The rapid cell turnover of the intestinal epithelium is achieved from small numbers of stem cells located in the base of glandular crypts. These stem cells have been variously described as rapidly ...cycling or quiescent. A functional arrangement of stem cells that reconciles both of these behaviours has so far been difficult to obtain. Alternative explanations for quiescent cells have been that they act as a parallel or reserve population that replace rapidly cycling stem cells periodically or after injury; their exact nature remains unknown. Here we show mouse intestinal quiescent cells to be precursors that are committed to mature into differentiated secretory cells of the Paneth and enteroendocrine lineage. However, crucially we find that after intestinal injury they are capable of extensive proliferation and can give rise to clones comprising the main epithelial cell types. Thus, quiescent cells can be recalled to the stem-cell state. These findings establish quiescent cells as an effective clonogenic reserve and provide a motivation for investigating their role in pathologies such as colorectal cancers and intestinal inflammation.
Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers in part because it is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that is refractory to the ...clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by coadministration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibition of the Hedgehog cellular signaling pathway. The combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to transient stabilization of disease. Thus, inefficient drug delivery may be an important contributor to chemoresistance in pancreatic cancer.
Tumour cells sustain their high proliferation rate through metabolic reprogramming, whereby cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis, even under normal oxygen ...levels. Hypoxia‐inducible factor 1A (HIF1A) is a major regulator of this process, but its activation under normoxic conditions, termed pseudohypoxia, is not well documented. Here, using an integrative approach combining the first genome‐wide mapping of chromatin binding for an endocytic adaptor, ARRB1, both in vitro and in vivo with gene expression profiling, we demonstrate that nuclear ARRB1 contributes to this metabolic shift in prostate cancer cells via regulation of HIF1A transcriptional activity under normoxic conditions through regulation of succinate dehydrogenase A (SDHA) and fumarate hydratase (FH) expression. ARRB1‐induced pseudohypoxia may facilitate adaptation of cancer cells to growth in the harsh conditions that are frequently encountered within solid tumours. Our study is the first example of an endocytic adaptor protein regulating metabolic pathways. It implicates ARRB1 as a potential tumour promoter in prostate cancer and highlights the importance of metabolic alterations in prostate cancer.
Synopsis
Global chromatin occupancy‐ and gene expression data, together with ‘pseudohypoxic’ regulation of HIF1alpha stability establish a predominantly metabolic function of nuclear ARRB1 in prostate cancer.
Unbiased, genome‐wide occupancy map of nuclear ARRB1
Integrated expression data that highlight ARRB1‐regulated gene networks
Discovery of ARRB1 as novel metabolic and cell cycle control regulator
Evidence for pseudohypoxic stabilisation of HIFA by ARRB1
Global chromatin occupancy‐ and gene expression data, together with ‘pseudohypoxic’ regulation of HIF1alpha stability establish a predominantly metabolic function of nuclear ARRB1 in prostate cancer.
Over expression of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in vascular smooth muscle cells (VSMCs) induces apoptosis and reduces neointima formation occurring after saphenous vein ...interposition grafting or coronary stenting. In studies to address the mechanism of TIMP-3-driven apoptosis in human VSMCs we find that TIMP-3 increased activation of caspase-8 and apoptosis was inhibited by expression of Cytokine response modifier A (CrmA) and dominant negative FAS-Associated protein with Death Domain (FADD). TIMP-3 induced apoptosis did not cause mitochondrial depolarisation, increase activation of caspase-9 and was not inhibited by over-expression of B-cell Lymphoma 2 (Bcl2), indicating a mitochondrial independent/type-I death receptor pathway. TIMP-3 increased levels of the First Apoptosis Signal receptor (FAS) and depletion of FAS with shRNA showed TIMP-3-induced apoptosis was FAS dependent. TIMP-3 induced formation of the Death-Inducing Signalling Complex (DISC), as detected by immunoprecipitation and by immunofluorescence. Cellular-FADD-like IL-1 converting enzyme-Like Inhibitory Protein (c-FLIP) localised with FAS at the cell periphery in the absence of TIMP-3 and this localisation was lost on TIMP-3 expression with c-FLIP adopting a perinuclear localisation. Although TIMP-3 inhibited FAS shedding, this did not increase total surface levels of FAS but instead increased FAS levels within localised regions at the cell surface. A Disintegrin And Metalloproteinase 17 (ADAM17) is inhibited by TIMP-3 and depletion of ADAM17 with shRNA significantly decreased FAS shedding. However ADAM17 depletion did not induce apoptosis or replicate the effects of TIMP-3 by increasing localised clustering of cell surface FAS. ADAM17-depleted cells could activate caspase-3 when expressing levels of TIMP-3 that were otherwise sub-apoptotic, suggesting a partial role for ADAM17 mediated ectodomain shedding in TIMP-3 mediated apoptosis. We conclude that TIMP-3 induced apoptosis in VSMCs is highly dependent on FAS and is associated with changes in FAS and c-FLIP localisation, but is not solely dependent on shedding of the FAS ectodomain.
