New biomarkers for primary mitral regurgitation.

BACKGROUND: Mitral regurgitation is a frequent valvular heart disease affecting around 2.5 % of the population with prevalence directly related to aging. Degeneration of mitral valve is broadly considered as a passive ongoing pathophysiological process and little is known about its physiological deregulation. The purpose of this study was to highlight new biomarkers of mitral regurgitation in order to decipher the underlying pathological mechanism as well as to allow the diagnosis and the monitoring of the disease. RESULTS: Modulation of various blood proteins expression was examined in patients suffering from different grades of mitral regurgitation (mild, moderate and severe) compared to healthy controls. To this end, several routine clinical assays and the multi analyte profile technology targeting 184 proteins were used. High-density lipoprotein, apolipoprotein-A1, haptoglobin and haptoglobin-α2 chain levels significantly decreased proportionally to the degree of mitral regurgitation when compared to controls. High-density lipoprotein and apolipoprotein-A1 levels were associated with effective regurgitant orifice area and regurgitant volume. Apolipoprotein-A1 was an independent predictor of severe mitral regurgitation. Moreover, with ordinal logistic regression, apolipoprotein-A1 remained the only independent factor associated with mitral regurgitation. In addition, myxomatous mitral valves were studied by immunocytochemistry. We observed an increase of LC3, the marker of autophagy, in myxomatous mitral valves compared with healthy mitral valves. CONCLUSION: These potential biomarkers of mitral regurgitation highlighted different cellular processes that could be modified in myxomatous degenerescence: reverse cholesterol transport, antioxidant properties and autophagy.

Comprehensive plasma profiling for the characterization of graft-versus-host disease biomarkers.

Acute graft-versus-host disease (aGVHD) remains a life-threatening complication of hematopoietic stem cell transplantation (HSCT) therefore limiting its application. To optimize the management of aGVHD and reduce therapy-related toxicity, early specific markers are needed. The main objective of this study was to uncover diagnostic biomarkers by comparing plasma protein profiles of patients at the time of acute GVHD diagnosis with those of patients undergoing HSCT without aGVHD. Additional analysis of samples taken 15 days before aGVHD diagnosis was also performed to evaluate the potential of our newly discovered biomarkers for early diagnosis. To get complementary information from plasma samples, we used three different proteomic approaches, namely 2D-DIGE, SELDI-TOF-MS and 2D-LC-MS(E). We identified and confirmed by the means of independent techniques, the differential expression of several proteins indicating significantly increased inflammation response and disturbance in the coagulation cascade. The variation of these proteins was already observed 15 days before GVHD diagnosis, suggesting the potential early detection of the disease before symptoms appearance. Finally, logistic regression analysis determined a composite biomarker panel comprising fibrinogen, fragment of fibrinogen beta chain, SAA, prothrombin fragments, apolipoprotein A1 and hepcidin that optimally discriminated patients with and without GVHD. The area under the receiver operating characteristic curve distinguishing these 2 groups was 0.95.

New role for EMD (emerin), a key inner nuclear membrane protein, as an enhancer of autophagosome formation in the C16-ceramide autophagy pathway.

To date, precise roles of EMD (emerin) remain poorly described. In this paper, we investigated the role of EMD in the C16-ceramide autophagy pathway. Ceramides are bioactive signaling molecules acting notably in the regulation of cell growth, differentiation, or cell death. However, the mechanisms by which they mediate these pathways are not fully understood. We found that C16-ceramide induces EMD phosphorylation on its LEM domain through PRKACA. Upon ceramide treatment, phosphorylated EMD binds MAP1LC3B leading to an increase of autophagosome formation. These data suggest a new role of EMD as an enhancer of autophagosome formation in the C16-ceramide autophagy pathway in colon cancer cells.

Kinetics of IL-7 and IL-15 levels after allogeneic peripheral blood stem cell transplantation following nonmyeloablative conditioning.

