RESUMO
Indoleamine-2,3-dioxygenase (IDO) has been established as a normal mechanism of peripheral tolerance and immunosuppression. Besides, malignant tumors release microvesicles (MV) related with tumor dissemination. The aims of this study were to determine the expression of IDO in breast cancer and circulating microvesicles from breast cancer patients and to perform an in silico analysis to find genes co-expressed to IDO. One hundred and twenty-two tissue and serum breast samples (91 malignant, 21 benign, and 10 normal), and MCF7, MDA-MB-231, and T47D breast cancer cell lines were included. Standard immunohistochemistry (IHC), immunocytochemistry (ICC), Western blot (WB), and RT-PCR were employed. Microvesicle isolation from plasma samples was obtained by serial centrifugation and ultracentrifugation. By IHC, 60 % breast cancer, 43 % benign, and 20 % normal samples were positive. Significant differences were found among normal, benign, and malignant samples. Breast cancer stages I, II, and III expressed IDO in 42, 66, and 71 % of samples, respectively, while breast cancer cell lines also reacted; by WB, 9/25 microvesicles fractions showed bands at 42 kD. In silico analysis of IDO 1 gene expression in breast cancer showed its association with several genes related to immune response and apoptosis. Moreover, IDO and co-expressed genes were found predominately in basal and erbB2 subtypes. The cumulative data indicate a high expression of IDO in breast cancer which increased with higher stages. Furthermore, IDO was found in association with circulating breast cancer MV, while experimental and in silico gene expression revealed that IDO was mainly expressed in a triple-negative subgroup.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Indolamina-Pirrol 2,3,-Dioxigenase/biossíntese , Neoplasias de Mama Triplo Negativas/genética , Apoptose/genética , Linhagem Celular Tumoral , Simulação por Computador , Feminino , Humanos , Imuno-Histoquímica , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Células MCF-7 , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
In previous studies, we identified rhomboid domain containing 2 (RHBDD2) gene to be markedly overexpressed in breast cancer patients that developed recurrence of the disease. In this study, we evaluated for the first time RHBDD2 gene expression in colorectal cancer (CRC). Five public available DNA microarray studies were compiled in a homogeneous dataset of 906 colorectal samples. The statistical analysis of these data showed a significant increase of RHBDD2 expression in the advanced stages of CRC (p < 0.01). We validated these findings by immunohistochemistry on 130 colorectal tissue samples; RHBDD2 protein overexpression was also observed in the advanced stages of the disease (p < 0.001). In addition, we investigated RHBDD2 expression in response to the chemotherapy agent 5-fluorouracile (5FU). We detected a significant increase of RHBDD2 mRNA and protein after 5FU treatment (20-40 µM; p < 0.001). Overall, these results showed that RHBDD2 overexpression might play a role in colorectal cancer progression.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Fluoruracila/uso terapêutico , Proteínas de Neoplasias/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Estudos de Casos e Controles , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Proteínas de Membrana , Proteínas de Neoplasias/metabolismo , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reto/metabolismo , Reto/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
In the course of breast cancer global gene expression studies, we identified an uncharacterized gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly over-expressed in primary tumors from patients with recurrent disease. In this study, we identified RHBDD2 mRNA and protein expression significantly elevated in breast carcinomas compared with normal breast samples as analyzed by SAGE (n=46) and immunohistochemistry (n=213). Interestingly, specimens displaying RHBDD2 over-expression were predominantly advanced stage III breast carcinomas (p=0.001). Western-blot, RT-PCR and cDNA sequencing analyses allowed us to identify two RHBDD2 alternatively spliced mRNA isoforms expressed in breast