Biomedical subjects

T J O'Brien

Publications and source records attributed to T J O'Brien.

156 records · Page 9Linked to original sources

PubMedDate not supplied

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 beta-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.

Biomarkers, Tumor↗

PubMedDate not supplied

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 (32)P and a phosphoimager was used to determine the relative expression of the protease M gene compared to internal control beta-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 ovary 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.

Adenocarcinoma, Clear Cell↗

PubMedDate not supplied

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.

Amino Acid Sequence↗

PubMedDate not supplied

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.

Amino Acid Sequence↗

PubMedDate not supplied

Epitopes on CA 125 from cervical mucus and ascites fluid and characterization of six new antibodies. Third report from the ISOBM TD-1 workshop.

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.

Antibodies, Monoclonal↗

PubMedDate not supplied

Arthroscopic anterior labral reconstruction using a transglenoid suture technique. Results in active-duty military patients.

We report the clinical outcome of arthroscopic labral reconstruction using a transglenoid suture technique in a young, active-duty military population. Forty-eight patients (49 shoulders) with varying degrees of glenohumeral instability underwent arthroscopic labral reconstruction using a transglenoid suture technique. All patients had traumatic injuries to their shoulders and all patients had magnetic resonance imaging scans demonstrating anterior labral tears. Postoperatively, the patients' shoulders were immobilized for up to 6 weeks. At a mean followup of 30 months (range, 12 to 49), 17 of the 41 patients (41%) with preoperative dislocation or subluxation had recurrent instability. Nine of these patients subsequently underwent open reconstruction procedures for recurrent instability. On the basis of the Rowe rating system, 53% had excellent or good results and 47% had fair or poor results. The overall perioperative complication rate was 14%. Suprascapular nerve palsy occurred in three cases (6%). Using the Fisher exact test, we determined that immobilization for 6 weeks postoperatively correlated with a lower recurrence rate in the patients with a history of glenohumeral dislocation (P = 0.007). The results of arthroscopic labral reconstruction using transglenoid sutures in the military patient are inferior to the reported 3% to 5% recurrence rate with open Bankart procedures, and the transglenoid pin technique jeopardizes the suprascapular nerve.

Adolescent↗

PubMedDate not supplied

Epidermal growth factor enhances secretion of the ovarian tumor-associated cancer antigen CA125 from the human amnion WISH cell line.

OBJECTIVE: We studied the relation between epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) and CA125 production in WISH cells. METHODS: We investigated quantitatively and immunohistochemically EGF-stimulated CA125 release from WISH cells and the effect of EGF on CA125 phosphorylation. RESULTS: Immunohistochemical staining demonstrated that CA125 and EGFR expression on the plasma membrane of the WISH cells was closely correlated with cell density. The WISH cell monolayers (day 4) stained for CA125 in both the cytoplasm and plasma membrane. By day 8, cells began to form clumps in the surrounding monolayer that were positive for membrane-associated CA125 and EGFR, while the monolayer was almost negative for both molecules. Four-day and 8-day cells exposed to EGF demonstrated a loss of both CA125 and EGFR staining. Epidermal growth factor increased the secreted CA125 levels by 50% in day-4 cells but had no effect on day-8 cells. CA125 from WISH cells was phosphorylated, and EGF further enhanced this phosphorylation.

Amnion↗

PubMedDate not supplied

More than 15 years of CA 125: what is known about the antigen, its structure and its function.

In 1997 CA 125 celebrated its 15th anniversary. Since the discovery of OC 125, an antibody that recognizes CA 125, by Bob Bast and his colleagues, considerable progress has been made toward the development of more sensitive and more precise assay systems. However, a great deal of mystery still remains about the CA 125 molecule and further enlightenment will probably not come until the gene for CA 125 is cloned and the complete open reading frame for the peptide core identified. In the meantime, we have learned some structural features of the CA 125 molecule as well as a little about its regulation and the requirements for its secretion or release from epithelial derived cells in cultures. The CA 125 molecule is almost certainly a glycoprotein with a predominance of O-linkages. It is heterogeneous with regard to both size and charge, most likely due to continuous deglycosylation of side chains during its life-span in bodily fluids. It exists as a very large complex (perhaps as much as 4 million daltons) under natural conditions. The core CA 125 subunit is in excess of 200,000 daltons and it retains the capacity to bind both OC 125 class antibodies and M 11 class antibodies. As a denatured purified subspecies the CA 125 molecule appears to autoproteolyse presumably due to an endogenous protease activity inherent to the molecule. Release or secretion of CA 125 appears directly linked to the epithelial growth factor receptor signal transduction pathway. Prior to its release from cultured cells, CA 125 is phosphorylated (at either/both serine and threonine) and dephosphorylated when released. To stimulate discussion on the regulation of CA 125 synthesis, its secretion and its structural configuration, we have presented a model of a theoretical CA 125 molecule. Perhaps it will provide a focus of attention until the CA 125 gene is cloned and the real molecule is described.

Antibodies↗

PubMedDate not supplied

Differential expression of matrix metalloprotease-7 in each component of uterine carcinosarcoma.

