搜索结果: 'NeuroCult Proliferation'

BACKGROUND Rhodiola crenulata is a Tibetan mountainous plant,commonly used in Eastern alternative medicine. Many phytochemicals possess estrogenic activity,a critical regulator of proliferation in mammary epithelial cells. We have previously characterized anti-cancer properties of R. crenulata in aggressive triple negative breast cancer cells,lacking the expression of estrogen receptor. Currently,it is unknown whether R. crenulata exerts estrogenic effects and as such consumption may be a concern for women with estrogen receptor positive breast cancer that use Rhodiola sp. to relieve mild to moderate depression. PURPOSE In this study,we wished to determine whether a hydroalcoholic fraction of the R. crenulata root extract exhibits estrogenic activity in estrogen receptor positive (ER+) breast cancer cells in vitro and whether it affects normal mammary epithelial ER target gene expression in vivo. METHODS ER transcriptional activity was analyzed in MCF7 cells expressing an ERE reporter construct and confirmed via qPCR of endogenous ER target genes. We also monitored cellular proliferation over time. Additionally,to assess stem-like properties in MCF7 cells,we performed a tumorsphere formation assay under anchorage independent conditions. We examined whether R. crenulata treatment reduced $$-catenin levels via Western blotting and measured $$-catenin transcriptional activity by a reporter assay. To examine the effects of R. crenulata on normal mammary epithelial cells,we performed immunohistochemical staining of ER and PR in the mammary glands of mice fed R. crenulata for 12 weeks. RESULTS We show an initial activation of ER transcriptional activity by dual reporter assay,qPCR and proliferation of MCF7 ER+ cells in response to 24 h of R. crenulata treatment. However,upon longer treatment basal and R. crenulata induced transcriptional activity was suppressed. There was a decrease in cell doubling times and a decrease in tumorsphere formation. In association with these changes,ER$$ transcript levels were decreased and active $$-catenin levels were reduced in the cells treated for 2 weeks. Finally,we show no change in estrogen targets in normal mammary cells in vivo. CONCLUSION These data suggest that the R. crenulata extract contains components with estrogenic activity. However,R. crenulata treatment could still be protective in ER+ breast cancer cells,as longer treatment reduced the transcriptional activity of $$-catenin and ER responses leading to reduced proliferation and tumorsphere formation. Furthermore,administration of 20 mg/kg/day R. crenulata to mice did not have an observable effect on mammary epithelial ER$$ target gene expression in vivo. View Publication

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells,and overexpression of Hoxa-9 markedly expands hematopoietic stem cells,suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin(-)c-kit(+)flk-2(-)Sca-1+ [KLFS] cells). However,sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia,indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays,Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability,a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures,Hoxa-9-deficient Lin(-)Sca-1(+) cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation,with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo. View Publication

Recently,a number of clinical trials used either mesenchymal stem cells (MSCs) or natural killer (NK) cells in an attempt to improve the effectiveness of hematopoietic stem cell transplantation (HSCT). In view of the relevant role of both MSCs and NK cells in HSCT,we have recently explored the result of possible interactions between the 2 cell types. We found that activated NK cells could kill MSCs,whereas MSCs strongly inhibited interleukin-2 (IL-2)-induced NK-cell proliferation. In this study,we further analyzed the inhibitory effect exerted by MSCs on NK cells. We show that MSCs not only inhibit the cytokine-induced proliferation of freshly isolated NK cells but also prevent the induction of effector functions,such as cytotoxic activity and cytokine production. Moreover,we show that this inhibitory effect is related to a sharp down-regulation of the surface expression of the activating NK receptors NKp30,NKp44,and NKG2D. Finally,we demonstrate that indoleamine 2,3-dioxygenase and prostaglandin E2 represent key mediators of the MSC-induced inhibition of NK cells. View Publication

