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Live and Let Die: Critical regulation of survival in normal and malignant hematopoietic stem and progenitor cells
Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology. Linköping University, Faculty of Health Sciences. (Jan-Ingvar Jönsson)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The hematopoietic stem cell (HSC) is characterized by its ability to self-renew and produce all mature blood cells throughout the life of an organism. This is tightly regulated to maintain a balance between survival, proliferation, and differentiation. The HSCs are located in specialized niches in the bone marrow thought to be low in oxygen, which is suggested to be involved in the regulation of HSC maintenance, proliferation, and migration. However, the importance of hypoxia in the stem cell niche and the molecular mechanisms involved remain fairly undefined. Another important regulator of human HSCs maintenance is the tyrosine kinase receptor FLT3, which triggers survival of HSCs and progenitor cells. Mutations in FLT3 cause constitutively active signaling. This leads to uncontrolled survival and proliferation, which can result in development of acute myeloid leukemia (AML). One of the purposes with this thesis is to investigate how survival, proliferation and self-renewal in normal HSCs are affected by hypoxia. To study this, we used both in vitro and in vivo models with isolated Lineage-Sca-1+Kit+ (LSK) and CD34-Flt3-LSK cells from mouse bone marrow. We found that hypoxia maintained an immature phenotype. In addition, hypoxia decreased proliferation and induced cell cycle arrest, which is the signature of HSCs with long term multipotential capacity. A dormant state of HSCs is suggested to be critical for protecting and preventing depletion of the stem cell pool. Furthermore, we observed that hypoxia rescues HSCs from oxidative stress-induced cell death, implicating that hypoxia is important in the bone marrow niche to limit reactive oxidative species (ROS) production and give life-long protection of HSCs. Another focus in this thesis is to investigate downstream pathways involved in tyrosine kinase inhibitor-induced cell death of primary AML cells and cell lines expressing mutated FLT3. Our results demonstrate an important role of the PI3K/AKT pathway to mediate survival signals from FLT3. We found FoxO3a and its target gene Bim to be key players of apoptosis in cells carrying oncogenic FLT3 after treatment with tyrosine kinase inhibitors. In conclusion, this thesis highlights hypoxic-mediated regulation of normal HSCs maintenance and critical effectors of apoptosis in leukemic cells expressing mutated FLT3.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2009. , 93 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1160
Keyword [en]
hematopoietic stem cells, hypoxia, self-renewal, survival, acute myeloid leukemia, FLT3
National Category
Biochemistry and Molecular Biology Hematology
Identifiers
URN: urn:nbn:se:liu:diva-52932ISBN: 978-91-7393-470-1 (print)OAI: oai:DiVA.org:liu-52932DiVA: diva2:286085
Public defence
2009-12-11, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Note

On the day of the defence date the title of article II was "Hypoxia, via hypoxia-inducible factor (HIF)-1, mediates low cell cycle activity and preserves the engraftment potential of mouse hematopoietic stem cells" and one of the authors is no longer included in the article.

Available from: 2010-01-19 Created: 2010-01-13 Last updated: 2017-01-12Bibliographically approved
List of papers
1. Hypoxia Expands Primitive Hematopoietic Progenitor Cells from Mouse Bone Marrow During In Vitro Culture and Preserves the Colony-Forming Ability
Open this publication in new window or tab >>Hypoxia Expands Primitive Hematopoietic Progenitor Cells from Mouse Bone Marrow During In Vitro Culture and Preserves the Colony-Forming Ability
2006 (English)In: Journal of Stem Cells, Vol. 1, no 4, 247-257 p.Article in journal (Refereed) Published
Abstract [en]

Self-renewal is a prerequisite for the maintenance of hematopoietic stem cells (HSCs) in the bone marrow throughout adult life. Cytokines are mainly providing pro-survival signals of HSC, whereas low oxygen levels (hypoxia) were recently shown to influence self-renewal. In contrast, the effects on other progenitor cell types is not clear. In the present work, we have analyzed whether hypoxia has any effects on mouse multipotent progenitors. When bone marrow-derived Lin-Sca1+c-kit+ (LSK) cells were kept in hypoxic cultures (1% O2 ) for 4 days together with cytokines, the numbers of colony forming high-proliferative progenitors (HPP-CFC) and precursors for cobble-stone forming cells (CAFC) were increased compared to normoxic conditions. A similar effect was seen with pre-CFCmulti from unfractionated bone marrow, whereas more committed progenitors (CFU-GM) were expanded better in normoxia compared to hypoxia. The observed increase in numbers of primitive colony-forming progenitor cells was associated with maintenance of the c-kit/Sca-1 phenotype and a preferential expansion of immature  blast-like appearing cells. The results suggest that a major function of hypoxia is to regulate differentiation by increased self-renewal. Furthermore, in cultures of limited cytokine supply, survival of the stem cell-like cell line FDCP-mix was increased during hypoxia. Thus, hypoxia allows for better survival and self-renewal of multipotent progenitors and HSCs from adult bone marrow. Such culture conditions may have beneficial clinical implications for ex vivo purposes and may improve the yields of stem cells and early progenitors.