BACKGROUND: We analysed kinetics of IL-7 and IL-15 levels in 70 patients given peripheral blood stem cells after nonmyeloablative conditioning. METHODS: EDTA-anticoagulated plasma and serum samples were obtained before conditioning and about once per week after transplantation until day 100. Samples were aliquoted and stored at -80°C within 3 hours after collection until measurement of cytokines. IL-7 and IL-15 levels were measured by ELISAs. RESULTS: Median IL-7 plasma levels remained below 6 pg/L throughout the first 100 days, although IL-7 plasma levels were significantly higher on days 7 (5.1 pg/mL, P=0.002), 14 (5.2 pg/mL, P<0.001), and 28 (5.1 pg/mL, P=0.03) (but not thereafter) than before transplantation (median value of 3.8 pg/mL). Median IL-15 serum levels were significantly higher on days 7 (12.5 pg/mL, P<0.001), 14 (10.5 pg/mL, P<0.001), and 28 (6.2 pg/mL, P<0.001) than before transplantation (median value of 2.4 pg/mL). Importantly, IL-7 and IL-15 levels on days 7 or 14 after transplantation did not predict grade II-IV acute GVHD. CONCLUSIONS: These data suggest that IL-7 and IL-15 levels remain relatively low after nonmyeloablative transplantation, and that IL-7 and IL-15 levels early after nonmyeloablative transplantation do not predict for acute GVHD.

BCL-3 degradation involves its polyubiquitination through a FBW7-independent pathway and its binding to the proteasome subunit PSMB1.

The oncogenic protein BCL-3 activates or represses gene transcription through binding with the NF-kappaB proteins p50 and p52 and is degraded through a phospho- and GSK3-dependent pathway. However, the mechanisms underlying its degradation remain poorly understood. Yeast two-hybrid analysis led to the identification of the proteasome subunit PSMB1 as a BCL-3-associated protein. The binding of BCL-3 to PSMB1 is required for its degradation through the proteasome. Indeed, PSMB1-depleted cells are defective in degrading polyubiquitinated BCL-3. The N-terminal part of BCL-3 includes lysines 13 and 26 required for the Lys(48)-linked polyubiquitination of BCL-3. Moreover, the E3 ligase FBW7, known to polyubiquitinate a variety of substrates phosphorylated by GSK3, is dispensable for BCL-3 degradation. Thus, our data defined a unique motif of BCL-3 that is needed for its recruitment to the proteasome and identified PSMB1 as a key protein required for the proteasome-mediated degradation of a nuclear and oncogenic IkappaB protein.

The proapoptotic C16-ceramide-dependent pathway requires the death-promoting factor Btf in colon adenocarcinoma cells.

Ceramides are central molecules in sphingolipid metabolism. They are involved in the regulation of cancer-cell growth, differentiation, senescence and apoptosis. To better understand how these secondary messengers induce their biological effects, adenocarcinoma cells (HCT116) were treated with exogenous long-chain ceramides (C16-ceramide) in order to mimic endogenous sphingolipids. This treatment induced a decrease of cell viability partly due to apoptosis as shown by PARP cleavage and a decrease of pro-caspase 3. Two-dimensional differential in-gel electrophoresis (2D-DIGE) revealed the differential expression of 51 proteins in response to C16-ceramide. These proteins are notably involved in cell proliferation, apoptosis, protein transport and transcriptional regulation. Among them, the cell death-promoting factor Btf was found to be implicated in the apoptotic signal triggered by ceramide. In adenocarcinoma cells, Btf regulates apoptosis related proteins such as Mdm2, p53, BAX and pBcl-2 and thus plays an important role in the ceramide mediated cell death. These findings bring new insight into the proapoptotic ceramide-dependent signaling pathway.

New biomarkers of Crohn's disease: serum biomarkers and development of diagnostic tools.

Crohn's disease (CD) is a complex and heterogeneous inflammatory disorder that is part of inflammatory bowel diseases. Its diagnosis is performed on clinical presentation and results of radiography, endoscopy and histological findings. New noninvasive diagnostic biomarkers are needed to allow rapid and accurate CD discrimination. Blood-derived biomarkers correlating with disease activity, supported by genetic evidences and valid for all CD patients subtypes are still missing. Hence, no biomarkers and no related diagnostic tests are recommended or used alone for CD diagnosis in clinical practice. This review describes diagnosis tests based on the detection/quantification of specific acute-phase reactant proteins, enzymes and derived antibody response developed by inflammatory bowel disease patients for pathogens or symbiotic flora determinant, as well as autoantibodies. Their power as diagnostic tools is discussed, as well as new high-throughput techniques, such as microarrays and proteomics, for the discovery of new CD clinical biomarkers and for the development of specific CD diagnostic tests. Some rapidly evolving nanotechnologies, mathematical analysis and bioinformatics methods are also mentioned to highlight their importance for further accurate CD diagnosis.

