Signal transducer and activator of transcription (STAT) 3 is overexpressed or activated in most types of human tumors and has been classified as an oncogene. In the present study, we investigated the ...contribution of the STAT3s to the proinvasive activity of trefoil factors (TFF) and vascular endothelial growth factor (VEGF) in human colorectal cancer cells HCT8/S11 expressing VEGF receptors. Both intestinal trefoil peptide (TFF3) and VEGF, but not pS2 (TFF1), activate STAT3 signaling through Tyr(705) phosphorylation of both STAT3alpha and STAT3beta isoforms. Blockade of STAT3 signaling by STAT3beta, depletion of the STAT3alpha/beta isoforms by RNA interference, and pharmacologic inhibition of STAT3alpha/beta phosphorylation by cucurbitacin or STAT3 inhibitory peptide abrogates TFF- and VEGF-induced cellular invasion and reduces the growth of HCT8/S11 tumor xenografts in athymic mice. Differential gene expression analysis using DNA microarrays revealed that overexpression of STAT3beta down-regulates the VEGF receptors Flt-1, neuropilins 1 and 2, and the inhibitor of DNA binding/differentiation (Id-2) gene product involved in the neoplastic transformation. Taken together, our data suggest that TFF3 and the essential tumor angiogenesis regulator VEGF(165) exert potent proinvasive activity through STAT3 signaling in human colorectal cancer cells. We also validate new therapeutic strategies targeting STAT3 signaling by pharmacologic inhibitors and RNA interference for the treatment of colorectal cancer patients.
To define genetic determinants of tumor cell resistance to the cytotoxic action of tumor necrosis factor alpha (TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 ...cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis of available literature data, the striking finding is the association of some differentially expressed genes involved in the phosphatidylinositol-3-kinase/Akt signaling pathway. More notably, we found that the PRNP gene coding for the cellular prion protein (PrP(c)), was 17-fold overexpressed in the 1001 cell line as compared with the MCF7 cell line. This differential expression was confirmed at the cell surface by immunostaining that indicated that PrP(c) is overexpressed at both mRNA and protein levels in the TNF-resistant derivative. Using recombinant adenoviruses expressing the human PrP(c,) our data demonstrate that PrP(c) overexpression converted TNF-sensitive MCF7 cells into TNF-resistant cells, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondria and nuclear condensation.
Introduction and aim
A national survey was initiated by representatives of French patients with haemophilia (AFH) and the French reference centre for haemophilia, in order to appreciate the awareness ...and knowledge of these patients regarding haemophilia gene therapy (HGT) and understand better their position about this innovative treatment that will soon become available.
Results
Of 143 answers received, 137 could be analysed, representing about 3.5% of patients with severe or moderate haemophilia over 16year‐old. They were 80.3% with haemophilia A and 19.7 % with haemophilia B, with a severe form of the disease for 80.3 % of them. Curiosity for HGT was formulated by 64.2% of the participants, 33.6 % being interested by this approach as soon as it will be available and 38.7 % preferring to wait until more patients have been treated. Only 3.6 % of the participants would never consider receiving HGT. The level of awareness and knowledge was estimated to be limited by 39.5 % of the patients. More than 60 % of them declared having never or almost never discussed HGT with the team of their haemophilia centre. Before deciding to get HGT, 54.4 % of the participants considered that it will be very important to compare it with their current treatment and 53.7 % would like to be better informed by their care providers.
Conclusions
These results highlight the need for training and education for patients, but also for professionals at haemophilia centres, about HGT and the shared decision‐making process. Objective, unbiased and transparent information must be available for patients about this very promising therapy which nonetheless carries more uncertainty and unknowns compared to other haemophilia treatments.
