Enteric glial cell heterogeneity regulates intestinal stem cell niches

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Combinations of genetic mutations in the adult neural stem cell compartment determine brain tumour phenotypes

It has been suggested that intrinsic brain tumours originate from a neural stem/progenitor cell population in the subventricular zone of the post‐natal brain. However, the influence of the initial genetic mutation on the phenotype as well as the contribution of mature astrocytes to the formation of brain tumours is still not understood. We deleted Rb/p53, Rb/p53/PTEN or PTEN/p53 in adult subventricular stem cells; in ectopically neurografted stem cells; in mature parenchymal astrocytes and in transplanted astrocytes. We found that only stem cells, but not astrocytes, gave rise to brain tumours, independent of their location. This suggests a cell autonomous mechanism that enables stem cells to generate brain tumours, whereas mature astrocytes do not form brain tumours in adults. Recombination of PTEN/p53 gave rise to gliomas whereas deletion of Rb/p53 or Rb/p53/PTEN generated primitive neuroectodermal tumours (PNET), indicating an important role of an initial Rb loss in driving the PNET phenotype. Our study underlines an important role of stem cells and the relevance of initial genetic mutations in the pathogenesis and phenotype of brain tumours.     

Down-regulation of WNK1 protein kinase in neural progenitor cells suppresses cell proliferation and migration

WNK1, a Ser/Thr protein kinase, is widely expressed in many tissues. Its biological functions are largely unknown. Disruption of the WNK1 gene in mice leads to embryonic lethality at day 13, implicating a critical role of WNK1 in embryonic development. To investigate this potential function, we used antisense strategy to knock down the expression of WNK1 in a mouse neural progenitor cell line, C17.2. Down-regulation of WNK1 in C17.2 cells greatly reduced cell growth. Addition of epidermal growth factor (EGF), a mitogen for C17.2 cells, had no effect on growth. The WNK1-knockdown cells showed a flat and rounded morphology, characteristic of the immature and non-differentiated phenotype of the progenitor cells; this was further demonstrated by immunostaining for the progenitor and neuronal markers. Migration of the WNK1-knockdown C17.2 cells was reduced as tested in culture dishes or Matrigel-covered chambers. Moreover, activation of extracellular signal-regulated kinase (ERK1)/2 and ERK5 by EGF in the WNK1-knockdown cells was suppressed. These results demonstrate a novel function of WNK1 in proliferation, migration, and differentiation of neural progenitor cells, likely by mechanisms involving activation of the mitogen-activated protein (MAP) kinase ERK1/2 and/or ERK5 pathways.     

DLC-1:a Rho GTPase-activating protein and tumour suppressor

The deleted in liver cancer 1 (DLC-1) gene encodes a GTPase activating protein that acts as a negative regulator of the Rho family of small GTPases. Rho proteins transduce signals that influence cell morphology and physiology, and their aberrant up-regulation is a key factor in the neoplastic process, including metastasis. Since its discovery, compelling evidence has accumulated that demonstrates a role for DLC-1 as a bona fide tumour suppressor gene in different types of human cancer. Loss of DLC-1 expression mediated by genetic and epigenetic mechanisms has been associated with the development of many human cancers, and restoration of DLC-1 expression inhibited the growth of tumour cells in vivo and in vitro. Two closely related genes, DLC-2 and DLC-3, may also be tumour suppressors. This review presents the current status of progress in understanding the biological functions of DLC-1 and its relatives and their roles in neoplasia.     

Blood progenitor cell separation from clinical leukapheresis product by magnetic nanoparticle binding and magnetophoresis

Positive selection of CD34+ blood progenitor cells from circulation has been reported to improve patient recovery in applications of autologous transplantation. Current magnetic separation methods rely on cell capture and release on solid supports rather than sorting from flowing suspensions, which limits the range of therapeutic applications and the process scale up. We tested CD34+ cell immunomagnetic labeling and isolation from fresh leukocyte fraction of peripheral blood (leukapheresis) using the continuous quadrupole magnetic flow sorter (QMS), consisting of a flow channel (SHOT, Greenville, IN) and a quadrupole magnet with a maximum field intensity (B(o)) of 1.42 T and a mean force field strength (S(m)) of 1.45 x 10(8) TA/m(2). Both the sample magnetophoretic mobility (m) and the inlet and outlet flow patterns highly affect the QMS performance. Seven commercial progenitor cell labeling reagent combinations were quantitatively evaluated by measuring magnetophoretic mobility of a high CD34 expression cell line, KG-1a, using the cell tracking velocimeter (CTV). The CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) showed the strongest labeling of KG-1a cells and was selected for progenitor cell enrichment from 11 fresh and 11 cryopreserved clinical leukapheresis samples derived from different donors. The CD34+ cells were isolated with a purity of 60-96%, a recovery of 18-60%, an enrichment rate of 12-169, and a throughput of (1.7-9.3) x 10(4) cells/s. The results also showed a highly regular dependence of the QMS performance on the flow conditions that agreed with the theoretical predictions based on the CD34+ cell magnetophoretic mobility.     