cancer cell lines. We further investigated the occurrence and frequency of gene amplification and over-expression affecting RHBDD2 in 131 breast samples. RHBDD2 gene amplification was detected in 21% of 98 invasive breast carcinomas analyzed. However, no RHBDD2 amplification was detected in normal breast tissues (n=17) or breast benign lesions (n=16) (p=0.014). Interestingly, siRNA-mediated silencing of RHBDD2 expression results in a decrease of MCF7 breast cancer cells proliferation compared with the corresponding controls (p=0.001). In addition, analysis of publicly available gene expression data showed a strong association between high RHBDD2 expression and decreased overall survival (p=0.0023), relapse-free survival (p=0.0013), and metastasis-free interval (p=0.006) in patients with primary ER-negative breast carcinomas. In conclusion, our findings suggest that RHBDD2 over-expression behaves as an indicator of poor prognosis and may play a role facilitating breast cancer progression.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Serina Endopeptidases/genética , Processamento Alternativo/genética , Sequência de Bases , Biomarcadores Tumorais , Mama/patologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Análise Mutacional de DNA , Células Epiteliais/enzimologia , Feminino , Seguimentos , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina Endopeptidases/metabolismoRESUMO
Breast cancer spreading to different organs have been related to different molecules and mechanisms, but cutaneous metastasis remains unexplored. Increasing evidence showed that MUC1 and some of its carbohydrate associated antigens may be implicated in breast cancer metastasis. In this study we analyzed these tumor markers in order to identify breast cancer cutaneous metastatic profiles. A cohort of 26 primary tumors from breast cancer patients with cutaneous metastases were included; also, cutaneous and lymphatic node metastatic samples and primary tumors from breast cancer patients without metastases were analysed. Immunohistochemical (IHC) studies demonstrated that both underglycosylated MUC1 (uMUC1) and sialyl Lewis x (sLex) to be positively associated with cutaneous metastatic primary tumors (pâ¯<â¯0.05). Notably, a high percentage of tumors with cutaneous metastases were characterized as triple negative and Her2+ tumors (37.5 % and 29 %, respectively). Some discordant results were found between primary tumors and their matched cutaneous metastases. To determine if MUC1 variants may be carriers of carbohydrate antigens, subcellular fractions from a cutaneous metastatic lesion were obtained, immunoprecipitated and analyzed by Western blot. We found that the isolated uMUC1 with a molecular weight of>200â¯kDa was also the site for binding of anti-sLex MAb; in coincidence, a high correlation of positive IHC expression of both markers was observed. Our findings confirm that breast cancer cutaneous metastases were associated to highly malignant primary tumors and sustain the hypothesis that u-MUC1 and sLe x may drive breast cancer cutaneous metastases.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Mucina-1/metabolismo , Antígeno Sialil Lewis X/metabolismo , Neoplasias Cutâneas/secundário , Adulto , Idoso , Biomarcadores Tumorais/análise , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Secreted mucins constitute a crucial part of the gel that protects respiratory and digestive epithelia, being MUC2/Muc2 the predominant gel-forming mucin of the intestine while MUC5AC/Muc5ac is one of the gel-forming mucins most expressed at the airways. In this study, we have analyzed Muc2 and Muc5ac during rat development by using immunohistochemistry, Western blotting and RT-PCR. We demonstrated that rat Muc2 was expressed in fetal intestinal goblet cells of surface epithelium of villi and developing Lieberkühn crypts. In neonates and adults, Muc2 was expressed at luminal goblet cells of small and large intestine and at gastric mucous and glandular cells. Muc5ac protein was observed in embryonic gastric and lung samples; expression increased during development and postnatal and adult life. After birth, a low reaction was detected at the tracheal surface epithelium and glands, which increased in adults.
