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Co-expression of human cancer-associated epitopes on mucin molecules.

This work describes the immunochemical comparison of mucin-like high-molecular-weight glycoproteins associated with human malignancies that are defined by murine MAbs, DU-PAN-2, 19-9, OC-125, B 72.3, and Mov 2. These MAbs were originally elicited to human adenocarcinomas from different organ sites such as pancreas, colon, ovary, and breast, respectively. Although each antibody recognizes an antigen on tumors of the same type as the tumor used for immunization, there is often considerable cross-reactivity with adenocarcinomas from other organ sites. Additionally, these antigens have common properties of large molecular size, high carbohydrate content, and molecular size heterogeneity. Three antigens, CA 19-9, Mov 2, and DU-PAN-2, demonstrated size heterogeneity on agarose gel immunoblotting with their respective antibodies and gave different migration patterns from sera, bile, and pancreatic juice of patients with pancreatic adenocarcinoma. Data from inhibitory double determinant analyses indicated that the DU-PAN-2, CA 19-9, CA 125, TAG-72, and Mov 2 epitopes are distinct and non-cross-reactive. However, immunoblotting and affinity chromatography indicated that both 19-9 and DU-PAN-2 epitopes may be co-expressed on the same mucin molecule in varying proportions.

Adenocarcinoma↗

Immunochemical characterization and radioimmunometric detection of molecules shed by human ovarian cancer.

Monoclonal antibodies (MAbs) OC125, MOv2 and MOv8 recognize the CA125, CAMOv2 and CAMOv8 epitopes, respectively, which are associated with human ovarian carcinomas and are shed by these tumors. The biochemical analysis of the recognized antigens and epitopes has been performed to investigate the possibility of exploiting their expression, if any, on the same molecules. Cross-competition experiments clearly indicated that the 3 epitopes are distinct. Biochemical analysis was performed on a cyst fluid from an ovarian cystadenoma. Heat treatment and periodate oxidation indicated that CAMOv2 and CAMOv8 are saccharidic and suggested that CA125 is a conformational epitope to which both saccharides and the protein backbone could contribute. By gel-filtration chromatography CA125 activity was eluted into 2 peaks of high molecular weight, whereas CAMOv2 and CAMOv8 activities were associated with a single broad peak which included CA125-positive fractions. Isopycnic centrifugation showed that CA125, CAMOv2 and CAMOv8 carrying molecules had the same high density (1.46 g/ml) in the first peak, whereas CA125 molecules had a lower density (1.41 g/ml) in the second peak. Double-determinant immunoradiometric assays, carried out using different combinations of the MAbs, indicated that in the cyst fluid molecules expressing both CAMOv2 and CAMOv8 could be detected, whereas CA125 was carried by different molecules. CAMOv2 and CA125 could however be expressed on the same molecule in 2 out of 9 ascitic fluids from ovarian carcinoma patients. Taken together, these data indicate that CA125 and CAMOv2-CAMOv8 were only occasionally expressed on the same molecules. Therefore, the use of the CA125-CAMOv2 combination could not increase the sensitivity achievable by using each respective simultaneous immunoradiometric assay.

Antigens, Neoplasm↗

Heterogeneity of antigen expression in benign and malignant breast and ovarian epithelial cells.

Expression of II distinct antigen families has been studied in normal and malignant epithelial cells from breast and ovary by avidin-biotin immunoperoxidase staining of frozen tissue sections. Each of the antigens was expressed in a fraction of the normal and malignant breast tissues from different individuals. Among breast and ovarian cancers, a similar fraction of tumors expressed each of the determinants. An epitope on a 42-kDa glycoprotein was expressed significantly more often by ovarian carcinomas than by benign ovarian epithelium. Several determinants on 66-, 240-kDa or high-molecular-weight proteins or glycoproteins were expressed significantly more often on benign breast epithelium than on normal ovarian epithelium. Substantial heterogeneity in antigen expression was observed among different tumor specimens. Each of 14 epithelial ovarian cancers expressed a distinctive combination of antigens. Among 18 breast cancers, 16 distinct antigenic phenotypes were observed. Interestingly, comparable heterogeneity was observed in the expression of epitopes on benign breast and ovarian epithelium, compatible with the possibility that the monoclonal reagents recognized polymorphic determinants. Substantial variations in antigen expression were also observed among cells within each neoplasm. However, when antibodies against 5 different determinants were used in combination, a majority of cells could be stained intensely in all 13 breast carcinomas tested and more than 95% of tumor cells could be stained in 10 of the 13.

