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1.
Sci Rep ; 12(1): 14662, 2022 08 29.
Article in English | MEDLINE | ID: mdl-36038621

ABSTRACT

Ancient charcoal fragments, produced by the use of wood as fuel in archaeological contexts or during natural or anthropic forest fires, persist in soil and sediments over centuries to millennia. They thus offer a unique window to reconstruct past climate, especially palaeo-precipitation regimes thanks to their stable carbon isotope composition. However, the initial δ13C of wood is slightly modified as a function of the carbonisation temperature. Carbonisation-induced 13C fractionation is classically investigated through a transfer function between experimental carbonisation temperatures and the carbon content. This approach assumes that the carbon content is conservative through time in ancient charcoals and neglects the potential impact of post-depositional oxidation occurring in soils and sediments. In the present study, we first show that post-depositional oxidation can lead to a large underestimation of past carbonisation temperatures, thereby minimising the estimation of carbonisation-induced 13C fractionations and possibly biasing δ13C-based climate reconstructions. Secondly, by combining carbon content, Fourier-transform infrared and Raman spectroscopy, we propose a new framework to assess the carbonisation temperatures registered in ancient charcoals. This new framework paves the way to reassessing δ13C-based climate reconstruction.


Subject(s)
Charcoal , Soil , Carbon , Carbon Isotopes/analysis , Charcoal/chemistry , Climate , Temperature
2.
Oncogene ; 26(5): 633-40, 2007 Feb 01.
Article in English | MEDLINE | ID: mdl-16909123

ABSTRACT

Recently, we have shown that RhoB suppresses EGFR-, ErbB2-, Ras- and Akt-mediated malignant transformation and metastasis. In this paper, we demonstrate that the novel antitumor agents farnesyltransferase inhibitors (FTIs) and geranylgeranyltransferase I inhibitors (GGTIs) upregulate RhoB expression in a wide spectrum of human cancer cells including those from pancreatic, breast, lung, colon, bladder and brain cancers. RhoB induction by FTI-277 and GGTI-298 occurs at the transcriptional level and is blocked by actinomycin D. Reverse transcription-PCR experiments documented that the increase in RhoB protein levels is due to an increase in RhoB transcription. Furthermore, treatment with FTIs and GGTIs of cancer cells results in HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter. Thus, promoter acetylation is a novel mechanism by which RhoB expression levels are regulated following treatment with the anticancer agents FTIs and GGTIs.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Farnesyltranstransferase/antagonists & inhibitors , Histone Acetyltransferases/metabolism , Histone Deacetylases/metabolism , Histones/metabolism , Promoter Regions, Genetic , rhoB GTP-Binding Protein/genetics , Acetylation , Alkyl and Aryl Transferases/metabolism , Antineoplastic Agents , Benzamides/pharmacology , Enzyme Inhibitors/pharmacology , Farnesyltranstransferase/metabolism , Histone Deacetylase 1 , Humans , Methionine/analogs & derivatives , Methionine/pharmacology , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Protein Processing, Post-Translational , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Up-Regulation , rhoB GTP-Binding Protein/metabolism
3.
Cell Death Differ ; 9(7): 702-9, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12058275

ABSTRACT

Recently, we have shown that the farnesyltransferase inhibitor FTI-2153 induces accumulation of two human lung cancer cell lines in mitosis by inhibiting bipolar spindle formation during prometaphase. Here we investigate whether this mitotic arrest depends on transformation, Ras and/or p53 mutation status. Using DAPI staining (DNA) and immunocytochemistry (microtubules), we demonstrate that in normal primary foreskin fibroblasts (HFF), as well as in several cancer cell lines of different origins including human ovarian (OVCAR3), lung (A-549 and Calu-1) and fibrosarcoma (HT1080), FTI-2153 inhibits bipolar spindle formation and induces a rosette morphology with a monopolar spindle surrounded by chromosomes. In both malignant cancer cell lines and normal primary fibroblasts, the percentage of prometaphase cells with bipolar spindles decreases from 67-92% in control cells to 2-28% in FTI-2153 treated cells. This inhibition of bipolar spindle formation correlates with an accumulation of cells in prometaphase. The ability of FTI-2153 to inhibit bipolar spindle formation is not dependent on p53 mutation status since both wild-type (HFF, HT1080 and A-549) and mutant (Calu-1 and OVCAR3) p53 cells were equally affected. Similarly, both wild-type (HFF and OVCAR3) and mutant (HT1080, Calu-1 and A-549) Ras cells accumulate monopolar spindles following treatment with FTI-2153. However, two cell lines, NIH3T3 (WT Ras and WT p53) and the human bladder cancer cell line, T-24 (mutant H-Ras and mutant p53) are highly resistant to FTI-2153 inhibition of bipolar spindle formation. Finally, the ability of FTI-2153 to inhibit tumor cell proliferation does not correlate with inhibition of bipolar spindle formation. Taken together these results demonstrate that the ability of FTI-2153 to inhibit bipolar spindle formation and accumulate cells in mitosis is not dependent on transformation, Ras or p53 mutation status. Furthermore, in some cell lines, FTIs inhibit growth by mechanisms other than interfering with the prophase/metaphase traverse.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Mitosis/drug effects , Spindle Apparatus/drug effects , Tumor Suppressor Protein p53/metabolism , ras Proteins/metabolism , 3T3 Cells , Animals , Cell Division/drug effects , Farnesyltranstransferase , Humans , Metaphase , Mice , Mitosis/physiology , Mutagenesis , Spindle Apparatus/physiology , Transformation, Genetic , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , ras Proteins/genetics
4.
Transplantation ; 72(7): 1256-61, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11602852

