Biomedical subjects

M P Merville

Publications and source records attributed to M P Merville.

At least 19 recordsLinked to original sources

PubMed2003

Pharmacological evaluation of the novel thromboxane modulator BM-567 (II/II). Effects of BM-567 on osteogenic sarcoma-cell-induced platelet aggregation.

Evidence exists that a large number of tumor cells such as osteosarcoma cells stimulate platelet aggregation, which can be an early step in the metastatic processes of these tumors. Thromboxane A(2) (TXA(2)) is released during platelet aggregation, and it has been suggested that this release may be pathogenic for tumor metastasis for several reasons:Some tumors release large amounts of TXA(2) compared to normal tissue.TXA(2) potentiates tumor growth in culture and increases metastasis in animals.TXA(2) is a potent stimulant of platelet aggregation and causes vascular injuries that may promote implantation of tumor cell-platelet aggregates. If TXA(2) participates in tumor metastasis, it may be hypothesized that TXA(2) inhibitors should decrease tumor metastasis. So, we have evaluated the effects of the original TXA(2) synthase inhibitor and TXA(2) receptor antagonist BM-567 on platelet aggregation induced by osteosarcoma cells using MG-63 tumor cells. Results obtained showed that this drug inhibited both MG-63 tumor-cell-induced platelet aggregation and platelet TXA(2) release following the tumor cell stimulation with IC(50) values of 3.04x10(-7) and 2.51x10(-8)M, respectively.

Blood Platelets↗

PubMed2001

Impaired accumulation of granulocytes in the lung during ozone adaptation.

Respiratory alterations induced by an acute exposure to ozone (O(3)) paradoxically resolve during multiday exposure. This adaptation is characteristically accompanied by a gradual attenuation of lung neutrophilia. As maintenance of neutrophilia at the site of inflammation is due to cytokine-mediated delayed neutrophil apoptosis, which is associated with reduced levels of Bax, a proapoptotic protein, we sought to determine whether defects in these mechanisms could account for O(3) adaptation. Lung granulocytes obtained at different time points from calves exposed to 0.75 ppm O(3) for 12 h/d for 7 consecutive days neither showed enhancement of survival nor Bax deficiency, when compared to blood granulocytes. To further investigate the effects of an exogenous oxidative stress on neutrophil survival, human granulocytes were treated with hydrogen peroxide alone, or in combination with granulocyte/macrophage colony-stimulating factor, an antiapoptotic cytokine. Both treatments led to rapid apoptosis associated with downregulation of Bcl-x(L) and Bcl-2, two antiapoptotic proteins. This study shows that O(3) adaptation is associated with a failure in the mechanisms leading to accumulation of neutrophils at the site of inflammation, and suggests that this defect is due to direct proapoptotic effects of exogenous oxidative stress on granulocytes.

Adaptation, Physiological↗

PubMed2001

Inhibition of the NF-kappa B transcription factor increases Bax expression in cancer cell lines.

The NF-kappa B transcription factor has been shown to inhibit apoptosis in several experimental systems. We therefore investigated whether the expression of the Bax proapoptotic protein could be influenced by NF-kappa B activity. Increased Bax protein expression was detected in HCT116, OVCAR-3 and MCF7 cells stably expressing a mutated unresponsive I kappa B-alpha inhibitory protein that blocks NF-kappa B activity. Northern blots showed that bax mRNA expression was increased as a consequence of mutated I kappa B-alpha expression in HCT116 cells. A careful examination of the human bax gene promoter sequence showed three putative binding sites for NF-kappa B, and the kappa B2 site at position -687 could indeed bind NF-kappa B complexes in vitro. Transient transfection of a bax promoter luciferase construct in HCT116 cells showed that NF-kappa B proteins could partially inhibit the transactivation of the bax promoter by p53. Mutations or deletions of the kappa B sites, including kappa B2, indicated that this NF-kappa B-dependent inhibitory effect did not require NF-kappa B DNA-binding, and was thus an indirect effect. However, cotransfection of expression vectors for several known cofactors failed to identify a competition between p53 and NF-kappa B for a transcription coactivator. Our findings thus demonstrate for the first time that NF-kappa B regulates, through an indirect pathway, the bax gene expression.

Binding Sites↗

PubMed2001

Rat gap junction connexin-30 inhibits proliferation of glioma cell lines.

