RESUMEN
Tumor-associated protein survivin is the bifunctional protein which can participate either in cell division regulation or in apoptosis inhibition depending on its localization and structure state. The aim of this work was to obtain monospecific antibodies useful for investigation of protein structure and functional features. Six affinity purified antibodies directed to different protein regions were obtained. The ability of antibodies obtained to detect survivin in tumor cells and breast cancer tissues was studied. It was shown that antibodies to (1-22) and (95-105) survivin fragments have the highest specific activity. In western-blot antibodies to (1-22) region predominantly binds with survivin-containing complex, which may be the survivin dimer as we suppose, while antibodies to (95-105) region detects only monomeric form of the protein. Breast cancer tissues study demonstrated that survivin monomer presents only in the tumor core tissues, while survivin-containing complex is expressed both in tumor core and tumor periphery tissues. It was shown that antibodies to (1-22) fragment detect predominantly nuclear survivin, which participates in mitosis regulation, while antibodies to (95-105) fragment gave nucleoplasm and cytoplasm staining at all stages of cell cycle. Thereby antibodies obtained are the useful tool for structure-functional study of survivin.
Asunto(s)
Anticuerpos/inmunología , Neoplasias de la Mama/inmunología , Proteínas Inhibidoras de la Apoptosis/inmunología , Péptidos/inmunología , Anticuerpos/química , Afinidad de Anticuerpos/inmunología , Neoplasias de la Mama/patología , Femenino , Células HeLa , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Péptidos/química , Péptidos/aislamiento & purificación , SurvivinRESUMEN
Whether the expression of the apoptosis inhibitor survivin was correlated with the degree of differentiation and the stage of transitional cell carcinoma of the urinary bladder was studied. Sixty samples of surgical specimens from patients with urothelial carcinomas of various degrees of differentiation and different stages were examined. An immunohistochemical study using the monoclonal antibodies obtained by the authors was conducted. The high expression of survivin was shown to be correlated with the lower-grade differentiation of a tumor and its higher stage.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Transicionales/patología , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Apoptosis , Carcinoma de Células Transicionales/metabolismo , Núcleo Celular/patología , Citoplasma/patología , Expresión Génica , Humanos , Estadificación de Neoplasias , Pronóstico , Survivin , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Survivin, an endogenous protein, is a promising marker for the diagnosis of cancer. The aim of the present work was to obtain antibodies specific to survivin and capable of detecting this protein in tumor tissues. Four peptides corresponding to fragments (1-22), (54-74), (80-88)-(153-165), and (118-144) of the survivin-2B sequence were selected and synthesized. Rabbits were immunized with the synthetic peptides. It has been shown that all peptides in a free state, without conjugation with a high-molecular-weight carrier, stimulate the production of antibodies capable of binding with recombinant survivin. Antipeptide antibodies were isolated from sera and their performance in the immunohistochemical detection of survivin in human tumor tissues was studied. It was shown that only antibodies to the (80-88)-(153-165) peptide bind to the survivin present in breast and bladder tumors. The ability of antibodies to this peptide to detect survivin in tumor tissue lysates was demonstrated by immunoblotting. The part of the sequence targeted by the antibodies against the (80-88)-(153-165) peptide was localized using truncated peptide fragments.
Asunto(s)
Anticuerpos/aislamiento & purificación , Neoplasias de la Mama/química , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/inmunología , Oligopéptidos/inmunología , Fragmentos de Péptidos/inmunología , Neoplasias de la Vejiga Urinaria/química , Secuencia de Aminoácidos , Animales , Biomarcadores de Tumor/análisis , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/química , Datos de Secuencia Molecular , Oligopéptidos/química , Fragmentos de Péptidos/química , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , SurvivinRESUMEN
Immunoactive fragments corresponding to the N-terminal (19-36) and C-terminal (283-294) regions of the NPM1.1 isoform of nucleophosmin and their shortened fragments were chosen and synthesized. Rabbits were immunized with free full-size peptides and their protein conjugates. Antibodies produced against the 19-36 and 283-294 peptides were purified by affinity chromatography on bromocyanogen-activated sepharose that was preliminary conjugated with the synthetic peptides. An analysis of immunoblots of lysates of the HeLa and Ramos cells demonstrated that the antibodies produced against the 19-36 peptide detected the monomeric form of nucleophosmin, whereas the antibodies against the 283-294 peptide predominantly revealed its oligomeric form. It was established by immunocytochemical analysis that the antibodies induced by the 19-36 peptide stained the nucleoplasm and perinuclear space of the cytoplasm of the HeLa and Ramos cells, but did not stain the nucleoli, while the antibodies against the 283-294 peptide stained only the nucleoli of the same cells. On the basis of these results, one could propose that the monomeric and oligomeric forms of nucleophosmin were located in the nucleoplasm and nucleoli of the examined cells, respectively. Thus, antibodies which can predominantly detect monomeric and oligomeric forms of nucleophosmin were produced for the first time. An analysis of the monomeric-oligomeric state and the location of the nucleophosmin in tumor cells could be performed using these antibodies.