Keyword
Hematopoiesis, Stem cells, Progenitor, Hypoxia, Survival, Self-renewal
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-17713 (URN)
Note
Original Publication: Pernilla Eliasson, Richard Karlsson and Jan-Ingvar Jönsson, Hypoxia Expands Primitive Hematopoietic Progenitor Cellsfrom Mouse Bone Marrow During In Vitro Culture and Preserves the Colony-Forming Ability, 2006, Journal of Stem Cells, (1), 4, 247-257. https://www.novapublishers.com/catalog/editorial.php?products_id=3730 Copyright: Nova Science Publishers https://www.novapublishers.com/ Available from: 2009-04-16 Created: 2009-04-16 Last updated: 2017-01-12Bibliographically approved
2. Hypoxia mediates low cell-cycle activity and increases the proportion of long-term reconstituting hematopoietic stem cells during in vitro culture
Open this publication in new window or tab >>Hypoxia mediates low cell-cycle activity and increases the proportion of long-term reconstituting hematopoietic stem cells during in vitro culture
Show others...
2010 (English)In: Experimental Hematology, ISSN 0301-472X, E-ISSN 1873-2399, Vol. 38, no 4, 301-310 p.Article in journal (Refereed) Published
Abstract [en]

Objective. Recent evidence suggests that hematopoietic stem cells (HSCs) in the bone marrow (BM) are located in areas where the environment is hypoxic. Although previous studies have demonstrated positive effects by hypoxia, its role in HSC maintenance has not been fully elucidated, neither has the molecular mechanisms been delineated. Here, we have investigated the consequence of in vitro incubation of HSCs in hypoxia prior to transplantation and analyzed the role of hypoxia-inducible factor (HIF)-1 alpha. Materials and Methods. HSC and progenitor populations isolated from mouse BM were cultured in 20% or 1% O-2, and analyzed for effects on cell cycle, expression of cyclin-dependent kinase inhibitors genes, and reconstituting ability to lethally irradiated mice. The involvement of HIF-1 alpha was studied using methods of protein stabilization and gene silencing. Results. When long-term FLT3(-)CD34(-)Lin(-)Sca-1(+)c-Kit(+) (LSK) cells were cultured in hypoxia, cell numbers were significantly reduced in comparison to normoxia. This was due to a decrease in proliferation and more cells accumulating in G(0). Moreover, the proportion of HSCs with long-term engraftment potential was increased. Whereas expression of the cyclin-dependent kinase inhibitor genes p21(cip1), p27(Kip1), and p57(Kip2) increased in LSK cells by hypoxia, only p21(cip1) was upregulated in FLT3(-)CD34(-)LSK cells. We could demonstrate that expression of p27(KiP1) and p57(Kip2) was dependent of HIF-1 alpha. Surprisingly, overexpression of constitutively active HIF-1 alpha or treatment with the HIF stabilizer agent FG-4497 led to a reduction in HSC reconstituting ability. Conclusions. Our results imply that hypoxia, in part via HIF-1 alpha, maintains HSCs by decreasing proliferation and favoring quiescence.

Place, publisher, year, edition, pages
Elsevier, 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-54780 (URN)10.1016/j.exphem.2010.01.005 (DOI)000276054300005 ()
Note

Original Publication: Pernilla Eliasson, Matilda Rehn, Petter Hammar, Peter Larsson, Oksana Sirenko, Lee A Flippin, Jorg Cammenga and Jan-Ingvar Jönsson, Hypoxia mediates low cell-cycle activity and increases the proportion of long-term reconstituting hematopoietic stem cells during in vitro culture, 2010, EXPERIMENTAL HEMATOLOGY, (38), 4, 301-310. http://dx.doi.org/10.1016/j.exphem.2010.01.005 Copyright: Elsevier Science B.V., Amsterdam. http://www.elsevier.com/

On the day of the defence date the title of this article was "Hypoxia, via hypoxia-inducible factor (HIF)-1, mediates low cell cycle activity and preserves the engraftment potential of mouse hematopoietic stem cells" and one of the authors is no longer included in the article.