Connexins, the structural components of gap junctions, control cell growth and differentiation and are believed to belong to a family of tumour suppressor genes. Studies on connexin localization in brain showed that several of these proteins were expressed in distinct compartments of the brain in a cell-type specific manner, indicating that different gap junctions play specific roles in the physiology of the mammalian brain. In this report, we first cloned rat connexin-30 cDNA from brain and showed that it was expressed in long-term primary culture of rat astrocytes. In order to examine the potential role of connexin-30 in tumour cell proliferation, we transfected the connexin-30 cDNA into two rat glioma cell lines (9L and C6) which have lost its expression. Transfected clones adequately expressed membrane-bound connexin-30 protein. Connexin-30-expressing clones showed slower growth, lower DNA synthesis and reduced proliferation in soft agar as compared with the parental and control cells. We concluded that connexin-30 may also probably be considered as a tumour suppressor in rat gliomas.

Amino Acid Sequence↗

PubMed2000

Mechanisms of persistent NF-kappa B activity in the bronchi of an animal model of asthma.

In most cells trans-activating NF-kappaB induces many inflammatory proteins as well as its own inhibitor, IkappaB-alpha, thus assuring a transient response upon stimulation. However, NF-kappaB-dependent inflammatory gene expression is persistent in asthmatic bronchi, even after allergen eviction. In the present report we used bronchial brushing samples (BBSs) from heaves-affected horses (a spontaneous model of asthma) to elucidate the mechanisms by which NF-kappaB activity is maintained in asthmatic airways. NF-kappaB activity was high in granulocytic and nongranulocytic BBS cells. However, NF-kappaB activity highly correlated to granulocyte percentage and was only abrogated after granulocytic death in cultured BBSs. Before granulocytic death, NF-kappaB activity was suppressed by simultaneous addition of neutralizing anti-IL-1beta and anti-TNF-alpha Abs to the medium of cultured BBSs. Surprisingly, IkappaB-beta, whose expression is not regulated by NF-kappaB, unlike IkappaB-alpha, was the most prominent NF-kappaB inhibitor found in BBSs. The amounts of IkappaB-beta were low in BBSs obtained from diseased horses, but drastically increased after addition of the neutralizing anti-IL-1beta and anti-TNF-alpha Abs. These results indicate that sustained NF-kappaB activation in asthmatic bronchi is driven by granulocytes and is mediated by IL-1beta and TNF-alpha. Moreover, an imbalance between high levels of IL-1beta- and TNF-alpha-mediated IkappaB-beta degradation and low levels of IkappaB-beta synthesis is likely to be the mechanism preventing NF-kappaB deactivation in asthmatic airways before granulocytic death.

Airway Obstruction↗

PubMed2000

Nuclear factor-kappa B, cancer, and apoptosis.

The role of nuclear factor (NF)-kappa B in the regulation of apoptosis in normal and cancer cells has been extensively studied in recent years. Constitutive NF-kappa B activity in B lymphocytes as well as in Hodgkin's disease and breast cancer cells protects these cells against apoptosis. It has also been reported that NF-kappa B activation by tumor necrosis factor (TNF)-alpha, chemotherapeutic drugs, or ionizing radiations can protect several cell types against apoptosis, suggesting that NF-kappa B could participate in resistance to cancer treatment. These observations were explained by the regulation of antiapoptotic gene expression by NF-kappa B. However, in our experience, inhibition of NF-kappa B activity in several cancer cell lines has a very variable effect on cell mortality, depending on the cell type, the stimulus, and the level of NF-kappa B inhibition. Moreover, in some experimental systems, NF-kappa B activation is required for the onset of apoptosis. Therefore, it is likely that the NF-kappa B antiapoptotic role in response to chemotherapy is cell type- and signal-dependent and that the level of NF-kappa B inhibition is important. These issues will have to be carefully investigated before considering NF-kappa B as a target for genetic or pharmacological anticancer therapies.

Animals↗

PubMed2000

Additive effect between NF-kappaB subunits and p53 protein for transcriptional activation of human p53 promoter.

The tumor suppressor p53 plays a pivotal role in the cellular response to DNA damage as it controls DNA repair, cell cycle arrest and apoptosis. We studied the autoregulation of human p53 gene transcription in colon cancer cell lines. Wild-type p53 has been shown to autoregulate its own transcription either positively or negatively and probably in a cell-type-specific manner. Indeed, a p53 binding site has been described in the human and murine p53 promoters, but a direct binding of wild-type p53 protein to this site has never been reported. In this study, we demonstrated a transactivation of human p53 promoter by wild-type p53 in human colon cancer cells. We identified in the human p53 promoter a novel potential p53-responsive element that binds wild-type p53. Moreover, wild-type p53 protein transactivated a reporter plasmid containing a luciferase gene driven by a minimal promoter harboring this p53 binding site. Finally, as the p53 promoter contains an NF-kappaB binding site, we demonstrated an additive effect when NF-kappaB subunits and p53 protein combined to transactivate the human p53 promoter.