Myotonic dystrophy type 1 (DM1) is a multisystem disorder affecting a variety of organs, including the central nervous system. By using neuronal progeny derived from human embryonic stem cells ...carrying the causal DM1 mutation, we have identified an early developmental defect in genes involved in neurite formation and the establishment of neuromuscular connections. Differential gene expression profiling and quantitative RT-PCR revealed decreased expression of two members of the
SLITRK family in DM1 neural cells and in DM1 brain biopsies. In addition, DM1 motoneuron/muscle cell cocultures showed alterations that are consistent with the known role of
SLITRK genes in neurite outgrowth, neuritogenesis, and synaptogenesis. Rescue and knockdown experiments suggested that the functional defects can be directly attributed to
SLITRK misexpression. These neuropathological mechanisms may be clinically significant for the functional changes in neuromuscular connections associated with DM1.
► Neural differentiation of hESC lines from myotonic dystrophy type 1 (DM1) embryos ► Coculture assays reveal defects in neuritis and neuromuscular connections ► Gene expression analysis highlights reduction in
SLITRK family proteins ► SLITRK knockdown mimics the phenotype, and SLITRK overexpression rescues it
Macro-arrays, on which 1339 human skeletal muscle cDNA clone inserts had been spotted as PCR products, were used to make large-scale measurement of gene expression in bovine muscles during ...ontogenesis. Ten complex cDNA targets derived from two mixed muscle samples, Rectus abdominis (rather red oxidative muscle, RA) and Semitendinosus (rather white glycolytic muscle, ST), were taken from foetuses at 4 different stages (110, 180, 210, and 260 days post-conception) and from 15-month-old young bulls to generate differential expression patterns. Each sample analysed was prepared from a pool of RNA extracted from muscle tissues sampled from at least 6 different animals. Approximately 200 expression signals were validated and taken into account to provide a first “bovine” muscle gene repertoire. Despite the relatively small number of probes and the heterologous approach, this made it possible to identify up to 7 genes differentially expressed between RA and ST, depending on age. From 110 days post-conception to 15 months of age, differences in the expression levels of 110 genes were detected in the four comparisons between two consecutive ages. By comparing 260 days post-conception foetal muscles and adult muscles, up to 87 genes were overexpressed, whereas only 7 genes were shown to be down-regulated. Among these genes, 33% have unknown biological functions. Taken together, the results reported here underline the importance of the last three months of gestation in muscle myogenesis, and highlight new genes involved in this process.
Mesenchymal stem cells (MSCs) are present in a wide variety of tissues during development of the human embryo starting as early as the first trimester. Gene expression profiling of these cells has ...focused primarily on the molecular signs characterizing their potential heterogeneity and their differentiation potential. In contrast, molecular mechanisms participating in the emergence of MSC identity in embryo are still poorly understood. In this study, human embryonic stem cells (hESs) were differentiated toward MSCs (ES-MSCs) to compare the genetic patterns between pluripotent hESs and multipotent MSCs by a large genomewide expression profiling of mRNAs and microRNAs (miRNAs). After whole genome differential transcriptomic analysis, a stringent protocol was used to search for genes differentially expressed between hESs and ES-MSCs, followed by several validation steps to identify the genes most specifically linked to the MSC phenotype. A network was obtained that encompassed 74 genes in 13 interconnected transcriptional systems that are likely to contribute to MSC identity. Pairs of negatively correlated miRNAs and mRNAs, which suggest miRNA-target relationships, were then extracted and validation was sought with the use of Pre-miRs. We report here that underexpression of miR-148a and miR-20b in ES-MSCs, compared with ESs, allows an increase in expression of the EPAS1 (Endothelial PAS domain 1) transcription factor that results in the expression of markers of the MSC phenotype specification.
