Phosphorylation of NF-kappa B and I kappa B proteins: implications in cancer and inflammation

peer reviewedNuclear factor-kappaB (NF-kappaB) is a transcription factor that has crucial roles in inflammation, immunity, cell proliferation and apoptosis. Activation of NF-kappaB mainly occurs via IkappaB kinase (IKK)-mediated phosphorylation of inhibitory molecules, including IkappaBalpha. Optimal induction of NF-kappaB target genes also requires phosphorylation of NF-kappaB proteins, such as p65, within their transactivation domain by a variety of kinases in response to distinct stimuli. Whether, and how, phosphorylation modulates the function of other NF-kappaB and IkappaB proteins, such as B-cell lymphoma 3, remains unclear. The identification and characterization of all the kinases known to phosphorylate NF-kappaB and IkappaB proteins are described here. Because deregulation of NF-kappaB and IkappaB phosphorylations is a hallmark of chronic inflammatory diseases and cancer, newly designed drugs targeting these constitutively activated signalling pathways represent promising therapeutic tools.Insight into the oncogenic potential of BCL-

Regulation of major histocompatibility complex class I expression by NF-kB-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 kB site or on a longer promoter is transactivated by NF-kB complexes contain- ing p65 or RelB. p100 as well as IkB-a 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-g or TNF-a. Stable transfection of a unresponsive mutated IkB-a Ser 32-36 expression vector showed that TNF-a induced MHC Cl I expression in an NF-kB-dependent way while IFN-g did it independently of any NF-kB activation

Challenges for Biomarker Discovery in Body Fluids Using SELDI-TOF-MS

Protein profiling using SELDI-TOF-MS has gained over the past few years an increasing interest in the field of biomarker discovery. The technology presents great potential if some parameters, such as sample handling, SELDI settings, and data analysis, are strictly controlled. Practical considerations to set up a robust and sensitive strategy for biomarker discovery are presented. This paper also reviews biological fluids generally available including a description of their peculiar properties and the preanalytical challenges inherent to sample collection and storage. Finally, some new insights for biomarker identification and validation challenges are provided

Proteomic mass spectra classification using decision tree based ensemble methods.

MOTIVATION: Modern mass spectrometry allows the determination of proteomic fingerprints of body fluids like serum, saliva or urine. These measurements can be used in many medical applications in order to diagnose the current state or predict the evolution of a disease. Recent developments in machine learning allow one to exploit such datasets, characterized by small numbers of very high-dimensional samples. RESULTS: We propose a systematic approach based on decision tree ensemble methods, which is used to automatically determine proteomic biomarkers and predictive models. The approach is validated on two datasets of surface-enhanced laser desorption/ionization time of flight measurements, for the diagnosis of rheumatoid arthritis and inflammatory bowel diseases. The results suggest that the methodology can handle a broad class of similar problems

Caspase-8-dependent HER-2 cleavage in response to tumor necrosis factor alpha stimulation is counteracted by nuclear factor kappa B through c-FLIP-L expression

The oncoprotein HER-2/neu is a prosurvival factor, and its overexpression has been correlated with poor prognosis in patients with breast cancer. We report that HER-2 is a new substrate for caspase-8 and that tumor necrosis factor alpha (TNF-alpha) stimulation leads to an early caspase-8-dependent HER-2 cleavage in MCF7 A/Z breast adenocarcinoma cells defective for nuclear factor kappaB (NFkappaB) activation. We show that the antiapoptotic transcription factor NFkappaB counteracts this cleavage through induction of the caspase-8 inhibitor c-FLIP. Our results also demonstrate that this HER-2 cleavage contributes to the TNF-alpha-induced apoptosis pathway because ectopic expression of an uncleavable HER-2 protects NFkappaB-defective cells against TNF-alpha-mediated cell death. Therefore, we propose an original model in which NFkappaB exerts a new antiapoptotic function by counteracting TNF-alpha-triggered cleavage of the HER-2 survival factor

