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| 2. |
- Kucharzewska, Paulina, et al.
(författare)
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Establishment of heparan sulphate deficient primary endothelial cells from EXT-1(flox/flox) mouse lungs and sprouting aortas.
- 2010
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Ingår i: In Vitro Cellular & Developmental Biology - Animal. - : Springer Science and Business Media LLC. - 1071-2690 .- 1543-706X. ; May 4, s. 577-584
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Tidskriftsartikel (refereegranskat)abstract
- Angiogenesis is a hallmark of expanding tissue e.g. during embryogenesis and wound healing in physiology as well as in diseases such as cancer and atherosclerosis. Key steps of the angiogenic process involve growth factor-mediated stimulation of endothelial cell sprouting and tube formation. Heparan sulphate proteoglycans (HSPGs) have been implicated as important co-receptors of several pro-angiogenic proteins. The importance of HSPGs in physiology was underscored by the finding that knockout of the gene encoding HS polymerase, EXT-1, resulted in early embryonic lethality. Here, we describe the establishment of HS-deficient endothelial cells from sprouting aortas as well as from the lungs of EXT-1(flox/flox) mice. Recombination of the loxP-flanked EXT-1 locus by Cre-expressing adenovirus was demonstrated at the mRNA level. Moreover, depletion of HS polysaccharides was verified by flow cytometry and fluorescence microscopy methodology using phage display-derived anti-HS antibodies. In summary, we provide a genetic model to unravel the functional role of HSPGs specifically in primary endothelial cells during early steps of angiogenesis. Our studies are applicable to most loxP-based transgenic mouse strains, and may thus be of general importance in the angiogenesis field.
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| 3. |
- Kucharzewska, Paulina, et al.
(författare)
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Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development.
- 2013
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 110:18, s. 7312-7317
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Tidskriftsartikel (refereegranskat)abstract
- Hypoxia, or low oxygen tension, is a major regulator of tumor development and aggressiveness. However, how cancer cells adapt to hypoxia and communicate with their surrounding microenvironment during tumor development remain important questions. Here, we show that secreted vesicles with exosome characteristics mediate hypoxia-dependent intercellular signaling of the highly malignant brain tumor glioblastoma multiforme (GBM). In vitro hypoxia experiments with glioma cells and studies with patient materials reveal the enrichment in exosomes of hypoxia-regulated mRNAs and proteins (e.g., matrix metalloproteinases, IL-8, PDGFs, caveolin 1, and lysyl oxidase), several of which were associated with poor glioma patient prognosis. We show that exosomes derived from GBM cells grown at hypoxic compared with normoxic conditions are potent inducers of angiogenesis ex vivo and in vitro through phenotypic modulation of endothelial cells. Interestingly, endothelial cells were programmed by GBM cell-derived hypoxic exosomes to secrete several potent growth factors and cytokines and to stimulate pericyte PI3K/AKT signaling activation and migration. Moreover, exosomes derived from hypoxic compared with normoxic conditions showed increased autocrine, promigratory activation of GBM cells. These findings were correlated with significantly enhanced induction by hypoxic compared with normoxic exosomes of tumor vascularization, pericyte vessel coverage, GBM cell proliferation, as well as decreased tumor hypoxia in a mouse xenograft model. We conclude that the proteome and mRNA profiles of exosome vesicles closely reflect the oxygenation status of donor glioma cells and patient tumors, and that the exosomal pathway constitutes a potentially targetable driver of hypoxia-dependent intercellular signaling during tumor development.
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| 4. |
- Kucharzewska, Paulina, et al.
(författare)
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Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells.
- 2010
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 316, s. 2683-2691
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Tidskriftsartikel (refereegranskat)abstract
- The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by alpha-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo.
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| 5. |
- Kucharzewska, Paulina, et al.
(författare)
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The polyamines regulate endothelial cell survival during hypoxic stress through PI3K/AKT and MCL-1.
- 2009
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 380:2, s. 413-418
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Tidskriftsartikel (refereegranskat)abstract
- Hypoxia-dependent angiogenesis is an inherent feature of solid tumors, and a better understanding of the molecular mechanisms of hypoxic cell-death should provide additional targets for cancer therapy. Here, we show a novel role of the polyamines in endothelial cell (EC) survival during hypoxia. Polyamine depletion by specific inhibition of ornithine decarboxylase was shown to protect ECs from hypoxia-induced apoptosis. Inhibition of the polyamines resulted in a significant induction of PI3K/AKT and its down-stream target MCL-1, i.e. an anti-apoptotic member of the BCL-2 family. Specific inhibitors of PI3K reversed the decrease of hypoxia-induced apoptosis as well as the induction of MCL-1 in polyamine-deprived cells. Moreover, siRNA-mediated down-regulation of MCL-1 was found to counter-act the protective effect of polyamine inhibition. We conclude that the polyamines regulate hypoxia-induced apoptosis in ECs through PI3K/AKT and MCL-1 dependent pathways. Our results may have important implications for the modulation of hypoxia-driven neovascularization.
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| 6. |
- Magzoub, Mazin, et al.
(författare)
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N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis
- 2006
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 348:2, s. 379-385
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Tidskriftsartikel (refereegranskat)abstract
- A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1–24) and a basic domain (KKRPKP, residues 25–30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp–DNA–gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide’s ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases.
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| 7. |
- Mani, Katrin, et al.
(författare)
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HIV-Tat protein transduction domain specifically attenuates growth of polyamine deprived tumor cells.