Antigens, Neoplasm↗

Synergistic cytotoxicity of different alkylating agents for epithelial ovarian cancer.

Alkylating agents have been used individually and in combination to treat epithelial ovarian carcinoma. In this study, the cytotoxicity of 7 alkylating agents has been measured using a serial dilution clonogenic assay. When individual agents were evaluated, markedly different activity was observed against several ovarian cancer cell lines. Among 4 cell lines tested, OVCA 432 was the most sensitive to cisplatin, thiotepa and melphalan. When alkylating agents were used in combination against OVCA 432, synergistic activity was observed with cisplatin and each of several other alkylating agents including thiotepa, melphalan, 4-hydroperoxycyclophosphamide (4HC) and carboplatin. The combination of cisplatin and thiotepa also exerted synergistic activity against the OVCA 420, 429 and 433 cell lines, but had only additive or subadditive activity against the NIH:OVCAR-3 cell line. Sequential treatment of tumor cell lines with the different alkylating agents was as effective as simultaneous treatment. Synergistic anti-tumor activity in cell culture is consistent with clinical observations that alkylating agents in combination appear more effective than single agents for treatment of advanced epithelial ovarian cancer. In addition, our study suggests that cisplatin in combination with thiotepa, 4HC or melphalan might prove useful for high-dose therapy with autologous bone-marrow support.

Alkylating Agents↗

Antibody-induced growth inhibition is mediated through immunochemically and functionally distinct epitopes on the extracellular domain of the c-erbB-2 (HER-2/neu) gene product p185.

Over-expression of the c-erbB-2 (HER-2/neu) gene product p185 occurs in 30% of breast and ovarian cancers. The p185 protein might serve as a target for serotherapy in that antibodies against different epitopes on the extracellular domain of p185 can inhibit growth of tumor cells in the absence of cellular or humoral effector mechanisms. To define epitopes of functional relevance, 11 monoclonal antibodies (MAbs) were evaluated for their ability to bind to the extracellular domain of p185. Results of competition studies with 125I-labeled and non-labeled antibodies indicated that 10 of 11 epitopes were grouped in a linear array. Antibodies against 7 epitopes inhibited anchorage-independent growth and antibodies against 2 of these epitopes also inhibited anchorage-dependent growth of SKBr3 breast-cancer cells that over-expressed p185. Treatment with antibodies exerted cytotoxic rather than cytostatic effects. When antibodies were used in combination, additive or supra-additive inhibition of anchorage-independent and anchorage-dependent growth was observed between pairs of antibodies. Growth inhibition did not relate to the affinity of the antibody or its isotype. Two antibodies that inhibited both anchorage-dependent and anchorage-independent growth also blocked binding of the HER-2/neu ligand, whereas 5 antibodies that inhibited only anchorage-independent growth had no effect on ligand binding. Inhibition of cell growth did not correlate with internalization of p185 or down-regulation of p185 on the cell surface. Fab fragments of active antibodies could also inhibit anchorage-independent growth of SKBr3. Thus, murine MAbs and their fragments recognized both immunochemically distinct and functionally distinct epitopes on the p185 molecule. Whereas inhibition of anchorage-dependent growth correlated with the ability of antibodies to block ligand binding, inhibition of anchorage-independent growth did not correlate with effects on ligand binding, internalization, cell-surface expression or cross-linking of p185.

Antibodies, Monoclonal↗

Secretion of extracellular matrix-degrading proteinases is increased in epithelial ovarian carcinoma.