Geldanamycin inhibits tyrosine phosphorylation-dependent NF-kappaB activation.

Hsp90 is a protein chaperone regulating the stability and activity of many signalling molecules. The requirement of Hsp90 activity in the NF-kappaB pathway has been recently reported by several authors using the Hsp90 ATPase inhibitor geldanamycin (GA), an anti-tumor drug. Hsp90 inhibition blocks the synthesis and activation of the IKK complex, the major kinases complex responsible for IkappaBalpha phosphorylation on serine 32 and 36, a key step for its degradation and the nuclear translocation of NF-kappaB. However, the effect of GA on other IkappaBalpha kinases, including tyrosine kinases, is unknown. In the present study, we investigated the effect of GA on NF-kappaB activation induced by sodium pervanadate (PV), a tyrosine phosphatase inhibitor triggering c-Src-mediated tyrosine phosphorylation of IkappaBalpha. We report for the first time that GA inhibits PV-induced IkappaBalpha tyrosine phosphorylation and degradation. Using an in vitro kinase assay, we demonstrated that GA inhibits the activity of c-Src as an IkappaBalpha tyrosine kinase, but not its cellular expression. As a result, GA blocked PV-induced NF-kappaB DNA-binding activity on an exogenous kappaB element and on the endogenous ikappabalpha promoter, thereby inhibiting ikappabalpha transcription. Finally, we demonstrated that, despite NF-kappaB inhibition, pre-treatment with GA does not potentiate PV-induced apoptosis. We conclude that c-Src requires Hsp90 for its tyrosine kinase activity, and its inhibition by GA blocks c-Src-dependent signalling pathways, such as NF-kappaB activation induced by sodium pervanadate. The effect of GA on PV-induced apoptosis is discussed in the light of recent publications in the literature.

Proteomics for prediction and characterization of response to infliximab in Crohn's disease: a pilot study.

OBJECTIVES: Infliximab is the first anti-TNFalpha accepted by the Food and Drug Administration for use in inflammatory bowel disease treatment. Few clinical, biological and genetic factors tend to predict response in Crohn's disease (CD) patient subcategories, none widely predicting response to infliximab. DESIGN AND METHODS: Twenty CD patients showing clinical response or non response to infliximab were used for serum proteomic profiling on Surface Enhanced Lazer Desorption Ionisation-Time of Flight-Mass Spectrometry (SELDI-TOF-MS), each before and after treatment. Univariate and multivariate data analysis were performed for prediction and characterization of response to infliximab. RESULTS: We obtained a model of classification predicting response to treatment and selected relevant potential biomarkers, among which platelet aggregation factor 4 (PF4). We quantified PF4, sCD40L and IL-6 by ELISA for correlation studies. CONCLUSIONS: This first proteomic pilot study on response to infliximab in CD suggests association between platelet metabolism and response to infliximab and requires validation studies on a larger cohort of patients.

Deregulated expression of pro-survival and pro-apoptotic p53-dependent genes upon Elongator deficiency in colon cancer cells.

Elongator, a multi-subunit complex assembled by the IkappaB kinase-associated protein (IKAP)/hELP1 scaffold protein is involved in transcriptional elongation in the nucleus as well as in tRNA modifications in the cytoplasm. However, the biological processes regulated by Elongator in human cells only start to be elucidated. Here we demonstrate that IKAP/hELP1 depleted colon cancer-derived cells show enhanced basal expression of some but not all pro-apoptotic p53-dependent genes such as BAX. Moreover, Elongator deficiency causes increased basal and daunomycin-induced expression of the pro-survival serum- and glucocorticoid-induced protein kinase (SGK) gene through a p53-dependent pathway. Thus, our data collectively demonstrate that Elongator deficiency triggers the activation of p53-dependent genes harbouring opposite functions with respect to apoptosis.

Monomeric calgranulins measured by SELDI-TOF mass spectrometry and calprotectin measured by ELISA as biomarkers in arthritis.