ABSTRACT

BACKGROUND: Plasminogen activator inhibitor type 1 (PAI-1) exerts antifibrinolytic and profibrotic activities. Inside the glomerulus, PAI-1 is mainly synthesized by mesangial cells. We hypothesized that thrombin, via its receptor protease activated receptor type 1 (PAR-1), present on the membrane of glomerular cells, is an important mediator of PAI-1 synthesis. METHODS: Using the technique of Peten et al., we microdissected the glomeruli of 23 kidney transplanted patients admitted in our department from 1993 to 1997, and we followed-up these patients for up to 5 years, with sometimes iterative renal biopsies. With this technique, we also microdissected the glomeruli of three patients who have had a nephrectomy for cancer (control patients). We investigated mRNA expression of the PAI-1, the thrombin receptor PAR-1, the alpha2 chain of type IV (alpha2 IV) collagen, and of a housekeeping gene (cyclophilin) by reverse transcription-polymerase chain reaction. The results were correlated with the renal function and the histological findings classified into acute rejection (9 biopsies), chronic rejection (22 biopsies), or normal (8 biopsies). RESULTS: A significant up-regulation of PAI-1 and alpha2 IV collagen mRNA was observed in acute rejection (P<0.05) when compared to normal kidneys. A positive correlation exists between alpha2 IV collagen mRNA level and the degree of cellular infiltration. A negative correlation was found between the level of mRNA of PAR-1 and the degree of vascular thrombosis (P=0.005) and glomerulosclerosis (P=0.04). A positive correlation was found between the degradation of renal function and the mRNA level of PAI-1 at the time of the renal biopsy (P<0.05). CONCLUSIONS: These results suggest that glomerular PAI-1 mRNA may be predictive of the long-term renal graft function.


Subject(s)
Kidney Glomerulus/metabolism , Kidney Transplantation , Kidney/metabolism , Plasminogen Activator Inhibitor 1/genetics , RNA, Messenger/metabolism , Adolescent , Adult , Dissection , Female , Humans , Kidney/physiopathology , Kidney Glomerulus/surgery , Male , Microsurgery , Middle Aged , Polymerase Chain Reaction , Prognosis , Time Factors
5.
Oncogene ; 20(45): 6531-7, 2001 Oct 04.
Article in English | MEDLINE | ID: mdl-11641777

ABSTRACT

While both nitric oxide synthase-2 (NOS-2) and low molecular weight GTPases, such as Ras and Rho, have been implicated in malignant transformation, the cross talk between these important proteins is ill understood. In this study we examined the ability of H-Ras, RhoA, RhoB and Rac1 to modulate cytokine-induced NOS2. In the normal human liver AKN-1 cell line and in the human non-small cell lung carcinoma cell line, A-549, the ability of the cytokines (INF-gamma, IL-1beta and TNF-alpha) to activate NOS-2 was blocked by activated L61-H-Ras whereas dominant negative N17-H-Ras enhanced NOS-2 activation. Consistent with this dominant negative Erk2 as well as a MEK inhibitor also enhanced cytokine activation of NOS-2. Furthermore, activated L63-RhoA blocked whereas activated V14-RhoB enhanced cytokine NOS-2 activation. Activated I115-Racl did not affect NOS-2 activation. These results demonstrate that the Ras/Erk and the Ras/RhoA pathways negatively regulate whereas RhoB enhances cytokine-induced NOS-2. This is the first demonstration that genes that promote malignant transformation such as Ras and RhoA inhibit, whereas genes with tumor suppressor activity such as RhoB enhance NOS2 induction.


Subject(s)
Cytokines/pharmacology , Liver/metabolism , Lung Neoplasms/genetics , Nitric Oxide Synthase/genetics , Proto-Oncogene Proteins p21(ras)/physiology , rho GTP-Binding Proteins/physiology , Cell Line , Genes, Reporter , Humans , Liver/drug effects , Lung Neoplasms/enzymology , Mitogen-Activated Protein Kinase Kinases/physiology , Mitogen-Activated Protein Kinases/physiology , Mutation , Nitric Oxide Synthase Type II , Proto-Oncogene Proteins p21(ras)/genetics , Transcriptional Activation , Transfection , Tumor Cells, Cultured , rac1 GTP-Binding Protein/physiology , rhoA GTP-Binding Protein/physiology , rhoB GTP-Binding Protein/physiology
6.
Int J Mol Med ; 7(6): 653-8, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11351280

