Separation of C/EBPalpha-mediated proliferation arrest and differentiation pathways.
(73/8706)
Cell proliferation and terminal differentiation are mutually exclusive in most cell lineages. The b-zip transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) induces proliferation arrest and differentiation in many cell types, suggesting that both activities are linked. Here we show that C/EBPalpha-mediated proliferation arrest and differentiation pathways can be separated by the E7 oncoprotein of the "high-risk" human papilloma virus 16. The E7 oncoprotein overrides C/EBPalpha-mediated cell cycle withdrawal without compromising the transactivation activity of C/EBPalpha or its ability to participate in differentiation. Uncoupling of both pathways depends on the casein kinase II site of the oncoprotein but not on its ability to neutralize pocket proteins or the cyclin-dependent kinase inhibitor protein p21. Our results suggest a bifurcation of C/EBPalpha-mediated proliferation arrest and differentiation pathways. (+info)
Conditional requirement for the Flk-1 receptor in the in vitro generation of early hematopoietic cells.
(74/8706)
Genetic studies in mice have previously demonstrated an intrinsic requirement for the vascular endothelial growth factor (VEGF) receptor Flk-1 in the early development of both the hematopoietic and endothelial cell lineages. In this study, embryonic stem (ES) cells homozygous for a targeted null mutation in flk-1 (flk-1 (-/-)) were examined for their hematopoietic potential in vitro during embryoid body (EB) formation or when cultured on the stromal cell line OP9. Surprisingly, in EB cultures flk-1 (-/-) ES cells were able to differentiate into all myeloid-erythroid lineages, albeit at half the frequency of heterozygous lines. In contrast, although flk-1 (-/-) ES cells formed mesodermal-like colonies on OP9 monolayers, they failed to generate hematopoietic clusters even in the presence of exogenous cytokines. However, flk-1 (-/-) OP9 cultures did contain myeloid precursors, albeit at greatly reduced percentages. This defect was rescued by first allowing flk-1 (-/-) ES cells to differentiate into EBs and then passaging these cells onto OP9 stroma. Thus, the requirement for Flk-1 in early hematopoietic development can be abrogated by alterations in the microenvironment. This finding is consistent with a role for Flk-1 in regulating the migration of early mesodermally derived precursors into a microenvironment that is permissive for hematopoiesis. (+info)
Effect of p21waf1/cip1 transgene on radiation induced apoptosis in T cells.
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The cyclin kinase inhibitor p21WAF1/Cip1 is upregulated by the tumor suppressor p53. While p21 is central for the G-1 arrest mediated by p53, it is still unclear if p21 also functions as a downstream effector of p53 dependent apoptosis. Apoptosis induced by DNA damage but not dexamethasone is p53 dependent in thymocytes. To investigate the physiological role of p21 in apoptosis, we have generated transgenic mice in which the p21 transgene is targeted for restricted expression in the T cell lineage. Thymocytes from p21 transgenic mice were hypersensitive to cell death induced by DNA damaging agents such as ionizing radiation and UV, but not be dexamethasone. Irradiated p21 transgenic thymocytes had approximately twofold more apoptotic cells as compared to irradiated age matched littermate control mice. Radiation induced death is comparable in thymocytes from p21 + Bcl2 + double transgenic mice and age matched littermate controls, indicating that the Bcl2 transgene rescues the radiation hypersensitivity imposed by p21. However, thymocytes from p53-/- mice even when they expressed the p21 transgene, were resistant to death induced by radiation. Together these results show that thymocytes from p21 transgenic mice are hypersensitive to radiation induced programmed cell death and suggest that the radiation hypersensitivity of p21 transgenic thymocytes involves p53 dependent pathway and signals in addition to p21. (+info)
Inhibition of microglial cell RANTES production by IL-10 and TGF-beta.
(76/8706)
Using human fetal microglial cell cultures, we found that the gram-negative bacterial cell wall component lipopolysaccharide (LPS) stimulated RANTES (regulated upon activation of normal T cell expressed and secreted) production through the protein kinase C signaling pathway and that activation of transcription nuclear factor (NF)-kappaB was required for this effect. Similarly, the proinflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha dose-dependently stimulated microglial cell RANTES production via NF-kappaB activation. Anti-inflammatory cytokines, IL-10, and transforming growth factor (TGF)-beta sequentially inhibited LPS- and cytokine-induced microglial cell NF-kappaB activation, RANTES mRNA expression, and protein release. Proinflammatory cytokines but not LPS also stimulated RANTES production by human astrocytes. These findings demonstrate that human microglia synthesize RANTES in response to proinflammatory stimuli, and that the anti-inflammatory cytokines IL-10 and TGF-beta down-regulate the production of this beta-chemokine. These results may have important therapeutic implications for inflammatory diseases of the brain. (+info)
Loss of FancC function results in decreased hematopoietic stem cell repopulating ability.
