Papel de las proteínas quinasas activadas por mitógenos (MAPK) en el carcinoma de células renales esporádico / Role of Mitogen-Activated Protein Kinase (MAPK) in the Sporadic Renal Cell Carcinoma

Contexto: Últimamente, basándose en la implicación del gen supresor vhl en los casos de carcinoma de células renales (CCR), se ha evaluado la implicación de la ruta de señalización entre pVHL y el factor inducible por hipoxia 1 alfa (HIF-1α), ante la necesidad de encontrar nuevos marcadores diagnósticos, pronósticos y de respuesta a fármacos. Síntesis de evidencia: La sobreexpresión de HIF-1α confiere mejor pronóstico en pacientes afectos de CCR de tipo células claras (ccRCC). Además HIF-1α regula otros genes, concretamente el de la anhidrasa carbónica IX (CA-IX), cuya sobreexpresión es prácticamente exclusiva de los ccRCC y su determinación útil para el diagnóstico de este subtipo. Sin embargo, no se ha demostrado la implicación de CA-IX ni en el pronóstico ni en la respuesta a inmunomoduladores o antiangiogénicos. Ello hace necesario la evaluación global de toda esta ruta: pVHL → HIF-1α → CA-IX, e incluso el análisis de otras proteínas y vías de señalización que también controlan la actividad de HIF-1α. En este último caso, las MAPK, son críticas en la activación de HIF-1α, existiendo evidencias a nivel experimental del control sobre su actividad, aunque no se ha establecido su papel clínico como biomarcador. Si bien está demostrado el papel de las MAPK en los fenómenos de resistencia a quimio y radioterapia convencional, no lo está en la respuesta a sorafenib, dato llamativo si tenemos en cuenta que es inhibidor de varias proteín quinasas. Recientemente se ha observado que las MAPK pueden estar implicadas en la respuesta a distintas terapias, incluidas las basadas en inhibidores de tirosín quinasa. Conclusiones: La confirmación de estos datos supondrá una explicación a la variación observada entre pacientes, que con una misma alteración funcional del gen vhl presentan un distinto comportamiento biológico y clínico, y a una mejor selección de terapias no quirúrgicas (AU)

Context: Only on the basis of the involvement of the vhl suppressor gene in the cases of renal cell carcinomas (RCC), the involvement of the signaling pathway between the pVHL and the Hypoxia inducible factor 1, alpha (HIF-1α) has been evaluated because of the need to find new diagnostic and prognostic and response to drugs markers. Evidence synthesis: The overexpression of HIF-1α confers better prognosis in clear cell type RCC (ccRCC). Furthermore, HIF-1α regulates other genes, specifically that of the carbon anhydrase IX (CA-IX), whose overexpression is practically only of the ccRCC and its determination is useful for this subtype. However, the involvement of the CA-IX has not been demonstrated in the prognosis or in the response to immunomodulators or antiangiogenics. Therefore, it is necessary to make a global evaluation of all this pathway: pVHL → HIF-1α → CA-IX, and even the analysis of other proteins and signaling pathways that also control the HIF-1α activity. In the latter case, the MAPK are critical in the HIF-1α activation, there being evidence on the experimental level of the control on its activity. although its clinical role as a biomarkers has not been established. Although the role of the MAPK in the phenomena of resistance to conventional chemotherapy and radiotherapy has been demonstrated, it has not been demonstrated in response to sorafenib, an important piece of information if we consider that it is an inhibitor of several protein kinases. Recently, it has been observed that the MAPK may be involved in the responses to different therapies, included those based on tyrosine kinase inhibitors. Conclusions: The confirmation of these data would suppose an explanation of the variation observed between patients who, with the same functional alteration of the vhl gene, have a different biological, clinical behavior and better selection of non-surgical therapies (AU)

Preliminary biological evaluation of ¹²5I-labeled anti-carbonic anhydrase IX monoclonal antibody in the mice bearing HT-29 tumors.