ABSTRACT

To analyse the mechanisms of PAR-1 internalisation, we constructed several PAR-1 mutants and stably expressed them in CHO cells. Our study shows that the Ser(306)-->Ala mutation (S306A), which eliminates a potential site of phosphorylation by PKC in the third intracellular loop of PAR-1, did not change the rate of phosphorylation but reduced the rate of thrombin-induced internalisation of the PAR-1 mutant (58 versus 78% of membrane PAR-1 in 15 min, p<0.005). Deletion of the last 43 amino acid residues of the PAR-1 cytoplasmic tail completely suppressed the thrombin phosphorylation of the mutated receptor and significantly reduced its internalisation upon activation. This deletion also inhibited the PMA-induced and the agonist-independent internalisation of the receptor. The Tyr(371)--> Ala mutation (Y371A), in a NPXXY motif of the seventh transmembrane domain of the receptor had no effect on the receptor behaviour. Our results indicate that both the C-tail and the third intracellular loop are involved in PAR-1 internalisation induced by thrombin while only the C-tail plays a role in the PMA-induced and in the agonist-independent PAR-1 internalisation.


Subject(s)
Receptors, Thrombin/chemistry , Receptors, Thrombin/metabolism , Alanine/chemistry , Animals , Binding Sites , CHO Cells , Cricetinae , Cytoplasm/metabolism , Gene Deletion , Mutation , Phosphorylation , Plasmids/metabolism , Precipitin Tests , Protein Kinase C/metabolism , Protein Structure, Tertiary , Receptor, PAR-1 , Receptors, Thrombin/genetics , Thrombin/metabolism , Time Factors , Transfection , Tyrosine/chemistry
7.
Nat Biotechnol ; 18(10): 1065-70, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11017044

ABSTRACT

We have designed a molecule, GFB-111, that binds to platelet-derived growth factor (PDGF), prevents it from binding to its receptor tyrosine kinase, and blocks PDGF-induced receptor autophosphorylation, activation of Erk1 and Erk2 kinases, and DNA synthesis. GFB-111 is highly potent (IC50 = 250 nM) and selective for PDGF over EGF, IGF-1, aFGF, bFGF, and HRGbeta (IC50 values > 100 microM), but inhibits VEGF-induced Flk-1 tyrosine phosphorylation and Erk1/Erk2 activation with an IC50 of 10 microM. GFB-111 treatment of nude mice bearing human tumors resulted in significant inhibition of tumor growth and angiogenesis. The results demonstrate the feasibility of designing novel growth factor-binding molecules with potent anticancer and antiangiogenic activity.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Antineoplastic Agents/therapeutic use , Drug Design , Glioblastoma/drug therapy , Peptides, Cyclic/pharmacology , Platelet-Derived Growth Factor/antagonists & inhibitors , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/metabolism , Angiogenesis Inhibitors/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Cell Division/drug effects , Cell Line , DNA/biosynthesis , Endothelial Growth Factors/antagonists & inhibitors , Endothelial Growth Factors/pharmacology , Enzyme Activation/drug effects , Glioblastoma/blood supply , Glioblastoma/pathology , Humans , Inhibitory Concentration 50 , Lymphokines/antagonists & inhibitors , Lymphokines/pharmacology , Mice , Mice, Nude , Mitogen-Activated Protein Kinases/metabolism , Neoplasm Transplantation , Neovascularization, Pathologic/drug therapy , Peptides, Cyclic/chemistry , Peptides, Cyclic/metabolism , Peptides, Cyclic/therapeutic use , Phosphorylation/drug effects , Platelet-Derived Growth Factor/metabolism , Platelet-Derived Growth Factor/pharmacology , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Receptors, Platelet-Derived Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor , Substrate Specificity , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
8.
Bioorg Med Chem ; 8(8): 2007-16, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11003145

ABSTRACT

New compounds have been synthesized based on the structure of the anti-tumoral drug tamoxifen and its diphenylmethane derivative, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine, HCl (DPPE). These new compounds have no affinity for the estrogen receptor (ER) and bind with various affinity to the anti-estrogen binding site (AEBS). Compounds 2, 10, 12, 13, 20a, 20b, 23a, 23b, 29 exhibited 1.1-69.5 higher affinity than DPPE, and compounds 23a and 23b have 1.2 and 3.5 higher affinity than tamoxifen. Three-dimensional structure analysis, performed using the intersection of the van der Waals volume occupied by tamoxifen in its crystallographic state and the van der Waals volume of these new compounds in their calculated minimal energy conformation, correlated well with their pKi for AEBS (r = 0.84, P<0.0001, n = 18). This is the first structure-affinity relationship (SAR) ever reported for AEBS ligands. Moreover in this study we have reported the synthesis of new compounds of higher affinity than the lead compounds and that are highly specific for AEBS. Since these compounds do not bind ER they will be helpful to study AEBS mediated cytotoxicity. Moreover our study shows that our strategy is a new useful guide to design high affinity and selective ligands for AEBS.