The biochemical events associated with tumor invasion involve localized degradation of the basement membrane by tumor-associated proteinases. In this study, we have characterized the proteinase secretion profiles of 5 ovarian epithelial carcinoma cell lines (DOV 13, OVCA 420, OVCA 429, OVCA 432, OVCA 433) as well as normal ovarian epithelial cells. Immunocapture assays demonstrated that all 5 carcinoma cell lines produce both secreted and surface-associated plasminogen activator. Urinary-type plasminogen activator (u-PA) production was one order of magnitude greater than production of tissue-type plasminogen activator (t-PA). Furthermore, t-PA secretion by normal ovarian epithelial cells was not detectable, whereas u-PA production was 17- to 38-fold lower than in ovarian carcinoma cells. Western-blotting analysis demonstrated that u-PA was secreted as the single chain form (scu-PA) when cells were cultured in serum-free medium. Incubation of plasminogen with ovarian carcinoma cell-conditioned medium resulted in direct activation of the zymogen to plasmin. Furthermore, following incubation of cells with plasminogen, plasmin was eluted from the cell surface, indicating that ovarian carcinoma cells contain binding sites for plasminogen/plasmin which are accessible to surface-associated plasminogen activators. In addition to plasminogen activators, metalloproteinases were also produced by DOV 13, OVCA 429 and OVCA 433 cells. DOV 13 cells produce a 68-kDa metalloproteinase similar to matrix metalloproteinase 2 (MMP-2) whereas a 92-kDa enzyme similar to MMP-9 is secreted by OVCA 429 and 433. Together, ovarian carcinoma-associated plasminogen activators and metalloproteinases catalyze the hydrolysis of the major basement membrane protein components, type-IV collagen, type-IV gelatin, laminin and fibronectin. The enhanced proteolytic capability of ovarian carcinoma cells relative to normal ovarian epithelium suggests a biochemical mechanism by which invasion and spread of ovarian epithelial carcinoma may be mediated.

Blotting, Western↗

The tyrosine kinase activity of the C-erbB-2 gene product (p185) is required for growth inhibition by anti-p185 antibodies but not for the cytotoxicity of an anti-p185-ricin-A chain immunotoxin.

Our previous studies have demonstrated that 7 of 10 IgG antibodies against distinct epitopes on the extracellular domain of the c-erbB-2 gene product (p185) inhibit the anchorage-independent growth of SKBr3 human breast-cancer cells that overexpress this transmembrane tyrosine kinase growth-factor receptor. Two of 7 growth-inhibitory antibodies also block the binding and function of the gp30 and p75 c-erbB-2 ligands. In this report we have studied phosphorylation of p185 and different intracellular substrates after binding of antibodies that do or do not inhibit tumor-cell growth. A correlation has been found between antibodies that inhibit growth and the intensity of tyrosine phosphorylation of p185. At late intervals, serine phosphorylation of at least 3 intracellular substrates is increased preferentially by growth-inhibitory antibodies. To test the importance of p185 kinase activity more critically, NIH3T3 cells were transfected with an expression vector containing the full-length human c-erbB-2 gene (cell line 17313), c-erbB-2 with deletion of the kinase region from codons 751-979 (cell line 9309) or c-erbB-2 with deletion of most of the intracellular domain from codons 684-1255 (cell line 9310). Unconjugated antibodies inhibited anchorage-independent growth of 17313 cells as well as SKBr3 cells, but did not inhibit growth of either 9309 or 9310 cells. In contrast, the cytotoxic effect of anti-p185-ricin A chain (RTA) conjugates was comparable for 17313, 9309 and 9310. The tyrosine-kinase activity of p185 is required for growth inhibition mediated by unconjugated anti-p185 antibodies, but not for the cytotoxic activity of anti-p185-RTA immunotoxins.

3T3 Cells↗

Molecular approaches to prevention and detection of epithelial ovarian cancer.