Extracellular nucleotide inhibits cell proliferation and negatively regulates Toll-like receptor 4 signalling in human progenitor endothelial cells

Extracellular nucleotides mediate a wide range of physiological effects by interacting with plasma membrane P2 purinergic receptors. P2 receptors are expressed in certain kinds of stem cells, and function to induce cytokine expression and to modulate cell proliferation. We have analysed the expression and the function of P2 receptors in human umbilical cord blood-derived EPCs (endothelial progenitor cells). EPCs expressed P2X4,6,7 and P2Y2,4,11,13,14 receptors and extracellular ATP inhibited EPCs proliferation. As in a previous study, EPCs expressed functional TLR4 (Toll-like receptor 4) and activation of TLR4 by LPS (lipopolysaccharide) evoked a pro-inflammatory immune response. When human EPCs were stimulated with LPS and nucleotides, ATP or UTP inhibited the expression of pro-inflammatory cytokines including MCP-1 (monocyte chemoattractant protein-1), IFNα (interferon α), TNFα (tumour necrosis factor α) and adhesion molecule VCAM-1 (vascular cell adhesion molecule 1) induced by LPS. ATP and UTP also down-regulated the gene expression of TLR4, CD14 and MyD88 (myeloid differentiation factor 88), a TLR adaptor molecule, and protein expression of CD14 and MyD88. Moreover, the phosphorylation of NF-κB (nuclear factor κB) p65 induced by TLR4 activation was inhibited partly by ATP or UTP at concentrations of 1-5 μM. These results suggest that extracellular nucleotides negatively regulate EPCs proliferation and TLR4 signalling.     

Adiponectin promotes endothelial progenitor cell number and function

Obesity-linked diseases are associated with suppressed endothelial progenitor cell (EPC) function. Adiponectin is an adipose-derived protein that is downregulated in obese and diabetic subjects. Here, we investigated the effects of adiponectin on EPCs. EPC levels did not increase in adiponectin deficient (APN-KO) in response to hindlimb ischemia. Adenovirus-mediated delivery of adiponectin increased EPC levels in both WT and APN-KO mice. Incubation of human peripheral blood mononuclear cells with adiponectin led to an increase of the number of EPCs. Adiponectin induced EPC differentiation into network structures and served as a chemoattractant in EPC migration assays. These data suggest that hypoadiponectinemia may contribute to the depression of EPC levels that are observed in patients with obesity-related cardiovascular disorders.     

Betacellulin regulates the proliferation and differentiation of retinal progenitor cells in vitro

Retinal progenitor cells (RPCs) hold great potential for the treatment of retinal degenerative diseases. However, their proliferation capacity and differentiation potential towards specific retinal neurons are limited, which limit their future clinical applications. Thus, it is important to improve the RPCs’ ability to proliferate and differentiate. Currently, epidermal growth factor (EGF) is commonly used to stimulate RPC growth in vitro. In this study, we find that betacellulin (BTC), a member of the EGF family, plays important roles in the proliferation and differentiation of RPCs. Our results showed that BTC can significantly promote the proliferation of RPCs more efficiently than EGF. EGF stimulated RPC proliferation through the EGFR/ErbB2‐Erk pathway, while BTC stimulated RPC proliferation more powerfully through the EGFR/ErbB2/ErbB4‐Akt/Erk pathway. Meanwhile, under differentiated conditions, the BTC‐pre‐treated RPCs were preferentially differentiated into retinal neurons, including photoreceptors, one of the most important types of cells for retinal cell replacement therapy, compared to the EGF‐pre‐treated RPCs. In addition, knockdown of endogenous BTC expression can also obviously promote RPC differentiation into retinal neuronal cells. This data demonstrate that BTC plays important roles in promoting RPC proliferation and differentiation into retinal neurons. This study may provide new insights into the study of RPC proliferation and differentiation and make a step towards the application of RPCs in the treatment of retinal degenerative diseases.     

