搜索结果: 'NeuroCult Proliferation'

BACKGROUND Neonatal hypoxia-ischemia induces massive brain damage during the perinatal period,resulting in long-term consequences to central nervous system structural and functional maturation. Although neural progenitor cells (NPCs) migrate through the parenchyma and home in to injury sites in the rodent brain,the molecular mechanisms are unknown. We examined the role of chemokines in mediating NPC migration after neonatal hypoxic-ischemic brain injury. METHODS Nine-day-old mice were exposed to a 120-minute hypoxia following unilateral carotid occlusion. Chemokine levels were quantified in mouse brain extract. Migration and proliferation assays were performed using embryonic and infant mouse NPCs. RESULTS The neonatal hypoxic-ischemic brain injury resulted in an ipsilateral lesion,which was extended to the cortical and striatal areas. NPCs migrated toward an injured area,where a marked increase of CC chemokines was detected. In vitro studies showed that incubation of NPCs with recombinant mouse CCL11 promoted migration and proliferation. These effects were partly inhibited by a CCR3 antagonist,SB297006. CONCLUSIONS Our data implicate an important effect of CCL11 for mouse NPCs. The effective activation of NPCs may offer a promising strategy for neuroregeneration in neonatal hypoxic-ischemic brain injury. View Publication

The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes,mTORC1 and mTORC2,which regulate cell growth,survival,and autophagy. Allosteric inhibitors of mTORC1,such as rapamycin,have been extensively used to study tumor cell growth,proliferation,and autophagy but have shown only limited clinical utility. Here,we describe AZD8055,a novel ATP-competitive inhibitor of mTOR kinase activity,against all class I phosphatidylinositol3-kinase (PI3K) and other members of the PI3K-like kinase family. The study was to determine the effect of AZD8055 on proliferation and apoptosis on Hep-2,a human laryngeal cancer cell line and to investigate the underlying mechanism(s) of action. Hep-2 cells were treated with AZD8055 for 24,48 or 72 h. MTT was used to determine cell proliferation. Rhodamine 123 and TUNEL staining were used to determine mitochondrial membrane potential and cell apoptosis analyzed by fluorescence-activated cell sorting (FACS). Protein expressions were examined by western blotting. Treatment with AZD8055 inhibited proliferation and induced apoptosis in Hep-2 cells in a dose- and time-dependent manner. During the prolonged treatment with AZD8055,AZD8055 inhibits the mammalian target of rapamycin mTOR. Further experiments showed which signaling cascade p-4EBP1 and substrate EIF4E as well as downstream proteins were down regulated. Furthermore,our study showed that the expression profiles of various BH3-only proteins including Bid,Bad,and Bim,apoptosis regulatory protein cleaved caspase3 was up regulated in a time-dependent manner in Hep-2 cells treated with AZD8055. Thus,in vitro,AZD8055 potently inhibits proliferation and induces apoptosis in head and neck squamous cell carcinoma. View Publication

Histone deacetylase inhibitors have attracted considerable attention because of their ability to overcome the differentiation block in leukemic blasts,an effect achieved either alone or in combination with differentiating agents,such as all-trans retinoic acid. We have previously reported favorable effects of the potent histone deacetylase inhibitor valproic acid in combination with all-trans retinoic acid in patients with advanced acute myeloid leukemia leading to blast cell reduction and improvement of hemoglobin. These effects were accompanied by hypergranulocytosis most likely due to an enhancement of nonleukemic myelopoiesis and the suppression of malignant hematopoiesis rather than enforced differentiation of the leukemic cells. These data prompted us to investigate the effect of valproic acid on normal hematopoietic stem cells (HSC). Here we show that valproic acid increases both proliferation and self-renewal of HSC. It accelerates cell cycle progression of HSC accompanied by a down-regulation of p21(cip-1/waf-1). Furthermore,valproic acid inhibits GSK3beta by phosphorylation on Ser9 accompanied by an activation of the Wnt signaling pathway as well as by an up-regulation of HoxB4,a target gene of Wnt signaling. Both are known to directly stimulate the proliferation of HSC and to expand the HSC pool. In summary,we here show that valproic acid,known to induce differentiation or apoptosis in leukemic blasts,stimulates the proliferation of normal HSC,an effect with a potential effect on its future role in the treatment of acute myeloid leukemia. View Publication

A liquid culture system is described whereby proliferation of haemopoietic stem cells (CFU-S),production of granulocyte precursor cells (CFU-C),and extensive granulopoiesis can be maintained in vetro for several months. Such cultures consist of adherent and non-adherent populations of cells. The adherent population contains phagocytic mononuclear cells,epithelial" cells� View Publication