Subject(s)
Microsomes/metabolism , Phenyl Ethers/chemistry , Receptors, Drug/metabolism , Selective Estrogen Receptor Modulators/chemistry , Selective Estrogen Receptor Modulators/chemical synthesis , Tamoxifen/analogs & derivatives , Animals , Binding Sites , Humans , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , Phenyl Ethers/metabolism , Radioligand Assay , Rats , Receptors, Drug/chemistry , Selective Estrogen Receptor Modulators/metabolism , Structure-Activity Relationship , Tamoxifen/chemistry , Tamoxifen/metabolism
9.
Biochem Pharmacol ; 57(6): 657-61, 1999 Mar 15.
Article in English | MEDLINE | ID: mdl-10037451

ABSTRACT

The antiestrogen binding site (AEBS) is a membranous protein complex that has been shown to be intimately linked with the antiproliferative and antiretroviral effects of certain antiestrogenic compounds such as tamoxifen (Tx). Various specific ligands of AEBS derived from benzylphenoxy ethanamine and a new benzoyl structure were synthesized either by modification of the aminoether side chain or by halogen substitution at the meta-, ortho-, and para position on the benzoyl group. Using the MCF-7 cellular strain and its RTx6 variant (a clone selected for its antigrowth resistance to tamoxifen), it was shown that under high drug concentrations the cytotoxicity of the ligands was directly correlated with their affinity for AEBS. In agreement with previous observations made on triphenylethylenic ligands, modification of the basic ethanamine side chain modulated the ligand affinities. Chloride in meta increased ligand efficacy, whereas chloride substitution in ortho and para decreased it. Effects on AEBS-positive MCF-7 cells were drug concentration- and time-dependent, whereas they were unspecific on the AEBS-negative RTx6 cell line. These cytotoxic effects were confirmed in the absence of estrogen receptor on human AEBS-positive uterine cervix cell carcinoma HeLa cells, but were non-specific on rat fibroblastic AEBS-negative (low concentration) NRK cells. The cytotoxicities of these ligands are related to their affinities for AEBS.


Subject(s)
Antineoplastic Agents/pharmacology , Binding Sites/drug effects , Estrogen Antagonists/pharmacology , Tamoxifen/analogs & derivatives , Animals , Binding, Competitive , Cell Line/metabolism , Cell Survival/drug effects , HeLa Cells/metabolism , Humans , Rats , Receptors, Estrogen/analysis
10.
Nephrologie ; 19(7): 411-6, 1998.
Article in French | MEDLINE | ID: mdl-9857376

ABSTRACT

Most proteases a receptor or a binding site that serves to concentrate the proteolytic activity on the cell surface and to mediate cellular effects. We looked for such a receptor for renin, an aspartyl protease. The binding of recombinant human renin labelled with 125I was studied on primary and immortalized human mesangial cells. The binding of renin was specific, saturable and was characterized by Kd = 0.4 nM and 8,000 sites/cell and Kd = 1 nM and 2,000 sites/cell for primary and immortalized cells, respectively. The binding did not depend on the active site of the enzyme, was not followed by internalization and degradation of renin and did not modify intracellular Ca2+. Stimulation of primary cells with 100 nM induced a significant increase of 3H thymidine incorporation but was not associated with an increase of the cell number. Furthermore, incubation of mesangial cells 24 h with 100 nM renin provoked an increase of tPA and of PAI1 in the conditioned medium. This increase was not modified neither by captopril nor by angiotensin II receptors antagonists. The tPA antigen elevation was confirmed by fibrin zymography showing an increase of tPA/PAI1 complexes. But, surprisingly, the reverse zymogram showed that PAI antigen increase was associated with decreased PAI activity which was due to PAI clivage in an inactive form. PAI clivage by renin required the presence of the cells and could not be obtained by incubating renin and recombinant human PAI alone. When primary mesangial cells were cultured in the presence of a specific inhibitor of renin active site, RO 42-5982, PAI accumulation in the conditioned medium was reduced by 50-60%, suggesting that endogenous renin plays a role in PAI synthesis and/or secretion. The binding of renin does not induce cAMP and cGMP generation. However, in the presence of renin (100 nM and 1 microM) the extent of cGMP generated by CNP (10 and 100 nM) was reduced by 50%. Preliminary results of the renin receptor purification by affinity chromatography indicate that the receptor Mr is about 57 kDa.