BACKGROUND: SELDI-TOF mass spectrometry (MS) is a high-throughput proteomic approach with potential for identifying novel forms of serum biomarkers of arthritis. METHODS: We used SELDI-TOF MS to analyze serum samples from patients with various forms of inflammatory arthritis. Several protein profiles were collected on different Bio-Rad Laboratories ProteinChip arrays (CM10 and IMAC-Cu(2+)) and were evaluated statistically to select potential biomarkers. RESULTS: SELDI-TOF MS analyses identified several calgranulin proteins [S100A8 (calgranulin A), S100A9 (calgranulin B), S100A9*, and S100A12 (calgranulin C)], serum amyloid A (SAA), SAA des-Arg (SAA-R), and SAA des-Arg/des-Ser (SAA-RS) as biomarkers and confirmed the results with other techniques, such as western blotting, immunoprecipitation, and nano-LC-MS/MS. The S100 proteins were all able to significantly differentiate samples from patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) from those of patients with inflammatory bowel diseases used as an inflammatory control (IC) group, whereas the SAA, SAA-R, and SAA-RS proteins were not, with the exception of AS. The 4 S100 proteins were coproduced in all of the pathologies and were significantly correlated with the plasma calprotectin concentration; however, these S100 proteins were correlated with the SAA peak intensities only in the RA and IC patient groups. In RA, these S100 proteins (except for S100A12) were significantly correlated with the serum concentrations of C-reactive protein, matrix metalloproteinase 3, and anti-cyclic citrullinated peptide and with the Disease Activity Score (DAS(28)). CONCLUSIONS: The SELDI-TOF MS technology is a powerful approach for analyzing the status of monomeric, truncated, or posttranslationally modified forms of arthritis biomarkers, such as the S100A8, S100A9, S100A12, and SAA proteins. The fact that the SELDI-TOF MS data were correlated with results obtained with the classic calprotectin ELISA test supports the reliability of this new proteomic technique.

Further insights in the mechanisms of interleukin-1beta stimulation of osteoprotegerin in osteoblast-like cells.

UNLABELLED: The mechanisms of IL-1beta stimulation of OPG were studied in more detail. Whereas p38 and ERK activation was confirmed to be needed, NF-kappaB was not necessary for this regulation. We also found that OPG production after IL-1beta stimulation was not sufficient to block TRAIL-induced apoptosis in MG-63 cells. INTRODUCTION: Osteoprotegerin (OPG) plays a key role in the regulation of bone resorption and is stimulated by interleukin (IL)-1beta. Herein, we defined the mechanisms of IL-1beta stimulation of OPG focusing on the potential involvement of MAPK and NF-kappaB. We also examined whether OPG production in response to IL-1beta influences TRAIL-induced apoptosis in MG-63 cells. MATERIALS AND METHODS: OPG mRNA levels in MG-63 cells were quantified by real-time RT-PCR and protein levels of OPG and IL-6 by ELISA. Cell viability was assessed using the methyltetrazidium salt (MTS) reduction assay. The role of the MAPK pathway was studied by both Western blotting and the use of specific chemical inhibitors. NF-kappaB function was studied using BAY 11-7085 and by siRNA transfection to inhibit p65 synthesis. Transcription mechanisms were analyzed by transiently transfecting MG-63 cells with OPG promoter constructs. Post-transcriptional effects were examined by using cycloheximide and actinomycin D. RESULTS: MG-63 cells treatment with IL-1beta resulted in the phosphorylation of c-Jun NH(2)-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). The use of the specific inhibitors showed that p38 and ERK but not JNK were needed for IL-1beta-induced OPG production. In contrast, NF-kappaB was not essential for IL-1beta induction of OPG. We also showed a small transcriptional and a possible post-transcriptional or translational regulation of OPG by IL-1beta. Exogenous OPG blocked TRAIL-induced apoptosis, but IL-1beta induction of OPG did not influence TRAIL-induced cell death. CONCLUSIONS: IL-1beta stimulates OPG production by mechanisms dependent on p38 and ERK. In contrast, NF-kappaB was not essential for this regulation. Although the relevance of IL-1beta stimulation of OPG is still not fully understood, our data showed that IL-1beta stimulation of OPG does not modify TRAIL-induced cell death.

Raloxifene-induced myeloma cell apoptosis: a study of nuclear factor-kappaB inhibition and gene expression signature.