Activated neutrophil-derived exosomes reportedly contribute to the proliferation of airway smooth muscle cells (ASMCs),thereby aggravating the airway wall remodeling during asthma; however,the specific mechanism remains unclear. Lipopolysaccharide (LPS)-EXO and si-CRNDE-EXO were extracted from the media of human neutrophils treated with LPS and LPS??+??si-CRNDE (a siRNA targets long non-coding RNA CRNDE),respectively. Human ASMCs were co-cultured with LPS-EXO or si-CRNDE-EXO,and cell viability,proliferation and migration were measured. The interplay of colorectal neoplasia differentially expressed (CRNDE),inhibitor of nuclear factor kappa B kinase subunit beta (IKK$\beta$) and nuclear receptor subfamily 2 group C member 2 (TAK1) was explored using RNA immunoprecipitation (RIP) and Co-IP assays. A mouse model of asthma was induced using ovalbumin. CRNDE was upregulated in LPS-EXO and successfully transferred from LPS-treated neutrophils to ASMCs through exosome. Mechanically,CRNDE loaded in LPS-EXO reinforced TAK1-mediated IKK$\beta$ phosphorylation,thereby activating the nuclear factor kappa B (NF-$\kappa$B) pathway. Functionally,silencing CRNDE in LPS-EXO,an IKK$\beta$ inhibitor,and an NF-$\kappa$B inhibitor all removed the upregulation of cell viability,proliferation and migration induced by LPS-EXO in ASMCs. In the end,the in vivo experiment demonstrated that CRNDE knockdown in neutrophils effectively reduced the thickness of bronchial smooth muscle in a mouse model for asthma. Activated neutrophils-derived CRNDE was transferred to ASMCs through exosomes and activated the NF-$\kappa$B pathway by enhancing IKK$\beta$ phosphorylation. The latter promoted the proliferation and migration of ASMCs and then contributed to airway remodeling in asthma. View Publication

During lung cancer metastasis,tumor cells undergo epithelial-to-mesenchymal transition (EMT),enabling them to intravasate through the vascular barrier and enter the circulation before colonizing secondary sites. Here,a human in vitro microphysiological model of EMT-driven lung cancer intravasation-on-a-chip was developed and coupled with machine learning (ML)-assisted automatic identification and quantification of intravasation events. A robust EMT-inducing cocktail (EMT-IC) was formulated by augmenting macrophage-conditioned medium with transforming growth factor-β1. When introduced into microvascular networks (MVNs) in microfluidic devices,EMT-IC did not affect MVN stability and physiologically relevant barrier functions. To model lung cancer intravasation on-a-chip,EMT-IC was supplemented into co-cultures of lung tumor micromasses and MVNs. Wihin 24 h of exposure,EMT-IC facilitated the insertion of membrane protrusions of migratory A549 cells into microvascular structures,followed by successful intravasation. EMT-IC reduced key basement membrane and vascular junction proteins - laminin and VE-Cadherin - rendering vessel walls more permissive to intravasating cells. ML-assisted vessel segmentation combined with co-localization analysis to detect intravasation events confirmed that EMT induction significantly increased the number of intravasation events. Introducing metastatic (NCI-H1975) and non-metastatic (BEAS-2B) cell lines demonstrated that both,baseline intravasation potential and responsiveness to EMT-IC,are reflected in the metastatic predisposition of lung cancer cell lines,highlighting the model's universal applicability and cell-specific sensitivity. The reproducible detection of intravasation events in the established model provides a physiologically relevant platform to study processes of cancer metastasis with high spatio-temporal resolution and short timeframe. This approach holds promise for improved drug development and informed personalized patient treatment plans. View Publication

