Farnesyltransferase and geranylgeranyltransferase I inhibitors upregulate RhoB expression by HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter.

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.

The farnesyltransferase inhibitor, FTI-2153, inhibits bipolar spindle formation during mitosis independently of transformation and Ras and p53 mutation status.

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.

Prognostic value of plasminogen activator inhibitor type 1 mRNA in microdissected glomeruli from transplanted kidneys.

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.

Ras and RhoA suppress whereas RhoB enhances cytokine-induced transcription of nitric oxide synthase-2 in human normal liver AKN-1 cells and lung cancer A-549 cells.

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.

Design of GFB-111, a platelet-derived growth factor binding molecule with antiangiogenic and anticancer activity against human tumors in mice.

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.

Synthesis, binding and structure-affinity studies of new ligands for the microsomal anti-estrogen binding site (AEBS).

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.

Modifications of benzylphenoxy ethanamine antiestrogen molecules: influence affinity for antiestrogen binding site (AEBS) and cell cytotoxicity.

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.

Estrogens modulate bovine vascular endothelial cell permeability and HSP 25 expression concomitantly.

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.

Ligands of the antiestrogen-binding site are able to inhibit virion production of human immunodeficiency virus 1-infected lymphocytes.

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.

Integrin-mediated interactions between primary/T-sv40 immortalized human glomerular epithelial cells and type IV collagen.

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.

Heparin selectively inhibits synthesis of tissue type plasminogen activator and matrix deposition of plasminogen activator inhibitor 1 by human mesangial cells.

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.

Induction of MHC class II molecules HLA-DR, -DP and -DQ and ICAM 1 in human podocytes by gamma-interferon.

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.

Functional endothelin 1 receptors on human glomerular podocytes and mesangial cells.

To determine if endothelin 1 (Et1) receptors are present in human glomeruli, and which glomerular cells possess these receptors, 125I Et1 binding to isolated glomeruli and cultured glomerular mesangial and epithelial cells was studied. The latter were identified as podocytes. We demonstrated that Et1 binds specifically and reversibly to isolated human glomeruli and to cultured glomerular mesangial and epithelial cells. Scatchard analysis of competitive inhibition of 125I Et1 binding gave the following results (m +/- SEM, n = 3): isolated glomeruli, Kd = 4.2 +/- 2.1 x 10(-10) M, Bmax = 8.1 +/- 1.2 x 10(10) sites/mg protein; mesangial cells, Kd = 5.2 +/- 1.5 x 10(-10) M, Bmax = 1.87 +/- 0.49 x 10(4) sites/cell; epithelial cells, Kd = 7.2 +/- 1.5 x 10(-10) M, Bmax = 2.46 +/- 0.15 x 10(4) sites/cell. These receptors seem to be functional, since in both mesangial and epithelial cells Et1 induces a rapid and transient increase in intracellular [Ca2+]i. All these results indicate that Et1 may regulate glomerular filtration rate through an autocrine-paracrine pathway on mesangial cells and on podocytes.

[TNF-alpha synthesis by circulating mononuclear cells in patients undergoing kidney transplantation]. / Synthèse de TNF-alpha par les cellules mononucléées circulantes chez les transplantés rénaux.

In 7 patients who received renal transplant, systematic blood samples and cytoaspiration of the graft were performed every 3 days after grafting. In vitro TNF-alpha generation by circulating mononuclear cells and by cells infiltrating the graft were measured under basal conditions and after stimulation by recombinant IL-2 (50 U/ml). TNF-alpha concentration was determined by immunoradiometric assay (IRMA). Not enough cells could be collected by cytoaspiration to measure TNF-alpha concentration. In contrast, the generation of TNF-alpha by circulating mononuclear cells was detectable. It increased 24 to 48 hours before the rejection crisis and decreased after successful treatment and return of creatinin level to initial value. IL-2 increased TNF-alpha production and was more effective under normal conditions (10 to 15 fold increase) than during rejection episodes (1.3 to 2.4 fold). These results suggest that TNF-alpha is produced by mononuclear cells during rejection episodes and could be used as a marker of rejection. Further studies are required to determine its sensitivity and specificity.