Assuntos
Trato Gastrointestinal/metabolismo , Expressão Gênica , Mucina-5AC/genética , Mucina-2/genética , Ratos/genética , Sistema Respiratório/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Trato Gastrointestinal/crescimento & desenvolvimento , Mucina-5AC/metabolismo , Mucina-2/metabolismo , Ratos/crescimento & desenvolvimento , Ratos/metabolismo , Sistema Respiratório/crescimento & desenvolvimentoRESUMO
OBJECTIVES: 1- to detect alpha 1-acid glycoprotein (AGP) and sialyl Lewis x (sLex) in colorectal malignant, benign and normal samples; 2- to isolate AGP from colorectal cancer and 3- to study its immunoreactivity with an anti-sLex monoclonal antibody (MAb). MATERIALS AND METHODS: tissue and serum samples from 88 patients with colorectal cancer, 22 adenomas and 23 normal were included. Expression of AGP and sLex was studied by immunohistochemistry (IHC); isolation approach: AGP was precipitated with ammonium sulphate and immunoprecipitated with anti-AGP MAb. The immune complex formed was isolated by protein A-Sepharose CL-4B affinity chromatography and further eluted; fractions were analysed by SDS-PAGE and Western-blot. Statistical analysis was performed by means of Principal Component Analysis. RESULTS: By Western blot employing anti-AGP MAb and sLex MAbs, isolated fractions from malignant samples showed a band at about 45 kD. IHC revealed that AGP was expressed in 70% of colorectal carcinoma samples, 50% of benign and 35% of normals. SLex was detected in 31% of malignant samples, 41% of benign and in one normal sample. In malignant samples, AGP reaction comprised the whole specimen with a strong and homogeneous staining while normal and benign samples showed a restricted reaction. In cancer, sLex expression consisted in an intense reactivity in membrane, cellular debris and some cytoplasmic foci while normal and benign samples were occasionally stained. A statistically significant positive correlation was found between AGP and sLex expression. Serum AGP levels were measured by radial immunodiffusion and statistical comparative analysis with tissue expression did not show a correlation between both parameters. CONCLUSION: AGP may constitute a carrier of sLex in colorectal cancer.
Assuntos
Carcinoma/metabolismo , Neoplasias Colorretais/metabolismo , Oligossacarídeos/metabolismo , Orosomucoide/metabolismo , Adenoma/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/imunologia , Carcinoma/imunologia , Colo/imunologia , Colo/metabolismo , Neoplasias Colorretais/imunologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Oligossacarídeos/imunologia , Orosomucoide/imunologia , Antígeno Sialil Lewis XRESUMO
UNLABELLED: Some mucin genes have been detected during human embryonic and fetal organ development; however, little is known about mucin expression in epidermal development, neither in humans nor in other species. The present research was developed to explore Muc5ac skin expression during prenatal and postnatal rat development. Immunohistochemistry (IHC), Western blotting (WB) and RT-PCR were employed. By IHC, Muc5ac protein was found early in embryonic epidermis from day 13 of gestation until seven days after birth when the surface epidermis became negative and the reaction was restricted to secreting sebum cells. In coincidence with IHC findings, WB analysis showed a band at approximately 200KDa at the same periods of development. Results were also confirmed by RT-PCR. Muc5ac expression in rat embryonic epidermis suggests that Muc5ac may play a protective role in embryonic skin previous to birth which may be replaced by pile covering. To our knowledge, this is the first report which confirmed Muc5ac expression during skin development. CONCLUSION: Muc5ac expression in rat embryonic epidermis suggests that Muc5ac may play a protective role in embryonic skin previous to birth which may be replaced by pile covering. To our knowledge, this is the first report which confirmed Muc5ac expression during skin development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Mucina-5AC/genética , Pele/embriologia , Animais , Eletroforese em Gel de Poliacrilamida , Feminino , Imuno-Histoquímica , Mucina-5AC/metabolismo , Ratos , Pele/crescimento & desenvolvimentoRESUMO
