Cíl: Cílem práce bylo prokázat přítomnost solubilní formy CA IX proteinu (s-CA IX) v kultivačním médiu nádorových buněk in vitro a v tělních tekutinách in vivo u CA IX protein pozitivních nádorů. Následně přizpůsobit současné diagnostické metody ke stanovení s-CA IX a pokusit se zjistit jeho možný význam jako markeru u adenokarcinomu ledviny (RCC) a uroteliálních karcinomů (TCC). Materiál a metoda: Do studie jsme zahrnuli dvě skupiny: 1. nemocní s RCC, 2. nemocní s TTC vývodných močových cest. V první skupině bylo testováno 31 pacientů s RCC a 61 kontrol, ve druhé 32 pacientů s endoskopickým podezřením na TCC a 16 kontrol. K průkazu CA IX antigenu ve vzorcích nádorů jsme použili imunohistochemické barvení pomocí monoklonální protilátky M75. V extraktech z nádorů a v tělních tekutinách jsme přítomnost CA IX stanovovali metodou "Western blot" nebo ELISA. Výsledky: V 1. skupině jsme v séru přítomnost s-CA IX zjistili pouze v podskupině s prokázaným RCC. Z 31 vzorků jich bylo 13 (41,9%) pozitivních. V moči v podskupině s RCC byl s-CA IX protein prokázán u 19 (70 %) z 27 vzorků. V kontrolní skupině byly všechny vzorky s-CA IX negativní. Ve 2. skupině byl TCC histologicky prokázán u 23 nemocných. Pozitivní nález s-CA IX v moči byl u 16 z nich (69,6 %). Negativní nález s-CA IX byl u 7 (30, 4%) nemocných. U žádného z TCC pozitivních pacientů jsme v séru s-CA IX antigen neprokázali. Závěr: Naše práce prokázala uvolňování CA IX do tělních tekutin u nemocných s RCC a TCC a možnost jeho detekce. Western blot v kombinaci s použitím zvýšené chemiluminiscence je specifickou a vysoce citlivou metodou pro laboratorní hodnocení, avšak je to technika pracná a časově náročná. Současné výsledky ukazují, že by se dalo uvažovat o vyvinutí vhodné kvantitativní metody pro stanovení s-CA IX, jež by umožnila zpracování větších souborů nemocných ke zhodnocení klinického významu jeho exprese u nemocných s RCC a TCC.
Aim: The aim of our study was to investigate the presence of soluble CA IX protein (s-CA IX) in tumor culture media in vitro and body fluids of patients carrying CA IX protein positive tumors. The final purpose was to improve present diagnostic methods to s-CA IX detection and to determine its possible use as a marker of renal clear cell carcinoma (RCC) and transitional cell carcinoma (TCC). Material and methods: In the preliminary study were two groups of patients: 1. RCC patients, 2. TCC patients. In the first group were included 31 RCC patients and 61 controles, in the second group 32 patients with endoscopic suspicion for TCC and 16 controles. We used immunohistochemical staining employing MAb M75 for CA IX identification in tumor tissue. In tumor extracts and body fluids we used Western blotting and ELIS A for quantitation of the s-CA IX protein. Results: In the RCC group s-CA IX protein was detected only in patients with proven RCC. In the blood 13 (41,9%) from 31 were positive. In urine 19 (70%) from 27 were CA IX protein positive. Control specimens were all s-CA IX negative. In the TCC group the tumor was identified in 23 patients histologically Positive s-CA IX was in 16 (69,6%) of them. Negative finding of s-CA IX was in 7 (30,4%) patients. Serum from all TCC patients was s-CA IX negative, even the serum from patients with CA IX positive urine. Conclusion: We demonstrated shedding of CA IX into body fluids in patients with RCC and TCC, although in exremely low concentrations. Nevertheless, Western blotting combined with enhanced chemiluminiscence is specific and extremely sensitive method allowing s-CA IX detection in body fluids. Our present data suggest that suitable quantitave method for s-CA IX detection should be developed, which in turn will enable evaluation of clinical significance of CA IX expression in RCC and TCC patients.
- Keywords
- uroteliální nádory vývodných močových cest,
- MeSH
- Antigens, Neoplasm isolation & purification blood urine MeSH
- Enzyme-Linked Immunosorbent Assay utilization MeSH
- Financing, Organized MeSH
- Carbonic Anhydrases isolation & purification blood urine MeSH
- Carcinoma, Transitional Cell diagnosis enzymology etiology MeSH
- Carcinoma, Renal Cell diagnosis MeSH
- Humans MeSH
- Biomarkers, Tumor isolation & purification blood urine MeSH
- Neoplasm Proteins isolation & purification blood urine MeSH
- Kidney Neoplasms diagnosis MeSH
- Urinary Bladder Neoplasms diagnosis MeSH
- Case-Control Studies MeSH
- Urothelium pathology MeSH
- Blotting, Western utilization MeSH
- Check Tag
- Humans MeSH
We investigated the expression of cell-associated CAIX protein in histological sections of the transitional cell carcinoma (TCC) of the urinary tract and of the soluble form of CAIX (s-CAIX) shed by the tumor into the serum and urine of TCC patients. A total of 23 patients with histologically confirmed TCC or squamous cell carcinoma (SCC) were enrolled in the pilot study. Sixteen healthy individuals served as controls. Membrane-bound CAIX was present in the tumor cells near the endoluminal surface. Necrosis was observed in only 4 samples. Using Western blots, s-CAIX concentrated from urine was visualized as a double band at 50 and 54 kDa. In most cases, the presence of s-CAIX in the urine correlated with CAIX expression in the tumor. On the other hand, s-CAIX did not exceed the normal level in the serum of TCC patients. Urine from patients with TCC of the urinary bladder and renal pelvis contained s-CAIX, allowing the detection of tumors in approximately 70% of the patients. Moreover, two additional patients with suspected, but unconfirmed bladder tumor, with s-CAIX detected in urine, developed tumors identified as TCC within six months. We suggest that after a simple, rapid and sensitive test, monitoring s-CAIX levels in urine will be developed, it may be useful for early detection of relapse in patients following transurethral tumor resection.