More than 90% of epithelial ovarian cancers arise from single cells. Malignant transformation can be associated with a number of molecular alterations including upregulation of tyrosine kinases and phosphatases, physiologic activation o ras, mutation of p53, amplification of myc, and increased activity of matrix metalloproteinases 2 and 9. Proliferation of transformed epithelial cells can be enhanced through the persistence of autocrine growth stimulation by TGF-alpha, loss of autocrine growth inhibition by TGF-beta, as well as paracrine growth stimulation by macrophage derived cytokines and OCAF, a novel lyso-phospholipid. Ascites tumor cells retain responsiveness to growth inhibition by TGF-beta which induces apoptosis in malignant ovarian epithelial cells, but not in normal ovarian surface epithelium. Proliferation of surface epithelial cells following ovulation may contribute to the pathogenesis of ovarian cancer. Use of oral contraceptives that suppress ovulation has been associated with reduced risk of ovarian cancer in later life. Retinoids also deserve further evaluation for chemoprevention. Treatment with fenretinide was associated with decreased incidence of ovarian cancer. Additive or synergistic inhibition of ovarian tumor cell proliferation has been observed with TGF-beta in combination with all-trans-retinoic acid. Early detection of ovarian cancer could improve survival. Transvaginal sonography (TVS) and serum markers such as CA-125 have been evaluated in multiple clinical trials. The former lacks adequate specificity, whereas the latter is not sufficiently sensitive. Use of multiple serum markers can improve sensitivity. A combination of CA-125, M-CSF and OVX-1 has detected > 95% of Stage I ovarian cancers. If similar results are obtained with different data sets, multiple serum markers could be used to trigger the performance of TVS, providing a potentially cost effective screening strategy. Prospective trials will be required to demonstrate that screening for early stage ovarian actually impacts on survival.

Diagnosis, Differential↗

Homologous and heterologous desensitization of proto-oncogene cfos expression in murine peritoneal macrophages.

Treatment of murine peritoneal macrophages for 30 min with lipopolysaccharide (LPS) resulted in a transient increase in c-fos proto-oncogene mRNA levels (Introna et al., 1986). After 2 h from the initial treatment, c-fos mRNA could no longer be detected and its expression could not be restimulated either by LPS or by other signals including colony stimulating factor-1 (CSF-1) and phorbol myristate acetate (PMA), both of which are able to induce expression of the c-fos gene in unstimulated macrophages. When LPS was removed after an initial 30 min incubation, responsiveness to a second exposure to LPS began to reappear after 3 h and was completely restored by 20 h. The same pattern of desensitization of c-fos induction was observed when CSF-1 stimulated macrophages were subsequently exposed to LPS. The loss of sensitivity to PMA following pretreatment with LPS was selective for c-fos expression as LPS treated macrophages remained responsive to PMA with respect to the ability to stimulate secretion of H2O2. The mechanism of desensitization was localized, at least in part, at the level of transcription as demonstrated by analysis of c-fos transcripts in nuclei isolated from macrophages pretreated and restimulated with LPS.

Animals↗

Relationship between molecular structure and cytochrome P450-metabolic intermediate complex formation, studied with orphenadrine analogues.

Complexation of ferrous cytochrome P450 by metabolic intermediates formed during NADPH-catalyzed metabolism of compounds structurally related to orphenadrine was studied. This so-called metabolic intermediate complexation was determined in rat liver microsomes, obtained from phenobarbital-pretreated rats, at 455 nm using 33 microM of the orphenadrine derivatives. Using secondary amine derivatives with various N-alkyl substituents, a parabolic relationship between the logarithm of percentage of cytochrome P450 complexation and hydrophobic fragmental constant was observed. The derivative with a bulky tertiary butyl group, however, was devoid of metabolic intermediate-complexing activity. This indicates that steric factors besides lipid solubility may govern the complexing activity; also substitution at the phenyl group affects metabolic intermediate complex formation.

Animals↗

Fluorescence spectroscopy for in vivo characterization of ovarian tissue.

BACKGROUND AND OBJECTIVE: The objective of this study was to explore whether fluorescence spectroscopy signatures differed between normal variations within the ovary, benign neoplasms, and ovarian cancer. STUDY DESIGN/MATERIALS AND METHODS: Ovarian tissue fluorescence emission spectra were collected sequentially at 18 excitation wavelengths ranging from 330 to 500 nm from 11 patients undergoing oophorectomy and assembled into fluorescence excitation emission matrices (EEMs); biopsies corresponded to the area interrogated. Spectral areas that could differentiate normal ovary, benign neoplasms, and cancers were evaluated, using histopathology as the reference standard. RESULTS: The most promising measurements are (1) the integrated fluorescence intensity from 400 to 430 nm excitation at 460 nm emission, and (2) the ratios of fluorescence intensities at 330 nm excitation, 385 and 500 nm emission, and at 375 and 415 nm excitation, 460 nm emission. Simple systems to visualize these optical signatures at laparoscopy could be designed. CONCLUSION: Fluorescence spectroscopy may have the ability to distinguish ovarian cancers from normal ovarian structures and benign neoplasms, as well as differentiate between normal variations and metaplastic structures and should be further explored as a device for the early detection of ovarian cancers.