Human pluripotent stem cells offer a limitless source of cells for regenerative medicine. Neural derivatives of human embryonic stem cells (hESCs) are currently being used for cell therapy in 3 ...clinical trials. However, hESCs are prone to genomic instability, which could limit their clinical utility. Here, we report that neural differentiation of hESCs systematically produced a neural stem cell population that could be propagated for more than 50 passages without entering senescence; this was true for all 6 hESC lines tested. The apparent spontaneous loss of evolution toward normal senescence of somatic cells was associated with a jumping translocation of chromosome 1q. This chromosomal defect has previously been associated with hematologic malignancies and pediatric brain tumors with poor clinical outcome. Neural stem cells carrying the 1q defect implanted into the brains of rats failed to integrate and expand, whereas normal cells engrafted. Our results call for additional quality controls to be implemented to ensure genomic integrity not only of undifferentiated pluripotent stem cells, but also of hESC derivatives that form cell therapy end products, particularly neural lines.
It is well established that biological aging is associated with functional deficits at the cellular, tissue, organ and system levels, but the molecular mechanisms that control lifespan and ...age-related phenotypes are still not well understood. In order to investigate the molecular mechanisms underlying myoblast aging, we have used quantitative hybridization of a cDNA array of 2016 clones from a human skeletal muscle 3′-end cDNA library to monitor gene expression patterns of myoblasts of individuals with different ages (5 days old, 52 years old and 79 years old) and at different stages of proliferation (early, presenescent and senescent). We have shown that expression profiles in satellite cells vary with donor age, with an up-regulation of genes involved in muscle structure, muscle differentiation and in metabolism in the newborn, and a down-regulation of genes involved in protein renewal in adults. We have also observed that myoblasts isolated from subjects of different ages have typical expression profiles at the beginning of their proliferative lifespan. However, this phenomenon progressively disappears as the cells approach senescence. In addition, even though some of the modifications are similar to those observed in other cell types, we have observed that many changes in gene expression are characteristic of the myoblasts, confirming the hypothesis that the program of replicative senescence is specific for each cell type. Finally, we have identified four potential new markers of presenescence for human myoblasts, which could be useful in developing therapeutic strategies.
Malignant tumor cell invasion is determinant for metastasis to occur. E2 and C5 colon carcinoma cells that were derived from the parental Lovo line and that differ experimentally in spontaneous ...metastatic ability have been monitored for gene expression by cDNA arrays. Among genes found differentially expressed, the CD63 tetraspanin, not previously recognized in colon cancer progression, and the α3 integrin chain were both up-regulated in low metastatic E2 cells and were analyzed for their functional role using adhesion, migration, and invasion assays. Cell surface expression of CD63 and α3 integrin was about 2-fold higher in E2 than in C5 cells and confocal microscopy showed that CD63 and α3 integrin colocalized evenly on C5 cells whereas they concentrated at elongated tips of the low-metastatic more substrate-adhesive E2 cells. Antibody-interference experiments identified laminin-5 (LN-5) as a ligand interacting with the α3β1/CD63 complex. Substrate-immobilized anti-CD63 antibodies enhanced tumor cell migration and invasion and induced prominent cell surface protrusions that were repressed by the PI3-kinase LY294002 inhibitor. Our results suggest that changes in the expression of surface CD63 and α3β1 integrin interacting with LN-5 could affect migratory signals and the progression of the metastatic disease.
Patients with myotonic dystrophy type 1 exhibit a diversity of symptoms that affect many different organs. Among these are cognitive dysfunctions, the origin of which has remained elusive, partly ...because of the difficulty in accessing neural cells. Here, we have taken advantage of pluripotent stem cell lines derived from embryos identified during a pre-implantation genetic diagnosis for mutant-gene carriers, to produce early neuronal cells. Functional characterization of these cells revealed reduced proliferative capacity and increased autophagy linked to mTOR signaling pathway alterations. Interestingly, loss of function of MBNL1, an RNA-binding protein whose function is defective in DM1 patients, resulted in alteration of mTOR signaling, whereas gain-of-function experiments rescued the phenotype. Collectively, these results provide a mechanism by which DM1 mutation might affect a major signaling pathway and highlight the pertinence of using pluripotent stem cells to study neuronal defects.
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