Subject(s)
Cyclic GMP/biosynthesis , Glomerular Mesangium/chemistry , Plasminogen Activator Inhibitor 1/metabolism , Receptors, Cell Surface/analysis , Receptors, Cell Surface/metabolism , Renin/metabolism , Calcium/metabolism , Glomerular Mesangium/metabolism , Humans , Iodine Radioisotopes , Recombinant Proteins/metabolism , Renin/pharmacology , Tissue Plasminogen Activator/metabolism
11.
Kidney Int ; 54(6): 1976-84, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9853262

ABSTRACT

BACKGROUND: Matrix metalloprotease 2 (MMP2) is secreted in a latent inactive form (pro-MMP2) that is activated on the cell surface by a membrane-type 1 MMP (MT1-MMP) in the presence of the tissue inhibitor of MMP (TIMP2). In spite of evidence for the synthesis of MT1-MMP shown by immunoblotting, immunocytochemistry and RT-PCR, and of TIMP2, MMP2 was found exclusively in a latent form in human mesangial cells (HMC) serum-free culture medium. METHODS AND RESULTS: On purified membranes of HMC, MT1-MMP was found in a 63 kD latent form and as a faint band of 55 kD. The 55 kD band was also present in the ultracentrifuged conditioned medium and likely represented MT1-MMP cleaved from its transmembrane domain, since Northern blot analysis showed only one transcription product. The addition of urokinase plasminogen activator (uPA, 100 nM) to HMC membranes induced the activation of pro-MMP2 via the activation of latent membrane-associated MT1-MMP as reflected by the cleavage of the 63 and 55 kD forms. In addition, when the conditioned medium was successively incubated with uPA and alpha 2-macroglobulin and analyzed by immunoblotting, MT1-MMP decreased, indicating that the soluble MT1-MMP was in a latent form and was activated by uPA. CONCLUSION: Our results provide the first evidence, to our knowledge, of the existence of a soluble latent form of MT1-MMP secreted by primary human cells in culture, confirming that MT1-MMP is an ectoenzyme, and show that uPA can regulate MT1-MMP activity in a soluble phase.


Subject(s)
Gelatinases/metabolism , Glomerular Mesangium/metabolism , Metalloendopeptidases/metabolism , Urokinase-Type Plasminogen Activator/pharmacology , Cell Membrane/metabolism , Culture Media, Conditioned/metabolism , Enzyme Activation/physiology , Enzyme Precursors/metabolism , Gelatinases/drug effects , Glomerular Mesangium/cytology , Glomerular Mesangium/drug effects , Humans , Matrix Metalloproteinase 2 , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/drug effects , Protein Precursors/metabolism , Solubility
12.
Am J Physiol ; 275(3): H1011-5, 1998 09.
Article in English | MEDLINE | ID: mdl-9724307

ABSTRACT

The atheroprotective properties of estrogens are supported by clinical data from postmenopausal women who use estrogen replacement therapy. However, the mechanisms mediating activity remain unknown, and it has been suggested that estrogens may help to modulate endothelial permeability to atherogenic lipoproteins. In these studies we used bovine vascular endothelial cells as an in vitro model to show that estrogens were able to regulate low-density lipoprotein transport and permeability of the endothelial monolayer. Macromolecular transport was observed to be a second-order polynomial function of estrogen concentration. Moreover, this regulation was correlated with expression of heat shock protein (HSP) 25, which is known to influence fluid phase pinocytosis and cytoskeleton remodeling, thus suggesting a role for HSP 25 in the estrogenic control of transcellular permeability of the endothelium monolayer.


Subject(s)
Cell Membrane Permeability/drug effects , Endothelium, Vascular/metabolism , Ethinyl Estradiol/pharmacology , Gene Expression Regulation/drug effects , Heat-Shock Proteins , Neoplasm Proteins/genetics , Animals , Aorta , Biological Transport/drug effects , Blotting, Western , Cattle , Cells, Cultured , Cytoskeleton/physiology , HSP27 Heat-Shock Proteins , Humans , Kinetics , Lipoproteins, LDL/metabolism , Male , Molecular Chaperones , Pinocytosis
13.
Biochem J ; 334 ( Pt 1): 107-12, 1998 Aug 15.
Article in English | MEDLINE | ID: mdl-9693109

ABSTRACT

A tritiated photoaffinity labelling analogue of tamoxifen, [(2-azido-4-benzyl)-phenoxy]-N-ethylmorpholine (azido-MBPE), was used to identify the anti-oestrogen-binding site (AEBS) in rat liver tissue [Poirot, Chailleux, Fargin, Bayard and Faye (1990) J. Biol. Chem. 265, 17039-17043]. UV irradiation of rat liver microsomal proteins incubated with tritiated azido-MBPE led to the characterization of two photolabelled proteins of molecular masses 40 and 50 kDa. The amino acid sequences of proteolytic products from the 50 kDa protein were identical with those from rat microsomal epoxide hydrolase (mEH). Treatment of hepatocytes with anti-sense mRNA directed against mEH abolished AEBS in these cells. In addition we found that tamoxifen and N-morpholino-2-[4-(phenylmethyl)phenoxy]ethanamine, a selective ligand of AEBS, were potent inhibitors of the catalytic hydration of styrene oxide by mEH. However, functional overexpression of the human mEH did not significantly modify the binding capacity of [3H]tamoxifen. Taken together, these results suggest that the 50 kDa protein, mEH, is necessary but not sufficient to reconstitute AEBS.