The aim of this study was to investigate the expression of matrix metalloprotease-7 (MMP-7) in each component of uterine carcinosarcoma. Surgical specimens of primary tumors of uterine carcinosarcomas were obtained from 13 patients. The carcinomatous component consisted of adenocarcinoma with or without squamous differentiation. The sarcomatous component consisted of spindle cell sarcoma, chondrosarcoma and rhabdomyosarcoma, either alone or in combination. The immunohistochemical expression of MMP-7 protein was examined using the avidin-biotin peroxidase complex technique employing the anti-MMP-7 monoclonal antibody. Expression of MMP-7 protein was detected in 9 (69.2%) of 13 adenocarcinoma components, while no staining was observed in any of the sarcomatous components examined. There was a statistically significant difference of the positive rate for MMP-7 between epithelial components and sarcomatous components of uterine carcinosarcoma (p<0.01). In some cases, MMP-7 was abundantly expressed at the invasive front of adenocarcinomas. It is concluded that MMP-7 protein is differentially expressed between the carcinomatous component and the sarcomatous component of uterine carcinosarcoma. Each component of carcinosarcoma may have its own potential for invasion and metastasis. MMP-7 may contribute to the invasive nature or growth capacity of the carcinomatous component of uterine carcinosarcoma, while it may not have a relation to that of the sarcomatous components.

Adenocarcinoma↗

PubMedDate not supplied

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

OBJECTIVE: In a continued effort to identify and characterize 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 27 carcinomas) and seven normal ovaries. The testisin mRNA expression level relative to internal control, beta-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 diseases (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.

Adenoma↗

PubMedDate not supplied

Cyclin E mRNA overexpression in epithelial ovarian cancers: inverse correlation with p53 protein accumulation.

OBJECTIVE: We investigated the relationship between cyclin E mRNA overexpression and p53 protein accumulation in epithelial ovarian cancers. METHODS: mRNA was isolated and cDNA was prepared from 36 epithelial ovarian tumors (three adenomas, three low malignant potential tumors, and 30 carcinomas), and six normal ovaries. The cyclin E mRNA expression levels relative to an internal control, beta-tubulin, were determined by semiquantitative polymerase chain reaction (PCR). Cyclin E and p53 protein expression in ovarian cancer tissues were examined by immunohistochemistry using the same series of samples. Fisher exact test of significance and an unpaired t test were used for statistical analysis. RESULTS: Considerable levels of cyclin E mRNA were detected in all normal ovaries and ovarian tumor samples examined by semiquantitative PCR amplification. mRNA levels of cyclin E were significantly higher in nine of 30 (30%) ovarian cancers compared with those in normal ovaries. The immunohistochemical expression of cyclin E protein was confirmed in the nuclei of tumor cells in 13 of 30 (43%) ovarian cancers. p53 protein accumulation was detected in 12 of 30 (40%) ovarian cancers examined. There was a significant inverse correlation between cyclin E mRNA overexpression and p53 protein accumulation (P <.01, Fisher exact test). CONCLUSIONS: Cyclin E mRNA overexpression frequently occurs in ovarian cancers without p53 protein accumulation. Cyclin E might have an important effect on the development of a limited number of ovarian cancers.

Adenoma↗

PubMedDate not supplied

p16 overexpression: a potential early indicator of transformation in ovarian carcinoma.

OBJECTIVE: The recently cloned gene p16 (MST1) has been identified as a putative tumor suppressor gene that binds to CDK4 and CDK6 (cyclin-dependent kinases), preventing their interaction with cyclin D1 and thereby preventing cell cycle progression at the G1 stage. In addition, the p16 gene has been shown to have a high frequency of mutation in some tumor cell lines; however, it has also been shown that a much lower frequency of mutation occurs in primary tumors. This study investigated the mRNA expression level and mutation status of the p16 gene in ovarian tumors. METHODS: We performed quantitative polymerase chain reaction and direct cDNA sequencing analysis. To confirm the p16 protein level in ovarian tumors, Western blotting and immunohistochemical staining were performed. Expression levels of mRNA for the p16 gene relative to the beta-tubulin gene were examined in 32 ovarian tumors (24 carcinomas, six low malignant potential tumors, and two benign tumors) and six normal ovaries. RESULTS: The mRNA expression level of p16 was significantly elevated in 28 ovarian tumors (22 carcinomas, five low malignant potential tumors, and one benign tumor) compared with that of normal ovaries. Western blotting analysis and immunohistochemical staining confirmed elevated p16 protein levels in ovarian tumor samples. Among 32 ovarian tumors, cDNA sequencing of the p16 gene showed no p16 mutation resulting in a coding error, although one silent mutation and three polymorphisms were found. CONCLUSIONS: Although p16 is seldom mutated in ovarian tumors, the overexpression of p16 in most ovarian tumor cases indicates a dysfunction in the regulatory complex for G1 arrest. Therefore, overexpression of p16 may be an important early event in the neoplastic transformation of the ovarian epithelium.

Biomarkers, Tumor↗