Expression of Hypoxic Marker Carbonic Anhydrase IX Predicts Poor Prognosis in Resectable Hepatocellular Carcinoma

Carbonic anhydrase IX (CA-IX), a hypoxia marker, correlates with tumor progression in a variety of human cancers. However, the role of CA-IX in hepatocellular carcinomas (HCCs) remains largely unknown. We examined the expression of 277 unifocal, resectable, primary HCC tumors using immunohistochemistry. The CA-IX protein was expressed in 110 of the 227 (48.5%) HCC tumors. The expression of CA-IX correlated with younger age (P = 0.0446), female sex (P = 0.0049), high serum α-fetoprotein levels (P<1x10^-6), larger tumor size (P = 0.0031), high tumor grade P<1x10^-6) and high tumor stage (P = 1.5x10^-6). Patients with HCC tumors that expressed CA-IX were more likely to have lower 5-year disease-free survival (DFS; P = 0.0001) and 5-year overall survival (OS; P<1x10^-6). The multivariate analysis indicated that CA-IX expression was an independent predictor for high tumor stage (P = 0.0047) and DFS (P = 0.0456), and a borderline predictor for OS (P = 0.0762). Furthermore, CA-IX expression predicted poor DFS and OS in patients with high tumor stage (P = 0.0004 and P<1x10^-6, respectively). Interestingly, CA-IX expression might contribute to the worse prognosis of female patients with advanced HCCs. Our study indicates the expression of the CA-IX protein is a crucial predictor of poor prognosis in resectable HCC, and it is also an unfavorable prognostic predictor in HCC patients with high tumor stage.     

Carbonic Anhydrase IX Expression is Associated with Favorable Prognostic Factors in Small Intestinal Carcinoma

Tumor hypoxia is associated with more aggressive behavior and resistance to chemotherapy and radiotherapy. Carbonic anhydrase IX (CA9) level increases under hypoxia and is related to poor prognostic factors. The aim of this study was to evaluate the expression of CA9 and to identify its prognostic significance in small intestinal carcinomas (SICs). CA9 expression was observed in 36% (63/175) of SICs. CA9 expression showed significant correlation with well- and moderately differentiated tumors compared with poorly differentiated tumors (p=0.039), tumors with no lymph node metastasis (p=0.005), and lower stage carcinomas (p=0.009). CA9 expression showed an inverse correlation with perineural invasion (p=0.021) and lymphatic invasion (p=0.022). No significant correlation was observed between CA9 expression and gross type, histological type, pathological tumor (pT) classification, vascular invasion, pancreas invasion, and retroperitoneal seeding. SICs with CA9 overexpression showed better overall survival compared with those with no or weak CA9 expression (p=0.048). In the multivariate analysis, poorly differentiated SICs (p<0.001) and SICs with lymph node metastasis (p=0.002) were independent poor prognostic factors. CA9 expression in SICs is more frequently associated with good prognostic markers and better overall survival; however, it is not an independent prognostic factor.     

Carbonic Anhydrase in Chlamydomonas reinhardtii II. Requirements for Carbonic Anhydrase Induction

Carbonic anhydrase (CA) activity in wild type cells of Chlamydomonasreinhardtii was low when cells were cultured under 2% CO₃ inthe light. When the gas phase was changed to air, CA activityincresaed as much as 20 fold over the next 24 hours. In contrast,CA activity did not change markedly in cells of the mutantspet 20-8 (PS II-negative), lip 10-2 (photophosphorylation-negative),and F60 (phosphoribulokinase-negative), when they were subjectedto the same induction regimen. DCMU (10^–5 M) and cydoheximide(3 µg/ml) severely inhibited the induction in wild typecells. No induction occured when CO₂ concentration was loweredin darkness. ³Present adress: Photoconversion Research Branch, Solar EnergyResearch Institute, Golden, Colorado 80401, USA. (Received June 7, 1982; Accepted December 25, 1982)     

Redox State and Carbonic Anhydrase Isozyme IX Expression in Human Renal Cell Carcinoma: Biochemical and Morphological Investigations

