Identification of protein biomarkers associated with cardiac ischemia by a proteomic approach.

Angina is chest pain induced by ischemia of the heart muscle, generally due to obstruction or spasm of the coronary arteries. People that suffer from average to severe cases of angina have an increased percentage of death before the age of 55, usually around 60%. Therefore, prevention of major complications, optimizing diagnosis, prognosis and therapeutics are of primary importance. The main objective of this study was to uncover biomarkers by comparing serum protein profiles of patients suffering from stable or unstable angina and controls. We identified by non-targeted proteomic approach and confirmed by the means of independent techniques, the differential expression of several proteins indicating significantly increased vascular inflammation response, disturbance in the lipid metabolism and in atherogenic plaques stability.

[Proteomics studies on arthritis by SELDI-TOF-MS: identification of the S100 proteins family as proteins of interest]. / Protéomique par SELDI-TOF-MS des maladies inflammatoires articulaires: identification des protéines S100 comme protéines d'intérêt.

Clinical proteomics is a technical approach studying the entire proteome expressed by cells, tissues or organs. It describes the dynamics of cell regulation by detecting molecular events related to diseases development. Proteomic techniques focus mainly on identification of new biomarkers or new therapeutic targets. It is a multidisciplinary approach using medical, biological, bioanalytical and bioinformatics knowledges. A strong collaboration between these fields allowed SELDI-TOF-MS proteomics studies to be performed at the CHU and the University of Liège, in GIGA-Research facilities. The aim of these studies was driven along three main axes of research related to the identification of biomarkers specific to a studied pathology, to a common biological pathway and, finally, to a treatment response. This work was presented in the setting of the "Synthèse CHU 2009" meeting.

Transcriptional activation of cyclooxygenase-2 by tumor suppressor p53 requires nuclear factor-kappaB.

Overexpression of cyclooxygenase-2 (Cox-2) is thought to exert antiapoptotic effects in cancer. Here we show that the tumor suppressor p53 upregulated Cox-2 in esophageal and colon cancer cell lines by inducing the binding of nuclear factor-kappaB (NF-kappaB) to its response element in the COX-2 promoter. Inhibition of NF-kappaB prevented p53 induction of Cox-2 expression. Cooperation between p53 and NF-kappaB was required for activation of COX-2 promoter in response to daunomycin, a DNA-damaging agent. Pharmacological inhibition of Cox-2 enhanced apoptosis in response to daunomycin, in particular in cells containing active p53. In esophageal cancer, there was a correlation between Cox-2 expression and wild-type TP53 in Barrett's esophagus (BE) and in adenocarcinoma, but not in squamous cell carcinoma (P<0.01). These results suggest that p53 and NF-kappaB cooperate in upregulating Cox-2 expression, promoting cell survival in inflammatory precursor lesions such as BE.

Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity.

We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells.

Expression of bone sialoprotein, a bone matrix protein, in human breast cancer.

Microcalcifications are often associated with human mammary lesions, particularly with breast carcinomas. To date, the molecular mechanism that leads to the deposition of hydroxyapatite in the mammary tissue has not been elucidated. Bone sialoprotein (BSP) is a glycoprotein the expression of which coincides with the appearance of the first hydroxyapatite crystals during bone development. In this study, we report the observation that BSP, a bone matrix protein, is expressed in human mammary cancer cells. Using an immunoperoxidase technique, we studied the expression of BSP in 79 breast lesions, including 28 benign and 51 malignant specimens. Two polyclonal antibodies, one directed against intact human BSP and the other against a synthetic peptide of BSP (residues 277-294), were used and gave identical results. Normal mammary glands expressed undetectable or barely detectable amounts of BSP, and the majority of the benign lesions examined were generally unstained (0) or weakly stained (1+). Most of the breast carcinoma specimens (around 87%) showed a significant increase (P = 0.0001) in BSP expression. Breast carcinomas with microcalcifications had the highest immunoreactivity (2+ or 3+) to BSP antibodies. This is the first demonstration that BSP expression is significantly increased in breast cancer. Expression of BSP by breast cancer cells could play a major role in the deposition of microcalcifications and in the preferred bone homing of breast cancer cells.

Stable inhibition of nuclear factor kappaB in cancer cells does not increase sensitivity to cytotoxic drugs.

