John B. Beard

The CA 125 Gene: An Extracellular Superstructure Dominated by Repeat Sequences

CA 125 has long presented problems to both clinicians and investigators because there was no definitive information on its structure and function. Here, we describe our work on cloning the CA 125 gene with the anticipation that such information will provide the basis for understanding its structure and its physiologic role in both normal and malignant tissues. The CA 125 protein core is composed of a short cytoplasmic tail, a transmembrane domain and an extraordinarily large glycosylated extracellular structure. This structure is dominated by a repeat domain composed of 156 amino acid repeat units which encompass the epitope binding sites. The molecule also includes an amino terminal domain of serine/threonine-rich sequences which would account for most of the O-glycosylation known to be present in CA 125. CA 125 is an unusually large transmembrane glycoprotein. Its release from the surface of the cell is most probably dependent on cytoplasmic phosphorylation followed by proteolytic cleavage. The extracellular domain is characterized by a large number of repeat units (probably 60+) which encompass an interactive disulfide bridged cysteine-loop and the site of OC125 and M11 binding. Sequencing the gene provides us with the ability to initiate the quest to understand the biological function of CA 125.

The CA 125 gene: A newly discovered extension of the glycosylated N-terminal domain doubles the size of this extracellular superstructure

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.

Transmembrane serine protease TADG-15 (ST14/Matriptase/MT-SP1): expression and prognostic value in ovarian cancer

Tumour-associated differentially expressed gene-15 (TADG-15/ST14/matriptase/MT-SP1) is a novel member of the transmembrane serine proteases. Previous studies indicated that TADG-15 is overexpressed in ovarian tumours; however, relationships between expression of TADG-15 and clinical characteristics of ovarian cancer remain unclear. The purpose of this study was to examine TADG-15 expression in ovarian cancers and determine any associations with clinicopathological characteristics or patient survival. Immunohistochemical study revealed that TADG-15 was expressed in 50 (56.2%) of 89 ovarian carcinomas, whereas it was not detected in normal ovaries. TADG-15 expression was significantly more common in patients with early stage disease compared with patients with advanced stage diseases (namely, stage I, 24 out of 33: 72.7%; stage II/III/IV, 26 out of 56: 46.4%; P=0.0157). Kaplan–Meier survival curves demonstrated that patients with TADG-15-positive tumours have had substantially longer survival (P=0.0480). The mean value of relative TADG-15 mRNA expression ratio was significantly higher in stage I tumours than in stage II/III/IV tumours (P=0.0053). Increased expression of TADG-15 is frequently detected in early stage cancers, with expression level downregulated during progression of disease. TADG-15 is associated with early stage ovarian cancer and longer patient survival; therefore, it may be a favourable prognostic marker for this malignancy.

Ovarian tumor cells express a novel multi-domain cell surface serine protease

Serine proteases serve many functions in normal biological processes. These functions are often usurped by cancer cells to allow progression of tumors by increasing the growth and metastatic potential of the neoplasia. Here, we have used a polymerase chain reaction (PCR)-based strategy to clone Tumor Associated Differentially-expressed Gene-12 (TADG-12), a new serine protease from ovarian carcinoma. This technique also revealed a variant splicing form of TADG-12 that could lead to a truncated protein product. Semi-quantitative PCR showed that TADG-12 is overexpressed in 41 of 55 ovarian cancer specimens relative to normal expression, and the variant form, TADG-12V is found at increased levels in 8 of 22 carcinomas examined. Northern blot revealed three transcripts, the largest of which is approximately 2.4 kb. An ovarian tumor cDNA library was screened, and the entire cDNA of TADG-12 has been identified. This sequence encodes a putative protein of 454 amino acids which includes a potential transmembrane domain, an LDL receptor-like domain, a scavenger receptor cysteine-rich domain, and a serine protease domain. These features imply that TADG-12 will be at the cell surface, and it may be useful as a molecular target for therapy or a diagnostic marker.

Overexpression of testisin, a serine protease expressed by testicular germ cells, in epithelial ovarian tumor cells.

Objective: In a continued effort to identify and characterized secreted proteases that are overexpressed in ovarian carcinomas, we discovered the testisin protease as such a candidate. When this discovery was originally made, no data existed in the literature or in the GenBank database that identified such a gene. Our main objective was to determine whether this gene was overexpressed exclusively in ovarian tumor tissues compared with normal ovary and whether it was expressed in any other normal tissues. Methods: mRNA was isolated and cDNA was prepared from 34 ovarian tumors (four adenomas, three low malignant potential tumors, and 37 carcinomas) and seven normal ovaries. The testisin mRNA expression level relative to internal control, β-tubulin, was determined by Northern blot analysis and semiquantitative polymerase chain reaction (PCR). Results: Northern blot hybridization showed that the testisin transcript was abundant in ovarian carcinoma but was not detected in normal ovary. On examination of Northern blots from normal fetal and adult tissues, only adult testis showed abundant transcripts of testisin. Semiquantitative PCR examination showed that the testisin mRNA levels in ovarian tumors of low malignant potential and in ovarian carcinomas were significantly higher than in normal ovaries (P < .01). Testisin mRNA level in ovarian carcinomas was also significantly higher than in ovarian adenomas (P < .05). Testisin overexpression rates in advanced stage (stage 2 or 3) diseases were significantly higher than that in early stage disease (stage 1) in ovarian carcinoma samples (P < .05). Conclusions: The induction of the testisin transcript might contribute to the development, progression, and invasive or metastatic capacity of ovarian carcinomas.

The Transmembrane Protease Serine (TMPRSS3/TADG-12) D Variant: A Potential Candidate for Diagnosis and Therapeutic Intervention in Ovarian Cancer

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.

Expression and epitope characterization of a recombinant CA 125 repeat: fourth report from the ISOBM TD-1 workshop.

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 × 107 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.