Adenocarcinoma↗

PubMed2000

Growth regulation of astrocytes and C6 cells by TGFbeta1: correlation with gap junctions.

Transforming growth factor (TGF) beta1 enhanced in vitro [3H]thymidine incorporation into C6 cells and reduced that of astrocytes in the presence of a high serum concentration. It concomitantly raised the gap junction intercellular communication (GJIC) in normal astrocytes but reduced the coupling of C6 cells, and respectively increased or decreased the proportion of P2-phosphorylated connexin (Cx) 43 isoform in these cells. Finally, octanol, which inhibited GJIC in both cell types, increased the thymidine incorporation in C6 cells, but neither altered the proliferation of astrocytes nor their response to TGFbeta1. These data indicate that an inhibition of gap junction intercellular communication, due to an altered phosphorylation of connexin 43, may contribute to the proliferative response of C6 glioblastoma cells to TGFbeta1.

Animals↗

PubMed2000

Cell type-specific role for reactive oxygen species in nuclear factor-kappaB activation by interleukin-1.

The role of reactive oxygen intermediates (ROIs) in nuclear factor-kappaB (NF-kappaB) activation remains a matter of controversy. We have studied whether ROIs played any role in NF-kappaB induction by interleukin-1beta (IL-1beta) in different cell types. Our studies indicated three different pathways. IL-1beta stimulation of lymphoid cells generates ROIs, which are required for IkappaB-alpha degradation and NF-kappaB activation. The source of these ROIs is the 5-lipoxygenase (5-LOX) enzyme. In monocytic cells, ROIs are also produced in response to IL-1beta and necessary for NF-kappaB induction, but their source appears to be the NADPH oxidase complex. Finally, epithelial cells do not generate ROIs after IL-1beta stimulation, but do rapidly activate NF-kappaB. Interestingly, transfection of epithelial cells with the 5-LOX and 5-LOX activating protein expression vectors restored ROI production and ROI-dependent NF-kappaB activation in response to IL-1beta. Our data thus indicate that ROIs are cell type-specific second messengers for NF-kappaB induction by IL-1beta.

Animals↗

PubMed2000

Cytosine deaminase suicide gene therapy for peritoneal carcinomatosis.

Gene therapy is a novel therapeutic approach that might soon improve the prognosis of some cancers. We investigated the feasibility of cytosine deaminase (CD) suicide gene therapy in a model of peritoneal carcinomatosis. DHD/K12 colorectal adenocarcinoma cells transfected in vitro with the CD gene were highly sensitive to 5-fluorocytosine (5-FC), and a bystander effect could also be observed. Treating CD+ cells with 5-FC resulted in apoptosis as detected by terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling. In vitro, several human cell lines derived from ovarian or colorectal carcinomas, as well as the rat glioblastoma 9 L cell line, responded to CD/5-FC and showed a very strong bystander effect. 5-FC treatment of peritoneal carcinomatosis generated in syngeneic BDIX rats by CD-expressing DHD/K12 cells led to a complete and prolonged response and to prolonged survival. Our study thus demonstrated the efficacy of CD suicide gene therapy for the treatment of peritoneal carcinomatosis.

Animals↗

PubMed2000

Antitumoral vaccination with granulocyte-macrophage colony-stimulating factor or interleukin-12-expressing DHD/K12 colon adenocarcinoma cells.

Immunomodulating gene therapy for the treatment of malignant diseases is under extensive investigation. In this study, we induced an antitumoral immune response with murine interleukin-12 (mIL-12) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cells in a model of peritoneal carcinomatosis. Intraperitoneal injection of DHD/K12 tumoral cells engineered to produce IL-12 or GM-CSF did not generate any tumors, whereas untransduced DHD/K12 cells gave rise to peritoneal carcinomatosis. IL-12-expressing DHD/K12 cells also protected against tumors derived from coinjected parental cells. To test whether cytokine-producing cells could elicit a memory antitumoral immune response, animals received a challenge with parental DHD/K12 cells 35 days after the injection of proliferating or irradiated DHD/K12 engineered cells. Under our experimental conditions, irradiated tumor cells did not generate any antitumoral immunity. In contrast, tumor development was delayed and survival increased in the animals vaccinated with cytokine-secreting proliferating cells. A specific cytotoxic T-lymphocyte response against DHD/K12 parental cells was observed after vaccination with GM-CSF-expressing cells. Our results demonstrated that intraperitoneal vaccination with IL-12- or GM-CSF-expressing adenocarcinoma cells induced a systemic immune antitumoral response that may be useful as an adjuvant therapy after surgical resection of colorectal cancer.