Because multiple myeloma remains associated with a poor prognosis, novel drugs targeting specific signaling pathways are needed. The efficacy of selective estrogen receptor modulators for the treatment of multiple myeloma is not well documented. In the present report, we studied the antitumor activity of raloxifene, a selective estrogen receptor modulator, on multiple myeloma cell lines. Raloxifene effects were assessed by tetrazolium salt reduction assay, cell cycle analysis, and Western blotting. Mobility shift assay, immunoprecipitation, chromatin immunoprecipitation assay, and gene expression profiling were performed to characterize the mechanisms of raloxifene-induced activity. Indeed, raloxifene, as well as tamoxifen, decreased JJN-3 and U266 myeloma cell viability and induced caspase-dependent apoptosis. Raloxifene and tamoxifen also increased the cytotoxic response to vincristine and arsenic trioxide. Moreover, raloxifene inhibited constitutive nuclear factor-kappaB (NF-kappaB) activity in myeloma cells by removing p65 from its binding sites through estrogen receptor alpha interaction with p65. It is noteworthy that microarray analysis showed that raloxifene treatment decreased the expression of known NF-kappaB-regulated genes involved in myeloma cell survival and myeloma-induced bone lesions (e.g., c-myc, mip-1alpha, hgf, pac1,...) and induced the expression of a subset of genes regulating cellular cycle (e.g., p21, gadd34, cyclin G2,...). In conclusion, raloxifene induces myeloma cell cycle arrest and apoptosis partly through NF-kappaB-dependent mechanisms. These findings also provide a transcriptional profile of raloxifene treatment on multiple myeloma cells, offering the framework for future studies of selective estrogen receptor modulators therapy in multiple myeloma.

TNFalpha- and IKKbeta-mediated TANK/I-TRAF phosphorylation: implications for interaction with NEMO/IKKgamma and NF-kappaB activation.

Pro-inflammatory cytokines trigger signalling cascades leading to NF-kappaB (nuclear factor-kappaB)-dependent gene expression through IKK [IkappaB (inhibitory kappaB) kinase]-dependent phosphorylation and subsequent degradation of the IkappaB proteins and via induced phosphorylation of p65. These signalling pathways rely on sequentially activated kinases which are assembled by essential and non-enzymatic scaffold proteins into functional complexes. Here, we show that the pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) promotes TANK [TRAF (TNF receptor-associated factor) family member associated NF-kappaB activator] recruitment to the IKK complex via a newly characterized C-terminal zinc finger. Moreover, we show that TANK is phosphorylated by IKKbeta upon TNFalpha stimulation and that this modification negatively regulates TANK binding to NEMO (NF-kappaB essential modulator). Interestingly, reduced TANK expression by RNA interference attenuates TNFalpha-mediated induction of a subset of NF-kappaB target genes through decreased p65 transactivation potential. Therefore the scaffold protein TANK is required for the cellular response to TNFalpha by connecting upstream signalling molecules to the IKKs and p65, and its subsequent IKKbeta-mediated phosphorylation may be a mechanism to terminate the TANK-dependent wave of NF-kappaB activation.

Discovery of new rheumatoid arthritis biomarkers using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry ProteinChip approach.

OBJECTIVE: To identify serum protein biomarkers specific for rheumatoid arthritis (RA), using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology. METHODS: A total of 103 serum samples from patients and healthy controls were analyzed. Thirty-four of the patients had a diagnosis of RA, based on the American College of Rheumatology criteria. The inflammation control group comprised 20 patients with psoriatic arthritis (PsA), 9 with asthma, and 10 with Crohn's disease. The noninflammation control group comprised 14 patients with knee osteoarthritis and 16 healthy control subjects. Serum protein profiles were obtained by SELDI-TOF-MS and compared in order to identify new biomarkers specific for RA. Data were analyzed by a machine learning algorithm called decision tree boosting, according to different preprocessing steps. RESULTS: The most discriminative mass/charge (m/z) values serving as potential biomarkers for RA were identified on arrays for both patients with RA versus controls and patients with RA versus patients with PsA. From among several candidates, the following peaks were highlighted: m/z values of 2,924 (RA versus controls on H4 arrays), 10,832 and 11,632 (RA versus controls on CM10 arrays), 4,824 (RA versus PsA on H4 arrays), and 4,666 (RA versus PsA on CM10 arrays). Positive results of proteomic analysis were associated with positive results of the anti-cyclic citrullinated peptide test. Our observations suggested that the 10,832 peak could represent myeloid-related protein 8. CONCLUSION: SELDI-TOF-MS technology allows rapid analysis of many serum samples, and use of decision tree boosting analysis as the main statistical method allowed us to propose a pattern of protein peaks specific for RA.

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 BclXL. 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.

Sodium nitroprusside-induced osteoblast apoptosis is mediated by long chain ceramide and is decreased by raloxifene.