Subject(s)
Epoxide Hydrolases/chemistry , Epoxide Hydrolases/metabolism , Estrogen Antagonists/metabolism , Liver/metabolism , Microsomes, Liver/enzymology , Tamoxifen/metabolism , Affinity Labels , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Cloning, Molecular , Cycloheximide/pharmacology , DNA, Complementary , Epoxide Hydrolases/genetics , Epoxide Hydrolases/isolation & purification , Female , Humans , Kinetics , Liver/drug effects , Macromolecular Substances , Molecular Sequence Data , Molecular Weight , Morpholines/metabolism , Morpholines/pharmacology , Open Reading Frames , Ovariectomy , Peptide Fragments/chemistry , RNA, Antisense/pharmacology , Rats , Rats, Sprague-Dawley
14.
Kidney Int ; 50(6): 1897-903, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8943472

ABSTRACT

Some proteases possess a membrane receptor that focalizes their proteolytic activity on the cell surface and may mediate a proliferative effect, such as urokinase on glomerular epithelial cells. Since some hypertensive states are associated with high concentrations of renin and proliferation of arteriolar smooth muscle cells, we asked whether renin, an aspartyl-protease, would bind to mesangial cells that are smooth-muscle derived cells, which would induce their proliferation. The binding of 125I labeled recombinant human renin (125I-R) was studied on human primary mesangial cells and mesangial cells immortalized by transfection with SV40-T antigen. At 37 degrees C, the binding of 125I-R was time dependent and reached a plateau after two hours. 125I-R was found to bind in a saturable and specific manner with a Kd = 0.4 nM and 1 nM and 8,000 and 2,000 binding sites/cell, for primary and immortalized cells, respectively. When binding experiments were performed in the presence RO 42-5892, a synthetic inhibitor of renin, RO 42-5892 could inhibit the specific binding of labeled renin only at concentrations 1,000 times superior to the IC 50, indicating that the renin-mesangial receptor interaction did not depend on the active site of renin. Analysis by SDS-PAGE and autoradiography of cross-linking experiments of 125I-R bound to a membrane preparation showed a band of approximately 110 to 120 kDa, suggesting a Mr of 70 to 80 kDa for the renin receptor. Incubation of mesangial cells with 100 nM renin for 24 hours provoked a 100% increase of 3H thymidine incorporation that was not accompanied by an increase of the cell number, even after a seven day period of incubation. However, the incubation of mesangial cells with renin for 24 hours induced a significant increase (170% of control, P = 0.04) of plasminogen activator inhibitor-1 (PAI1) antigen in the conditioned medium. In conclusion, we have shown that human mesangial cells in culture express a specific receptor for renin, and that the binding of renin increases 3H thymidine incorporation independently of renin enzymatic activity. The absence of cell proliferation, the increase of 3H thymidine incorporation and the increase of PAI1 antigen suggest that the binding of renin can induce mesangial cell activation, which is reflected by a change in the fibrinolytic capacity of the cells. The role of this receptor remains to be determined in nephropathies and hypertensive states associated with high plasma/tissue renin concentrations, hypertrophy of mesangial or smooth muscle cells and extracellular matrix remodeling.


Subject(s)
Glomerular Mesangium/metabolism , Plasminogen Activator Inhibitor 1/analysis , Receptors, Cell Surface/metabolism , Renin/metabolism , Cell Division , Cells, Cultured , Glomerular Mesangium/cytology , Humans , Thymidine/metabolism
15.
Mol Pharmacol ; 50(1): 75-9, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8700122

ABSTRACT

Since the discovery of human immunodeficiency retrovirus, the drug arsenal against retrovirus has rapidly increased. Concomitantly, new challenges in the therapy of acquired immune deficiency syndrome have arisen, including drug toxicities, drug resistance, and the development of various cancers as effective therapies prolong survival. Tamoxifen, a nonsteroidal antiestrogen with a low incidence of side effects, is widely used in cancer therapy; it is known to exert pleiotropic activities by binding essentially to the estrogen receptor and other unidentified proteins. In the present work, quantification of the p24 core protein of human immunodeficiency virus 1 produced by infected lymphocytes shows an inhibitory effect of tamoxifen on virion production. Moreover, we assume that this effect is not mediated by the estrogen receptor because antiestrogen ligands interacting with the antiestrogen-binding site exhibit efficacy related to their affinity for this site, although specific antiestrogens of the estrogen receptor are ineffective.


Subject(s)
Antiviral Agents/pharmacology , Estrogen Antagonists/pharmacology , HIV-1/physiology , Lymphocytes/virology , Receptors, Drug/metabolism , Tamoxifen/pharmacology , Virus Replication/drug effects , Binding Sites , Cells, Cultured , Estrogen Antagonists/metabolism , Ethanol/pharmacology , HIV Core Protein p24/biosynthesis , HIV-1/drug effects , Humans , Interleukin-2/pharmacology , Kinetics , Ligands , Lymphocyte Activation , Lymphocytes/drug effects , Lymphocytes/immunology , Neomycin/pharmacology , Tamoxifen/metabolism , Virion/drug effects , Virion/physiology , Zidovudine/pharmacology
16.
Lab Invest ; 74(3): 650-7, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8600316

