搜索结果: 'NeuroCult Proliferation'

PURPOSE Nonexudative (dry) age-related macular degeneration (AMD),a leading cause of blindness in the elderly,is associated with the loss of retinal pigmented epithelium (RPE) cells and the development of geographic atrophy,which are areas devoid of RPE cells and photoreceptors. One possible treatment option would be to stimulate RPE attachment and proliferation to replace dying/dysfunctional RPE and bring about wound repair. Clinical trials are underway testing injections of RPE cells derived from pluripotent stem cells to determine their safety and efficacy in treating AMD. However,the factors regulating RPE responses to AMD-associated lesions are not well understood. Here,we use cell culture to investigate the role of RhoA coiled coil kinases (ROCKs) in human embryonic stem cell-derived RPE (hESC-RPE) attachment,proliferation,and wound closure. METHODS H9 hESC were spontaneously differentiated into RPE cells. hESC-RPE cells were treated with a pan ROCK1/2 or a ROCK2 only inhibitor; attachment,and proliferation and cell size within an in vitro scratch assay were examined. RESULTS Pharmacological inhibition of ROCKs promoted hESC-RPE attachment and proliferation,and increased the rate of closure of in vitro wounds. ROCK inhibition decreased phosphorylation of cofilin and myosin light chain,suggesting that regulation of the cytoskeleton underlies the mechanism of action of ROCK inhibition. CONCLUSIONS ROCK inhibition promotes attachment,proliferation,and wound closure in H9 hESC-RPE cells. ROCK isoforms may have different roles in wound healing. TRANSLATIONAL RELEVANCE Modulation of the ROCK-cytoskeletal axis has potential in stimulating wound repair in transplanted RPE cells and attachment in cellular therapies. View Publication

OBJECTIVE Pancreatic cancer continues to be an urgent clinical problem. We used the novel DNA methyltransferase inhibitor Zebularine and the histone deacetylase inhibitor SAHA to investigate the epigenetic influence on viability and differentiation of the pancreatic cancer cell lines YAP C,DAN G and Panc-89 in vitro and in vivo. MATERIAL AND METHODS Cell vitality,proliferation and expression of PDX-1,cytokeratin 7 and 20,chromogranin A,vimentin,bax and bcl-2 were determined on the protein and mRNA level in vitro and in a subcutaneous xenograft model. RESULTS A time- and dose-dependent increase of apoptosis,paralleled by decreased proliferation,was observed after incubation with single agents or a combination therapy with lower concentrations. This was associated with up-regulation of pro-apoptotic bax and a phenotypic stabilization by the enhanced expression of cytokeratin 7. In vivo,growth of xenografts was delayed with the most pronounced effect in Panc-89 after 1 week of daily intraperitoneal injections of Zebularine paralleled with CK7 up-regulation and down-regulation of dedifferentiation markers. CONCLUSIONS Epigenetic modulation via inhibition of DNA methyltransferase and histone deacetylase induces apoptosis in human pancreatic cancer cells in vitro and delays xenograft growth in vivo,which is associated with a morphological/molecular phenotypic stabilization. These compounds may therefore be suitable as adjunctive therapeutic agents in the treatment of pancreatic cancer. View Publication

Fbxo7 is an unusual F-box protein because most of its interacting proteins are not substrates for ubiquitin-mediated degradation. Fbxo7 directly binds p27 and Cdk6,enhances the level of cyclin D-Cdk6 complexes,and its overexpression causes Cdk6-dependent transformation of immortalised fibroblasts. Here,we test the ability of Fbxo7 to transform haematopoietic pro-B (Ba/F3) cells which,unexpectedly,it was unable to do despite high levels of Cdk6. Instead,reduction of Fbxo7 expression increased proliferation,decreased cell size and shortened G1 phase. Analysis of cell cycle regulators showed that cells had decreased levels of p27,and increased levels of S phase cyclins and Cdk2 activity. Also,Fbxo7 protein levels correlated inversely with those of CD43,suggesting direct regulation of its expression and,therefore,of B cell maturation. Alterations to Cdk6 protein levels did not affect the cell cycle,indicating that Cdk6 is neither rate-limiting nor essential in Ba/F3 cells; however,decreased expression of Cdk6 also enhanced levels of CD43,indicating that expression of CD43 is independent of cell cycle regulation. The physiological effect of reduced levels of Fbxo7 was assessed by creating a transgenic mouse with a LacZ insertion into the Fbxo7 locus. Homozygous Fbxo7(LacZ) mice showed significantly increased pro-B cell and pro-erythroblast populations,consistent with Fbxo7 having an anti-proliferative function and/or a role in promoting maturation of precursor cells. View Publication

