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Biomedical subjects

M Pagé

Publications and source records attributed to M Pagé.

At least 19 recordsLinked to original sources
PubMed2003

Levels of vascular endothelial growth factor (VEGF) in serum of patients with endometriosis.

BACKGROUND: Elevated concentrations of vascular endothelial growth factor (VEGF) have been detected in the peritoneal fluid of patients with endometriosis. Furthermore, it was postulated that VEGF is involved in the development of endometriotic lesions. The present study is aimed at determining whether high levels of VEGF could also be found in the serum of patients with endometriosis. METHODS: VEGF levels were measured by enzyme-linked immunosorbent assay (ELISA) in serum from 131 subjects with surgically confirmed endometriosis and 146 controls with no clinical evidence of the disease or detectable endometriotic lesions at the time of surgical examination. Parameters such as demographics, personal habits, menstrual characteristics and clinical profile were collected from each subject included in this study. RESULTS: The mean VEGF levels were not significantly modulated in serum samples of cases compared with controls in a crude general linear model and in a model adjusted for possible confounders. VEGF serum levels did not correlate with the score, stage of endometriosis or the presence of benign gynaecological disorders. However, a correlation was found between circulating concentrations of VEGF and body mass index. CONCLUSION: Although VEGF seems to play a pivotal role locally in the implantation and development of endometriotic lesions, the disease is not associated with a significant modulation in the levels of circulating VEGF.

Adolescent↗
PubMed2001

Localization of membrane-type 1 matrix metalloproteinase in caveolae membrane domains.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-associated MMP that has been recently reported to have a central role in tumour cell invasion. Here we report that both the native and overexpressed recombinant forms of MT1-MMP are highly enriched in low-density Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin 1. Moreover, the MT1-MMP-dependent activation of proMMP-2 induced by concanavalin A and cytochalasin D was correlated with the processing of MT1-MMP to its proteolytically inactive 43 kDa fragment in U-87 glioblastoma and HT-1080 fibrosarcoma tumour cell lines; this processing was also preferentially observed within the caveolar fraction. Interestingly, whereas the expression of caveolin 1 had no effect on the MT1-MMP-dependent activation of proMMP-2, its co-expression with MT1-MMP antagonized the MT1-MMP-increased migratory potential of COS-7 cells. Taken together, our results provide evidence that MT1-MMP is preferentially compartmentalized and proteolytically processed in caveolae of cancer cells. The inhibition of MT1-MMP-dependent cell migration by caveolin 1 also suggests that the localization of MT1-MMP to caveolin-enriched domains might have an important function in the control of its enzymic activity.

Animals↗
PubMed2000

Rapid activation of matrix metalloproteinase-2 by glioma cells occurs through a posttranslational MT1-MMP-dependent mechanism.

Matrix metalloproteinase-2 (MMP-2) has been suggested to play a crucial role in tumor invasion and angiogenesis. In order to understand the mechanisms underlying proMMP-2 activation, we compared the biochemical and cellular events triggered by two potent MMP-2 activators, the lectin concanavalin A (ConA) and the cytoskeleton disrupting agent cytochalasin D (CytoD). Incubation of U87 human glioma cells for 24 h in the presence of ConA or CytoD induced a marked activation of proMMP-2 and this activation was correlated in both cases with an increase in the mRNA levels of MT1-MMP. At the protein level, proMMP-2 activation induced by CytoD or ConA strongly correlated with the appearance of a 43-kDa MT1-MMP proteolytic breakdown product in cell lysates. Interestingly, CytoD also induced a very rapid (2 h) activation of proMMP-2 that was independent of protein synthesis. Under these conditions, CytoD also promoted the rapid proteolytic breakdown of the 63 kDa pro form of MT1-MMP, resulting in the appearance of the 43 kDa MT1-MMP processed form. Overexpression of a recombinant full-length MT1-MMP protein in glioma cells resulted in the activation of proMMP-2 that was correlated with the generation of the 43 kDa fragment of the protein. By contrast, overexpression of the protein in COS-7 cells promoted proMMP-2 activation without inducing the production of the 43 kDa fragment. These results thus suggest that activation of proMMP-2 occurs through both translational and post-translational mechanisms, both involving proteolytic processing of membrane-associated MT1-MMP. This processing of MT1-MMP is, however, not essential to proMMP-2 activation but may represent a regulatory mechanism to control the activity of MT1-MMP.

Concanavalin A↗
PubMed2000

Matrix metalloproteinase inhibition by green tea catechins.