Challenges in T cell receptor gene therapy

The function of T lymphocytes as orchestrators and effectors of the adaptive immune response is directed by the specificity of their T cell receptors (TCRs). By transferring into T cells the genes encoding antigen-specific receptors, the functional activity of large populations of T cells can be redirected against defined targets including virally infected or cancer cells. The potential of therapeutic T cells to traffic to sites of disease, to expand and to persist after a single treatment remains a major advantage over the currently available immunotherapies that use monoclonal antibodies. Here we review recent progress in the field of TCR gene therapy, outlining challenges to its successful implementation and the strategies being used to overcome them. We detail strategies used in the optimization of affinity and surface expression of the introduced TCR, the choice of T cell subpopulations for gene transfer, and the promotion of persistence of gene-modified T cells in vivo. We review the safety concerns surrounding the use of gene-modified T cells in patients, discussing emerging solutions to these problems, and describe the increasingly positive results from the use of gene-modified T cells in recent clinical trials of adoptive cellular immunotherapy. The increasing sophistication of measures to ensure the safety of engineered T cells is accompanied by an increasing number of clinical trials: these will be essential to guide the effective translation of cellular immunotherapy from the laboratory to the bedside.     

The CRAL/TRIO and GOLD domain protein TAP-1 regulates RAF-1 activation

The activation of the protein kinase Raf at the cell membrane is a critical step in cell signaling during development, but the mechanisms that regulate Raf activity remain incompletely defined. We previously demonstrated that the C. elegans cgr-1 gene encodes a CRAL/TRIO domain-containing protein that is a critical modulator of Ras-dependent cell fate specification during C. elegans development. Here we identify the mammalian α-tocopherol associated protein-1 (TAP-1) as a functional ortholog of cgr-1. TAP-1 mRNA was expressed in many tissues, and TAP-1 protein colocalized with Ras and Raf at the cell membrane. Reducing TAP-1 expression by RNA interference increased Ras/ERK signaling in multiple cell types. These functional studies demonstrate that CRAL/TRIO domain proteins play a conserved role in regulating Ras signaling. Biochemical analyses indicated that TAP-1 operates at the level of Raf, since TAP-1 function negatively regulated the amount of Raf-1 recruited to GTP-bound Ras at the cell membrane. TAP-1 plays a significant physiological role in controlling cell division, since reducing TAP-1 expression increased the oncogenic capacity of Ras transformed human cancer cell lines. These studies identify TAP-1 as a critical modulator of Ras-mediated cellular signaling.     

miR-25-5p regulates endothelial progenitor cell differentiation in response to shear stress through targeting ABCA1

The importance of flow shear stress (SS) on the differentiation of endothelial progenitor cells (EPCs) has been demonstrated in various studies. Cholesterol retention and microRNA regulation have been also proposed as relevant factors involved in this process, though evidence regarding their regulatory roles in the differentiation of EPCs is currently lacking. In the present study on high shear stress (HSS)-induced differentiation of EPCs, we investigated the importance of ATP-binding cassette transporter 1 (ABCA1), an important regulator in cholesterol efflux, and miR-25-5p, a potential regulator of endothelial reconstruction. We first revealed an inverse correlation between miR-25-5p and ABCA1 expression levels in EPCs under HSS treatment; their direct interaction was subsequently validated by a dual-luciferase reporter assay. Further studies using flow cytometry and quantitative polymerase chain reaction demonstrated that both miR-25-5p overexpression and ABCA1 inhibition led to elevated levels of specific markers of endothelial cells, with concomitant downregulation of smooth muscle cell markers. Finally, knockdown of ABCA1 in EPCs significantly promoted tube formation, which confirmed our conjecture. Our current results suggest that miR-25-5p might regulate the differentiation of EPCs partially through targeting ABCA1, and such a mechanism might account for HSS-induced differentiation of EPCs.     

Ptch1-mediated dosage-dependent action of Shh signaling regulates neural progenitor development at late gestational stages

Sonic hedgehog (Shh) signaling regulates cell differentiation and proliferation during brain development. However, the role of Shh in neurogenesis during late gestation (embryonic day 13.5–18.5) remains unclear. Herein, we used a genetic approach and in utero electroporation to investigate the role of mouse Shh and patched homolog 1 (Ptch1), the putative receptor for Shh. Proliferating cortical intermediate (basal) progenitor cells (IPCs) were severely reduced in Shh mutant mice, suggesting that endogenous Shh signaling could play an essential role in cortical IPC development. During cortical neurogenesis, strong upregulation of Shh signaling enhanced the transition from ventricular zone (VZ) progenitors to ventralized IPCs, while low levels of signaling enhanced the generation and proliferation of cortical IPCs in the subventricular zone. The effects of Shh upregulation in this study were consistent with a phenotype of conditional loss of function of Ptch1, and the phenotype of a hypomorphic allele of Ptch1, respectively. These data indicated that endogenous Ptch1 mediates the broad effects of Shh on the transition from VZ progenitors to IPCs and activation of proliferation of the IPCs in the cortex during late gestational stages.     