Recent work investigating exercise induced changes in immunocompetence suggests that some of the ambiguity in the literature is resultant from different cell isolation protocols and mitogen selection. To understand this effect,we compared post-exercise measures of T cell activation and proliferation using two different stimulation methods (costimulation through CD28 or stimulation with phytohaemagglutinin [PHA]). Further,we investigated whether exercise induced changes are maintained when T cell isolation from whole blood is delayed overnight in either a room temperature or chilled (4°C) environment. As expected,an increased proliferation response was observed post-exercise in T cells isolated from whole blood of previously trained individuals immediately after blood collection. Also,cells stimulated with PHA after resting overnight in whole blood were not adversely impacted by the storage conditions. In contrast,allowing cells to rest overnight in whole blood prior to stimulation through CD28,lessened the proliferation observed by cells following exercise rendering both the room temperature and chilled samples closer to the results seen in the control condition. Changes in early markers of activation (CD25),followed a similar pattern,with activation in PHA stimulated cells remaining fairly robust after overnight storage; whereas cell activation following stimulation through CD3+CD28 was disproportionately decreased by the influence of overnight storage. These findings indicate that decisions regarding cell stimulation methods need to be paired with the timeline for T cell isolation from whole blood. These considerations will be especially important for field based studies of immunocompetence where there is a delay in getting whole blood samples to a lab for processing as well as clinical applications where a failure to isolate T cells in a timely manner may result in loss of the response of interest. View Publication

BACKGROUND: Previous studies have shown that patients with Bcr-Abl-positive acute lymphoblastic leukemia (ALL) either have primary disease that is refractory to imatinib mesylate or develop disease recurrence after an initial response. METHODS: The authors investigated the effects of a newly designed Bcr-Abl inhibitor,AMN107,by comparing its in vitro inhibitory potency on p190 Bcr-Abl ALL cell lines with that of imatinib. RESULTS: In two Philadelphia (Ph)-positive ALL cell lines,AMN107 was found to be 30-40 times more potent than imatinib in inhibiting cellular proliferation. AMN107 was also more effective than imatinib in inhibiting phosphorylation of p190 Bcr-Abl tyrosine kinase in cell lines and primary ALL cells. The inhibition of cellular proliferation was associated with the induction of apoptosis in only one of the cell lines. No activity was observed in cell lines lacking the BCR-ABL genotype. CONCLUSIONS: The results of the current study suggest the superior potency of AMN107 compared with imatinib in Ph-positive ALL and support clinical trials of AMN107 in patients with Ph-positive ALL. View Publication

INTRODUCTION: The physiological signals that direct the division and differentiation of the zygote to form a blastocyst,and subsequent embryonic stem cell division and differentiation during early embryogenesis,are unknown. Although a number of growth factors,including the pregnancy-associated hormone human chorionic gonadotropin (hCG) are secreted by trophoblasts that lie adjacent to the embryoblast in the blastocyst,it is not known whether these growth factors directly signal human embryonic stem cells (hESCs).backslashnbackslashnMETHODS: Here we used hESCs as a model of inner cell mass differentiation to examine the hormonal requirements for the formation of embryoid bodies (EB's; akin to blastulation) and neuroectodermal rosettes (akin to neurulation).backslashnbackslashnRESULTS: We found that hCG promotes the division of hESCs and their differentiation into EB's and neuroectodermal rosettes. Inhibition of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) signaling suppresses hESC proliferation,an effect that is reversed by treatment with hCG. hCG treatment rapidly upregulates steroidogenic acute regulatory protein (StAR)-mediated cholesterol transport and the synthesis of progesterone (P4). hESCs express P4 receptor A,and treatment of hESC colonies with P4 induces neurulation,as demonstrated by the expression of nestin and the formation of columnar neuroectodermal cells that organize into neural tubelike rosettes. Suppression of P4 signaling by withdrawing P4 or treating with the P4-receptor antagonist RU-486 inhibits the differentiation of hESC colonies into EB's and rosettes.backslashnbackslashnCONCLUSIONS: Our findings indicate that hCG signaling via LHCGR on hESC promotes proliferation and differentiation during blastulation and neurulation. These findings suggest that trophoblastic hCG secretion and signaling to the adjacent embryoblast could be the commencement of trophic support by placental tissues in the growth and development of the human embryo. View Publication