Several reports indicated that nuclear factor kappaB (NF-kappaB) activation by cytokines, cytotoxic drugs, or ionizing radiation protects cells against apoptosis. Therefore, we investigated the consequence of NF-kappaB inhibition on the efficiency of antineoplastic agents. HPB, HCT116, MCF7, and OVCAR-3 cells stably expressing a dominant negative IkappaBalpha inhibitor showed a decreased NF-kappaB activation following treatment with tumor necrosis factor a and various chemotherapeutic agents. However, there was no difference in survival between parental cells and cells expressing mutated IkappaBalpha. These studies suggest that, at least in these cell lines, stable NF-kappaB inhibition did not modify the response to cytotoxic drugs.

Impact of injection solvent composition on protein identification in column-switching chip-liquid chromatography/mass spectrometry.

In shotgun proteomics, the gold standard technique is reversed-phase liquid chromatography coupled to mass spectrometry. Many researches have been carried out to study the effects on identification performances of chromatographic parameters such as the stationary phase and column dimensions, mobile phase composition and flow rate, as well as the gradient slope and length. However, little attention is usually paid to the injection solvent composition. In this study, we investigated the effect of the injection solvent on protein identification parameters (number of distinct peptides, amino acid coverage and MS/MS search score) as well as sensitivity. Tryptic peptides from six different proteins, covering a wide range of physicochemical properties, were employed as training set. Design of experiments was employed as a tool to highlight the factors related to the composition of the injection solvent that significantly influenced the obtained results. Optimal results for the training set were applied to analysis of more complex samples. The experiments pointed out optimising the composition of the injection solvent had a strong beneficial effect on all the considered responses. On the basis of these results, an approach to determine optimal conditions was proposed to maximise the protein identification performances and detection sensitivity.

Nuclear factor - kappaB-dependent regulation of p53 gene expression induced by daunomycin genotoxic drug.

Anthracycline drugs are widely used for the treatment of solid tumors and leukemia, but the molecular basis of their biological effect is still poorly understood. In the HCT116 colon carcinoma cell line, which retains a wild-type inducible p53 gene, we show that the anthracycline daunomycin is a potent inducer of p53 and NF-kappaB transcription factors. Nuclear accumulation of p53 protein occurred because of increased protein stability and enhanced gene expression. In addition, daunomycin induced the p53 promoter through the binding of p50/p65 NF-kappaB heterodimers to the kappaB site in the p53 promoter. Under our conditions, the free radical scavengers NAC and PDTC were not able to block NF-kappaB activation or p53 induction, indicating that reactive oxygen intermediates were not involved in the cellular response to daunomycin stimulation. Overexpression of a stable unresponsive IkappaBalpha mutant in HCT116 cells resulted in a complete inhibition of the NF-kappaB activation but only a partial impairment of the p53 protein accumulation induced by daunomycin. We conclude that the p53-activating signal generated by daunomycin is partially regulated by NF-kappaB.

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.

Highly-expressed p100/p52 (NFKB2) sequesters other NF-kappa B-related proteins in the cytoplasm of human breast cancer cells.

Several observations have suggested that NF-kappa B transcription factors could be involved in carcinogenesis. To investigate the possibility that members of the NF-kappa B family participate in the molecular control of the transformed phenotype, we examined the expression of these proteins in human breast cancer cell lines as well as in primary tumors. Western Immunoblots demonstrated high expression of the p52 precursor p100 (NFKB2) in several breast cancer cell lines while human mammary epithelial cells express this protein only faintly. Eighteen primary breast tumors out of 24 displayed significant expression of the p100/p52 protein. In MDA-MB-435 cells, overexpressed p100 and p52 are predominantly cytoplasmic and coimmunoprecipitation experiments demonstrated that p100 sequesters the heterodimer p50/p65 in the cytoplasm. We demonstrate that most p65 protein is complexed with p100 in these cells while it is complexed predominantly with I kappa B-alpha in cell lines expressing less p100. Our data strengthen the hypothesis that NF-kappa B could be involved in carcinogenesis and suggest that the p100/p52 NF-kappa B subunit could play a role in the development of human breast cancers, possibly by sequestering other NF-kappa B-related proteins in the cytoplasm.

Regulation of major histocompatibility complex class I expression by NF-kappaB-related proteins in breast cancer cells.