- 2007
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Ingår i: Molecular Cancer Therapeutics. - 1538-8514. ; 6:2, s. 782-788
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Tidskriftsartikel (refereegranskat)abstract
- Polyamines are essential for tumor cell growth, and the polyamine pathway represents an attractive target for cancer treatment. Several polyamine transport proteins have been cloned and characterized in bacteria and yeast cells; however, the mechanism of polyamine entry into mammalian cells remains poorly defined, although a role for proteoglycans has been suggested. Here, we show that the HIV-Tat transduction peptide, which is known to enter cells via a proteoglycan-dependent pathway, efficiently inhibits polyamine uptake. Polyamine uptake–deficient mutant cells with intact proteoglycan biosynthesis (CHO MGBG) displayed unperturbed HIV-Tat uptake activity compared with wild-type cells, supporting the notion that HIV-Tat peptide interferes with polyamine uptake via competition for proteoglycan binding sites rather than a putative downstream transporter. HIV-Tat specifically inhibited growth of human carcinoma cells made dependent on extracellular polyamines by treatment with the polyamine biosynthesis inhibitor {alpha}-difluoromethylornithine; accordingly, the Tat peptide prevented intracellular accumulation of exogenous polyamines. Moreover, combined treatment with {alpha}-difluoromethylornithine and HIV-Tat efficiently blocked tumor growth in an experimental mouse model. We conclude that HIV-Tat transduction domain and polyamines enter cells through a common pathway, which can be used to target polyamine-dependent tumor growth in the treatment of cancer.
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| 8. |
- Svensson, Katrin, et al.
(författare)
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Hypoxia-mediated induction of the polyamine system provides opportunities for tumor growth inhibition by combined targeting of vascular endothelial growth factor and ornithine decarboxylase.
- 2008
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Ingår i: Cancer Research. - 1538-7445. ; 68:22, s. 9291-9301
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Tidskriftsartikel (refereegranskat)abstract
- Hypoxia is a hallmark of solid tumors, which may offer opportunities for targeted therapies of cancer; however, the mechanisms that link hypoxia to malignant transformation and tumor progression are not fully understood. Here, we show that up-regulation of the polyamine system promotes cancer cell survival during hypoxic stress. Hypoxia was found to induce polyamine transport and the key enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), in a variety of cancer cell lines. Increased ODC protein expression was shown in hypoxic, GLUT-1-expressing regions of tumor spheroids and experimental tumors, as well as in clinical tumor specimens. Hypoxic induction of the polyamine system was dependent on antizyme inhibitor (i.e., a key positive regulator of ODC and polyamine transport), as shown by RNA interference experiments. Interestingly, depletion of the polyamines during hypoxia resulted in increased apoptosis, which indicates an essential role of the polyamines in cancer cell adaptation to hypoxic stress. These results were supported by experiments in an in vivo glioma tumor model, showing significantly enhanced antitumor effects of the antiangiogenic, humanized anti-vascular endothelial growth factor (VEGF) antibody bevacizumab when used in combination with the well-established, irreversible inhibitor of ODC, alpha-difluoromethylornithine. Our results provide important insights into the hypoxic stress response in malignant cells and implicate combined targeting of VEGF and ODC as an alternative strategy to treat cancer disease.
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| 9. |
- Welch, Johanna E, et al.
(författare)
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Single chain fragment anti-heparan sulfate antibody targets the polyamine transport system and attenuates polyamine-dependent cell proliferation.
- 2008
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Ingår i: International Journal of Oncology. - 1019-6439. ; 32:4, s. 749-756
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Tidskriftsartikel (refereegranskat)abstract
- The growth-promoting polyamines are polybasic compounds that efficiently enter cancer cells by as yet incompletely defined mechanisms. Strategies to inhibit their internalization may have important implications in the management of tumor disease. Here, we show that cellular binding and uptake of polyamines are inhibited by a single chain variable fragment anti-heparan sulfate (HS) antibody. Polyamine uptake was inhibited in a dose-dependent manner, and was associated with compensatory up-regulation of ornithine decarboxylase (ODC), i.e. the key enzyme of the polyamine biosynthesis pathway. Conversely, depletion of intracellular polyamines by the specific ODC-inhibitor alpha-difluoromethylornithine (DFMO) resulted in increased cellular binding of polyamine and anti-HS antibody. Importantly, anti-HS antibody also efficiently targeted DFMO-induced polyamine uptake, and combined polyamine biosynthesis inhibition by DFMO, and uptake inhibition by anti-HS antibody attenuated tumor cell proliferation in vitro. In conclusion, cell-surface HS proteoglycan is a relevant target for antibody-mediated inhibition of the uptake of polyamines, and polyamine-dependent cell proliferation.
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| 10. |
- Welch, Johanna, et al.
(författare)
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Heparan sulfate proteoglycan-mediated polyamine uptake.
- 2011
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 720, s. 327-338
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Tidskriftsartikel (refereegranskat)abstract
- The polyamines are polycationic compounds essential for cellular proliferation and transformation. In addition to a well-defined biosynthesis pathway, polyamines are internalized into cells by as yet incompletely defined mechanisms. Numerous reports have shown that efficient polyamine uptake depends on the presence of polyanionic, cell surface-associated heparan sulfate proteoglycans (HSPGs). In this chapter, we provide protocols for studying HSPG-mediated uptake of polyamines in various cell lines, and provide instructions for the use of two different genetic models of HSPG deficiency. We describe the enzymatic reduction of cell surface HSPG through Heparinase III lyase treatment as well as the use of phage display-derived single chain variable fragment (scFv) anti-HS antibodies to block HSPGs at the cell surface. Finally, we provide a protocol for the quantitative verification of loss or reduction of cell surface HSPGs and a detailed description of polyamine uptake measurement.
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