Four-color multiplex real-time PCR assay prototype targeting azithromycin resistance mutations in Mycoplasma genitalium.

BACKGROUND: The worldwide expansion of macrolide-resistant Mycoplasma genitalium (MG) in cases of genital infections has led to an increased recurrence rate of these infections after first-line azithromycin treatment. By detecting the presence of azithromycin-resistant MG, the patient's antibiotic treatment can be targeted and the spread of resistance prevented. With this aim in mind, macrolide-resistance detection kits are helpful tools for the physician. METHODS: Azithromycin resistance mutations in MG are targeted using a four-color multiplex real-time RT-PCR assay. Tested targets include plasmid DNA (as positive controls) as well as macrolide-sensitive and macrolide-resistant genomic DNA from characterized cell lines and clinical samples. RESULTS: The analytical data presented here were generated from plasmid DNA and genomic RNA/DNA and include adaptation to an internal control, specificity between targets, specificity vs non-MG species, limit of detection (LoD) and interference studies (co-infection and endogenous substances). The clinical data were based on the application of the assay to clinical samples characterized by sequencing. CONCLUSIONS: A new NAAT (nucleic acid amplification test) prototype has been developed in collaboration with the Diagenode s.a. company, this prototype targets MG and azithromycin-resistance mutations in that pathogen.

Biomarkers for detection and prognosis of breast cancer identified by a functional hypermethylome screen.

Breast cancer (BC) is a disease with diverse tumor heterogeneity, which challenges conventional approaches to develop biomarkers for early detection and prognosis. To identify effective biomarkers, we performed a genome-wide screen for functional methylation changes in BC, i.e., genes silenced by promoter hypermethylation, using a functionally proven gene expression approach. A subset of candidate hypermethylated genes were validated in primary BCs and tested as markers for detection and prognosis prediction of BC. We identified 33 cancer specific methylated genes and, among these, two categories of genes: (1) highly frequent methylated genes that detect early stages of BC. Within that category, we have identified the combination of NDRG2 and HOXD1 as the most sensitive (94%) and specific (90%) gene combination for detection of BC; (2) genes that show stage dependent methylation frequency pattern, which are candidates to help delineate BC prognostic signatures. For this category, we found that methylation of CDO1, CKM, CRIP1, KL and TAC1 correlated with clinical prognostic variables and was a significant prognosticator for poor overall survival in BC patients. CKM [Hazard ratio (HR) = 2.68] and TAC1 (HR = 7.73) were the strongest single markers and the combination of both (TAC1 and CKM) was associated with poor overall survival independent of age and stage in our training (HR = 1.92) and validation cohort (HR = 2.87). Our study demonstrates an efficient method to utilize functional methylation changes in BC for the development of effective biomarkers for detection and prognosis prediction of BC.

Connective tissue growth factor enhances osteoblastogenesis in vitro.

Connective tissue growth factor (CTGF), a member of the CCN family of proteins, is expressed by osteoblasts, but its function in cells of the osteoblastic lineage has not been established. We investigated the effects of CTGF overexpression by transducing murine ST-2 stromal cells with a retroviral vector, where CTGF is under the control of the cytomegalovirus promoter. Overexpression of CTGF in ST-2 cells increased alkaline phosphatase activity, osteocalcin and alkaline phosphatase mRNA levels, and mineralized nodule formation. CTGF overexpression decreased the effect of bone morphogenetic protein-2 on Smad 1/5/8 phosphorylation and of Wnt 3 on cytosolic beta-catenin, indicating that the stimulatory effect on osteoblastogenesis was unrelated to BMP and Wnt signaling. CTGF overexpression suppressed Notch signaling and induced the transcription of hairy and E (spl)-1 (HES)-1, by Notch-independent mechanisms. CTGF induced nuclear factor of activated T cells (NFAT) transactivation by a calcineurin-dependent mechanism. Down-regulation of CTGF enhanced Notch signaling and decreased HES-1 transcription and NFAT transactivation. Similar effects were observed following forced CTGF overexpression, the addition of CTGF protein, or the transduction of ST-2 cells with a retroviral vector expressing HES-1. In conclusion, CTGF enhances osteoblastogenesis, possibly by inhibiting Notch signaling and inducing HES-1 transcription and NFAT transactivation.