The RNA binding protein Musashi1 regulates apoptosis,gene expression and stress granule formation in urothelial carcinoma cells

The RNA‐binding protein Musashi1 (MSI1) is a marker of progenitor cells in the nervous system functioning as a translational repressor. We detected MSI1 mRNA in several bladder carcinoma cell lines, but not in cultured normal uroepithelial cells, whereas the paralogous MSI2 gene was broadly expressed. Knockdown of MSI1 expression by siRNA induced apoptosis and a severe decline in cell numbers in 5637 bladder carcinoma cells. Microarray analysis of gene expression changes after MSI1 knockdown significantly up‐regulated 735 genes, but down‐regulated only 31. Up‐regulated mRNAs contained a highly significantly greater number and density of Musashi binding sites. Therefore, a much larger set of mRNAs may be regulated by Musashi1, which may affect not only their translation, but also their turnover. The study confirmed p21^CIP1 and Numb proteins as targets of Musashi1, suggesting additionally p27^KIP1 in cell‐cycle regulation and Jagged‐1 in Notch signalling. A significant number of up‐regulated genes encoded components of stress granules (SGs), an organelle involved in translational regulation and mRNA turnover, and impacting on apoptosis. Accordingly, heat shock induced SG formation was augmented by Musashi1 down‐regulation. Our data show that ectopic MSI1 expression may contribute to tumorigenesis in selected bladder cancers through multiple mechanisms and reveal a previously unrecognized function of Musashi1 in the regulation of SG formation.     

MicroRNA expression and its implications for diagnosis and therapy of tongue squamous cell carcinoma

Tongue squamous cell carcinoma (TSCC) is the most common type of oral squamous cell carcinomas and is well known for its high rate of lymph nodal metastasis. Despite the identification of many molecular mechanisms in TSCC, the number of deaths associated with TSCC increased during the past 5 years. MicroRNAs (miRNAs) are a family of small non‐coding RNA molecules, which regulate gene expression by either translational inhibition or mRNA degradation. miRNAs have been proven to be key regulators of various biological and pathological processes including cell proliferation, development and tumourigenesis. Increasing evidence has demonstrated that the deregulated miRNAs are implicated in the diagnosis and treatment of TSCC. In this review, we summarized the expressions and roles of miRNAs in TSCC and comment on the potential roles of miRNAs in diagnosis, prognosis and treatment of this malignancy.     

TCA-100 tumour chemosensitivity assay: Differences in sensitivity between cultured tumour cell lines and clinical studies

The BATLE LE TCA-100 tumour chemosensitivity assay has been used to evaluate chemotherapeutic drug sensitivity of cultured tumour cell lines. Studies were performed using test drug concentrations calibrated to discriminate sensitivity and resistance of clinical specimens. Strong sensitivity which appeared to be inconsistent with clinical experience was detected for some drugs and cell lines. Findings of strong sensitivity were consistent with basic differences between sensitivity testing cultured cell lines and clinical specimens. Results with cell lines frequently may not apply directly to clinical applications. Characterization of differences between cell lines and clinical specimens may assist in application of cell line findings to clinical trials.     

The apoptosis modulator and tumour suppressor protein RBM5 is a phosphoprotein

RBM5/LUCA-15/H37 is a nuclear SR-related RNA binding protein with the ability to modulate both apoptosis and the cell cycle, and retard tumour formation. How RBM5 functions to carry out these, potentially interrelated, biological activities is unknown. Since reversible phosphorylation has been shown to play an important role in the regulation of SR protein function, apoptosis and cell cycle control, in an attempt to elucidate the underlying mechanisms regulating RBM5 function, the phosphorylation status of RBM5 was investigated. Whole cell lysate from growing cell cultures was treated with the broad phosphatase spectrum of CIP, resulting in a decrease in the molecular mass of RBM5. A similar decrease in molecular mass, of a subset of RBM5 proteins, was observed during growth factor deprivation, in a manner consistent with partial dephosphorylation of RBM5. Molecular mass increased upon growth factor addition, demonstrating that this apoptosis-associated alteration in molecular mass was a reversible process. Immunoprecipitation and mutagenesis experiments strongly suggested that phosphotyrosines are not present in RBM5 under normal growth conditions, and that serine 69 is phosphorylated, but not by Akt kinase. Taken together, these results suggest that reversible phosphorylation of RBM5 is a mechanism capable of regulating RBM5 participation in modulating apoptosis, and perhaps tumour suppression.