When finally published online the title of this article changed name to Hypoxia mediates low cell-cycle activity and increases the proportion of long-term-reconstituting hematopoietic stem cells during in vitro culture.

Available from: 2010-04-09 Created: 2010-04-09 Last updated: 2017-01-12Bibliographically approved
3. Hypoxia rescues hematopoietic stem cells from oxidative stress-induced cell death and preserves the long-term repopulation ability
Open this publication in new window or tab >>Hypoxia rescues hematopoietic stem cells from oxidative stress-induced cell death and preserves the long-term repopulation ability
(English)Manuscript (preprint) (Other academic)
Abstract [en]

A balanced regulation of the ability of hematopoietic stem cells (HSCs) to undergo self-renewal and give rise to new blood cells is crucial for blood homeostasis. Recent studies utilizing genetically modified mice have demonstrated that reactive oxygen species (ROS) damage cellular functions and decrease the lifespan of long-term (LT) HSCs. These LT-HSCs are predominately located in a low-oxygen, or hypoxic, niche, essential for maintaining stem cell capacities. Here, we show that hypoxic culturing rescues HSCs from oxidative stress-induced cell death. Hypoxia inducible factor (HIF)-1 and its target gene pyruvate dehydrogenase kinase 1 (PDK1) were both crucial for survival and long term repopulating ability of HSCs, but less important for hypoxic resistance towards oxidative stress. Moreover, hypoxia increased the expression of Foxo3a, a transcription factor important in adaption to stress stimuli. In conclusion, hypoxia protects LT-HSCs from oxidative stress, possibly by multiple mechanisms, where Foxo3a is likely to play a central role.

Keyword
Hematopoiesis, Stem cells, Progenitor, Hypoxia, Hypoxia-inducible factor 1 alpha, oxidative stress, Puruvate dehydrogenase kinase 1
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-52941 (URN)
Available from: 2010-01-13 Created: 2010-01-13 Last updated: 2017-01-12
4. BH3-only protein Bim more critical than Puma in tyrosine kinase inhibitor-induced apoptosis of human leukemic cells and transduced hematopoietic progenitors carrying oncogenic FLT3.
Open this publication in new window or tab >>BH3-only protein Bim more critical than Puma in tyrosine kinase inhibitor-induced apoptosis of human leukemic cells and transduced hematopoietic progenitors carrying oncogenic FLT3.
Show others...
2009 (English)In: Blood, ISSN 0006-4971, Vol. 113, no 10, 2302-2311 p.Article in journal (Refereed) Published
Abstract [en]

Constitutively activating internal tandem duplications (ITD) of FLT3 (FMS-like tyrosine kinase 3) are the most common mutations in acute myeloid leukemia (AML) and correlate with poor prognosis. Receptor tyrosine kinase inhibitors targeting FLT3 have developed as attractive treatment options. Because relapses occur after initial responses, identification of FLT3-ITD–mediated signaling events are important to facilitate novel therapeutic interventions. Here, we have determined the growth-inhibitory and proapototic mechanisms of 2 small molecule inhibitors of FLT3, AG1295 or PKC412, in hematopoietic progenitor cells, human leukemic cell lines, and primary AML cells expressing FLT3-ITD. Inactivation of the PI3-kinase pathway, but not of Ras–mitogen-activated protein (MAP) kinase signaling, was essential to elicit cytotoxic responses. Both compounds induced up-regulation of proapoptotic BH3-only proteins Bim and Puma, and subsequent cell death. However, only silencing of Bim, or its direct transcriptional activator FOXO3a, abrogated apoptosis efficiently. Similar findings were made in bone marrow cells from gene-targeted mice lacking Bim and/or Puma infected with FLT3-ITD and treated with inhibitor, where loss of Puma only provided transient protection from apoptosis, but loss of Bim preserved clonal survival upon FLT3-ITD inhibition.

 

Place, publisher, year, edition, pages
Washington, D.C: The American Society of Hematology, 2009
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-52728 (URN)10.1182/blood-2008-07-167023 (DOI)
Available from: 2010-01-11 Created: 2010-01-11 Last updated: 2017-01-12

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