Gene delivery by retroviruses.

Gene delivery by retroviruses is an easy and safe tool to stably over express a gene of interest and determine its role in a cell model. The gene of interest is cloned into the multiple cloning site of a retroviral vector that also contains a packaging signal and an antibiotic resistance marker for selection. Packaging cell lines, transfected with a retroviral vector containing the gene of interest or with a control vector, produce retroviral RNA packaged into infectious, replication incompetent virus. This is a consequence of the stable integration of the viral genes necessary for particle formation and replication into the packaging cell genome. Retrovirus-containing conditioned medium from packaging cells is used to transduce target cells. After antibiotic selection, the target cells are tested for overexpression of the gene of interest and its biological effects.

Twisted gastrulation, a bone morphogenetic protein agonist/antagonist, is not required for post-natal skeletal function.

Twisted gastrulation (Tsg) is a secreted glycoprotein that binds bone morphogenetic proteins (BMP)-2 and -4 and can display both BMP agonist and antagonist functions. Tsg promotes BMP-mediated endochondral ossification, but its activity in adult bone is not known. We created tsg null mice and examined the consequences of the tsg deletion on the skeleton in vivo and on osteoblast function in vitro. Analysis of the skeletal phenotype of 4-week-old tsg null mice revealed a 40% decrease in trabecular bone volume, but osteoblast and osteoclast number, and bone formation and resorption were not affected. The phenotype was transient, and at 7 weeks of age tsg null mice were not different from control wild-type mice. The decreased trabecular bone is congruent with a defect in endochondral bone formation. In osteoblasts isolated from tsg null mice, tsg gene inactivation decreased the BMP-2 stimulatory effects on osteocalcin expression and alkaline phosphatase activity, indicating that in the bone microenvironment endogenous Tsg enhances BMP activity. Accordingly, tsg null cells displayed impaired BMP signaling. These results were confirmed by Tsg down-regulation in primary osteoblasts from wild-type mice using RNA interference. In conclusion, endogenous Tsg is required for normal BMP activity in osteoblastic cells in vitro, but it plays a minor role in the regulation of adult bone homeostasis in vivo.

Role of the RAM domain and ankyrin repeats on notch signaling and activity in cells of osteoblastic lineage.

UNLABELLED: Notch proteins belong to a family of single pass transmembrane receptors that are activated after interactions with the membrane-bound ligands Delta and Jagged/Serrate. We determined the pathways responsible for the inhibitory effects of Notch on osteoblastogenesis and the contributions of the RAM domain and ankyrin repeats to this process in cells of the osteoblastic lineage. INTRODUCTION: Notch receptors play a role in osteoblast differentiation. Activation of Notch results in its cleavage and the release of its intracellular domain (NICD), which interacts with the CBF1/RBP-Jkappa, Suppressor of Hairless, Lag-1 (CSL) family of transcription factors. The interaction is presumably mediated by the RBP-Jkappa-associated module (RAM) of NICD, although the role of the ankyrin repeats is uncertain. MATERIALS AND METHODS: To determine the contributions of the RAM domain and ankyrin repeats to the inhibitory effects of Notch on osteoblastogenesis, ST-2 and MC3T3-E1 cells were transfected or transduced with vectors expressing NICD, RAM (NICD DeltaRAM), and ankyrin (NICD DeltaANK) deletion mutants. RESULTS: Notch increased the transactivation of transiently transfected 12xCSL-Luc constructs, containing 12 repeats of an RBP-Jkappa/CSL binding site, and of the hairy and E (spl) (HES)-1 promoter. Deletion of the ankyrin repeats resulted in the loss of 12xCSL-Luc and HES-1 promoter transactivation, whereas deletion of the RAM domain caused a partial loss of 12xCSL-Luc and sustained HES-1 promoter transactivation. NICD overexpression inhibited osteocalcin mRNA levels and alkaline phosphatase activity in ST-2 cells, and deletion of the ankyrin repeats, and to a lesser extent of the RAM domain, resulted in loss of the NICD inhibitory effect. NICD inhibited Wnt signaling and deletion of ankyrin repeats or the RAM domain restored Wnt signaling activity. CONCLUSIONS: The RAM domain and ankyrin repeats are required for Notch signaling and activity, and the CSL pathway is central to the inhibitory effect of Notch on osteoblastogenesis.