Seeing the sugar coating: N‐Acetyl‐glucosamine and mannosamine derivatives tagged with an isonitrile group are metabolically incorporated into cell‐surface glycans and can be detected with a ...fluorescent tetrazine. This bioorthogonal isonitrile–tetrazine ligation is also orthogonal to the commonly used azide‐cyclooctyne ligation, and so will allow simultaneous detection of the incorporation of two different sugars.
Two reagents have been synthesized for selective labeling of cell surface azidoglycans, an unusually stable version of a dibenzocyclooctyne (TMDIBO) and a third-generation difluorinated cyclooctyne ...(DIFO3). Both syntheses are efficient with minimal purification, and the dibenzocyclooctyne is stable under basic and acidic conditions. Flow cytometric measurements with azidosugar labeled cancer cells, in which these reagents were linked to the fluorophore Alexa Fluor 647, gave a signal-to-background ratio of up to 35 with TMDIBO as compared to ≈10 for DIFO3 and ≈5 for a phosphine reagent. TMDIBO-based probes should have applications in molecular imaging of cell surface glycans in vivo.
Imaging sialylated tumor cell glycans in vivo Neves, André A.; Stöckmann, Henning; Harmston, Rebecca R. ...
The FASEB journal,
August 2011, 2011-Aug, 2011-08-00, 20110801, Letnik:
25, Številka:
8
Journal Article
Recenzirano
ABSTRACT
Cell surface glycans are involved in numerous physiological processes that involve cell‐cell interactions and migration, including lymphocyte trafficking and cancer metastasis. We have used ...a bioorthogonal metabolic labeling strategy to detect cell surface glycans and demonstrate, for the first time, fluorescence and radionuclide imaging of sialylated glycans in a murine tumor model in vivo. Peracetylated azido‐labeled N‐acetyl‐man‐nosamine, injected intraperitoneally, was used as the metabolic precursor for the biosynthesis of 5‐azidoneuraminic, or azidosialic acid. Azidosialic acid‐labeled cell surface glycans were then reacted, by Staudinger ligation, with a biotinylated phosphine injected intraperitoneally, and the biotin was detected by subsequent intravenous injection of a fluorescent or radiolabeled avidin derivative. At 24 h after administration of NeutrAvidin, labeled with either a far‐red fluorophore or 111In, there was a significant azido‐labeled N‐acetyl‐mannosamine‐dependent increase in tumor‐to‐tissue contrast, which was detected using optical imaging or single‐photon‐emission computed tomography (SPECT), respectively. The technique has the potential to translate to the clinic, where, given the prognostic relevance of altered sialic acid expression in cancer, it could be used to monitor disease progression.—Neves, A. A., Stöckmann, H., Harmston, R. R., Pryor, H. J., Alam, I. S., Ireland‐Zecchini, H., Lewis, D. Y., Lyons, S. K., Leeper, F. J., Brindle, K. M. Imaging sialylated tumor cell glycans in vivo. FASEB J. 25, 2528–2537 (2011). www.fasebj.org
We report the first account of metabolically labelling N-acetylglucosamine, in conjunction with either N-acetylgalactosamine or N-acetylmannosamine using a combination of isonitrile- and azide-based ...chemistries. With the appropriately labelled fluorescent probe molecules, that react with either the azido or isonitrile groups, the method enabled co-visualisation of cancer cell glycoproteins.
Abstract
Background
Reporter genes are widely used in biology and only a limited number are available. We present a new reporter gene for the localization of mammalian cells and transgenic tissues ...based on detection of the
bglA
(
SYNbglA
) gene of
Caldocellum saccharolyticum
that encodes a thermophilic β-glucosidase.
Results
SYNbglA
was generated by introducing codon substitutions to remove CpG motifs as these are associated with gene silencing in mammalian cells.
SYNbglA
expression can be localized
in situ
or detected quantitatively in colorimetric assays and can be co-localized with
E. coli
β-galactosidase. Further, we have generated a Cre-reporter mouse in which
SYNbglA
is expressed following recombination to demonstrate the general utility of
SYNbglA
for
in vivo
analyses.
SYNbglA
can be detected in tissue wholemounts and in frozen and wax embedded sections.
Conclusions
SYNbglA
will have general applicability to developmental and molecular studies
in vitro
and
in vivo
.
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