[TNF-alpha synthesis by cells from bronchioloalveolar lavage and by circulating mononuclear cells in recipients of heart-lung transplantation]. / Synthèse de TNF-alpha par les cellules du lavage bronchiolo-alvéolaire et les cellules mononucléées circulantes chez les transplantés coeur-poumon.

In 10 patients who received a heart lung transplant, TNF-alpha generation by cells collected during bronchioloalveolar lavages (n = 30) and by circulating mononuclear cells was measured. Basal and recombinant IL-2-stimulated productions (50 U/ml) were measured. TNF-alpha concentration was determined by an immunoradiometric assay (IRMA). Circulating mononuclear cells produced at least 4 times less TNF-alpha than BAL cells. Rejection episodes or CMV diseases were not associated with significant changes in TNF-alpha generation. Recombinant IL-2 increased this production in both cell populations but the magnitude of this effect was smaller in BAL cells, suggesting an in vivo preactivation.

Stable cell line of T-SV40 immortalized human glomerular visceral epithelial cells.

Human subcultures (third passage) of glomerular visceral epithelial cells (VEC) isolated from one month old kidney were successfully transfected by two recombinant plasmids containing the cloned oncogenes from the simian virus 40 large T antigen and H-ras gene. One postcrisis cell clone (56/10 A1) was selected, propagated and characterized. One hundred percent of the 56/10 A1 cells (current passage greater than 100th; doubling time 30 hrs) expressed the nuclear T-SV40 antigen assayed by IF; the cells failed to express H-ras (RNA blot analysis). Immortalized cells were morphologically and phenotypically compared to parental cell type (third passage). Phenotypic characterization of the 56/10 A1 cells was achieved using indirect immunofluorescence (IF) and immunogold silver staining coupled to bright field and epipolarization microscopy. Both parental and 56/10 A1 cells displayed positivity for cytokeratin, CALLA and PHM5, whereas von Willebrand factor was not detected in the two cell types. Since we have previously shown that human glomerular epithelial cells in culture synthetize plaminogen activator (PA) related compounds, we investigated the secretion pattern of these products in parental and transfected cells. Zymographic analysis of secreted PA related compounds revealed production of free urokinase (u-PA) and type 1 plasminogen activator inhibitor (PAI-1) complexed to tissular plasminogen activator (t-PA). Finally, in the transfected cells, increased cGMP generation under atrial natriuretic factor (ANF) stimulation agreed with previous work performed on nontransfected human VEC. In conclusion, the establishment of a human permanent cell line which retains most of the phenotypic features of parental glomerular visceral epithelial cells should represent a new tool to study human glomerular cell functions.

Urokinase synthesis and binding by glomerular epithelial cells in culture.

Fibrin deposits are frequently observed in the course of proliferative extracapillary glomerulonephritis and could be related to a defective local fibrinolysis. We studied human glomerular epithelial cells in culture which were found to release mainly a urokinase-type plasminogen activator (u-PA) identified on zymography by its molecular weight (53 kD), its plasminogen activator activity, and its neutralization by specific polyclonal anti-u-PA IgG. Trace amounts of tissue-type plasminogen activator (t-PA) complexed to a plasminogen activator inhibitor type 1 (PAI-1) were identified with specific antibodies. Specific binding sites were found at the surface of glomerular epithelial cells (kD: 2.10(-9) M), partially occupied by secreted u-PA. The spontaneous u-PA activity of the culture medium conditioned by glomerular epithelial cells was very low, suggesting that u-PA was released in its inactive single chain proenzyme form (SC-u-PA). After activation of SC-u-PA by plasmin, u-PA activity of the culture medium was found to increase in a time- and dose-dependent manner when cells were incubated with phorbol myristic acetate (PMA). This effect was inhibited by H7, a protein kinase C inhibitor. Stimulation of u-PA synthesis by PMA was also observed in two different epithelial tubular cell lines. LLC-PK1 and MDCK cells. However, 8 bromo cyclic AMP which increased u-PA release by LLC-PK1 cells was found to inhibit u-PA release by PMA-stimulated glomerular epithelial cells and MDCK cells. By Northern blot analysis we found that PMA induced an increase of u-PA mRNA level in glomerular epithelial cells and that cyclic AMP had an opposite effect.(ABSTRACT TRUNCATED AT 250 WORDS)