Release of high levels of nitric oxide (NO) is associated with osteoblastic cell death. The mechanisms of NO-induced cytotoxicity are not well documented and it is presently not known if estrogenic compounds prevent this effect. We studied the role of ceramides in cell death induced by the NO donor sodium nitroprusside (SNP) and we tested the possibility that 17beta-estradiol, the anti-estrogen ICI 182.780 and two selective estrogen receptor modulators raloxifene and tamoxifen modify osteoblastic cell apoptosis. SNP dose-dependently decreased MC3T3-E1 osteoblast viability, increased NO production in the culture media and enhanced the release of intracellular ceramides C22 and C24. Cell death induced by SNP was partially inhibited when MC3T3-E1 cells were pretreated with raloxifene and tamoxifen but was not modified when the cells were pretreated with 17beta-estradiol or ICI 182.780. Cell death induced by SNP resulted from apoptosis as demonstrated by Annexin-V and propidium iodide labeling and a reduction of SNP-induced MC3T3-E1 apoptosis was confirmed in the presence of raloxifene and tamoxifen. SNP induction of C22 and C24 production was inhibited by a pretreatment with raloxifene but not with 17beta-estradiol. Moreover, the synthetic ceramide C24 (0.75 and 1microM) decreased MC3T3-E1 cell viability and osteoblast cell death induced by C24 was partially decreased by raloxifene and to a lesser extent by 17beta-estradiol. These data demonstrate that SNP-induced cell death is mediated by the long chain ceramides C22 and C24 and that raloxifene protected osteoblast from apoptosis induced by SNP, an effect that might be relevant to its pharmacological properties on bone remodeling.

Low daunomycin concentrations protect colorectal cancer cells from hypoxia-induced apoptosis.

Hypoxia, a common feature of solid tumors, is a direct stress that triggers apoptosis in many cell types. Poor or irregular tumor vascularization also leads to a decreased drug diffusion and cancer cells distant from blood vessels (hypoxic cells) are exposed to low drug concentrations. In this report, we show that low daunomycin concentrations protect HCT116 colorectal cancer cells from hypoxia-induced apoptosis. While hypoxia induced p53 accumulation without expression of its responsive genes (bax and p21), daunomycin treatment restored p53 transactivation activity and cell cycle progression. We also demonstrated a role for Akt activation in daunomycin-induced protection through phosphorylation and inactivation of the Bcl-2 family proapoptotic factor Bad. Our data therefore suggest that chemotherapy could possibly, because of low concentrations in poorly vascularized tumors, protect cancer cells from hypoxia-induced cytotoxicity.

Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation.

Nuclear 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.

Interleukin-6 receptor shedding is enhanced by interleukin-1beta and tumor necrosis factor alpha and is partially mediated by tumor necrosis factor alpha-converting enzyme in osteoblast-like cells.

OBJECTIVE: Interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) activation of gp130 represents an alternative pathway for osteoclast development in inflammatory conditions. The goal of the present study was to investigate changes in sIL-6R levels in response to the inflammatory cytokines IL-1beta and tumor necrosis factor alpha (TNFalpha) and to determine the role of TNFalpha-converting enzyme (TACE) in this process. METHODS: Levels of sIL-6R in the culture media of MG63 and SAOS-2 osteoblast-like cell lines after exposure to various agents were determined by immunoassay. TACE protein levels were measured by Western immunoblotting. Cells were transfected with small interfering RNA (siRNA) or with an expression plasmid for IL-6R and TACE to determine the potential involvement of TACE in IL-6R shedding. RESULTS: IL-1beta and TNFalpha increased the levels of sIL-6R in the culture media of MG63 osteoblast-like cells. This effect was not influenced by cycloheximide or 5,6-dichlorobenzimidazole riboside but was markedly inhibited by the calcium chelator EGTA and by the TACE and matrix metalloproteinase inhibitor hydroxamate (Ru36156). IL-1beta and TNFalpha had no influence on the alternatively spliced form of IL-6R RNA. Levels of sIL-6R were reduced when MG63 cells were transiently transfected with TACE siRNA. Transfection of SAOS-2 cells with expression plasmids for IL-6R and TACE produced a dose-dependent increase in sIL-6R levels. CONCLUSION: IL-1beta- and TNFalpha-mediated induction of IL-6R shedding in osteoblast-like cells is at least partly dependent on TACE activation.