Adenocarcinoma↗

PubMed2000

Combined suicide and cytokine gene therapy for peritoneal carcinomatosis.

BACKGROUND: Gene therapy is a novel approach for the treatment of cancers, and tumours disseminated in the peritoneal cavity are suitable for in situ delivery of a therapeutic gene. AIMS: The efficacy of a therapy combining a suicide gene (herpes simplex virus type I thymidine kinase (HSV-TK)) and cytokine genes was investigated in a model of peritoneal carcinomatosis induced by colon carcinoma cells in syngeneic rats. MATERIAL AND METHODS: Pre-established macroscopic tumours in BDIX rats were treated by intraperitoneal injections of retrovirus producing cells (FLYA13 TK, FLYA13 granulocyte macrophage-colony stimulating factor (GM-CSF), FLYA13 interleukin 12 (IL-12)) and ganciclovir (GCV). RESULTS: TK/GCV treated animals showed a slight increase in survival time (72 days) compared with the control group (63 days) while the association of cytokine and TK/GCV gene therapy resulted in significantly improved survival, with a large proportion of animals remaining tumour free on day 480 (60% and 40% for TK/GCV/GM-CSF and TK/GCV/IL-12 treated animals, respectively). Histological analysis of treated animals showed that the remaining tumour nodes were infiltrated by mononuclear cells but no major differences were observed between the various treatments. Immunohistochemical analysis revealed that lymphoid CD4(+) and CD8(+) T cells as well as macrophages accumulated outside untreated tumour nodes while CD8(+) and CD25(+) activated T cells and macrophages heavily infiltrated the tumours after the different treatments. CONCLUSIONS: Our data indicate that combined suicide and cytokine gene therapy is a powerful approach for the treatment of macroscopic peritoneal carcinomatosis.

Animals↗

PubMed2000

Correlation between nuclear factor-kappaB activity in bronchial brushing samples and lung dysfunction in an animal model of asthma.

Asthma is a chronic inflammatory disease of the airways, in which many inflammatory genes are overexpressed. Transcription factor, nuclear factor-kappaB (NF-kappaB), which is thought to control the transcriptional initiation of inflammatory genes, has been poorly investigated in asthma. In the present report, bronchial cells (BCs), recovered by bronchial brushing in healthy and heaves-affected horses (i.e., an animal model of asthma), were assessed for NF-kappaB activity. Small amounts of active NF-kappaB were present in BCs of healthy horses, whereas high levels of NF-kappaB activity was found during crisis (i.e., acute airway obstruction) in all heaves-affected horses. Three weeks after the crisis, the level of NF-kappaB activity found in BCs of heaves-affected horses was highly correlated (p < 0.01) to the degree of residual lung dysfunction. Unexpectedly, active NF- kappaB complexes found in BCs of heaves-affected horses were mainly p65 homodimers, rather than classic p65-p50 heterodimers. At last, intercellular adhesion molecule-1 (ICAM-1) expression paralleled p65 homodimers activity in these cells. These results demonstrate that the kinetics of NF-kappaB activity is strongly related to the course of the disease and confirm the relevance of NF-kappaB as a putative target in asthma therapy. Moreover, uncommon p65 homodimers could transactivate, in BCs, a subset of genes, such as ICAM-1, characteristic of chronic airway inflammation.

Animals↗

PubMed2000

Similar efficiency of DNA-liposome complexes and retrovirus-producing cells for HSV-tk suicide gene therapy of peritoneal carcinomatosis.

Several experimental approaches have been tested for suicide gene delivery into tumor cells, including viral and non-viral vectors. In this study, we compared the efficiency of Herpes Simplex Virus type 1 thymidine kinase gene (HSV-tk) delivery by retrovirus-producing cells and DNA/liposome complexes for the treatment of peritoneal carcinomatosis induced in syngeneic rats by DHD/K12 colorectal adenocarcinoma cells. After in vitro determination of the best transduction conditions, rats were treated with multiple intraperitoneal injections of plasmid DNA containing one or two copies of CMV-driven HSV-tk gene (pCMV-TK and p(CMV-TK)2, respectively) associated with LipofectAMINE, each injection being followed by a Ganciclovir (GCV) course. Animals treated by DNA/liposome complexes and GCV or with retrovirus-producing cells and GCV showed a similar increase of survival as compared to the control group. After DNA/ liposome injections, expression of the tk transgene was detected in tumor nodes (epiploon) and also in liver, lung, spleen, bowels and brain. The expression was not homogeneous throughout the different organs and most likely reflected the transfection of only a limited number of cells.