- MeSH
- Antigens, Neoplasm metabolism MeSH
- Cell Membrane enzymology pathology MeSH
- Adult MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Immunoenzyme Techniques MeSH
- Carbonic Anhydrases metabolism MeSH
- Carcinoma, Transitional Cell enzymology blood urine MeSH
- Kidney Pelvis enzymology pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Urinary Bladder enzymology pathology MeSH
- Biomarkers, Tumor metabolism MeSH
- Pilot Projects MeSH
- Prognosis MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Neoplasm Staging MeSH
- Case-Control Studies MeSH
- Urologic Neoplasms enzymology blood urine MeSH
- Blotting, Western MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
Specific antibodies interfere with the function of human tumor-associated carbonic anhydrase IX (CA IX), and show potential as tools for anticancer interventions. In this work, a correlation between structural elements and thermodynamic parameters of the association of antibody fragment Fab M75 to a peptide corresponding to its epitope in the proteoglycan-like domain of CA IX, is presented. Comparisons of the crystal structures of free Fab M75 and its complex with the epitope peptide reveal major readjustments of CDR-H1 and CDR-H3. In contrast, the overall conformations and positions of CDR-H2 and CDR-L2 remain unaltered, and their positively charged residues may thus present a fixed frame for epitope recognition. Adoption of the altered CDR-H3 conformation in the structure of the complex is accompanied by an apparent local stabilization. Analysis of domain mobility with translation-libration-screw (TLS) method shows that librations of the entire heavy chain variable domain (V(H)) decrease and reorient in the complex, which correlates well with participation of the heavy chain in ligand binding. Isothermal titration microcalorimetry (ITC) experiments revealed a highly unfavorable entropy term, which can be attributed mainly to the decrease in the degrees of freedom of the system, the loss of conformational freedom of peptide and partially to a local stabilization of CDR-H3. Moreover, it was observed that one proton is transferred from the environment to the protein-ligand complex upon binding. Molecular dynamics simulations followed by molecular mechanics/generalized Born surface area (MM-GBSA) calculations of the ligand (epitope peptide) binding energy yielded energy values that were in agreement with the ITC measurements and indicated that the charged residues play crucial role in the epitope binding. Theoretical arguments presented in this work indicate that two adjacent arginine residues (ArgH50 and ArgH52) are responsible for the observed proton transfer. 2007 Wiley-Liss, Inc.
- MeSH
- Antigens, Neoplasm chemistry immunology MeSH
- Epitopes chemistry immunology MeSH
- Financing, Organized MeSH
- Immunoglobulin Fab Fragments chemistry MeSH
- Isoenzymes chemistry immunology MeSH
- Calorimetry MeSH
- Carbonic Anhydrases chemistry immunology MeSH
- Crystallography, X-Ray MeSH
- Humans MeSH
- Molecular Sequence Data MeSH
- Antibodies, Monoclonal chemistry MeSH
- Cell Line, Tumor MeSH
- Computer Simulation MeSH
- Amino Acid Sequence MeSH
- Thermodynamics MeSH
- Binding Sites, Antibody MeSH
- Check Tag
- Humans MeSH
- Publication type
- Comparative Study MeSH
Carbonic anhydrases (CAs) are physiologically important enzymes that catalyze a reversible conversion of carbon dioxide to bicarbonate and participate in ion transport and pH control. Two human isoenzymes, CA IX and CA XII, are overexpressed in cancer and contribute to tumor physiology. Particularly CA IX is confined to only few normal tissues but is ectopically induced in many tumor types mainly due to its strong transcriptional activation by hypoxia accomplished via HIF-1 transcription factor. Therefore, CA IX can serve as a surrogate marker of hypoxia and a prognostic indicator. CA IX appears implicated in cell adhesion and in balance of pH disturbances caused by tumor metabolism. Both tumor-related expression pattern and functional involvement in tumor progression make it a suitable target for anticancer treatment. Here we summarize a current knowledge on CA IX and CA XII, and discuss possibilities of their exploitation for cancer detection, diagnostics, and therapy.
- MeSH
- Financing, Organized MeSH
- Carbonic Anhydrases metabolism MeSH
- Humans MeSH
- Neoplasms enzymology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
- MeSH
- Arenavirus genetics immunology growth & development MeSH
- Bioterrorism trends MeSH
- Coronavirus genetics immunology growth & development MeSH
- Orthohantavirus genetics immunology growth & development MeSH
- Hemorrhagic Fever, Ebola etiology genetics immunology MeSH
- HIV Infections genetics immunology virology MeSH
- Lassa Fever etiology genetics immunology MeSH
- Humans MeSH
- Severe acute respiratory syndrome-related coronavirus genetics immunology growth & development MeSH
- Viruses genetics immunology growth & development MeSH
- Virus Shedding genetics immunology MeSH
- Check Tag
- Humans MeSH