ABSTRACT

The use of human glomerular epithelial cells (HGEC) in research has been severely restricted by several obstacles, which have been circumvented by the generation of T-SV40 immortalized human visceral glomerular epithelial cells (Delarue et al, 1991). In this work, we compared the primary and immortalized HGEC for expression of integrin and some nonintegrin surface receptors. We also studied the adhesion of both types of HGEC to glomerular basement membrane (GBM), type IV collagen (tIV), and its major noncollagenous NC1 domain. The integrins mediating adhesion of HGEC to tIV were also examined. Expression of integrin and some nonintegrin cell surface receptors was analyzed by flow cytometry. Adhesion to GBM, tIV, and its major noncollagenous NC1 domain was studied by direct solid phase cell adhesion assays. Identification of integrins mediating adhesion of HGEC to tIV was achieved by inhibition of cell adhesion using monoclonal antibodies to integrin subunits. The primary and immortalized HGEC share phenotypic characteristics, and alpha3beta1 appeared to be the major integrin present on both HGEC types. The kinetics of binding to GBM, tIV, and its noncollagenous NCI domain were similar in both the primary and immortalized HGEC, although the latter displayed a somewhat weaker binding. Both the primary and immortalized HGEC displayed significantly better adhesion to NC1-alpha3 compared with NC1-alpha1, alpha3beta1 appears to be the major integrin mediating the adhesion of HGEC to tIV. Our studies suggest that alpha3beta1 is the major integrin present on HGEC. This has been confirmed by flow cytometric analysis. In addition, we demonstrated a functional role for this integrin in mediating attachment of HGEC to tIV. Our data also demonstrate a preference in binding of HGEC to alpha3 chains of NC1 compared with alpha1 chains of NC1. These findings were seen in both the primary and immortalized HGEC. The T-SV40 immortalized HGEC can therefore serve as a very useful tool to study glomerular visceral cell biology.


Subject(s)
Collagen/metabolism , Integrins/physiology , Kidney Glomerulus/physiology , Animals , Antigens, Polyomavirus Transforming , Cattle , Cell Adhesion/physiology , Cell Line, Transformed , Epithelial Cells , Humans , Integrin alpha3beta1 , Kidney Glomerulus/cytology , Mice
17.
Lab Invest ; 74(1): 253-8, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8569189

ABSTRACT

Preeclampsia is characterized by maternal hypercoagulable state and intravascular coagulation, microthromboses in several organs, and impairment of uteroplacental circulation. Excessive fibrin deposition occurs in the placenta, suggesting that disorders of placental coagulation and fibrinolysis physiologic systems may have a role in hemostasis activation. Term placentas were collected from 17 hypertensive/preeclamptic women and from 17 healthy pregnant women, and processed for both histologic and hemostasis studies. Placental fibrinoid deposition was visualized by cresyl-violet staining and quantified by histomorphometric analysis. The content in hemostasis factors was measured on extracts from homogenized placentas treated by a nonionic detergent. The percentage of villi with fibrinoid deposits was higher in the diseased placentas than in controls: 13.2 +/- 11.2 versus 6.75 +/- 2.7% (p < 0.001) for the total amount of deposits; 4.8 +/- 6.7 versus 1.5 +/- 1.0% (p = 0.04) for perivillous fibrinoid deposits, which are considered as histologic markers of intraplacental fibrin. The content in type 2 plasminogen activator inhibitor (PAI-2) antigen was higher in the diseased placentas than in controls: 124 +/- 8 versus 104 +/- 6 ng/mg placental protein (p = 0.046); there was a negative correlation between PAI-2 antigen and thrombomodulin activity (r = -0.57, p = 0.02) in the diseased placentas. No significant differences were found between the two groups for placental procoagulant tissue factor and anticoagulant thrombomodulin activities, and for the content in plasminogen activators and PAI-1 antigens. Placental antifibrinolytic potential is increased in pregnancy-induced hypertension and preeclampsia. This change, and the association of the highest PAI-2 placental concentrations with the lowest concentrations of thrombomodulin, may contribute to the prethrombotic state and to the excessive placental perivillous fibrin deposition observed in these situations.


Subject(s)
Fibrin/metabolism , Hypertension/physiopathology , Placenta/metabolism , Pre-Eclampsia/physiopathology , Pregnancy Complications, Cardiovascular/physiopathology , Adult , Female , Fibrinolysis/physiology , Hemostasis/physiology , Humans , Placenta/enzymology , Plasminogen Activator Inhibitor 1/metabolism , Pregnancy , Thrombomodulin/metabolism
19.
Lab Invest ; 71(6): 828-37, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7807964