Female↗

Gynecologic tumor markers.

The advent of monoclonal technology has increased the potential utility of antibody-dependent tumor marker assays in gynecologic oncology. The availability of unlimited quantities of several pure monoclonal antibodies directed against novel epitopes on tumor-associated antigens has permitted development of highly sensitive assays for serum markers. Traditional assays for human chorionic gonadotropin (hCG), NB/70K and TA-4 have been improved. CA 125 has provided a useful first-generation marker for monitoring ovarian cancer and triaging patients with pelvic masses, despite limitations in sensitivity and specificity. In the next decade, the challenge is to identify new markers that will complement CA 125 in monitoring ovarian cancer and facilitate screening for occult early-stage disease. Strategies involving multiple markers and modalities may be required. Some markers may emerge through a more fundamental knowledge of the biology of gynecologic neoplasms, including the expression of growth factors and their receptors. Finally, the application of monoclonal antibodies to immunohistochemistry and radionuclide imaging also may provide new areas of diagnostic application for monoclonal antibodies in gynecologic oncology.

Antibodies, Monoclonal↗

Lipoic acid favors thiolsulfinate formation after hypochlorous acid scavenging: a study with lipoic acid derivatives.

Lipoic acid, the oxidized form of 6,8-dimercapto-octanoic acid has a strained cyclic disulfide in a 1,2-dithiolane ring. Recently its antioxidant activity gained attention. Hypochlorous acid (HOCl) is an oxidant produced by neutrophils. A prominent effect of HOCl is the inactivation of alpha-1-antiproteinase. Due to this inactivation, the ability of alpha-1-antiproteinase to inhibit elastase is lost. The resulting higher activity of elastase is held responsible for tissue damage in lung emphysema. We studied the HOCl scavenging capability of three metabolites of lipoic acid: tetranor-, bisnor-, and beta-lipoic acid. To obtain some insight on the molecular basis of HOCl scavenging 1,2-dithiane-4,5-diol, cystine, lipoic acid methyl ester, and lipoamide were also included in the study. The extent of alpha-1-antiproteinase inactivation by HOCl in the presence of scavenger was taken as a parameter to quantify the scavenging activity. It was found that lipoic acid, tetranor- and bisnorlipoic acid, lipoic acid methyl ester, and lipoamide all showed the same activity toward HOCl. beta-Lipoic acid, 1,2-dithiane-4,5-diol and cystine were less active. The products of lipoic acid after reaction with HOCl were studied using GC/MS. Indications for thiolsulfinate formation were found by comparing these products with the GC/MS profile of beta-lipoic acid. Thiolsulfinate formation may also be suggested in the reaction of tetranor- and bisnorlipoic acid and lipoic acid methyl ester with HOCl. The present results show an antioxidant activity of the metabolites tetranor- and bisnorlipoic acid. The 1,2-dithiolane ring may enhance the reactivity toward HOCl compared to less strained disulfides, resulting in the formation a thiolsulfinate.

Free Radical Scavengers↗

A method for screening hypochlorous acid scavengers by inhibition of the oxidation of 5-thio-2-nitrobenzoic acid: application to anti-asthmatic drugs.

Neutrophils use a bactericidal mechanism through the release of the enzyme myeloperoxidase which catalyzes the formation of the powerful oxidant hypochlorous acid (HOCl) from H2O2 and Cl-. HOCl can inactivate alpha 1-antiproteinase (alpha 1-AP) which causes increased proteolytic activity at sites of pulmonary inflammation. The search for possible HOCl scavengers usually involves time-consuming enzyme assays (e.g., alpha 1-AP and elastase). We developed a method in which the compound 5-thio-2-nitrobenzoic acid could easily be oxidized by HOCl. The inhibition of this oxidation by a test compound is a measurement of its HOCl scavenging activity. To illustrate the method we tested some well-known HOCl scavengers such as S-methylated glutathione and oxidized lipoate. Finally several anti-asthmatic drugs such as terbutaline, isoproterenol, salbutamol, cromoglycate, theophylline, and dexamethasone were evaluated. Only the drug terbutaline acted as a HOCl scavenger.

