Kazushi Shigemasa

The Stratum Corneum Chymotryptic Enzyme That Mediates Shedding and Desquamation of Skin Cells Is Highly Overexpressed in Ovarian Tumor Cells

Proteases play essential roles in the process of tumor invasion and metastasis. The serine protease stratum corneum chymotryptic enzyme (SCCE) has been purified from human stratum corneum and is known to contribute to the cell shedding process by catalyzing the degradation of intercellular cohesive structures at the skin surface. The presence of SCCE on the surface of tumor cells suggests it also may contribute to the process of tumor cell shedding, resulting in early metastasis of carcinoma.

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.

Ovarian Tumor Cells Express a Transmembrane Serine Protease: A Potential Candidate for Early Diagnosis and Therapeutic Intervention

Proteases have been implicated in the extracellular modulation required for tumor growth and invasion. In an effort to categorize those proteases contributing to ovarian carcinoma progression, we have utilized redundant primers to conserved amino acid (AA) domains surrounding the catalytic triad of His, Asp and Ser to amplify serine proteases that are differentially expressed in carcinomas. Using this method, we have identified and cloned a serine protease named TADG-15 (tumor-associated differentially expressed gene 15) that is overexpressed in ovarian tumors. TADG-15 is a transmembrane multidomain serine protease which includes ligand binding domains and a serine protease in the extracellular space.

Increased Expression of Protease M in Ovarian Tumors

Proteases are known to play important roles in tumor invasion and metastasis. Protease M, which was originally identified by Anisowicz and colleagues in 1996, is a new member of the serine protease family. We also identified the protease M transcript in a differential PCR screen of ovarian tumors and have investigated its expression in 44 ovarian tumors (12 low malignant potential tumors, 32 carcinomas) and 10 normal ovaries using quantitative PCR. The PCR product was labeled with 32P and a phosphoimager was used to determine the relative expression of the protease M gene compared to internal control β-tubulin. mRNA expression levels of protease M were significantly elevated in 9 of 12 low malignant potential tumors and 30 of 32 carcinomas. Northern blot hybridization showed that the 1.7-kb protease M transcript was abundant in carcinoma but not detected in normal ovary. Immunohistochemical staining of normal overy and ovarian tumor tissue sections with antibodies generated to protease M derived peptides corroborated the semi-quantitative PCR and Northern analysis data. Our results suggest that protease M is frequently overexpressed in ovarian tumors and may therefore contribute to the invasive nature or growth capacity of ovarian carcinomas.

Increased MCL–1 Expression Is Associated with Poor Prognosis in Ovarian Carcinomas

To investigate the potential role of the BCL–2 gene family (BAX, BCL–2, MCL–1, and BCL‐XL) in ovarian cancer development and progression, mRNA expression levels of these genes were measured using semi‐quantitative PCR in epithelial ovarian tumor tissues and normal ovaries. The immunohistochemical expression of MCL–1 in ovarian tumors was also examined. The expression levels of BAX and MCL–1 mRNA were significantly higher in ovarian cancers and in adenomas than in normal ovaries (P<0.05). In contrast, the BCL–2 mRNA expression level in ovarian cancers was significantly lower than in ovarian adenomas and in normal ovaries (P<0.05). Expression of BCL‐XL mRNA was no different between normal ovaries and ovarian tumors. Log‐rank testing showed that low BAX mRNA expression and high MCL–1 mRNA expression significantly correlate with poor survival for patients with stage III ovarian carcinomas (BAX, P=0.05; MCL–1, P=0.02). Immunohistochemical analysis showed that diffuse‐positive expression of MCL–1 protein in mucinous carcinomas was significantly higher than in mucinous low malignant potential (LMP) tumors (P=0.03). In ovarian cancer cases, diffuse‐positive expression of MCL–1 protein significantly correlates with advanced clinical stage, high histologic grade, and poor survival (stage, P<0.01; grade, P=0.01; survival, P=0.01). These results suggest that increased MCL–1 expression may play an important role in replacing the functions of increased BAX and decreased BCL–2 in ovarian carcinoma cells, thereby promoting cell survival, and resulting in a poor prognosis for patients with ovarian cancer.

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.

The matrix metalloprotease pump-1 (MMP-7, matrilysin) : A candidate marker/target for ovarian cancer detection and treatment

Matrix metalloproteases are known to play an important role in tumor invasion by mediating degradation of extracellular matrix. In this study, we have investigated the expression of the matrix metalloprotease pump-1 gene (also referred to as MMP-7, Matrilysin) in 44 ovarian tumors (12 low malignant potential tumors, 32 carcinomas) and 10 normal ovaries using quantitative PCR. The PCR product was labelled with 32P and a phosphoimager was used to determine the relative expression of pump-1 compared to an internal control β-tubulin. mRNA expression levels of pump-1 were significantly elevated in 9 of 12 low malignant potential tumors and 26 of 32 carcinomas. Northern blot hybridization showed that the 1.1-kb pump-1 transcript was abundant in carcinoma but seldom expressed in normal adult tissues including normal ovary. Immunohistochemical localization of the pump-1 protein confirms its expression by ovarian tumor cells. Our results suggest that pump-1 is frequently overexpressed in ovarian tumors and may contribute to its invasive nature or growth capacity, therefore pump-1 may serve as a useful marker for early detection of disease and/or a target for therapeutic intervention in downregulation of tumor progression.

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.

Epitopes on CA 125 from Cervical Mucus and Ascites Fluid and Characterization of Six New Antibodies

CA 125 is found in body fluids in a variety of molecular weight forms. The largest species are found in normal abdominal fluid and cervical mucus. The present study therefore incorporated CA 125 derived from these sources as well as ascites fluid to investigate if the source of CA 125 influenced epitope characterization. Ascites-derived CA 125 varied in size from about 190 to about 2,700 kD. Cervical mucus-derived CA 125 treated with ultrasound changed its apparent size from more than 20,000 to 700 kD. Epitope mapping of antibodies was not grossly influenced by the size or source of CA 125 used as target. However, low-molecular-weight CA 125, i.e. ascites fractions CA 17/E, CA 17/F and CA 10/7, did show differences in certain assay combinations and cross-inhibition patterns which probably can be explained by steric effects due to the smaller size compared with the most abundant forms of CA 125 present in serum and other body fluids. The specificity of six new monoclonal antibodies to CA 125 was tested by cross-inhibition and immunometric assay combinations and compared to reference antibodies. One antibody, X306, belonged to the OC125-like antibodies. Four antibodies, X52, X75, X325 and VK8, were M11-like. The sixth antibody, 7C12, reacted with an epitope which was difficult to define. This antibody was inhibited by M11-like antibodies and OV197. However, used as an inhibitor, 7C12 inhibited only itself. We grouped it as an OV197-like antibody, but clearly different from OV197. The topography of epitopes was studied by analyzing all antibody pairs in immunoradiometric assays. These results confirmed the grouping of antibodies described above and are in accordance with previous findings that the highest signal is obtained using an OC125-like antibody or OV197 on the solid phase and an M11-like antibody as tracer. The composition of the sample in terms of high- and low-molecular-weight species of CA 125 was measured, with different responses depending on the antibody pair used. This might be one reason for discrepancies between assay results for CA 125 using different assays.