We have investigated the effects of different biologically active components from natural products, including green tea polyphenols (GTP), resveratrol, genistein and organosulfur compounds from garlic, on matrix metalloproteinase (MMP)-2, MMP-9 and MMP-12 activities. GTP caused the strongest inhibition of the three enzymes, as measured by fluorescence assays using gelatin or elastin as substrates. The inhibition of MMP-2 and MMP-9 caused by GTP was confirmed by gelatin zymography and was observed for MMPs associated with both various rat tissues and human brain tumors (glioblastoma and pituitary tumors). The activities of MMPs were also measured in the presence of various catechins isolated from green tea including (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate(ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC) and (+)-catechin (C). The most potent inhibitors of these activities, as measured by fluorescence and by gelatin or casein zymography, were EGCG and ECG. GTP and the different catechins had no effect on pancreatic elastase, suggesting that the effects of these molecules on MMP activities are specific. Furthermore, in vitro activation of proMMP-2 secreted from the glioblastomas cell line U-87 by the lectin concanavalin A was completely inhibited by GTP and specifically by EGCG. These results indicate that catechins from green tea inhibit MMP activities and proMMP-2 activation.

Animals↗
PubMed2000

Expression of endogenously activated secreted or cell surface carboxypeptidase A sensitizes tumor cells to methotrexate-alpha-peptide prodrugs.

Methotrexate (MTX) is one of the most commonly used agents in the treatment of solid malignancies; however, the toxicities of MTX to bone marrow and gastrointestinal tract complicate this therapy. We, therefore, propose a gene-dependent enzyme prodrug therapy to limit these toxicities by localizing the production of MTX to the site of the tumor. The combination of MTX-alpha-peptide prodrugs, which cannot be internalized by the cellular reduced folate carrier, with carboxypeptidase A (CPA), which can remove the blocking peptide, has been demonstrated previously in vitro using antibody-dependent enzyme prodrug therapy. CPA is normally synthesized as a zymogen that is inactive without proteolytic removal of its propeptide by trypsin. Therefore, to adapt this system to gene-dependent enzyme prodrug therapy, a mutant form of CPA was engineered, CPA(ST3), that does not require trypsin-dependent zymogen cleavage but is instead activated by ubiquitously expressed intracellular propeptidases. Purification, peptide sequencing, and kinetic analysis indicated that mature CPA(ST3) is structurally and functionally similar to the trypsin-activated, wild-type enzyme. In addition, CPA(ST3)-expressing tumors cells were sensitized to MTX prodrugs in a dose- and time-dependent manner. To limit diffusion of CPA, a cell surface localized form was generated by constructing a fusion protein between CPA(ST3) and the phosphatidylinositol linkage domain from decay accelerating factor. SDS-PAGE and flow cytometric analysis of infected tumor cells indicated that CPA(DAF) was cell surface localized. Finally, after retroviral transduction, this enzyme/prodrug strategy exhibited a potent bystander effect, even when <10% of the cells were transduced, because extracellular production of MTX sensitized both transduced and nontransduced cells.

Antineoplastic Agents↗
PubMed2000

A method for detecting antibody internalization in drug targeting.

BCM43/2E5 monoclonal antibody with a specificity for human ovarian carcinoma was evaluated for its binding and internalization behaviour on both fixed and live cells. Internalization of this antibody by the targeted cells was demonstrated by ELISA, flow cytometry and immunoperoxidase. At reduced temperatures or in the presence of electron transfer blockers, internalization of bound antibody was prevented. This method is rapid and reproducible and may be used efficiently for the selection of antibodies suitable for drug targeting in cancer therapy.

3T3 Cells↗
PubMed1998

Development of a fluorescence polarization immunoassay (FPIA) for the quantitative determination of paclitaxel.

We have developed a fluorescence polarization immunoassay (FPIA) for the quantitative determination of paclitaxel in serum, crude Taxus extracts and Erwinia taxi culture medium. To achieve this, we used an antipaclitaxel monoclonal antibody and an FITC-labeled paclitaxel. Paclitaxel was chemically modified by introducing an amino function to enable coupling with fluorescein isothiocyanate. Paclitaxel competitively inhibited the binding of the monoclonal antibody with FITC-paclitaxel causing a decrease in polarization. We were able to detect paclitaxel in a concentration as low as 2 nM. Fluorescence polarization immunoassay requires only the addition of the fluorescent probe to the antibody, followed by an incubation and measurement of polarization. Both the simplicity and the sensitivity of this method make it useful for estimating the paclitaxel content in yew tree crude extracts and culture medium of paclitaxel producing micro-organisms and a possible assay for monitoring paclitaxel level in patients under treatment.

