RESUMO
Serum from patients with different malignancies contain an abnormal concentration of a a1-acidic-glycoprotein (AAG) and also, increased levels of AAG are associated with the presence of tumor mass. In the present report, serum levels of AAG were measured by radial immunodiffusion in squamous cell carcinoma of the head and neck (SCCHN) patients taking into account disease status parameters such as tumor localization, stage and extension of disease. Immunohistochemical methods, SDS-PAGE and Western-blotting were employed to study the expression of AAG and a carbohydrate related antigen (sialyl Lewis x) in tumor tissues and derived fractions. AAG showed abnormal levels in 7/15 oral cavity tumor patients sera, 2/5 oropharynx and 5/10 larynx tumors; increased AAG serum levels belonged to patients with disseminated disease. On the other hand, the presence of AAG and sialyl Lewis x were demonstrated in carcinoma cells and in derived fractions from tumor tissues belonging to patients with elevated AAG serum levels. In the present study, we have found elevated levels of AAG in serum samples from SCCHN patients; these neoplastic cells are capable to express AAG.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/química , Neoplasias de Cabeça e Pescoço/química , Proteínas de Neoplasias/análise , Orosomucoide/análise , Adenocarcinoma/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/sangue , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias Colorretais/química , Células Epiteliais/química , Feminino , Neoplasias de Cabeça e Pescoço/sangue , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/sangue , Estadiamento de Neoplasias , Oligossacarídeos/análise , Especificidade de Órgãos , Orosomucoide/biossíntese , Antígeno Sialil Lewis XRESUMO
There is convincing epidemiological evidence that multiparity provides protection against the development of breast cancer. In the present study we evaluated the levels of MUC1 and MUC1 circulating immune complexes (MUC1-CIC) in 135 serum samples obtained from healthy women. The study population included 13 women who had never been pregnant, 31 primiparous pregnant women, 36 multiparous pregnant women who had lactated, 5 multiparous pregnant women who had never lactated, 24 multiparous non-pregnant women who were lactating at the time of the study, 24 multiparous non-pregnant women who had lactated, and 2 multiparous non-pregnant women who had never lactated. The purpose of this work was to detect MUC1 variations during pregnancy and lactation as well as to study the possible induction of a humoral immune response against MUC1 in these conditions. We employed ELISA techniques to measure MUC1 (CASA test) and MUC1-CIC (IgM and IgG) using two anti-MUC1 monoclonal antibodies (MAbs): C595 and SM3. Statistical analysis was performed using the ANOVA test. The pooled results pertaining to pregnant versus non-pregnant women were compared and significant differences were observed in MUC1 and MUC1-CIC-lgM levels detected with both MAbs; the MUC1-CIC-lgG levels detected with C595 were increased in the pregnant group while the MUC1-CIC-lgG levels detected with SM3 did not show any significant differences. When the results were compared between lactating and non-lactating women, no significant differences were found. In conclusion, MUC1 and MUC1-CIC-lgM, detected with both MAbs, and MUC1-CIC-4gG levels detected with the MAb C595 are apparently induced by pregnancy.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Mucina-1/imunologia , Adulto , Anticorpos Monoclonais , Antígenos de Neoplasias/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imunoglobulina G/análise , Imunoglobulina M/análise , Lactação , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes , Paridade , GravidezRESUMO
The purpose of this report was the initiation and further maintenance of tumor cells from a primary larynx squamous cell carcinoma. A tumor fragment was mechanically dissociated, the cells were grown in RPMI medium, being the primary culture dependent on the presence of epidermal growth factor and insulin; during subsequent passages the adaptation to conventional growth conditions was obtained. Cells grew in monolayer with an epitheliod shape, showing a pavement-like arrangement; at confluence, cells piled up without contact inhibition maintaining the same morphology. Population doubling time was about 48 h with a colony-forming efficiency of 10%. Immunocytochemical characterization was performed with a panel of monoclonal antibodies reactive against tumor associated antigens, including mucin glycoproteins and related carbohydrate antigens, carcinoembryonic antigen (CEA), p53 as well as cytokeratins, vimentin and desmin. T201 expressed CEA, sialyl Lewis x, Lewis x, Lewis y, MUC1 mucin, Tn hapten, p53, vimentin and cytokeratins. On the other hand, a modal chromosome diploid number of 46 occurring in 74% of cells was detected. Present data confirmed that the methodology employed was adequate for the establishment and characterization of a new cell line which can provide a useful model to study biological and immunological aspects of larynx squamous cell carcinoma.
Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Laríngeas/patologia , Células Tumorais Cultivadas/patologia , Animais , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais , Testes de Carcinogenicidade , Carcinoma de Células Escamosas/metabolismo , Divisão Celular , Análise Citogenética , Eletroforese em Gel de Poliacrilamida , Humanos , Técnicas Imunoenzimáticas , Neoplasias Laríngeas/metabolismo , Metástase Linfática/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Células Tumorais Cultivadas/metabolismo , Ensaio Tumoral de Célula-TroncoRESUMO
UNLABELLED: An immunological analysis to study MUC1 mucin core protein and carbohydrate associated antigens as tissue tumor markers in head and neck carcinoma was performed. Twenty nine patients with the following tumor localizations were included: tongue (n=10), larynx (n=8), oral cavity (n=4), maxillary sinus (n=3), tonsillar ring (n=3) and pharynx (n=1); seven samples of epithelium obtained from normal organs at the same localizations were studied as controls. Immunohistochemical analysis was performed following standard procedures and reaction was graded according to staining intensity and distribution. From each tissue section, membrane, cytoplasmic and nuclear moieties were obtained by differential centrifugation with subsequent fractionation by density gradient centrifugation (6M guanidium chloride-CsCl); subcellular moieties and CsCl derived fractions were analyzed by immunoblotting. Monoclonal antibodies (MAbs) reacting with the core protein of MUCI (C595) and associated carbohydrate antigens were: Tn, 83D4 MAb; Lewis y antigen (Le y), C14 MAb; Lewis x antigen (Le x), KM380 MAb and sialyl Lewis x (sLe x), KM93 MAb. Statistical analysis was undertaken by Spearman rank correlation. In tumor samples, the immunohistochemical identification of MUCl core protein and associated antigens was extended; differences were found in the pattern and intensity of expression; results were corroborated by immunoblotting although in a few samples there was not coincidence between both methods. Localization, tumor mass or node involvement did not show significant differences for any of the antigens studied. CONCLUSIONS: 1) head and neck carcinoma expressed MUCI and associated carbohydrate antigens in high levels; 2) no relationship between antigenic expression and tumor status was found.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Mucina-1/metabolismo , Adulto , Idoso , Antígenos Glicosídicos Associados a Tumores/metabolismo , Carcinoma de Células Escamosas/patologia , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-IdadeRESUMO
UNLABELLED: Serum levels of MUC1 and antibodies (Abs) against MUC1 (IgG and IgM-MUC1) were evaluated in healthy women related to pregnancy and lactation status. A total of 149 serum samples were obtained from: nulliparous, primiparous pregnant, multiparous pregnant that have lactated, multiparous pregnant without lactation, multiparous non-pregnant actual lactating, multiparous non-pregnant that have lactated and finally, multiparous non-pregnant women without lactation. In all assays, we included pre- and post-serum samples belonging to a breast cancer patient vaccinated with a MUC1 derived peptide. CASA test was employed to measure MUC1 while IgG- and IgM-MUC1 serum Abs were evaluated with an ELISA using a 100 mer peptide as catcher. In all groups, mean IgM levels were higher than IgG mean values; when samples were grouped in pregnants versus non-pregnants, a significant difference was detected with both Abs, being raised in non-pregnants. When samples were grouped in lactating versus non-lactating a significant difference was detected with IgG-MUC1, being raised in lactating women while no significant difference was found with IgM-MUC1. The evaluation of serum MUC1 levels confirmed previous results since a significant difference between pregnant versus non-pregnant groups was found while lactating versus non-lactating samples did not. CONCLUSIONS: (i) Increased MUC1 serum levels are apparently associated with pregnancy but not with lactation; (ii) MUC1 Abs are mainly associated with lactation and with non-pregnant status. These results may be considered a contribution on studies about protection against breast cancer induced by pregnancy and lactation.