The dissemination of a malignant neoplasia is a complex process, which requires a set of molecules that remains unknown. It has been suggested that mucins and their carbohydrate-associated antigens may be implicated in tumour spreading which may be also influenced by an anti-MUC1 immune response. In this pilot study, we report the pattern of carbohydrate and peptidic MUC1-associated epitopes on carcinoma cells isolated from bone marrow (BM), taking into account primary tumour histopathologic features. We also bring information about the anti-MUC1 humoral response in these patients. Seventeen patients with invasive breast carcinoma were included. A sample of the primary tumour, a serum sample and a BM aspirate were obtained from each patient. Clinical features studied were tumour size, number of metastatic nodes, histological type and disease stage. Standard immunohistochemistry was performed with antigenic retrieval using different monoclonal antibodies (MAbs): anti carbohydrate antigens: Lewis x (KM380), sLewis x (KM93), Lewis y (C14) and Tn, anti-MUC1 peptide core MAbs: C595, HMFG2 and SM3, anti-cytokeratins, anti-protoncogenes ErbB2 and ErbB3 (IgG) MAbs and also anti-CD34 and anti-CD45 MAbs. ELISA techniques were employed to study circulating MUC1 as well as free and complexed anti-MUC1 antibodies. Immunohistochemical results showed that carbohydrate antigenic expression increases in BM neoplastic cells compared to the original tumours. However, we were not able to demonstrate that a humoral immune response to MUC1 has been induced in these patients. Finally, the employed procedures allow the selective immortalisation of micrometastatic carcinoma cells since short-term cell lines were established.
Assuntos
Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/imunologia , Medula Óssea/imunologia , Neoplasias da Mama/imunologia , Mucina-1/imunologia , Células-Tronco Neoplásicas/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/sangue , Especificidade de Anticorpos , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Carcinoma Ductal/imunologia , Carcinoma Ductal/patologia , Carcinoma Intraductal não Infiltrante/imunologia , Carcinoma Intraductal não Infiltrante/patologia , Carcinoma Lobular/imunologia , Carcinoma Lobular/patologia , Carcinoma Papilar/imunologia , Carcinoma Papilar/patologia , Linhagem Celular Tumoral , Epitopos/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Neoplasias/imunologia , Oligossacarídeos/imunologia , Peptídeos/imunologia , Projetos PilotoRESUMO
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
We measured antibody responses to recombinant Mycobacterium leprae 65-kDa (rML65) and 10-kDa (rML10) by indirect ELISA in sera from leprosy patients, household contacts and healthy controls in a leprosy-endemic area in the north east of Argentina. Serum antibody levels to those antigens were correlated with IgM anti-phenolic glycolipid I (PGL-I) levels, with bacterial index and the period of time under chemotherapy. Bacterial index positive (BI+) patients showed higher mean values when compared with BI negatives (BI-). Among lepromatous patients a positive correlation was observed between IgG antibody responses to both recombinant antigens and IgM antibody response to PGL-I. Anti-rML10 test detected a higher percentage of positives/total than anti-rML65 in all leprosy groups and healthy contacts. Bacterial load, leprosy clinical form and the time under chemotherapy were factors which could influence levels of the antibody response. The contribution of these antibody studies for a precise and early diagnosis in leprosy is discussed.