OBJECTIVE: To evaluate ¹²5I-labeled anti-carbonic anhydrase IX monoclonal antibody (¹²5I-MAb) as a novel single-photon emission computed tomography tracer for imaging carbonic anhydrase IX in the mice bearing HT-29 tumors. METHODS: Anti-carbonic anhydrase IX monoclonal antibody was labeled with iodine-125 by the iodogen method. The radiochemical purity of ¹²5I-MAb was measured by radio-thin-layer chromatography. The in-vitro stability of ¹²5I-MAb was determined in PBS (0.05 mol/l, pH 7.4) or new-born calf serum at 37°C, and analyzed by radio-thin-layer chromatography. A biodistribution study and planar imaging were carried out in the mice bearing HT-29 tumors. The expression of CA IX in HT-29 tumors was analyzed by immunohistochemistry. RESULTS: ¹²5I-MAb was obtained with a radiolabeling efficiency of 98%, and showed high stability in PBS and new-born calf serum. Furthermore, the biodistribution study showed specific tumor uptake in the mice bearing HT-29 tumors, and planar imaging with ¹²5I-MAb 48 h post injection showed a high concentration of radioactivity in tumors and a much decreased concentration in tumors in the blocking group. An immunohistochemical analysis showed the expression of CA IX in HT-29 tumors. CONCLUSION: The preliminary biodistribution study and results from planar imaging showed the potential of ¹²5I-MAb as an agent for tumor diagnosis and encouraged further investigation.

Hypoxia can be detected in irradiated normal human tissue: a study using the hypoxic marker pimonidazole hydrochloride.

Chronic tissue hypoxia may play a role in the pathogenesis of late radiation fibrosis. In order to investigate this hypothesis, the immunohistochemical distribution of pimonidazole hydrochloride (n = 14 patients) and carbonic anhydrase IX (CAIX) (n = 38 patients) was studied in samples of previously irradiated normal human tissue. One sample of irradiated breast tissue, which also showed marked histological features of radiation injury, stained positive for pimonidazole hydrochloride. No CAIX staining was seen in irradiated tissue other than some evidence of physiological hypoxia in the epidermis of two samples of irradiated skin; both were positive for pimonidazole and one was focally positive for CAIX. Pimonidazole hydrochloride staining of tissue with morphological changes of radiation injury could support a role for hypoxia in the pathogenesis of late normal tissue fibrosis in humans.

Band-3 crosslinking-induced targeting of mouse carrier erythrocytes.

Mouse band-3 crosslinked carrier erythrocytes have been prepared. [125I]Carbonic anhydrase (CA) has been encapsulated into mouse erythrocytes. Then, loaded erythrocytes were labelled with 51Cr. Eventually, these doubly labelled cells were crosslinked with band-3 crosslinking reagents. [125I]CA was shown to have cytosolic localization in crosslinked carrier erythrocytes. Estimation of the action of band-3 crosslinkers on mouse carrier-erythrocyte membranes rendered values around 1721% of band-3 monomer reduction. Crosslinked carrier erythrocytes were in vivo targeted to liver, as shown by chromium-labelling localization. Also, encapsulated CA radioactivity was localized in vivo predominantly in liver, which is clearly in contrast with the behaviour shown by free CA injected into animals. These results support this model as a feasible system for the analysis of carrier-erythrocyte survival and targeting as well as the in vivo efficacy of release and targeting of encapsulated compounds.

Cross-linking treatment of loaded erythrocytes increases delivery of encapsulated substance to macrophages.

Previous investigation has shown that osmotically loaded erythrocytes can act as drug carriers in systemic circulation, whereas chemically modified erythrocytes can be targeted to organs of the mononuclear phagocytic system because of changes introduced in the membrane that are recognized by macrophage cells. In this study we have examined the delivery of 125I-labelled carbonic anhydrase (125I-CA) carried by mouse erythrocytes, either loaded, or loaded and cross-linked with bis(sulphosuccinimidyl)suberate (BS3) and 3,3'-dithiobis-(sulphosuccinimidyl propionate), into homologous peritoneal macrophages maintained in culture. The hypotonically loaded mouse erythrocytes show a slight recognition by macrophages, similar to native erythrocytes. CA loaded into erythrocytes is thus delivered to a limited extent into macrophages. Neither the number of recognized loaded 51Cr-labelled erythrocytes nor the amount of delivered 125I-CA is affected by the presence of serum components or IgG. In contrast, cross-linking these loaded erythrocytes results in a greater phagocytosis by macrophages as assessed by microscopic observations, producing a markedly increased amount of targeted enzyme. The amount of CA delivered into macrophages, after BS3 cross-linker treatment of erythrocytes, is dependent on the presence of serum components in the incubation medium. Thus these cross-linking treatments improve the capacity of loaded mouse erythrocytes to deliver significant amounts of targeted enzyme to macrophage cells, increasing the therapeutic potential of carrier erythrocytes.

Comparative distribution of carbonic anhydrase isozymes III and II in rodent tissues.