Wnt/$$-catenin signalling has been suggested to be active in basal-like breast cancer. However,in highly aggressive metastatic triple-negative breast cancers (TNBC) the role of $$-catenin and the underlying mechanism(s) for the aggressiveness of TNBC remain unknown. We illustrate that WNT10B induces transcriptionally active $$-catenin in human TNBC and predicts survival-outcome of patients with both TNBC and basal-like tumours. We provide evidence that transgenic murine Wnt10b-driven tumours are devoid of ER$$,PR and HER2 expression and can model human TNBC. Importantly,HMGA2 is specifically expressed during early stages of embryonic mammogenesis and absent when WNT10B expression is lost,suggesting a developmentally conserved mode of action. Mechanistically,ChIP analysis uncovered that WNT10B activates canonical $$-catenin signalling leading to up-regulation of HMGA2. Treatment of mouse and human triple-negative tumour cells with two Wnt/$$-catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation. We demonstrate that WNT10B has epistatic activity on HMGA2,which is necessary and sufficient for proliferation of TNBC cells. Furthermore,HMGA2 expression predicts relapse-free-survival and metastasis in TNBC patients. View Publication

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in insulin sensitivity,tissue homeostasis,and regulating cellular functions. We found high-level expression of PPARgamma in embryo mouse brain and neural stem cells (NSCs),in contrast to extremely low levels in adult mouse brain. Here,we show that PPARgamma mediates the proliferation and differentiation of murine NSCs via up-regulation of the epidermal growth factor receptor and activation of the ERK pathway. Cell growth rates of NSCs prepared from heterozygous PPARgamma-deficient mouse brains,PPARgamma-RNA-silenced NSCs,and PPARgamma dominant-negative NSCs were significantly decreased compared with those of wild-type NSCs. Physiological concentrations of PPARgamma agonists,rosiglitazone and pioglitazone,stimulated NSC growth,whereas antagonists caused cell death in a concentration-dependent manner via activation of the caspase cascade. The stimulation of cell growth by PPARgamma was associated with a rapid activation of the ERK pathway by phosphorylation and up-regulation of epidermal growth factor receptor and cyclin B protein levels. In contrast,activation of PPARgamma by agonists inhibited the differentiation of NSCs into neurons. The inhibition of differentiation was associated with an activation of STAT3. These data indicate that PPARgamma regulates the development of the central nervous system during early embryogenesis via control of NSC proliferation. View Publication

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor (EPOR). The presence of EPO and its receptor in the CNS suggests a different function for EPO other than erythropoiesis. The purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation of neonatal spinal cord-derived neural progenitor cells. The effect of EPO on cell cycle progression was also examined,as well as the signaling cascades involved in this process. Our results showed that EPOR was present in the neural progenitor cells and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated neural progenitor cells indicated a reduced percentage of cells in G0/G1 phase,whereas the cell proliferation index (S phase plus G2/M phase) was increased. EPO also increased the proportion of 5-bromo-2-deoxyuridine (BrdU)-positive cells. With respect to the cell cycle signaling,we examined the cyclin-dependent kinases D1,D2 and E,and cyclin-dependent kinase inhibitors,p21cip1,p27kip1 and p57kip2. No significant differences were observed in the expression of these transcripts after EPO administration. Interestingly,the anti-apoptotic factors,mcl-1 and bcl-2 were significantly increased twofold. Moreover,these specific effects of EPO were eliminated by incubation of the progenitor cells with anti-EPO neutralizing antibody. Those observations suggested that EPO may play a role in normal spinal cord development by regulating cell proliferation and apoptosis. View Publication

Adoptive immunotherapy is evolving to assume an increasing role in treating cancer. Most imaging studies in adoptive immunotherapy to date have focused primarily on locating tumor-specific T cells rather than understanding their effector functions. In this study,we report the development of a noninvasive imaging strategy to monitor T-cell activation in living subjects by linking a reporter gene to the Granzyme B promoter (pGB),whose transcriptional activity is known to increase during T-cell activation. Because pGB is relatively weak and does not lead to sufficient reporter gene expression for noninvasive imaging,we specifically employed 2 signal amplification strategies,namely the Two Step Transcription Amplification (TSTA) strategy and the cytomegalovirus enhancer (CMVe) strategy,to maximize firefly luciferase reporter gene expression. Although both amplification strategies were capable of increasing pGB activity in activated primary murine splenocytes,only the level of bioluminescence activity achieved with the CMVe strategy was adequate for noninvasive imaging in mice. Using T cells transduced with a reporter vector containing the hybrid pGB-CMVe promoter,we were able to optically image T-cell effector function longitudinally in response to tumor antigens in living mice. This methodology has the potential to accelerate the study of adoptive immunotherapy in preclinical cancer models. View Publication