Assuntos
Anticorpos Antibacterianos/análise , Antígenos de Bactérias , Proteínas de Bactérias , Chaperonina 10/imunologia , Chaperoninas/imunologia , Imunoglobulina G/análise , Imunoglobulina G/biossíntese , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Proteínas Recombinantes/imunologia , Adulto , Idoso , Anticorpos Monoclonais/imunologia , Argentina/epidemiologia , Chaperonina 60 , Quimioterapia Combinada , Ensaio de Imunoadsorção Enzimática , Glicolipídeos/imunologia , Humanos , Imunoglobulina M/análise , Imunoglobulina M/biossíntese , Hanseníase/diagnóstico , Hanseníase/epidemiologia , Hanseníase/transmissão , Pessoa de Meia-Idade , Estudos Soroepidemiológicos , Fatores de TempoRESUMO
The aim of the present work was to evaluate the levels of anti-PGL-I and anti-10-kDa heat shock protein antibodies in serum and immune complexes isolated from leprosy patients, convivients and controls. Leprosy patients with erythema nodosum leprosum or without it were included and a comparative study was done to investigate intergroup differences. Immune complexes were precipitated from serum by polyethylene glycol 3.5%; antibody levels were measured in sera and in dissociated immune complexes by ELISA. Serum antibody levels were then correlated with immune complex-associated antibody levels. The results showed that the erythema nodosum leprosum group differed from controls, contacts and non-erythema patients in their immune complex levels. IgM anti-PGL-I and IgG anti-10-kDa heat shock protein antibodies were constituents of the immune complexes in patients with erythema nodosum leprosum, who exhibited a significant difference in their immune complex composition compared with controls, contacts and non-erythema patients; while free antibody levels (anti-PGL-I and anti-10-kDa) did not differentiate between erythema and non-erythema patients, the measurement of immune complex-associated antibodies demonstrated a significant difference between the two clinical conditions. Furthermore, the measurement of immune complex-associated anti-PGL-I IgM made it possible to differentiate between contacts and controls. The significance of these results is discussed.
Assuntos
Anticorpos Antibacterianos/análise , Complexo Antígeno-Anticorpo/análise , Chaperonina 10/imunologia , Eritema Nodoso/imunologia , Glicolipídeos/imunologia , Hanseníase Virchowiana/imunologia , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/isolamento & purificação , Antígenos de Bactérias/imunologia , Chaperonina 10/genética , Ensaio de Imunoadsorção Enzimática , Eritema Nodoso/sangue , Humanos , Imunoglobulina G/análise , Imunoglobulina M/análise , Hanseníase Virchowiana/sangue , Pessoa de Meia-Idade , Polietilenoglicóis/farmacologia , Proteínas Recombinantes/imunologiaRESUMO
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
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
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
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
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
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
A serie of 29 human normal, benign and malignant breast tissues were cultivated in an attempt to isolate and propagate primary breast epithelial cells in vitro. Explants methodology as well as disaggregation techniques (enzymatic and mechanical) were employed to obtain better culture conditions. Cells derived from breast malignant tissue were propagated in an appropriate and survived in culture for at least 6 months, exhibiting a marked preference to grow in suspension (independent anchorage). Tumorigenicity assays in nude mice were performed with malignant cells obtained from primary culture cells as well as with cells achieved from successive passages. The rate of long time cell survival from malignant and non-malignant tissues demonstrated the accuracy of the methodology but it also emphasised the need for improving technology to obtain cell lines with long survival.
Assuntos
Mama/citologia , Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Animais , Doenças Mamárias/patologia , Neoplasias da Mama/patologia , Sobrevivência Celular , Células Cultivadas , Meios de Cultura , Feminino , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Manejo de Espécimes , Suspensões , Células Tumorais CultivadasRESUMO
We are dedicated to the study of circulating immune complexes (CIC) associated with different diseases: malignant tumors, leprosy and rheumatoid arthritis. Immune complexes were evaluated by various methods: 125I-Clq binding assay, 125I-IgG binding test, 125I-bovine conglutinin binding assay and polyethylene glycol precipitation test (3.5% and 2.5%). Techniques for the isolation and splitting of CIC in their components were performed in sera from patients with tumors and with leprosy. These methods consisted in the combination of CIC with protein A followed by elution with different buffers. CIC splitting techniques were first applied on immune complexes formed in vitro (BSA-aBSA, OVA-aOVA). The analysis of CIC fractions was done by SDS-PAGE, immunoelectrophoresis and immunoblotting techniques. Results were as follows: CIC levels correlated with active stages of disease, decreasing during remission so that CIC detection can be useful to evaluate response to treatment. The isolation and splitting of immune complexes into their components resulted in the obtention of immunologically active fractions, especially in sera from patients with gastrointestinal and breast cancer and with leprosy.