Assuntos
Formação de Anticorpos , Neoplasias da Mama/imunologia , Lactação/imunologia , Mucina-1/imunologia , Gravidez/imunologia , Adulto , Neoplasias da Mama/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/análise , Imunoglobulina M/análise , Pessoa de Meia-Idade , Mucina-1/análise , ParidadeRESUMO
UNLABELLED: The progression from uncontrolled cell proliferation to invasion and metastasis of epithelial tumors is partially understood. Alteration of epithelial mucin expression have been described in different malignant localizations but only few attempts have been made to identify mucin expression in malignant laryngeal tumors. In the present report, results are shown of studies on the expression of mucins and carbohydrate related antigens in laryngeal cancer and on the isolation of MUC1 mucin from this tumor tissue. Malignant laryngeal specimens were processed for immunohistochemical analysis and for extranuclear membrane fractions (ENM) which were obtained by ultracentrifugation. Subsequently, ENM samples were centrifuged in density-gradient; the analysis of fractions was performed by means of SDS-PAGE and Western-blotting. The panel of monoclonal antibodies (MAbs) included anti MUC1 mucin, anti Lewis x, anti sialyl Lewis x, anti Lewis y, anti MUC-5B, anti oral mucin (gp230), anti Tn hapten, anti p53 and anti cytokeratins. By immunohistochemistry, it was possible to detect MUC1 mucin, Lewis x and Lewis y showing strong reactions while sialyl-Lewis x and Tn antigen only reacted weakly in a few cells; cytokeratins were detected in all samples. In ENM derived fractions obtained by CsCl centrifugation, MUC1 was demonstrated by Western blotting. CONCLUSIONS: (1) laryngeal cancer antigenic expression comprises mostly MUC1 mucin, Lewis x, Lewis y as well as Tn antigen and (2) the methodology here employed is useful to isolate MUC1 from tumor samples.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Laríngeas/metabolismo , Mucina-1/isolamento & purificação , Mucinas/análise , Idoso , Antígenos Glicosídicos Associados a Tumores/análise , Western Blotting , Humanos , Imuno-Histoquímica , Antígenos do Grupo Sanguíneo de Lewis/análise , Antígenos CD15/análise , MasculinoRESUMO
The morphology, cell growth, antigenic expression and tumorigenicity of cell subpopulations from the A549 lung adenocarcinoma isolated by Percoll gradient separation have been analysed. Four subpopulations were obtained (subpopulations A, B, C and D). Immunocytochemical analysis of several antigens was performed with monoclonal antibodies (MAbs): MUC1 mucin (C595, HMFG1 and HMFG2), MUC5B (PANH2); gp230 (PANH4); carbohydrate antigens including sialyl Lewis x (KM93), Tn antigen (83D4), Lewis y (C14); 5, 6, 8, 17 and 19 cytokeratins and p53. The cell population D tended to form cell aggregates that piled up on the monolayer similar to overgrowth cultures of the A549 parental cell line, whereas A, B and C cell subpopulations formed well spread monolayers. Both parental A549 and subpopulation D secreted abundant mucus. The topographic distribution and secretion production were correlated with tumorigenic assays since only subpopulation D grew in nude mice exhibiting reduced latency period; these characteristics correlated with the fast growth of the subpopulation D in vitro. Immunocytochemical analysis demonstrated that subpopulation D showed greater expression of MUC1 mucin and carbohydrate antigens such as Tn antigen, sialyl Lewis x and Lewis y and less expression of cytokeratins, p53, MUC5B and gp230; conversely, subpopulations A, B and C showed the opposite antigenic profile. Our results illustrate heterogeneity in the A549 cell line; subpopulations A, B and C retained characteristics of more differentiated adenocarcinoma while subpopulation D displayed features of a less differentiated tumor line.