Notch 1 overexpression inhibits osteoblastogenesis by suppressing Wnt/beta-catenin but not bone morphogenetic protein signaling.

Notch proteins are transmembrane receptors that control cell-fate decisions. Upon ligand binding, Notch receptors undergo proteolytic cleavage leading to the release of their intracellular domain (NICD). Overexpression of NICD impairs osteoblastogenesis, but the mechanisms are not understood. We examined consequences of the constitutive activation of Notch 1 in ST-2 cells. Notch opposed the effects of bone morphogenetic protein (BMP)-2 and Wnt 3a on alkaline phosphatase activity (APA). BMP-2 induced the phosphorylation of Smad 1/5/8 and the transactivation of a BMP/Smad-responsive construct (12xSBE-Oc-pGL3), but the effect was not modified by Notch. BMP-2 had minimal effects on the phosphorylation of the mitogen-activated protein kinases ERK, p38, and JNK, in the absence or presence of NICD. Notch overexpression decreased the transactivating effect of Wnt 3a, cytoplasmic beta-catenin levels, and Wnt-dependent gene expression. Transfection of a mutant beta-catenin expression construct, or the use of a glycogen synthase kinase 3beta inhibitor to stabilize beta-catenin, partially blocked the inhibitory effect of NICD on Wnt signaling and on APA. HES-1 or Groucho1/TLE1 RNA interference enhanced basal and induced Wnt/beta-catenin signaling opposing NICD effects, but only HES-1 silencing enhanced Wnt 3a effects on APA. In conclusion, NICD overexpression prevents BMP-2 and Wnt biological effects by suppressing Wnt but not BMP signaling. HES-1 appears to mediate effects of Notch on osteoblastogenesis.

Overexpression of twisted gastrulation inhibits bone morphogenetic protein action and prevents osteoblast cell differentiation in vitro.

Twisted gastrulation (Tsg) is a secreted glycoprotein that binds bone morphogenetic protein-2 (BMP-2) and BMP-4 and can display both BMP agonist and antagonist functions. Tsg acts as a BMP agonist in chondrocytes, but its expression and actions on the differentiation of cells of the osteoblastic lineage are not known. We investigated the effects of Tsg overexpression by transducing murine ST-2 stromal and MC3T3 cells with a retroviral vector where Tsg is under control of the cytomegalovirus promoter and compared them to cells transduced with the parental vector alone. ST-2 cells were cultured in osteoblastic differentiating conditions in the presence or absence of BMP-2. Tsg overexpression precluded the appearance of mineralized nodules induced by BMP-2, led to a delay in the expression of osteoblastic gene markers, and decreased the responsiveness of ST-2 differentiating cells to PTH. BMP-2 induced the phosphorylation of signaling mothers against decapentaplegic-1/5/8, but not ERK, c-Jun N-terminal kinase, and p38. ST-2 cells overexpressing Tsg displayed an inhibition of BMP/signaling mother against decapentaplegic signaling. Tsg action was specific to BMP, because Tsg overexpression did not affect TGF-beta or Wnt/beta-catenin signaling pathways. Tsg also opposed MC3T3 cell differentiation and the expression of a mature osteoblast phenotype. In conclusion, Tsg overexpression inhibits BMP action in stromal and preosteoblastic cells and, accordingly, arrests their differentiation toward the osteoblastic pathway.