Biomarker discovery for inflammatory bowel disease, using proteomic serum profiling

peer reviewedCrohn's disease and ulcerative colitis known as inflammatory bowel diseases (IBD) are chronic immuno-inflammatory pathologies of the gastrointestinal tract. These diseases are multifactorial, polygenic and of unknown etiology. Clinical presentation is non-specific and diagnosis is based on clinical, endoscopic, radiological and histological criteria. Novel markers are needed to improve early diagnosis and classification of these pathologies. We performed a study with 120 serum samples collected from patients classified in 4 groups (30 Crohn, 30 ulcerative colitis, 30 inflammatory controls and 30 healthy controls) according to accredited criteria. We compared protein sera profiles obtained with a Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometer (SELDI-TOF-MS). Data analysis with univariate process and a multivariate statistical method based on multiple decision trees algorithms allowed us to select some potential biomarkers. Four of them were identified by mass spectrometry and antibody based methods. Multivariate analysis generated models that could classify samples with good sensitivity and specificity (minimum 80%) discriminating groups of patients. This analysis was used as a tool to classify peaks according to differences in level on spectra through the four categories of patients. Four biomarkers showing important diagnostic value were purified, identified (PF4, MRP8, FIBA and Hpalpha2) and two of these: PF4 and Hpalpha2 were detected in sera by classical methods. SELDI-TOF-MS technology and use of the multiple decision trees method led to protein biomarker patterns analysis and allowed the selection of potential individual biomarkers. Their downstream identification may reveal to be helpful for IBD classification and etiology understanding

Dexamethasone inhibits the HSV-tk/ ganciclovir bystander effect in malignant glioma cells

BACKGROUND: HSV-tk/ ganciclovir (GCV) gene therapy has been extensively studied in the setting of brain tumors and largely relies on the bystander effect. Large studies have however failed to demonstrate any significant benefit of this strategy in the treatment of human brain tumors. Since dexamethasone is a frequently used symptomatic treatment for malignant gliomas, its interaction with the bystander effect and the overall efficacy of HSV-TK gene therapy ought to be assessed. METHODS: Stable clones of TK-expressing U87, C6 and LN18 cells were generated and their bystander effect on wild type cells was assessed. The effects of dexamethasone on cell proliferation and sensitivity to ganciclovir were assessed with a thymidine incorporation assay and a MTT test. Gap junction mediated intercellular communication was assessed with microinjections and FACS analysis of calcein transfer. The effect of dexamethasone treatment on the sensitivity of TK-expressing to FAS-dependent apoptosis in the presence or absence of ganciclovir was assessed with an MTT test. Western blot was used to evidence the effect of dexamethasone on the expression of Cx43, CD95, CIAP2 and Bcl(XL). RESULTS: Dexamethasone significantly reduced the bystander effect in TK-expressing C6, LN18 and U87 cells. This inhibition results from a reduction of the gap junction mediated intercellular communication of these cells (GJIC), from an inhibition of their growth and thymidine incorporation and from a modulation of the apoptotic cascade. CONCLUSION: The overall efficacy of HSV-TK gene therapy is adversely affected by dexamethasone co-treatment in vitro. Future HSV-tk/ GCV gene therapy clinical protocols for gliomas should address this interference of corticosteroid treatment

Protein phosphorylation as a key mechanism for the regulation of BCL-3 activity

Constitutive NF-kappaB activation, a hallmark of many human cancers, upregulates anti-apoptotic gene expression and therefore disrupts the balance between apoptosis and proliferation. In some lymphomas, this constitutive NF-kappaB activity is the result of point mutations or translocations of the genes coding for NF-kappaB inhibitors, namely IkappaBalpha or p100. The BCL-3 protein is another member of the IkappaB family and is overexpressed in a subset of human B-cell chronic lymphocytic leukemias because of a chromosomal translocation. This oncoprotein is phosphorylated by multiple kinases including GSK3 and this phosphorylation regulates BCL-3 function by modulating its oncogenic potential and by regulating the expression of a subset of its target genes. Therefore, deciphering the NF-kappaB/IkappaB protein phosphorylations is critical in order to better understand the molecular mechanisms of NF-kappaB-mediated oncogenesis.Insight into the oncogenic potential of BCL-

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