary carbonic anhydrase isoenzyme VI.

The carbonic anhydrases (CAs) participate in the maintenance of pH homeostasis in various tissues and biological fluids of the human body by catalysing the reversible reaction CO2 + H2O HCO3- + H+ (Davenport & Fisher, 1938; Davenport, 1939; Maren, 1967). Carbonic anhydrase isoenzyme VI (CA VI) is the only secretory isoenzyme of the mammalian CA gene family. It is exclusively expressed in the serous acinar cells of the parotid and submandibular glands, from where it is secreted into the saliva. In this review, we will discuss recent advances in research focused on the physiological role of salivary CA VI in the oral cavity and upper alimentary canal.

A low concentration of carbonic anhydrase isoenzyme VI in whole saliva is associated with caries prevalence.

Carbonic anhydrases maintain pH homeostasis in various tissues of the human body by catalyzing the reversible reaction CO2 + H2O <=> HCO3- + H+. Carbonic anhydrase isoenzyme VI (CA VI) is secreted into human saliva by the serous acinar cells of the parotid and submandibular glands. Although it represents about 3% of the total protein in stimulated parotid saliva, its exact physiological significance in the saliva has not been established. In the present study, saliva samples were collected under strictly controlled conditions from young, healthy men and assayed for CA VI concentrations using a specific time-resolved immunofluorometric assay. Salivary secretion rate, pH, buffering capacity, alpha-amylase activity levels, lactobacillus and Streptococcus mutans counts were also determined, and the results were correlated with the dental status of the subjects. Salivary CA VI concentration, pH and buffering capacity values correlated negatively with the numbers of decayed, missing and filled teeth (DMFT index). The correlations between salivary CA VI concentration and DMFT index were most significant in subjects with poor oral hygiene. No correlation was found between salivary CA VI concentration and lactobacillus or Streptococcus mutans counts. As predicted, salivary lactobacillus and Streptococcus mutans counts showed a close positive correlation with the DMFT index. In contrast, no significant correlation was seen between salivary secretion rate or amylase activity and the DMFT index. The present results indicate that low salivary CA VI concentrations are associated with increased caries prevalence, particularly in subjects with neglected oral hygiene.

Salivary carbonic anhydrase isoenzyme VI is located in the human enamel pellicle.

Salivary carbonic anhydrase (CA VI) appears to protect teeth from caries via mechanisms other than direct regulation of salivary pH and buffering capacity. To elucidate whether CA VI acts in the local microenvironment of the tooth surface, we studied the location and activity of the enzyme in the human enamel pellicle. The study was performed using a specific rabbit antiserum to human CA VI in conjunction with immunostaining and immunoblot techniques. CA activity was demonstrated using a histochemical staining method. CA VI immunostaining of extracted teeth having in vivo formed pellicle showed that the enzyme is present in the enamel pellicle. Immunostaining for salivary alpha-amylase, which is known to be present in the pellicle, showed a similar staining pattern. The presence of CA VI in the enamel pellicle was confirmed by immunoblotting of in vivo formed pellicle proteins. In vitro studies showed that CA VI binds to polished enamel surfaces from both saliva and solutions of purified enzyme. The intensity of the CA VI immunostaining on the enamel surface was dependent on the concentration of the applied enzyme. The histochemical staining of in vitro formed enamel pellicle confirmed that the bound enzyme retains its enzymatic activity. The presence of active CA VI in the human enamel pellicle suggests that it may accelerate the removal of acid by functioning locally in the pellicle layer on dental surfaces.

Salivary carbonic anhydrase VI concentration and its relation to basic characteristics of saliva in young men.

Two successive saliva samples were collected under strictly standardized conditions from 209 healthy, selected male soldiers prior to and after breakfast in the morning and were assayed for carbonic anhydrase (CA) VI concentrations using a specific time-resolved immunofluorometric assay. Salivary secretion rates, pH and buffer capacity pH values, and amylase activity levels were also determined. CA VI concentrations correlated positively to salivary secretion rates and amylase activity levels. By contrast, no significant correlation was seen between CA VI concentrations and pH or buffer capacity pH values, nor between amylase activity levels and salivary secretion rates, pH or buffer capacity pH values. The smokers had unaltered salivary secretion rates, CA VI and amylase activity levels, but lower salivary pH and buffer capacity pH values than the non-smokers. Present results suggest that salivary CA VI is not directly involved in the regulation of pH in saliva.

Secretory carbonic anhydrase isoenzyme (CA VI) in human serum.

Carbonic anhydrase VI (CA VI) is a secretory isoenzyme that, by analogy to alpha-amylase, is produced in the salivary glands and delivered into saliva. To determine whether CA VI is transferred into the circulation and is detectable in human serum, we collected blood samples from four healthy subjects at 3-h intervals throughout a 24-h period and measured concentrations of CA VI by a specific time-resolved immunofluorometric assay. All serum samples contained CA VI, the concentrations being approximately 22 times lower in serum than in the corresponding saliva samples. The presence of CA VI in serum was confirmed by Western blotting, which under reducing conditions identified a 42-kDa polypeptide band corresponding to the monomeric CA VI. The described time-resolved immunofluorometric assay for CA VI might be useful to identify or exclude diseases of the salivary glands in the differential diagnosis of patients whose serum amylase concentrations are increased.

Serum immunoreactive gastrin concentrations in the dog: basal and postprandial values measured by radioimmunoassay.

A commercially available radioimmunoassay designed for quantitating human gastrin was evaluated for use in quantitating dog gastrin. Because of the similarities in molecular structures of the two, the assay appears to be useful for dogs. Serum gastrin concentration in 12 fasting dogs averaged 64.8 pg/ml. After dogs were fed, the concentration increased to 117.8 pg/ml within 30 minutes and returned to base line by 90 minutes. A standard feeding procedure may be useful in evaluating dogs with gastrinoma.