Downregulation of MHC Class I antigens has been observed in many cancers and usually results from a decreased gene transcription. A reporter CAT gene dependent on the MHC Class I kappaB site or on a longer promoter is transactivated by NF-kappaB complexes containing p65 or RelB. p100 as well as IkappaB-alpha are potent inhibitors of this transcription and p100 sequesters RelB and p65 complexes in the cytoplasm of breast cancer cells. However, although p100 is highly expressed in a number of breast cancer cell lines, MHC Class I antigen expression was observed on all the cell lines we analysed and could be further induced by stimulation with the cytokines IFN-gamma or TNF-alpha. Stable transfection of a unresponsive mutated IkappaB-alpha Ser 32-36 expression vector showed that TNF-alpha induced MHC Cl I expression in an NF-kappaB-dependent way while IFN-gamma did it independently of any NF-kappaB activation.

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.

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.

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.

NF- kappa B2/p100 induces Bcl-2 expression.

The NF-kappaB2/p100 and bcl-3 genes are involved in chromosomal translocations described in chronic lymphocytic leukemias (CLL) and non-Hodgkin's lymphomas, and nuclear factor kappaB (NF-kappaB) protects cancer cells against apoptosis. Therefore, we investigated whether this transcription factor could modulate the expression of the Bcl-2 antiapoptotic protein. Bcl-2 promoter analysis showed multiple putative NF-kappaB binding sites. Transfection assays of bcl-2 promoter constructs in HCT116 cells showed that NF-kappaB can indeed transactivate bcl-2. We identified a kappaB site located at position -180 that can only be bound and transactivated by p50 or p52 homodimers. As p50 and p52 homodimers are devoid of any transactivating domains, we showed that they can transactivate the bcl-2 promoter through association with Bcl-3. We also observed that stable overexpression of p100 and its processed product p52 can induce endogenous Bcl-2 expression in MCF7AZ breast cancer cells. Finally, we demonstrated that, in breast cancer and leukemic cells (CLL), high NF-kappaB2/p100 expression was associated with high Bcl-2 expression. Our data suggest that Bcl-2 could be an in vivo target gene for NF-kappaB2/p100.

A cell type-specific and gap junction-independent mechanism for the herpes simplex virus-1 thymidine kinase gene/ganciclovir-mediated bystander effect.

Tumor cells expressing the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene are killed by nucleoside analogues such as ganciclovir (GCV). GCV affects not only the cells expressing HSV-tk but also neighboring cells that do not express the gene; this phenomenon commonly is called "bystander effect." GCV metabolites transfer via gap junctional intercellular communication (GJIC) accounts for the bystander effect in different cell lines, but other mechanisms have also been described. In this study, we analyzed the mechanisms of the bystander effect in two cell lines exhibiting different capacities of communication (DHD/K12 and 9L). The 9L cells exhibited a very good bystander effect, which was completely blocked by a long-term inhibitor of GJIC, 18 alpha-glycyrrhetinic acid. DHD/K12 cells exhibited a moderate bystander effect that was not abolished by 18 alpha-glycyrrhetinic acid or 1-octanol, another strong inhibitor of GJIC. Interestingly, we also observed a bystander effect in cultures where HSV-tk-expressing DHD/K12 cells were physically separated from their untransfected counterparts but grown in the same medium. Moreover, the transfer of filtered conditioned medium from GCV-treated HSV-tk-expressing DHD/K12 cells to DHD/K12 parental cells induced a decrease of survival in a concentration-dependent manner, suggesting that the bystander effect in this cell line was mediated by a soluble factor.

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.

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.

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.

Varicella-zoster virus gene regulation.

The varicella-zoster virus genome contains 71 open reading frames (ORFs), five of which (ORF62, ORF4, ORF63, ORF61, and ORF10) encode regulatory proteins. ORF62 codes for the major immediate early protein of the virus exhibiting DNA-binding and regulatory functions. This protein, localized in the cell nucleus, is a functional homologue to ICP4 of herpes simplex virus type 1 (HSV-1). It trans-activates several varicella-zoster virus promoters of the various gene classes and autoregulates its own expression. ORF4 protein activates gene promoters provided they have basal activities, but it is not a functional homologue of HSV-1 ICP27. Gene regulation activity appears to be linked to its cysteine-rich C-terminal region. ORF63 codes for an immediate early protein mainly located in the cell nucleus. The regulatory functions it performs are still unclear. ORF61 protein is the functional homologue of HSV-1 ICP0. Its N-terminal region exhibits a RING domain responsible for trans-activating and trans-repressing activities. ORF10 protein exhibits similarities with HSV-1 VP16 and activates the ORF62 promoter.