Bronchodilator Agents↗

Flavonoids as scavengers of nitric oxide radical.

Flavonoids are a group of naturally occurring compounds used, e.g., in the treatment of vascular endothelial damage. They are known to be excellent scavengers of oxygen free radicals. Since the nitric oxide radical (.NO) probably plays a role in this pathology, the .NO scavenging capacity of the flavonoids was determined. It was found that the flavonoids are very potent .NO scavengers. The anthocyanidins were found to be more effective scavengers than the hydroxyethylrutosides, which correlated with their therapeutic activity. The values of their scavenging rate constants are only 30 times less active than the very potent endogenous .NO scavenger haemoglobin. It is speculated that .NO scavenging plays a role in the therapeutic effect of the flavonoids.

Flavonoids↗

Peroxynitrite scavenging by flavonoids.

The peroxynitrite scavenging activity of a series of structurally related flavonoids was tested. It was found that flavonoids are excellent scavengers of peroxynitrite. Compared to the known peroxynitrite scavenger ebselen, the most active flavonoids proved to be 10 times more effective. Indications were found that the catechol group (ring B) and the hydroxyl group at position 3 give the highest contribution to the peroxynitrite scavenging effect. The peroxynitrite scavenging is discussed in relation to the beneficial effect of flavonoid intake on the incidence of coronary heart disease.

Flavonoids↗

alpha-Tocopherol inhibits human glutathione S-transferase pi.

alpha-Tocopherol is the most important fat-soluble, chain-breaking antioxidant. It is known that interplay between different protective mechanisms occurs. GSTs can catalyze glutathione conjugation with various electrophiles, many of which are toxic. We studied the influence of alpha-tocopherol on the activity of the cytosolic pi isoform of GST. alpha-Tocopherol inhibits glutathione S-transferase pi in a concentration-dependent manner, with an IC(50)-value of 0.5 microM. At alpha-tocopherol additions above 3 microM there was no GST pi activity left. alpha-Tocopherol lowered the V(max) values, but did not affect the K(m) for either CDNB or GSH. This indicates that the GST pi enzyme is noncompetitively inhibited by alpha-tocopherol. An inhibition of GST pi by alpha-tocopherol may have far-reaching implications for the application of vitamin E.

Antioxidants↗

Expression of two interleukin 4 mRNA isoforms in B lymphoid cells.

Expression of an alternative spliced IL4 mRNA was found in in vitro activated T cells. In this study we show that the expression of IL4 mRNA, as well as the expression of this alternatively spliced form of IL4 mRNA, is not restricted to these cells. We analyzed different human lymphoid tissues and cell lines of different origin and found that the alternatively spliced IL4 transcripts are also expressed in human lymphoid tissues, in purified B cells, in the various B cell-derived cell lines, and even in nonlymphoid cell lines. Stimulation with the phorbol ester PMA enhanced expression of both transcripts in all cells studied. The two IL4 transcripts were cloned and sequenced from the B cell line Namalwa. In the alternatively spliced form, the same exon 2 is deleted as has been observed in in vitro activated T cells. In principle, such alternatively spliced mRNA may give rise to a truncated IL4 protein, as the deletion does not result in a frameshift. We tested supernatants of activated PBMC, cell lines, and cell extracts for the presence of IL4 protein. We found IL4 protein expression in activated PBMC, but not in any of the stimulated cell lines or in the purified B cells. Using a modified in situ hybridization method with Dig-labeled PCR products, however, these cells did express IL4 mRNA. This shows that transcription of both IL4 forms is not restricted to T cells and can be induced in other cell types as well. Using these non-T cells, no protein has been found in the supernatant, however. It is possible that transcription of the IL4 gene is not necessarily followed by translation and that translation into the IL4 protein requires an additional signal.

Alternative Splicing↗