BACKGROUND Alzheimer's disease (AD) is characterized by progressive memory loss and impaired cognitive function. Early-onset familial forms of the disease (FAD) are caused by inheritance of mutant genes encoding presenilin 1 (PS1) variants. We have demonstrated that prion promoter (PrP)-driven expression of human FAD-linked PS1 variants in mice leads to impairments in environmental enrichment (EE)-induced adult hippocampal neural progenitor cell (AHNPC) proliferation and neuronal differentiation,and have provided evidence that accessory cells in the hippocampal niche expressing PS1 variants may modulate AHNPC phenotypes,in vivo. While of significant interest,these latter studies relied on transgenic mice that express human PS1 variant transgenes ubiquitously and at high levels,and the consequences of wild type or mutant PS1 expressed under physiologically relevant levels on EE-mediated AHNPC phenotypes has not yet been tested. RESULTS To assess the impact of mutant PS1 on EE-induced AHNPC phenotypes when expressed under physiological levels,we exposed adult mice that constitutively express the PSEN1 M146V mutation driven by the endogenous PSEN1 promoter (PS1 M146V knock-in" (KI) mice) to standard or EE-housed conditions. We show that in comparison to wild type PS1 mice AHNPCs in mice carrying homozygous (PS1M146V/M146V) or heterozygous (PS1M146V/+) M146V mutant alleles fail to exhibit EE-induced proliferation and commitment towards neurogenic lineages. More importantly we report that the survival of newborn progenitors are diminished in PS1 M146V KI mice exposed to EE-conditions compared to respective EE wild type controls. CONCLUSIONS Our findings reveal that expression at physiological levels achieved by a single PS1 M146V allele is sufficient to impair EE-induced AHNPC proliferation survival and neuronal differentiation in vivo. These results and our finding that microglia expressing a single PS1 M146V allele impairs the proliferation of wild type AHNPCs in vitro argue that expression of mutant PS1 in the AHNPC niche impairs AHNPCs phenotypes in a dominant non-cell autonomous manner. View Publication

Tumors with mutant BRAF and some with mutant RAS are dependent upon ERK signaling for proliferation,and their growth is suppressed by MAPK/ERK kinase (MEK) inhibitors. In contrast,tumor cells with human EGF receptor (HER) kinase activation proliferate in a MEK-independent manner. These findings have led to the development of RAF and MEK inhibitors as anticancer agents. Like MEK inhibitors,the RAF inhibitor PLX4032 inhibits the proliferation of BRAF(V600E) tumor cells but not that of HER kinase-dependent tumors. However,tumors with RAS mutation that are sensitive to MEK inhibition are insensitive to PLX4032. MEK inhibitors inhibit ERK phosphorylation in all normal and tumor cells,whereas PLX4032 inhibits ERK signaling only in tumor cells expressing BRAF(V600E). In contrast,the drug activates MEK and ERK phosphorylation in cells with wild-type BRAF. In BRAF(V600E) tumor cells,MEK and RAF inhibitors affect the expression of a common set of genes. PLX4032 inhibits ERK signaling output in mutant BRAF cells,whereas it transiently activates the expression of these genes in tumor cells with wild-type RAF. Thus,PLX4032 inhibits ERK signaling output in a mutant BRAF-selective manner. These data explain why the drug selectively inhibits the growth of mutant BRAF tumors and suggest that it will not cause toxicity resulting from the inhibition of ERK signaling in normal cells. This selectivity may lead to a broader therapeutic index and help explain the greater antitumor activity observed with this drug than with MEK inhibitors. View Publication

Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes,the extracellular signal-regulated kinase (ERK),Jun NH2-terminal kinase,and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation,we used the inhibitor U0126,which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK),the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs,but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy,unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly,induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK,unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term,but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants. View Publication

OBJECTIVE: In this study,the alternative splicing product of vasohibin 1 (VASH1B) was analyzed in direct comparison to the major isoform (VASH1A) for antiangiogenic effects on endothelial colony forming cells (ECFCs) from peripheral blood and on human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Expression studies in primary human endothelial cells revealed that both vasohibin proteins,hVASH1A and hVASH1B,localized in the nucleus and cytoplasm. Adenoviruses carrying the cDNA for VASH1A/B and purified recombinant proteins were used to study the function of both molecules in ECFCs and HUVECs. Recombinant VASH1A protein did not inhibit cell proliferation,tube formation,or vessel growth in vivo in the chick chorioallantoic membrane (CAM) assay,but promoted endothelial cell migration in vitro. The VASH1B protein had an inhibitory effect on cell proliferation,migration,tube formation,and inhibited blood vessel formation in the CAM assay. Adenoviral overexpression of VASH1B,but not of VASH1A,resulted in inhibition of endothelial cell growth,migration,and capillary formation. Interestingly,overexpression of VASH1A and B induced apoptosis in proliferating human fibroblasts,but did not affect cell growth of keratinocytes. CONCLUSIONS: Our data point out that alternative splicing of the VASH1 pre-mRNA transcript generates a potent antiangiogenic protein. View Publication

Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain hematologic malignancies,but the risk of graft-versus-host disease (GVHD) is a major limitation for wider application. Ideally,strategies to improve allo-HCT would involve suppression of T lymphocytes that drive GVHD while sparing those that mediate graft-versus-malignancy (GVM). Recently,using a xenograft model,we serendipitously discovered that myxoma virus (MYXV) prevented GVHD while permitting GVM. In this study,we show that MYXV binds to resting,primary human T lymphocytes but will only proceed into active virus infection after the T cells receive activation signals. MYXV-infected T lymphocytes exhibited impaired proliferation after activation with reduced expression of interferon-?,interleukin-2 (IL-2),and soluble IL-2R?,but did not affect expression of IL-4 and IL-10. MYXV suppressed T-cell proliferation in 2 patterns (full vs partial) depending on the donor. In terms of GVM,we show that MYXV-infected activated human T lymphocytes effectively deliver live oncolytic virus to human multiple myeloma cells,thus augmenting GVM by transfer of active oncolytic virus to residual cancer cells. Given this dual capacity of reducing GVHD plus increasing the antineoplastic effectiveness of GVM,ex vivo virotherapy with MYXV may be a promising clinical adjunct to allo-HCT regimens. View Publication

The sympathetic (SNS) and parasympathetic (PNS) branches of the autonomic nervous system have been implicated in the modulation of the renewal of many tissues,including the intestinal epithelium. However,it is not known whether these mechanisms are direct,requiring an interaction between autonomic neurotransmitters and receptors on proliferating epithelial cells. To evaluate the existence of a molecular framework for a direct effect of the SNS or PNS on intestinal epithelial renewal,we measured gene expression for the main autonomic neurotransmitter receptors in this tissue. We separately evaluated intestinal epithelial regions comprised of the stem,progenitor,and mature cells,which allowed us to investigate the distinct contributions of each cell population to this proposed autonomic effect. Notably,we found that the stem cells expressed the receptors for the SNS-associated alpha2A adrenoreceptor and the PNS-associated muscarinic acetylcholine receptors (M1 and M3). In a separate experiment,we found that the application of norepinephrine or acetylcholine decreases the expression of cyclin D1,a gene necessary for cell cycle progression,in intestinal epithelial organoids compared with controls (P {\textless} 0.05). Together,these results provide evidence of a direct mechanism for the autonomic nervous system influence on intestinal epithelial stem cell proliferation. View Publication

Whether cytokines can modulate the fate of primitive hematopoietic progenitor cells (HPCs) through successive in vitro cell divisions has not been established. Single human marrow CD34+CD38-/lo cells in the G0 phase of cell cycle were cultured under 7 different cytokine combinations,monitored for proliferation on days 3,5,and 7,then assayed for long-term culture-initiating cell (LTC-IC) function on day 7. LTC-IC function was then retrospectively correlated with prior number of in vitro cell divisions to determine whether maintenance of LTC-IC function after in vitro cell division is dependent on cytokine exposure. In the presence of proliferation progression signals,initial cell division was independent of cytokine stimulation,suggesting that entry of primitive HPCs into the cell cycle is a stochastic property. However,kinetics of proliferation beyond day 3 and maintenance of LTC-IC function were sensitive to cytokine stimulation,such that LTC-IC underwent an initial long cell cycle,followed by more synchronized shorter cycles varying in length depending on the cytokine combination. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) transplantation studies revealed analogous results to those obtained with LTC-ICs. These data suggest that although exit from quiescence and commitment to proliferation might be stochastic,kinetics of proliferation,and possibly fate of primitive HPCs,might be modulated by extrinsic factors. View Publication

The phosphatidylinositol 3' kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten/Akt pathway,which is a critical regulator of cell proliferation and survival,is mutated or activated in a wide variety of cancers. Akt appears to be a key central node in this pathway and thus is an attractive target for targeted molecular therapy. We demonstrated that Akt is highly phosphorylated in thyroid cancer cell lines and human thyroid cancer specimens,and hypothesised that KP372-1,an Akt inhibitor,would block signalling through the PI3K pathway and inhibit cell proliferation while inducing apoptosis of thyroid cancer cells. KP372-1 blocked signalling downstream of Akt in thyroid tumour cells,leading to inhibition of cell proliferation and increased apoptosis. As thyroid cancer consistently expresses phosphorylated Akt and KP372-1 effectively blocks Akt signalling,further preclinical evaluation of this compound for treatment of thyroid cancer is warranted. View Publication