Antibodies, Monoclonal↗
PubMed1997

CD4+ CD8+ thymocytes are preferentially induced to die following CD45 cross-linking, through a novel apoptotic pathway.

Ligation of the protein tyrosine phosphatase CD45 on both mature and immature T cells modulates the amplitude of TCR-mediated signals. In this work, we have evaluated the consequences of CD45 ligation on immature T cells, in the absence of TCR engagement. Cross-linking of CD45 on thymocytes by mAbs led to the induction of cellular death, characterized by a reduction in mitochondrial membrane potential (delta psi(m)), production of reactive oxygen species, loss in membrane asymmetry, exposure of phosphatidylserine residues, and incorporation of vital dyes. In sharp contrast to most stimuli causing thymocyte death, CD45 cross-linking did not lead to DNA degradation. Cell death was not blocked by Bcl-2 overexpression or treatment with caspase inhibitor. However, death was inhibited by the addition of scavengers of reactive oxygen species. We also established that susceptibility to CD45-mediated death is acquired during the transition of early CD4- CD8- TCR- T cell precursors into CD4+ CD8+ TCR- thymocytes and is increased with further acquisition of surface TCR on these cells. Moreover, mature thymocytes were much less sensitive to CD45 cross-linking than CD4+ CD8+ cells. We propose that during T cell development, CD45 ligation could induce the death of those immature thymocytes that do not fulfill the requirements for positive selection.

Animals↗
PubMed1995

Measurement of tumor necrosis factor activity by flow cytometry.

Tumor necrosis factor-alpha (TNF-alpha) is a monokine of 17 kDa produced by activated macrophages and various cells involved in the immune system. We propose a new method for the measurement of TNF activity using flow cytometry. After an incubation with TNF, L929 cells were harvested and treated with a calcein-AM and ethidium homodimer-1 solution. Nonfluorescent calcein-AM is hydrolyzed by intracellular esterases to yield fluorescent calcein. The ethidium homodimer-1 is a high-affinity red fluorescent DNA dye that is internalized only through altered cell membranes. A very good correlation was observed between the calcein fluorescence intensity and the number of viable cells as well as the ethidium fluorescence and the number of cells with altered membranes. The assay is sensitive, inexpensive, and correlates with the already reported crystal violet assay while measuring membrane alteration by TNF. It allows the simultaneous measurement of total living and dead cells. There is no interference with culture medium components. This method is rapid and may be used for routine measurement of TNF activity.

Animals↗
PubMed1995

An M(r) 7-kDa membrane protein overexpressed in human multidrug-resistant ovarian cancer cells.

We have developed a monoclonal antibody (designated 1D7) which recognizes an M(r) 7-kDa plasma membrane protein overexpressed in ovarian MDR cancer cells. The expression of the M(r) 7-kDa protein in various human multidrug-resistant and drug-sensitive cell lines was analysed by Western blot and flow cytometry methods. The small molecular weight protein was overexpressed in the human ovarian carcinoma cell line, SKVLB which was selected for vinblastine resistance from SKOV3 cells and in OVCAR 4/ADR100 and OVCAR 4/VBL200 which were generated from NIH:OVCAR4 by stepwise selection against adriamycin and vinblastine, respectively. Only a minor amount of the M(r) 7-kDa protein was found in the parent cell line, SKOV3. It was not found in other drug-resistant human cell lines such as the vinblastine-resistant CEM cells (CEM/VLB300), the intrinsic MDR colon cell line HCT15 and the human MDR breast cancer cell line, MCF7/AdrVp. 1D7 specifically inhibited the proliferation of the resistant cells. Our results suggest that the expression of the M(r) 7-kDa protein on the plasma membrane of ovarian MDR cancer cells may be involved in a mechanism related to the proliferation of the drug resistant cancer cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Improved fluorescent bioassay for the detection of tumor necrosis factor activity.

Tumor necrosis factor alpha (TNF-alpha) is a monokine of 17 kDa produced by activated macrophages and various cells involved in the immune system. We propose a new method for the measurement of TNF activity on mouse L929 fibroblast cells. After an incubation with TNF, the cells were stained with a solution of ethidium homodimer-1, a high-affinity red fluorescent DNA dye that is internalized only through altered cell membranes. The assay is sensitive, inexpensive and correlates with the already reported TNF assays while measuring the membrane alteration by TNF and not the cell detachment. It requires no rinsing before dye addition which may cause cell loss; there is no interference with culture medium components since the assay is performed in PBS. This method is more rapid and precise for routine measurement of TNF activity.