ABSTRACT

BACKGROUND: Mesangial changes in a variety of pathologic conditions involve mesangial cell proliferation and mesangial matrix remodelling. Heparin has been shown to prevent these processes in vivo. In vitro, heparin interferes with cell growth, proto-oncogene expression, synthesis of specific proteins, and extracellular matrix composition. In some cell types, it seems to interact with intracellular protein kinase C-dependent pathways. The effect of heparin on the mesangial plasminogen activating system (tissue type plasminogen activator, t-PA, and plasminogen activator inhibitor type 1, PAI-1), which is thought to be involved in matrix remodelling, has not been previously reported. EXPERIMENTAL DESIGN: Cultured human mesangial cells were stimulated by 10% fetal calf serum (FCS) or 16 nM phorbol myristate acetate (PMA) in the presence or absence of anticoagulant or nonanticoagulant heparins. Cell proliferation, synthesis of t-PA and PAI-1, cell morphology, and PAI-1 matrix deposition were studied using cell counting, [3H]thymidine incorporation, specific t-PA and PAI-1 enzyme-linked immunosorbent assay, Northern blot analysis, light microscopy, immunofluorescence and immunogold silver staining with combined bright-field and epipolarization microscopy. RESULTS: Heparin partially inhibited FCS-stimulated cell growth but not PMA-induced thymidine incorporation. FCS and PMA stimulated t-PA (p < 0.05 and p < 0.01, respectively) and PAI-1 synthesis (p < 0.05 and p < 0.01 respectively). Heparin selectively and partially inhibited FCS-stimulated t-PA, but not PAI-1 synthesis. It has no effect on PMA-stimulated t-PA or PAI-1 synthesis but prevented cell shape-changes induced by PMA, suggesting that heparin inhibits some but not all protein kinase C (PKC)-dependent effects and that heparin block in t-PA synthesis is distal to PKC activation. Heparin decreased PAI-1 matrix accumulation. Similar distal to PKC activation. Heparin decreased PAI-1 matrix accumulation. Similar results were observed with anticoagulant and nonanticoagulant heparin fragments. CONCLUSIONS: In human mesangial cells, anticoagulant and nonanticoagulant heparin exert an antiproliferative effect and may prevent mesangial matrix changes by decreasing FCS-stimulated t-PA synthesis and PAI-1 deposition in the matrix. Heparin is able to inhibit PKC-dependent cell shape changes but not PKC-dependent t-PA or PAI-1 synthesis. It also inhibits PKC-independent cell proliferation and t-PA synthesis. These results suggest multiple intracellular sites of action for heparin, unrelated or distal to PKC activation.


Subject(s)
Glomerular Mesangium/drug effects , Heparin/pharmacology , Plasminogen Activator Inhibitor 1/biosynthesis , Tissue Plasminogen Activator/biosynthesis , Blotting, Northern , Cell Division/drug effects , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Glomerular Mesangium/cytology , Heparin/chemistry , Heparitin Sulfate/pharmacology , Humans , Immunohistochemistry , Microscopy, Polarization , Plasminogen Activator Inhibitor 1/immunology , Proto-Oncogene Mas , Tetradecanoylphorbol Acetate/pharmacology
20.
Exp Nephrol ; 2(5): 306-12, 1994.
Article in English | MEDLINE | ID: mdl-7812737

ABSTRACT

MHC class II-encoded molecules HLA-DR, -DP and -DQ play a pivotal role in the human immune response. Their constitutive expression is restricted to a number of immunocompetent cells referred to as antigen-presenting cells. However, gamma-interferon (gamma-IFN) has been shown to induce MHC class II molecule expression in several epithelia. Using flow cytometric analysis, we show here that normal and SV40-transformed human podocytes in culture constitutively expressed gamma-IFN receptors. We also show that MHC class I molecules are constitutively expressed in these cells and that HLA-DR, -DP and -DQ expression, which is not found in unstimulated cells, can be induced by gamma-IFN stimulation. This induction was a time-dependent event, a lag phase of 24-48 h being necessary for MHC class II molecules to become detectable at the cell surface by flow cytometric analysis. Induction of MHC class II molecules in human podocytes also showed a concentration dependence, a plateau being reached at a concentration of 500 IU of gamma-IFN/ml of culture medium. This effect was blunted by coincubation of the cells with an antihuman gamma-IFN receptor monoclonal antibody. HLA-DR expression was associated with specific mRNA accumulation, as detected by Northern blot analysis. By indirect immunofluorescence, the intercellular adhesion molecule 1 was also induced by gamma-IFN stimulation. Induction of DR, DP and DQ in human podocytes may be involved in the pathogenesis of immune glomerulonephritis in man.


Subject(s)
HLA-DP Antigens/analysis , HLA-DQ Antigens/analysis , HLA-DR Antigens/analysis , Intercellular Adhesion Molecule-1/analysis , Interferon-gamma/pharmacology , Kidney Glomerulus/chemistry , Kidney Glomerulus/cytology , Blotting, Northern , Cells, Cultured , Epithelial Cells , Epithelium/chemistry , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression Regulation , HLA-DP Antigens/genetics , HLA-DQ Antigens/genetics , HLA-DR Antigens/genetics , Humans , Intercellular Adhesion Molecule-1/genetics , Interferon-gamma/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , Receptors, Interferon/analysis , Receptors, Interferon/metabolism
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