Clear renal cell carcinomas (RCC) frequently express carbonic anydrase IX (CA IX) because of non-functional mutation of von Hippel Lindau (VHL) tumor suppressor gene. CA IX is a tumor-associated transmembrane antigen, which catalyzes the extracellular, reversible hydration of carbon dioxide to bicarbonate and proton and thereby contributes to acidification of extracellular milieu. Extracellular acidic pH facilitates tumor growth and progression. CA IX expression is upregulated by Hypoxia Inducible Factor-1 (HIF-1), which is negatively controlled by oxygen via wild type VHL protein and is also regulated by the cell redox state. We investigated the immunohistochemical pattern of distribution of CA IX in a small series (14 cases) of RCCs. CA IX expression was matched with the redox state of RCC, stratifying our series in relation to clinical and histopathological parameters, such as Fuhrman grade, staging, proliferation markers expression, and particularly, the presence of necrosis. Our results show for the first time the existence of a perivascular pattern of CA IX distribution in RCC. We also found a significant relationship between CA IX expression and the presence of necrosis. Tumors with higher CA IX expression exhibited higher degree of necrosis [Formula: See Text] Notably, an almost significant relationship between the redox state and CA IX expression was detected in RCC patients with 5 years disease-free survival, most of them showing organ-confined disease. Tumors with lower redox state showed an algebraically higher degree of CA IX expression. On the contrary, tumors with higher redox state exhibited an algebraically lower CA IX expression [Formula: See Text] The observed relationship of CA IX expression and necrosis suggests a role for CA IX in RCC. Further investigations are necessary to further establish the role of the redox state in regulation of CA IX expression in RCC.     

Carbonic Anhydrase in Marine Algae

An electrometric system for determination of carbonic anhydrase activity was constructed. Enzyme activity was assayed in homogenates of marine macroscopic Chlorophyta, Rhodophyta, and Phaeophyta. Plants surveyed included Ulva expansa (Setchell) Setchell and Gardner, Codium fragile (Suringar) Hariot, Enteromorpha sp., Chaetomorpha torta (Farlow) McClatchie (Chlorophyta); Laurencia papillosa (Greville). Plocamium coccineum var. pacificum (Kylin) Dawson, Pterocladia capillacea (Gmelin) Bornet and Thuret. Gigartina armata J. Agardh (Rhodophyta); Eisenia arborea Areschoug and Macrocystis pyrifera (Linnaeus) C. A. Agardh (Phaeophyta). Activity was present in all algae; in the Phaeophyta this could be demonstrated only after dialysis. p-Chloromercuriphenylsulfonic acid (10⁻⁴m) decreased activity in 1 species, Plocamium; this inhibition could be almost completely overcome with the addition of 10⁻³m dithiothreitol. In 2 green and 2 red algae assayed for sensitivity to acetazolamide (Diamox), inhibition was complete at 10⁻⁴m concentration of inhibitor. Dithiothreitol at a concentration of 10⁻³m did not enhance activity in any of the homogenates, and was not necessary for enzyme expression.     

Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H⁺ (mg Chl)^–1 h^–1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal -CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO₃ ^– dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H⁺ (mg Chl)^–1 h^–1) were observed in the presence of detergents (Triton X-100 or n-octyl--D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H⁺ (mg Chl)^–1 h^–1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.This revised version was published online in October 2005 with corrections to the Cover Date.     

Carbonic Anhydrase in Anoxygenic Phototrophic Bacteria

Microbiology - The genes encoding carbonic anhydrase (CA) were found in all anoxygenic purple bacteria. The genes of α- and β-CA were found in purple nonsulfur bacteria of the class...     

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii with a Carbonic Anhydrase-Directed Photoaffinity Label

A carbonic anhydrase (CA)-directed photoaffinity reagent, 125I-labeled p-aminomethylbenzenesulfonamide-4-azidosalicylamide,was synthesized and shown to derivatize periplasmic CA in the unicellular green alga Chlamydomonas reinhardtii. The photoderivatization of purified C. reinhardtii periplasmic CA or intact C. reinhardtii cells with the reagent resulted in the modification of the large (37 kD) subunit of the enzyme. Photoderivatization of proteins in lysed C. reinhardtii cells also resulted in the specific labeling of a polypeptide of 30 kD. Centrifugation of the cell extract prior to photoaffinity labeling revealed that the labeled peptide was present predominantly in a particulate fraction. The photoaffinity-labeled 30-kD polypeptide was not observed in extracts from a mutant of C. reinhardtii that is believed to be deficient in an intracellular form of CA. These results provide evidence that the 30-kD polypeptide, which is photoaffinity labeled in lysed C. reinhardtii cells, is an intracellular form of CA.     

碳酸酐酶与临床医学

碳酸酐酶是一类含锌的蛋白酶,共发现了10种同工酶及三种碳酸酐酶相关蛋白,广布于人体各组织,能可逆性地催化C02的水合反应,参与调节pH、离子运输等多种生理过程,碳酸酐酶的缺乏和异常将可能会导致一系列的疾病。     

Histochemical Demonstration of Carbonic Anhydrase Activity

Fresh tissue slices fixed in chilled acetone for 1 hour and washed in distilled water for 10-30 minutes were incubated for 30-45 minutes at 37°C. in the freshly prepared incubating mixture: filtrate of a mixture of 8% sodium bicarbonate, 100 ml., and MnCl₂·4H₂O, 1 g. After washing in distilled water for 1 hour, they were dehydrated and embedded in paraffin. Sections were cut 15-20μU, deparaffinized, rinsed in absolute alcohol and placed in a 0.1% solution of potassium periodate for 48 hours at 37°C. The mounted sections were counterstained (if desired), dehydrated in alcohol, cleared in xylene (not carbol-xylene) and mounted in balsam. Many brown granules were produced on the sites of enzyme activity by this procedure. The results obtained seem to be in good agreement with previous findings by biochemical determinations.     