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Fanconi anemia (FA) is a complex genetic disorder characterized by progressive bone marrow (BM) aplasia, chromosomal instability, and acquisition of malignancies, particularly myeloid leukemia. We used a murine model containing a disruption of the murine homologue of FANCC (FancC) to evaluate short- and long-term multilineage repopulating ability of FancC -/- cells in vivo. Competitive repopulation assays were conducted where "test" FancC -/- or FancC +/+ BM cells (expressing CD45.2) were cotransplanted with congenic competitor cells (expressing CD45.1) into irradiated mice. In two independent experiments, we determined that FancC -/- BM cells have a profound decrease in short-term, as well as long-term, multilineage repopulating ability. To determine quantitatively the relative production of progeny cells by each test cell population, we calculated test cell contribution to chimerism as compared with 1 x 10(5) competitor cells. We determined that FancC -/- cells have a 7-fold to 12-fold decrease in repopulating ability compared with FancC +/+ cells. These data indicate that loss of FancC function results in reduced in vivo repopulating ability of pluripotential hematopoietic stem cells, which may play a role in the development of the BM failure in FA patients. This model system provides a powerful tool for evaluation of experimental therapeutics on hematopoietic stem cell function. (+info)
Clustered microsatellite mutations in the pipefish Syngnathus typhle.
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Clustered mutations are copies of a mutant allele that enter a population's gene pool together due to replication from a premeiotic germline mutation and distribution to multiple successful gametes of an individual. Although the phenomenon has been studied in Drosophila and noted in a few other species, the topic has received scant attention despite claims of being of major importance to population genetics theory. Here we capitalize upon the reproductive biology of male-pregnant pipefishes to document the occurrence of clustered microsatellite mutations and to estimate their rates and patterns from family data. Among a total of 3195 embryos genetically screened from 110 families, 40% of the 35 detected de novo mutant alleles resided in documented mutational clusters. Most of the microsatellite mutations appeared to involve small-integer changes in repeat copy number, and they arose in approximately equal frequency in paternal and maternal germlines. These findings extend observations on clustered mutations to another organismal group and motivate a broader critique of the mutation cluster phenomenon. They also carry implications for the evolution of microsatellites with respect to mutational models and homoplasy among alleles. (+info)
Activation of the extracellular signal-related kinase/mitogen-activated protein kinase pathway discriminates CD4 versus CD8 lineage commitment in the thymus.
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We have investigated the role of the mitogen-activated protein kinase (MAPK) pathway in the differentiation of CD4+ and CD8+ T cells by looking specifically at the effects of inhibitors of MAPK-activating enzyme, MAPK/extracellular signal-related kinase (ERK) kinase (MEK), during the positive selection step from double-positive to single-positive (SP) thymocytes. Using a variety of transgenic/knockout mouse strain combinations that fail to differentiate individual lineages of SP thymocytes together with genetically engineered F(ab')2 reagents that induce maturation preferentially to either the CD4 or CD8 subpopulations, we show that induction of CD4 differentiation cells is highly sensitive to levels of MEK inhibition that have no effect on CD8 maturation. In addition, the presence of MEK inhibitor is able to modify signals that normally induce CD4 differentiation to instead promote CD8 differentiation. Finally, we show that continuous culture in the presence of inhibitor interferes with TCR up-regulation in SP thymocytes, suggesting that MAPK signaling may be involved in final maturation steps for both lineages. These data indicate that there is discrimination in the biochemical pathways that are necessary to specify CD4 and CD8 lineage commitment and can reconcile previously conflicting reports on the influence of MAPK activation in commitment and maturation of thymocytes. (+info)
Biphasic dispersion of clones containing Purkinje cells and glia in the developing chick cerebellum.
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The cerebellum is a highly conserved structure which exhibits patterns of gene expression and axonal connections that are organized into parasagittal domains. These aspects of the mature cerebellum are presaged during embryonic development by the expression patterns of vertebrate homologs of Drosophila segmentation genes. We wished to determine whether the parasagittal domains of gene expression are compartments of lineage restriction. To this end, a clonal analysis of the chick cerebellum was conducted with a complex retroviral library. From embryonic day (E) 8 to E12, clones derived from the more medial portion of the cerebellar ventricular zone (VZ) were observed to spread preferentially in the mediolateral direction, crossing the boundaries of the parasagittal domains of gene expression. In late embryonic and posthatch periods, VZ clones were found to comprise Purkinje cells, glial cells, or both types of cells. At these later times, clonally related glial cells formed tight parasagittal clusters, while clonally related Purkinje cells were scattered extensively in the anteroposterior direction. We propose that a subset of the cerebellar VZ clones, those with medial origins, undergoes a biphasic dispersion: an early phase of mediolateral dispersion and a late phase of anteroposterior dispersion. This novel pattern of clonal dispersion suggests that the cerebellar VZ is not partitioned into parasagittal domains of lineage restriction. It leaves open the possibility that the later dispersion along the anteroposterior axis results from the parasagittal patterns of gene expression in the developing cerebellar cortex. (+info)