Assuntos
Adenocarcinoma/patologia , Neoplasias Pulmonares/patologia , Adenocarcinoma/imunologia , Animais , Anticorpos Monoclonais , Separação Celular , Humanos , Imuno-Histoquímica , Queratinas/análise , Neoplasias Pulmonares/imunologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Oligossacarídeos/análise , Fenótipo , Antígeno Sialil Lewis X , Fatores de Tempo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/análiseRESUMO
The most common clinical form of lung cancer is a disseminated disease with distant metastases; several years of cancer progression precede presentation, and this ultimately limits the efficacy of curative therapy. In this immunohistochemical study, we examined a mucinous adenocarcinoma cell line, maintained by xenogeneic transplantation, and a spontaneous metastatic variant which produces distant tumors (in liver, spleen and kidney). The aim was to investigate possible parameters which characterize the metastatic process. Histopathological comparison between the two subcutaneous transplanted tumor lines showed that both lines presented a similar cellular morphology, a different pattern of cellular growth and an increased vascularization in the metastatic line with respect to its parent. All the tumor sections expressed differential immune reactivity with monoclonal antibodies against Lewis y (MAb C14), sialyl-Lewis x (MAb SNH3) and Lewis x (MAb FH2) determinants. Neither expressed MUC 1 mucins detectable with monoclonal antibodies reactive with the mucin protein core (MAbs C595 and SM3) nor was carcinoembryonic antigen (MAb C365) expressed. Neoplastic cells were reactive with an anti-pan cytokeratin monoclonal antibody confirming their epithelial histogenesis. Our findings have been evaluated with respect to defining metastatic phenotypes in lung cancer by examination of distinct histopathological and immunological parameters.
Assuntos
Adenocarcinoma Mucinoso/secundário , Neoplasias Renais/secundário , Neoplasias Hepáticas Experimentais/secundário , Neoplasias Pulmonares/patologia , Mucina-1 , Fragmentos de Peptídeos , Neoplasias Esplênicas/secundário , Adenocarcinoma Mucinoso/irrigação sanguínea , Adenocarcinoma Mucinoso/química , Adenocarcinoma Mucinoso/patologia , Animais , Anticorpos Monoclonais/imunologia , Apoptose , Biomarcadores Tumorais/análise , Antígeno Carcinoembrionário/análise , Núcleo Celular/ultraestrutura , Citoplasma/ultraestrutura , Gangliosídeos/análise , Humanos , Processamento de Imagem Assistida por Computador , Técnicas Imunoenzimáticas , Injeções Subcutâneas , Neoplasias Renais/irrigação sanguínea , Neoplasias Renais/química , Neoplasias Renais/patologia , Antígenos do Grupo Sanguíneo de Lewis/análise , Antígenos CD15/análise , Neoplasias Hepáticas Experimentais/irrigação sanguínea , Neoplasias Hepáticas Experimentais/química , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Pulmonares/química , Camundongos , Camundongos Nus , Mucinas/análise , Proteínas de Neoplasias/análise , Transplante de Neoplasias , Células-Tronco Neoplásicas/química , Células-Tronco Neoplásicas/ultraestrutura , Oligopeptídeos/análise , Fenótipo , Antígeno Sialil Lewis X , Neoplasias Esplênicas/irrigação sanguínea , Neoplasias Esplênicas/química , Neoplasias Esplênicas/patologia , Transplante Heterólogo , Células Tumorais Cultivadas/patologia , Células Tumorais Cultivadas/transplanteRESUMO
Breast carcinoma cells may express a variety of clinically relevant epitopes, some of which are associated with aberrant glycosylation of MUC1 mucin molecules, as well as determinants which are commonly expressed on their normal molecular counterparts. The present investigation is primarily an immunochemical analysis of MUC1 epitopes and other tumour associated antigenic determinants, as defined by their reaction with monoclonal antibodies and expressed in normal, benign and malignant epithelia. It was determined that malignant tissues of the breast expressed MUC1 mucin, as well as the Le(y) hapten and CEA, at different intensities, cellular distribution and patterns and percentages of positively stained cells. Conversely, benign tissues expressed a low intensity of MUC1 which was restricted to apical cell surface membranes and lumen debris; a similar pattern was found in some normal breast sections. It was concluded that MUC1 mucin exhibits heterogeneous antigenicity (as defined by its reactivity with a panel of related anti-MUC1 monoclonal antibodies) which is predominantly related to the progression of malignant disease. Le(y) is a marker of breast neoplasia, while CEA was found on only a small proportion of tumours. These immunohistochemical findings are considered in the context of improving breast cancer diagnosis and therapy.
