RESUMO
BACKGROUND: CA 125 antigenic domains appear to reside within a region containing 156-amino acid sequence repeats. Surprisingly, anti-CA 125 antibodies can be classified into three families (groups A, B and C) indicating limited epitope diversity. In this study we describe the heterologous expression of a CA 125 repeat unit (R11) and an analysis of its epitope topography. METHODS: R11 was expressed using a baculovirus approach and purified from culture supernatants by sequential ion exchange chromatography. Monoclonal antibody binding was assessed using antigen capture and cross-inhibition methods. RESULTS: The recombinant repeat was purified to 2.5 x 10(7) U/mg. Although a number of group A and B monoclonal antibodies were found to bind R11, the prototype antibody OC125 (group A) showed little reactivity. However, the prior binding of some group B monoclonal antibodies dramatically enhanced subsequent OC125 binding. Low monoclonal antibody reactivity to R11 correlated well with poor binding to SDS-denatured human ascites CA 125. CONCLUSION: The ability to 'activate' R11 epitopes indicates that some may not be displayed optimally on isolated repeats. This observation, together with the concordance between monoclonal antibody binding to R11 and denatured CA 125, suggests that a number of epitopes are preferentially displayed only when contained within multiple repeat domains.
Assuntos
Biomarcadores Tumorais/imunologia , Antígeno Ca-125/imunologia , Epitopos/imunologia , Expressão Gênica , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Baculoviridae/genética , Baculoviridae/metabolismo , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/isolamento & purificação , Antígeno Ca-125/química , Antígeno Ca-125/genética , Antígeno Ca-125/isolamento & purificação , Linhagem Celular , Epitopos/química , Epitopos/genética , Epitopos/isolamento & purificação , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , SpodopteraRESUMO
The purpose of this study was to examine the expression of splice variants of the TADG-12 (TMPRSS3) gene in normal ovarian epithelial tissue and ovarian carcinoma and further to associate the expression of TADG-12 variant with clinicopathologic characteristics if such an association exists. TADG-12D variant expression was examined by semiquantitative PCR in 50 ovarian tumors [41 adenocarcinomas, 3 low malignant potential (LMP) tumors, and 6 adenomas] and 7 normal ovaries. In carcinomas as well as LMP tumors and adenomas, TADG-12D variant mRNA expression was significantly elevated compared to that in normal ovary samples. TADG-12 has several splice variants, one of which we originally identified and 3 others identified by Scott et al. [Nat Genet 2001;27:59-63]. We previously examined the expression of TADG-12V variant and here we confirm the overexpression of TADG-12D variant in ovarian carcinomas. Moreover, TADG-12D variant mRNA expression level in carcinomas was significantly elevated compared to that in adenomas and TADG-12D variant mRNA expression level in advanced clinical stage diseases was significantly higher than that in early stage diseases in ovarian carcinomas. With regard to histological type, TADG-12D variant mRNA expression level in mucinous adenocarcinomas was significantly higher than those in the other tissue subtypes. These features imply that TADG-12D variant expression may play an important role in ovarian cancer development and progression, and this variant may be useful both as a molecular target for therapy and/or a diagnostic marker.
Assuntos
Adenocarcinoma Mucinoso/genética , Adenoma/genética , Carcinoma/genética , Perfilação da Expressão Gênica , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Adenocarcinoma Mucinoso/diagnóstico , Adenocarcinoma Mucinoso/terapia , Adenoma/diagnóstico , Adenoma/terapia , Sequência de Aminoácidos , Carcinoma/diagnóstico , Carcinoma/terapia , Transformação Celular Neoplásica , Feminino , Humanos , Dados de Sequência Molecular , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/terapia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossínteseRESUMO
CA 125 is a well-established marker for patients diagnosed with ovarian carcinoma. It is clearly elaborated in serous cystadenocarcinomas and less likely to be expressed in mucinous tumors. It has been 20 years since CA 125 was first recognized and it is only in recent years (the past 2) that some progress has been made toward cloning the gene, providing the basis for an understanding of the functional role of this molecule in embryonic development and neoplastic transformation. It is now clear that CA 125 is a large glycoprotein which is anchored to the epithelium by a transmembrane domain and is released into the extracellular space by enzymatic cleavage. Here, we describe a further major extension to the glycosylated extracellular amino terminal domain of this molecule. These additional data in association with our previous understanding of this molecule will provide the basis for our ability to understand the physiologic function of this molecule in biologic development and pathologic transformation.