OBJECTIVES: Recent data show that Imatinib mesylate (IM) also affects haematopoietic stem cells (HSC),T lymphocytes and dendritic cells that do not harbour constitutively active tyrosine kinases. MATERIALS AND METHODS: We evaluated possible effects of IM on human bone marrow-derived mesenchymal stem cells (MSC) in vitro. RESULTS: Screening the activity of 42 receptor tyrosine kinases revealed an exclusive inhibition of platelet-derived growth factor receptorbeta (PDGFRbeta). Analysis of downstream targets of PDGFRbeta demonstrated IM-mediated reduction of Akt and Erk1/2 phosphorylation. Culture of MSC with IM led to the reversible development of perinuclear multi-vesicular bodies. The proliferation and clonogenicity of MSC were significantly reduced compared to control cultures. IM favoured adipogenic differentiation of MSC whereas osteogenesis was suppressed. The functional deficits described led to a 50% reduction in the support of clonogenic haematopoietic stem cells,cultured for 1 month on a monolayer of MSC with IM. CONCLUSION: In summary,inhibition of PDGFRbeta and downstream Akt and Erk signalling by IM has a significant impact on proliferation and differentiation of human MSC in vitro. View Publication

The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes,the co-stimulation of CD4+ T cells,and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells,but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together,these data implicate HSA as a potent negative regulator of B cell development and activation. View Publication

FOXM1 is an important cell cycle regulator and regulates cell proliferation. In addition,FOXM1 has been reported to contribute to oncogenesis in various cancers. However,it is not clearly understood how FOXM1 contributes to acute myeloid leukemia (AML) cell proliferation. In this study,we investigated the cellular and molecular function of FOXM1 in AML cells. The FOXM1 messenger RNA (mRNA) expressed in AML cell lines was predominantly the FOXM1B isoform,and its levels were significantly higher than in normal high aldehyde dehydrogenase activity (ALDH(hi)) cells. Reduction of FOXM1 expression in AML cells inhibited cell proliferation compared with control cells,through induction of G(2)/M cell cycle arrest,a decrease in the protein expression of Aurora kinase B,Survivin,Cyclin B1,S-phase kinase-associated protein 2 and Cdc25B and an increase in the protein expression of p21(Cip1) and p27(Kip1). FOXM1 messenger RNA (mRNA) was overexpressed in all 127 AML clinical specimens tested (n = 21,56,32 and 18 for M1,M2,M4 and M5 subtypes,respectively). Compared with normal ALDH(hi) cells,FOXM1 gene expression was 1.65- to 2.26-fold higher in AML cells. Moreover,the FOXM1 protein was more strongly expressed in AML-derived ALDH(hi) cells compared with normal ALDH(hi) cells. In addition,depletion of FOXM1 reduced colony formation of AML-derived ALDH(hi) cells due to inhibition of Cdc25B and Cyclin B1 expression. In summary,we found that FOXM1B mRNA is predominantly expressed in AML cells and that aberrant expression of FOXM1 induces AML cell proliferation through modulation of cell cycle progression. Thus,inhibition of FOXM1 expression represents an attractive target for AML therapy. View Publication

Basic fibroblast growth factor (bFGF) is a crucial factor sustaining human pluripotent stem cells (hPSCs). We designed this study to search the substitutive factors other than bFGF for the maintenance of hPSCs by using human placenta-derived conditioned medium without exogenous bFGF (hPCCM-),containing chemokine (C-X-C motif) receptor 2 (CXCR2) ligands,including interleukin (IL)-8 and growth-related oncogene $\$(GRO$\$),which were developed on the basis of our previous studies. First,we confirmed that IL-8 and/or GRO$\$ independent roles to preserve the phenotype of hPSCs. Then,we tried CXCR2 blockage of hPSCs in hPCCM- and verified the significant decrease of pluripotency-associated genes expression and the proliferation of hPSCs. Interestingly,CXCR2 suppression of hPSCs in mTeSR™1 containing exogenous bFGF decreased the proliferation of hPSCs while maintaining pluripotency characteristics. Lastly,we found that hPSCs proliferated robustly for more than 35 passages in hPCCM- on a gelatin substratum. Higher CXCR2 expression of hPSCs cultured in hPCCM- than those in mTeSR™1 was observable. Our findings suggest that CXCR2 and its related ligands might be novel factors comparable to bFGF supporting the characteristics of hPSCs and hPCCM- might be useful for the maintenance of hPSCs as well as for the accurate evaluation of CXCR2 role in hPSCs without the confounding influence of exogenous bFGF. View Publication