Carbonic anhydrase (CA) III was demonstrated immunocytochemically in epithelium in some regions of salivary gland ducts, colon, bronchi, and male genital tract and in adipocytes, in addition to skeletal muscle and liver where the isozyme was previously localized. Basal cells beneath the submandibular gland's excretory ducts in guinea pig stained for CA III. Carbonic anhydrase III occurred alone in some and with CA II in other sites but was often absent from CA-II-containing types of cells. This was exemplified by CA III's abundance in CA-II-positive proximal colon and its sparsity in the CA-II-rich distal colon of the mouse. Striated ducts in guinea pig, but not mouse salivary glands, stained darker for CA and appeared accordingly to function more actively in ion transport compared with excretory ducts. Carbonic anhydrase content varied among genera in liver and pancreas and between mouse species and strains in salivary glands and kidney. Newly observed murine sites of CA II activity included Auerbach's plexus and a population of leukocytes infiltrating the lamina propria in small intestine, and several types of cells in the male genital tract. In immunoblot tests, antisera to CA III showed no cross reactivity with antisera to CA II, but those to CA II disclosed weak cross reactivity with CA III.

Binding of sulfonamide and acetamide to the active-site Zn2+ in carbonic anhydrase: a theoretical study.

Self-consistent field molecular orbital (SCF MO) calculations at both 4-31G and STO-3G levels have been used to examine the binding conformations of sulfonamide and acetamide compounds to the active site of carbonic anhydrase. The results are as follows: (1) sulfonamide binds to the Zn2+ ion in its deprotonated form through the sulfonamide nitrogen to the fourth coordination site of the metal ion; (2) acetamide as neutral species binds to the basic form of the enzyme through the carbonyl oxygen to the fifth coordination site of the metal ion; and (3) the acetamidate ion binds to the acid form of the enzyme through the amide nitrogen to form a tetracoordinated metal complex with three histidine ligands. Analysis of the effects of individual active-site residues on the binding conformations of these inhibitors suggests that metal alone favors bidentate coordination of sulfonamidate and acetamidate complexes and that electron donation from three histidine ligands to the metal ion determines the formation of a tetracoordinated metal complex, which is further stabilized by the presence of Thr 199, as it receives one hydrogen bond from the sulfonamide NH- or from the acetamide NH- and donates a backbone NH hydrogen bond to a sulfonamide oxygen. The calculated binding conformation of sulfonamide and the hydrogen-bonding interactions between sulfonamide and the enzyme are consistent with the X-ray diffraction study of the AMSulf-HCA II complex. However, no X-ray structures are available for amide-HCA II complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue distribution of 125I-labelled carbonic anhydrase isozymes I, II and III in the rat.

Carbonic anhydrase (CA) isozymes CA I and CA II were isolated from rat erythrocytes, and CA III from rat skeletal muscle. They were purified to homogeneity and labelled with 125I using the Bolton-Hunter method. The tissue distribution of these [125I]CA isozymes was studied in rats with whole-body autoradiography at various times after an intravenous injection. The distribution pattern showed a remarkable organ specificity. CA I and CA III were to a great extent localized to the renal cortex. This is compatible with renal uptake, secondary to glomerular filtration, of these isozymes. This would be expected from the renal handling of proteins with the following characteristics: molecular weight of 29,000; iso-electric points, pI, around 7.2 and 6.5 respectively. However, CA II of similar molecular shape and size, with a pI of 6.8, remained in the blood and was preferentially localized to the liver. Further studies are needed to clarify why such similar proteins are targeted to different organs.

Tissue and species distribution of the secreted carbonic anhydrase isoenzyme.

The secreted carbonic anhydrases, CA VI, are high molecular mass, oligomeric enzymes originally found in the sheep parotid gland and saliva. The enzymes have been purified from the saliva or parotid glands of several different species. All the CA VI enzymes studied have an apparent subunit Mr of about 45,000 as previously reported for the sheep enzyme. By Western analysis, CA VI from human, cow and dog cross-reacted with antibody raised against the purified sheep enzyme whereas that of the mouse did not. The N-terminal sequences of the sheep, human, cow and mouse enzymes are reported. The sheep, cow and human N-terminal sequences are similar to one another while the mouse sequence is substantially different. Nevertheless, the amino acids in the aromatic cluster I (Trp-5, Tyr-7, Trp-16 and Tyr/Phe-20) have all been conserved, as is the case with the cytoplasmic carbonic anhydrases. Eighteen tissues from the sheep have been examined for the presence of CA VI by Western analysis but it has been found only in the salivary glands. Northern analysis and hybridization histochemistry show that the mRNA for CA VI in sheep is expressed specifically in the acinar cells of the parotid and submandibular glands.