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Hypoxia inducible factor 1 alpha: dependent and independent regulation of hematopoietic stem cells and leukemia
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis has studied the role of low oxygen levels, or hypoxia, in hematopoietic stem cells (HSCs) and how, at the molecular level, it regulates stem cell maintenance and protects against oxidative stress induced by reactive oxygen species (ROS). HSCs reside within the bone marrow in specific niches created by a unique vascularized environment, which is suggested to be hypoxic and crucial for HSCs by maintaining a quiescent state of cell cycle and by redirecting metabolism away from the mitochondria to glycolysis. The niches are also believed to limit the production of ROS, which could damage DNA and disrupt the stem cell features. The hypoxia-responsive protein hypoxia-inducible factor 1 alpha (HIF-1α) is a major regulator of the hypoxic cell response in HSCs as well as in leukemic stem cells. Both these cells are thought to reside in the bone marrow where they are protected from stress and chemotherapy by niche cells and hypoxia.

The thesis demonstrates that pyruvate dehydrogenase kinase 1 regulates a metabolic shift to glycolysis, and maintains the engraftment potential of both HSCs and multipotent progenitors upon transplantation. Furthermore, we wanted to determine whether HIF-1α or other signaling pathways are involved in protecting HSCs from ROS-induced cell death. Overexpression, silencing or a knockout mouse model of Hif-1α could not identify HIF-1α as important for protecting HSCs from oxidative stress-induced cell death through inhibition of synthesis of the antioxidant glutathione. Gene expression analysis instead identified the transcription factor nuclear factor kappa B (NF-κB) as induced by hypoxia. By studying NF- κB signaling we found increased NF-κB activity in cells cultured in hypoxia compared to normoxia. Suppression of inhibitor of kappa B indicated a putative role of NF-κB signaling in hypoxia-induced protection against oxidative stress. The findings show that hypoxia-induced protection to elevated levels of ROS upon glutathione depletion seems to be attributed to activation of the NF-κB signaling pathway independently of HIF-1α.

To address the question whether hypoxic in vitro cultures support maintenance and promote HSC expansion we performed a limited dilution-transplantation assay. Our data indicate that hypoxic cultures maintain more long-term-reconstituting HSCs than normoxia, but this could not be confirmed statistically. Finally, we wanted to study the mechanisms by which hypoxia protect against chemotherapy. We could demonstrate that hypoxic culture protects leukemic cell lines against apoptosis induced by chemotherapy or inhibitors used for treatment of leukemia. This multidrug resistance seems to be mediated by ATP-binding cassette transporter genes, which are upregulated by hypoxia and whose inhibition has been shown to increase chemosensitivity. In addition, HIF-1α was upregulated in the leukemic cell lines in hypoxia and its inhibition increased the sensitivity to chemotherapy, indicating a role in inducing chemotherapy resistance.

Conclusively, the results presented in this thesis stress the importance of hypoxia in regulating metabolism, oxidative-stress response and maintenance of both HSCs as well as leukemic cells, especially through the critical transcription factors HIF-1α and NF-κB and their target genes.  

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2018. , p. 71
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1643
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-152129DOI: 10.3384/diss.diva-152129ISBN: 9789176852101 (print)OAI: oai:DiVA.org:liu-152129DiVA, id: diva2:1256752
Public defence
2018-12-14, Hasselquistsalen, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2018-10-29 Created: 2018-10-18 Last updated: 2018-11-27Bibliographically approved
List of papers
1. Pyruvate dehydrogenase kinase 1 is essential for transplantable mouse bone marrow hematopoietic stem cell and progenitor function
Open this publication in new window or tab >>Pyruvate dehydrogenase kinase 1 is essential for transplantable mouse bone marrow hematopoietic stem cell and progenitor function
2017 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 2, article id e0171714Article in journal (Refereed) Published
Abstract [en]

Background Accumulating evidence suggests that hypoxic areas in the bone marrow are crucial for maintenance of hematopoietic stem cells (HSCs) by supporting a quiescent state of cell cycle and regulating the transplantation capacity of long-term (LT)-HSCs. In addition, HSCs seem to express a metabolic profile of energy production away from mitochondrial oxidative phosphorylation in favor of glycolysis. At oxygen deprivation, hypoxia inducible factor 1 alpha (HIF-1 alpha) is known to induce glycolytic enzymes as well as suppressing mitochondrial energy production by inducing pyruvate dehydrogenase kinase 1 (Pdk1) in most cell types. It has not been established whether PDK1 is essential for HSC function and mediates hypoxia-adapting functions in HSCs. While the Pdk gene family contains four members (Pdk1-4), it was recently shown that Pdk2 and Pdk4 have an important role in regulating LT-HSCs. Principle findings Here we demonstrate that PDK1 activity is crucial for transplantable HSC function. Whereas Pdkl, Pdk2, and Pdk3 transcripts were expressed at higher levels in different subtypes of HSCs compared to differentiated cells, we could not detect any major differences in expression between LT-HSCs and more short-term HSCs and multipotent progenitors. When studying HIF-1 alpha-mediated regulation of Pdk activity in vitro, Pdk1 was the most robust target regulated by hypoxia, whereas Pdk2, Pdk3, and Pdk4 were not affected. Contrary, genetic ablation in a cre-inducible Hif-1 alpha knockout mouse did not support a link between HIF-1 alpha and Pdk1. Silencing of Pdk1 by shRNA lentiviral gene transfer partially impaired progenitor colony formation in vitro and had a strong negative effect on both long-term and short-term engraftment in mice. Conclusions Our study demonstrates that PDK1 has broad effects in hematopoiesis and is a critical factor for engraftment of both HSCs and multipotent progenitors upon transplantation to recipient mice. While Pdk1 was a robust hypoxia-inducible gene mediated by HIF-1 alpha in vitro, we could not find evidence of any in vivo links between Pdk1 and HIF-1 alpha.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2017
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-136062 (URN)10.1371/journal.pone.0171714 (DOI)000394231800095 ()28182733 (PubMedID)
Note

Funding Agencies|Swedish Research Council; Swedish Cancer Society; Swedish Childhood Cancer Foundation; County Council of Ostergotland; Faculty of Medicine at Linkoping University; Ollie and Elof Ericssons Foundation

Available from: 2017-03-27 Created: 2017-03-27 Last updated: 2018-10-18
2. Letter: Hypoxic and normoxic in vitro cultures maintain similar numbers of long-term reconstituting hematopoietic stem cells from mouse bone marrow
Open this publication in new window or tab >>Letter: Hypoxic and normoxic in vitro cultures maintain similar numbers of long-term reconstituting hematopoietic stem cells from mouse bone marrow
2012 (English)In: Experimental Hematology, ISSN 0301-472X, E-ISSN 1873-2399, Vol. 40, no 11, p. 879-881Article in journal, Letter (Other academic) Published
Abstract [en]

n/a

Place, publisher, year, edition, pages
Elsevier, 2012
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-85627 (URN)10.1016/j.exphem.2012.07.005 (DOI)000310182400001 ()
Available from: 2012-11-26 Created: 2012-11-26 Last updated: 2018-10-18

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Halvarsson, Camilla

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