Animals↗
PubMed1995

P-glycoprotein expression in ovarian cancer cell line following treatment with cisplatin.

Human ovarian cancer cell line SKOV3 was grown during a period of four months in the presence of increasing concentrations of cisplatin (25-100 ng/ml). In the course of this treatment, the cells exhibited dramatic changes in morphology, including reduction in cell size, loss of cellular projections and clustering. This was accompanied by the appearance of P-glycoprotein (Pgp) on the cell membrane, as detected by flow cytometry and immunochemistry methods using the anti-Pgp monoclonal antibodies MRK16 and C219. The new cell line, designated SKOV3/CIS, was also resistant to alkylating agents, such as chlorambucil, similarly to the parental SKOV3 cells. In addition, it also acquired resistance to classical multidrug resistance drugs, such as doxorubicin, taxol and actinomycin D. Verapamil enhanced the sensitivity of SKOV3/CIS to doxorubicin (260-fold), in conformity with the proposed mechanism of Pgp in multidrug resistance (MDR), but it did not potentiate cisplatin cytotoxicity in SKOV3/CIS cells. Our results suggest that cisplatin can cause Pgp expression, and that both cisplatin-resistance and Pgp-mediated MDR phenotypes can coexist in some tumor types. Although Pgp does not appear to be responsible for cisplatin resistance, exposure to cisplatin can lead to the development of MDR phenotype, a complication that should be considered in clinical situations, especially in the chemotherapy of ovarian cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Rapid purification method for human recombinant tumor necrosis factor alpha.

Human recombinant tumor necrosis factor alpha was purified in a single step to about 95% purity from Escherichia coli lysate by chromatography on hydroxyapatite. The last traces of contaminants were removed by fast protein liquid chromatography on a Mono Q column. The final product was found to be pure by gel electrophoresis with silver staining. A molecular mass of approximately 17,000 and a specific activity of 4.3 x 10(6) U/mg after a single purification step were found.

Amino Acids↗
PubMed1988

1-Aryl-3-(2-chloroethyl) ureas: synthesis and in vitro assay as potential anticancer agents.

1-Aryl-3-(2-chloroethyl) ureas and 1-aryl-3-nitroso-3-(2-chloroethyl) ureas, derived from 4-phenylbutyric acid and alkylanilines, were synthesized and their cytotoxicity was evaluated on human adenocarcinoma cells in vitro. Methyl 4-[p-[3-(2-chloroethyl)ureido]-phenyl]butyrate, 4-methyl [3-(2-chloroethyl)ureido]benzene, and 4-butyl[3-(2-chloroethyl)ureido]benzene were found to be at least as cytotoxic as 4-[p-[bis-(2-chloroethyl)amino]phenyl]butyric acid (chlorambucil), while their N-nitroso derivatives were inactive.

Adenocarcinoma↗
PubMed1988

Optimization of the tetrazolium-based colorimetric assay for the measurement of cell number and cytotoxicity.

We report some modifications of the semiautomated tetrazolium-based assay for the measurement of anchorage-dependent and -independent mammalian cells. The various factors affecting color production, such as the concentration of tetrazolium, incubation period, the type and volume of solvent, were optimized. Using KCN and daunorubicin as cytotoxic agents, the influence of dead cells was studied on the measurement. The assay was tested with mouse leukemia P388 cells, H69 small cell carcinoma cells growing in suspension and anchorage dependent colon adenocarcinoma cells (LoVo). Centrifugation of the microtitration plate was eliminated by the use of a Skatron supernatant collection system. Although the use of the MTT assay is rapid and precise, we found that care should be taken when using this assay for short-term cytotoxicity assays since non-viable cells also reduce the tetrazolium.

Animals↗
PubMed1986

Growth changes of 3T3 cells in the presence of mineral fibers.

The relationship between exposure to asbestos fibers and the development of mesothelioma or bronchial carcinoma prompted many countries to ban its use from commercial products. The biological mechanism by which asbestos induces or promotes mesothelioma or carcinoma is still unknown. In order to study the influence of fibers on the cell surface, 3T3 fibroblasts were cultured in the presence of various mineral fibers. The acute cytotoxicity produced by mineral fibers was evaluated by the trypan blue dye exclusion method; growth of 3T3 cells was measured as well as the maximum cell density at saturation. We found that growth of 3T3 cells was slower in the presence of chrysotile while light microscopy revealed an increased cellular chromogenicity and a modification of the cell-cell arrangement in the presence of this fiber. We describe an assay in which chrysotile causes an increase in the maximum cell density at saturation.

Animals↗