RESUMEN
The Tn determinant (GalNAcalpha-O-Ser/Thr), normally a cryptic structure in mucin-type O-glycans, is a tumor-associated marker which has attracted particular interest in cancer biology. We herein report the characterization of N-nitrosomethylurea (NMU)-induced breast cancer in rats as a new model for the study of aberrant O-glycosylation products. Tn-antigen expression is detectable not only in mammary carcinoma induced by NMU but also in carcinogen-initiated mammary epithelium, indicating that Tn could be a pre-cancerous biomarker in rats treated with NMU. Serum Tn levels were followed up longitudinally in 30 rats from the time of the first injection of NMU to the development of advanced breast cancer. Tn antigen increased in serum several weeks before tumor development, and became highly positive after 56 days of carcinogenesis (prior to breast-cancer occurrence), and the levels correlated with Tn expression in mammary tissues. However, during the follow-up after detection of mammary cancer, all animals displayed a significant decrease of serum Tn antigen, and low levels were observed in animals with advanced breast cancer. We have shown that the humoral immune response to cancer, with the production of anti-Tn antibodies, could hamper the detection of Tn antigen in animals with advanced breast cancer. These results suggest that NMU-induced rat mammary carcinogenesis is a useful experimental model to study the regulation of O-glycosylation at the cellular level during malignant transformation.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/análisis , Biomarcadores de Tumor/análisis , Glándulas Mamarias Animales/química , Neoplasias Mamarias Experimentales/metabolismo , Metilnitrosourea/toxicidad , Lesiones Precancerosas/metabolismo , Animales , Complejo Antígeno-Anticuerpo/sangre , Antígenos de Carbohidratos Asociados a Tumores/sangre , Biomarcadores de Tumor/sangre , Femenino , Glicosilación , Inmunohistoquímica , Neoplasias Mamarias Experimentales/inducido químicamente , Lesiones Precancerosas/inducido químicamente , Ratas , Ratas WistarRESUMEN
Using synthetic Tn (GalNAc-O-Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti-Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/inmunología , Lectinas de Plantas , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Carbohidratos Asociados a Tumores/química , Biomarcadores de Tumor/inmunología , Técnicas Biosensibles , Epítopos/química , Glicopéptidos/síntesis química , Glicopéptidos/inmunología , Glicosilación , Cinética , Lectinas/inmunología , Ratones , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
We report here the first amino acid sequence of an anti-Tn monoclonal antibody raised against human breast cancer cells and show that a single chain Fv fragment of this IgM retains the Tn-binding specificity as defined by functional assays with asialo-OSM and membrane extracts from MCF-7 cells. Sequence comparisons and molecular modeling of 83D4 indicate that the antibody combining site displays a cavity-like feature primarily defined by the CDR H1 and H2 loops. This pocket could accommodate a single Tn molecule, thus, suggesting a structural explanation for the predominant expression of a particular VH gene segment in a group of antibodies that recognize tumor-associated antigens arising from an aberrant O-glycosylation.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Inmunoglobulina M/inmunología , Región Variable de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Antineoplásicos/genética , Especificidad de Anticuerpos , Antígenos de Carbohidratos Asociados a Tumores/genética , Secuencia de Bases , Neoplasias de la Mama/inmunología , Carcinoma/inmunología , Clonación Molecular , Amplificación de Genes , Humanos , Hibridomas , Fragmentos de Inmunoglobulinas/genética , Inmunoglobulina M/genética , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
BACKGROUND: The detection of occult carcinoma cells in patients with breast cancer may aid determination of prognosis and the development of new therapeutic approaches. In this study, we report a new method to detect rare human breast cancer cells, which combines an immunomagnetic separation (IMS) procedure with cytokeratin 19 (CK 19) immunostaining. MATERIALS AND METHODS: Four monoclonal antibodies (MAb) previously characterized against cell surface antigens (1BE12, ED8, 7B10 and 83D4), were evaluated for IMS optimization. Immunoseparated epithelial cells were identified using a MAb against CK 19. We compared the IMS procedure with the immunocytochemistry (ICC) and the RT-PCR for CK 19 on an "in vitro" experimental model. RESULTS: The best results in IMS procedures were obtained using MAbs 1BE12 (directed against Lewis y antigen) and ED8 (directed against MUC 1). In reconstitution experiments, using several ratios of T47D cells mixed with peripheral-blood mononuclear (PBMN) cells, the IMS procedure reliably detects one mammary carcinoma cell in 5 x 10(5) PBMN cells, whereas the ICC detects up to one T47D cell per 10(5) PBMN cells. The best sensitivity was observed with the RT-PCR (up to one T47D cell per 10(6) PBMN cells). We found the same high specificity with the three methods evaluated. CONCLUSIONS: The IMS procedure using MAbs 1BE12 or ED8 associated with CK 19 immunostaining is a specific, sensitive, and feasible method for the detection of rare human breast cancer cells. This method proved to be better than the ICC staining but its sensitivity was lower than that of RT-PCR for CK 19.