Guanidine Hydrochloride Unfolding of a Carbonic Anhydrase Molten Globule

Guanidine hydrochloride-induced unfolding of a carbonic anhydrase molten globule was studied by high-resolution nuclear magnetic resonance spectroscopy. The study resulted in estimation of the number of water and denaturant molecules bound to the molten globule at various denaturant concentrations in solution. When compared with the data on unfolding of native carbonic anhydrase, these estimates indicate that the unfolding is underlain by an increased local concentration of the denaturant near the protein molecule, which results from the increased ratio between guanidine hydrochloride-bound and protein-bound waters.     

Carbonic Anhydrase Polymorphism in Cattle and Swine

Two electrophoretically different carbonic anhydrase (Ca) isoenzymes have been demonstrated in cattle where their presence is presumed to be controlled by codominant allelic genes (Sartore & Bernoco 1966). Previous reports of Ca polymorphism in swine are not available.     

Carbonic Anhydrase and the Regulation of Photosynthesis

THE role of CO₂ in the regulation of photosynthetic and respiratory metabolism in plants is little understood in the unicellular alga Chlorella pyrenoidosa; for example, after autotrophic growth in high CO₂ (5·5% by volume), transfer to a CO₂ concentration about ten times less than the concentration in air results initially in low rates of photosynthesis characterized by the virtual absence of the Calvin cycle¹ of CO₂ fixation². An induction period of about 2 h is necessary before normal photosynthetic rates are established. Cells grown in air (0.03% CO₂) do not show this effect and photosynthesize at comparatively high rates even in very low concentrations of CO₂.     

Carbonic Anhydrase of Chlamydomonas: Purification and Studies on its Induction Using Antiserum against Chlamydomonas Carbonic Anhydrase

Carbonic anhydrase (EC 4.2.1.1[EC]; CA) was purified by affinitychromatography from cells of the unicellular green alga Chlamydomonasreinhardtii which had been grown photoautotrophically in ordinaryair. Antiserum raised in rabbit against this purified CA crossreactedwith Chlamydomonas CA but not with spinach leaf CA nor bovineerythrocyte CA. When the CO₂ concentration provided to the algalcells was decreased from 4% to the ordinary air level (0.04%),CA activity and the content of CA protein determined by theimmunodiffusion test showed parallel increases. In contrast,when the CO₂ concentration was raised from air level to 4% CO₂CA activity and its content expressed on the basis of culturevolume remained rather constant. These results indicate thatsynthesis of the CA protein is induced when the CO₂ concentrationis lowered from 4 to 0.04% during algal growth. On the otherhand, the synthesis of CA stops when CO₂ concentration is raisedfrom air level to 4%. (Received June 30, 1984; Accepted October 8, 1984)     

Expression and Functional Study of Single Mutations of Carbonic Anhydrase 8 in Neuronal Cells

Cellular and Molecular Neurobiology - Carbonic anhydrase 8 (CA8), an isozyme of α-carbonic anhydrases, lacks the ability to catalyze the reversible hydration of CO2 to bicarbonate and proton....     

Carbonic Anhydrase Activity and CO2-Transfer Resistance in Zn-Deficient Rice Leaves

It has been reported that carbonic anhydrase (CA) activity in plant leaves is decreased by Zn deficiency. We examined the effects of Zn deficiency on the activity of CA and on photosynthesis by leaves in rice plants (Oryza sativa L.). Zn deficiency increased the transfer resistance from the stomatal cavity to the site of CO₂ fixation 2.3-fold and, consequently, the value of the transfer resistance relative to the total resistance in the CO₂-assimilation process increased from 10% to 21%. This change led to a reduced CO₂ concentration at the site of CO₂ fixation, resulting in an increased gradient of CO₂ between the stomatal cavity and this site. The present findings support the hypothesis that CA functions to facilitate the supply of CO₂ from the stomatal cavity to the site of CO₂ fixation. We also showed that the level of mRNA for CA decreased to 13% of the control level during Zn deficiency. This decrease resembled the decrease in CA activity, suggesting the possible involvement of the CA mRNA level in the regulation of CA activity.