Assuntos
Antígenos de Neoplasias/análise , Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Mama/citologia , Mama/patologia , Doença da Mama Fibrocística/patologia , Anticorpos Monoclonais , Antígeno Carcinoembrionário/análise , Células Epiteliais/citologia , Epitélio/patologia , Epitopos/análise , Feminino , Humanos , Imuno-Histoquímica , Antígenos do Grupo Sanguíneo de Lewis/análise , Mucina-1/análise , Valores de ReferênciaRESUMO
The aim of this study was to examine tissue from patients with breast carcinoma or benign breast disease for the presence of monoclonal-antibody-defined antigens, including the MUC1 mucin and carcinoembryonic antigen CEA. The tests were performed by sodium dodecyl sulphate/polyacrylamide gel electrophoretic separation of proteins, electrophoretic transfer to nitrocellulose membranes and immunostaining with the monoclonal antibodies. Some of the antigens identified are known to circulate at high levels in some but not necessarily all, breast carcinoma patients. Serum from a panel of ten breast cancer patients was subjected to a fractionation procedure designed to release antigen from immune complexes, and again these samples were analysed for the presence of monoclonal-antibody-defined antigens. A high frequency of positive reactions was detected by the anti-MUC1 monoclonal antibody C595 with both breast carcinoma subcellular membrane fractions as well as antigen fractions eluted from circulating immune complexes. No reactions were observed with equivalent materials from benign breast disease samples. The findings illustrate the variability in antigen expression between breast tumours. The data also indicate that a proportion of patients respond to their tumour by the production of antibodies that recognise the MUC1 antigen in their circulation.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Neoplasias da Mama/imunologia , Carcinoma/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Complexo Antígeno-Anticorpo/imunologia , Antígenos Glicosídicos Associados a Tumores/sangue , Western Blotting , Doenças Mamárias/imunologia , Neoplasias Colorretais/imunologia , Humanos , Membranas Intracelulares/imunologia , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/imunologia , Peso Molecular , Mucina-1 , Mucinas/sangue , Mucinas/imunologiaRESUMO
Two monoclonal antibodies (anti-791T/36 and anti-791T/48) prepared against an osteogenic sarcoma cell line (791T) following xenogeneic immunization, reacted against the immunizing tumor, but not against normal cells from the tumor-donor, using an indirect 125I-protein A binding assay. Both antibodies cross-reacted with a small number of other osteogenic sarcomas and a few unrelated cell lines from an extensive panel, but the specificity of these cross-reactions was different. Both antibodies were labelled with 125I to detect direct binding to target cells, and the specificity of their reactivity was essentially identical to that observed in the indirect assay. Direct binding of each labelled antibody was inhibited by pretreating target cells with its unlabelled counterpart, but the two antibodies could not inhibit each other. The binding of anti-791T/36 was also not inhibited by pretreating the target cells with sera from the 791-T-tumor donor, which were shown to contain antibody reacting with the autochthonous tumor. It is concluded that 791T has two distinct tumor-associated antigens recognized by the monoclonal antibodies, and furthermore that at least one of these antigens is independent of those recognized by the patient from which the tumor cell line was derived. The efficacy of anti-791T/36 antibody labelled with radioactive iodine was demonstrated for localizing tumor deposits growing in immunodeprived mice.