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.

Caspase-8-dependent HER-2 cleavage in response to tumor necrosis factor alpha stimulation is counteracted by nuclear factor kappaB 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.

Mechanisms involved in exogenous C2- and C6-ceramide-induced cancer cell toxicity.

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation, and programmed cell death. To determine whether ceramides can mediate the apoptosis of HCT116 and OVCAR-3 cancer cells, exogenous C2-, C6-, and C16-ceramides were used to mimic the endogenous lipid increase that follows a large variety of stresses. C2- and C6-ceramides (cell-permeable ceramide analogs), but not C16-ceramide, induced nuclear factor-kappaB (NF-kappaB) DNA-binding, caspase-3 activation, poly(ADP-ribose) polymerase degradation, and mitochondrial cytochrome c release, indicating that apoptosis occurs through the caspase cascade and the mitochondrial pathway. No difference in survival was observed between control cells and cells expressing mutated IkappaBalpha and treated with the permeable ceramides. This suggests that, at least in these cell lines, stable NF-kappaB inhibition did not modify the ceramide-induced cytotoxicity pathway. C6-ceramide also induced a double block in G1 and G2, thus emptying the S phase.

Identification of cytokine-induced nuclear factor-kappaB target genes in ovarian and breast cancer cells.

NF-kappaB is a pleiotropic transcription factor controlling the expression of many genes and viruses. NF-kappaB plays a role in immune response, cellular adhesion or acute phase response. It also inhibits apoptosis and favors cancer cell survival. We studied the expression of genes controlled by NF-kappaB in ovarian and breast adenocarcinoma cancer cells. We stably transfected OVCAR-3 and MCF7 A/Z cells with an expression vector coding for the mutated inhibitor IkappaBalpha, which sequesters NF-kappaB in the cytoplasm. We stimulated control and IkappaBalpha expressing cells with IL-1beta or TNF-alpha and extracted the RNA, which was reverse-transcribed and hybridized to DNA microarrays. Several of the genes identified were not known as NF-kappaB target genes. Among them, we confirmed the differential expression of ephrin-A1 and caveolin-1 by quantitative real-time polymerase chain reaction. Our results showed an NF-kappaB-dependent induction of ephrin-A1 and caveolin-1 mRNAs after stimulation with TNF-alpha and IL-1beta, confirming that NF-kappaB controls target genes implied in tumor angiogenesis and cell transformation.

NF-kappaB-dependent MnSOD expression protects adenocarcinoma cells from TNF-alpha-induced apoptosis.

NF-kappaB is known to exert a cytoprotective action against TNF-alpha-induced apoptosis. To study the role of NF-kappaB in various TNF-alpha-treated epithelial cell lines, we generated stable transfectants overexpressing a mutated unresponsive form of the IkappaBalpha inhibitor (MT cells). As NF-kappaB prevented TNF-alpha-induced apoptosis in various epithelial cancer cell lines, we searched for NF-kappaB target gene products responsible for this difference of sensitivity. We observed an increased Bcl-X(L) expression level in OVCAR-3 cells compared with OVCAR-3 cells expressing a mutated IkappaBalpha inhibitor (MT cells). Induction of the antioxidant enzyme MnSOD was detected only in TNF-alpha-treated OVCAR, MCF7A/Z and HCT116 cells but not in MT cells. Moreover, reactive oxygen species were involved in TNF-alpha-induced apoptosis, as various antioxidants partially protected these cells from apoptosis. At last, transfection of the MnSOD cDNA in MT cells, which do not express this protein after TNF-alpha stimulation, partially restored resistance to TNF-alpha-induced cell death, as observed by clonogenic assays. However, transfection of the Bcl-X(L) cDNA did not induce any protective effect. Therefore, MnSOD expression is induced by NF-kappaB in epithelial cancer cells in response to TNF-alpha, and is at least partially responsible for their resistance to TNF-alpha-induced apoptosis, presumably through the clearance of death-inducing ROS.