Adenocarcinoma↗

PubMed2000

Roles of nuclear factor-kappaB, p53, and p21/WAF1 in daunomycin-induced cell cycle arrest and apoptosis.

Daunomycin is a potent inducer of p53 and NF-kappaB transcription factors. It is also able to increase the amount of the p21 cyclin-dependent kinase inhibitor. The human p21 promoter harbors p53-responsive elements and an NF-kappaB binding site. We demonstrated, in human breast and colon carcinoma cells, the binding of NF-kappaB dimers to the kappaB site and the transcriptional activation of the human p21 promoter by daunomycin and by NF-kappaB subunits, thereby confirming the functionality of this kappaB binding site. However, using different tumor cell lines where p53 or NF-kappaB was inactive, we showed that p21 activation and cell cycle arrest induced by daunomycin was p53-dependent and NF-kappaB-independent, whereas daunomycin-induced apoptosis was p53- and NF-kappaB-independent.

Antibiotics, Antineoplastic↗

PubMed1999

The homeodomain-containing proteins: an update on their interacting partners.

Homeodomain-containing proteins are transcription regulators controlling the coordinated expression of genes involved in development, differentiation, and cellular transformation. They share a highly conserved 60-amino-acid region (the "homeodomain"), which allows them to bind DNA and modulate the expression of multiple target genes, whose identities remain largely unknown. Although each HOX gene product exhibits in vivo specificity, they harbor very similar DNA-binding affinities in vitro, suggesting that other mechanisms such as protein-protein interactions are critical to modulate their function. In this commentary, we describe the proteins that can interact with the HOX gene products, including newly identified partners such as CREB binding protein and the NF-kappaB/IkappaB-alpha proteins. We also outline the molecular programs that are regulated by the transcriptional complexes involving the HOX gene products and where new pharmacological tools could find interesting targets.

Acetylation↗

PubMed1999

CBP and histone deacetylase inhibition enhance the transactivation potential of the HOXB7 homeodomain-containing protein.

Homeodomain-containing proteins are transcription factors regulating the coordinated expression of multiple target genes involved in development, differentiation and cellular transformation. In this study, we demonstrated that HOXB7, one member of this family, behaved as a transactivator in breast cancer cells. Deletion of either the HOXB7 N-terminal domain or the C-terminal acidic tail abolished this transcriptional effect, suggesting a combination of distinct functional transactivating domains. HOXB7 physically interacted both in vitro and in vivo with the coactivator CREB-binding protein (CBP). This interaction led to an enhanced transactivating potential and required the N-terminal of HOXB7 as well as two domains located at the C-terminal part of CBP. Moreover, trichostatin A, a deacetylase inhibitor, strongly enhanced the transcriptional properties of HOXB7. Our data therefore indicate that HOX proteins can directly interact with CBP and that acetylation/deacetylation may regulate their transcriptional properties.

Breast Neoplasms↗

PubMed1999

Regulation of NF-kappaB activity by I kappaB-related proteins in adenocarcinoma cells.

Constitutive NF-kappaB activity varies widely among cancer cell lines. In this report, we studied the expression and the role of different I kappaB inhibitors in adenocarcinoma cell lines. High constitutive NF-kappaB activity and low I kappaB-alpha expression was found in a number of these cell lines. Moreover, some of these cells showed a high p100 expression, responsible for the cytoplasmic sequestration of most of p65 complexes. Treatment of these cells with TNF-alpha or other NF-kappaB activating agents induced only weakly nuclear NF-kappaB activity without significant p100 processing and led to a very weak transcription of NF-kappaB-dependent reporter gene. Induction of NF-kappaB activity can be restored by expression of the Tax protein or by treatment with antisense p100 oligonucleotides. In MCF7 A/Z cells stably transfected with a p100 expression vector, p65 complexes were sequestered in the cytoplasm by p100. These cells showed a reduced nuclear NF-kappaB induction and NF-kappaB-dependent gene transcription following TNF-alpha stimulation. As a consequence of a competition between I kappaB-alpha and p100, cells expressing high levels of p100 respond poorly to NF-kappaB activating stimuli as TNF-alpha.

Adenocarcinoma↗