Polymorphonuclear neutrophils (PMNs) are critical for the formation,maintenance,and resolution of bacterial abscesses. However,the mechanisms that regulate PMN survival and proliferation during the evolution of an abscess are not well defined. Using a mouse model of Staphylococcus aureus abscess formation within a cutaneous wound,combined with real-time imaging of genetically tagged PMNs,we observed that a high bacterial burden elicited a sustained mobilization of PMNs from the bone marrow to the infected wound,where their lifespan was markedly extended. A continuous rise in wound PMN number,which was not accounted for by trafficking from the bone marrow or by prolonged survival,was correlated with the homing of c-kit(+)-progenitor cells from the blood to the wound,where they proliferated and formed mature PMNs. Furthermore,by blocking their recruitment with an antibody to c-kit,which severely limited the proliferation of mature PMNs in the wound and shortened mouse survival,we confirmed that progenitor cells are not only important contributors to PMN expansion in the wound,but are also functionally important for immune protection. We conclude that the abscess environment provides a niche capable of regulating PMN survival and local proliferation of bone marrow-derived c-kit(+)-progenitor cells. View Publication

Inner ear hair cells are specialized sensory cells essential for auditory function. Previous studies have shown that the sensory epithelium is postmitotic,but it harbors cells that can behave as progenitor cells in vitro,including the ability to form new hair cells. Lgr5,a Wnt target gene,marks distinct supporting cell types in the neonatal cochlea. Here,we tested the hypothesis that Lgr5(+) cells are Wnt-responsive sensory precursor cells. In contrast to their quiescent in vivo behavior,Lgr5(+) cells isolated by flow cytometry from neonatal Lgr5(EGFP-CreERT2/+) mice proliferated and formed clonal colonies. After 10 d in culture,new sensory cells formed and displayed specific hair cell markers (myo7a,calretinin,parvalbumin,myo6) and stereocilia-like structures expressing F-actin and espin. In comparison with other supporting cells,Lgr5(+) cells were enriched precursors to myo7a(+) cells,most of which formed without mitotic division. Treatment with Wnt agonists increased proliferation and colony-formation capacity. Conversely,small-molecule inhibitors of Wnt signaling suppressed proliferation without compromising the myo7a(+) cells formed by direct differentiation. In vivo lineage tracing supported the idea that Lgr5(+) cells give rise to myo7a(+) hair cells in the neonatal Lgr5(EGFP-CreERT2/+) cochlea. In addition,overexpression of β-catenin initiated proliferation and led to transient expansion of Lgr5(+) cells within the cochlear sensory epithelium. These results suggest that Lgr5 marks sensory precursors and that Wnt signaling can promote their proliferation and provide mechanistic insights into Wnt-responsive progenitor cells during sensory organ development. View Publication

The aim of the present study was to determine the effect of AZD8055 on proliferation,apoptosis and glycolysis in the human cervical cancer cell line HeLa and to investigate the underlying mechanism(s) of action. HeLa human cervical cancer cells were treated with 10 nM AZD8055 for 24,48 or 72 h. MTT was used to determine cell proliferation. Annexin V/propidium iodide staining was used to determine cell apoptosis analyzed by fluorescence-activated cell sorting (FACS). Glycolytic activity was determined by measuring the activity of the key enzyme lactate dehydrogenase (LDH) and lactate production. RNA and protein expression were examined by qRT-PCR and western blotting,respectively. Treatment with AZD8055 inhibited proliferation and glycolysis,and induced apoptosis in HeLa cells in a time-dependent manner. During the prolonged treatment with AZD8055,the phosphorylation of mammalian target of rapamycin (mTOR) C1 substrates p70S6K and phosphorylation of the mTORC2 substrate Akt were deregulated,suggesting that the activity of mTOR was downregulated. Furthermore,our study showed that the expression of miR-143 was upregulated in a time-dependent manner in HeLa cells treated with AZD8055. In summary,the present study reveals a novel antitumor mechanism of AZD8055 in HeLa human cervical cancer cells. View Publication

Sprouty (Spry) genes encode negative regulators of receptor tyrosine kinase (RTK) signaling,which plays important roles in human embryonic stem cells (hESCs). SPRY2 and SPRY4 are the two most highly expressed Sprouty family members in hESCs,suggesting that they may influence self-renewal. To test this hypothesis,we performed siRNA-mediated knock down (KD) studies. SPRY2 KD resulted in increased cell death and decreased proliferation,whereas SPRY4 KD enhanced survival. In both cases,after KD the cells were able to differentiate into cells of the three germ layers,although after SPRY2 KD there was a tendency toward increased ectodermal differentiation. SPRY2 KD cells displayed impaired mitochondrial fusion and cell membrane damage,explaining in part the increased cell death. These data indicate that Sprouty genes regulate pathways involved in proliferation and cell death in hESCs. View Publication