AKT1 and AKT2 maintain hematopoietic stem cell function by regulating reactive oxygen species. Blood

Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Blood (Impact Factor: 10.45). 03/2010; 115(20):4030-8. DOI: 10.1182/blood-2009-09-241000
Source: PubMed


Although AKT is essential for multiple cellular functions, the role of this kinase family in hematopoietic stem cells (HSCs) is unknown. Thus, we analyzed HSC function in mice deficient in the 2 isoforms most highly expressed in the hematopoietic compartment, AKT1 and AKT2. Although loss of either isoform had only a minimal effect on HSC function, AKT1/2 double-deficient HSCs competed poorly against wild-type cells in the development of myeloid and lymphoid cells in in vivo reconstitution assays. Serial transplantations revealed an essential role for AKT1 and AKT2 in the maintenance of long-term HSCs (LT-HSCs). AKT1/2 double-deficient LT-HSCs were found to persist in the G(0) phase of the cell cycle, suggesting that the long-term functional defects are caused by increased quiescence. Furthermore, we found that the intracellular content of reactive oxygen species (ROS) is dependent on AKT because double-deficient HSCs demonstrate decreased ROS. The importance of maintaining ROS for HSC differentiation was shown by a rescue of the differentiation defect after pharmacologically increasing ROS levels in double-deficient HSCs. These data implicate AKT1 and AKT2 as critical regulators of LT-HSC function and suggest that defective ROS homeostasis may contribute to failed hematopoiesis.

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    • "Although low levels of ROS production are required for stem cells to undergo self-renewing proliferation and proper differentiation (Ezashi et al., 2005; Juntilla et al., 2010; Kinder et al., 2010; Lewandowski et al., 2010; Owusu-Ansah & Banerjee, 2009; Sauer & Wartenberg, 2005), increased production of ROS is detrimental to stem cells and has been implicated in the pathogenesis of many pathological conditions and diseases by causing damage to stem cells. The pathological effects of ROS on stem cells are dose-dependent. "
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    ABSTRACT: Reactive oxygen species (ROS) play an important role in determining the fate of normal stem cells. Low levels of ROS are required for stem cells to maintain quiescence and self-renewal. Increases in ROS production cause stem cell proliferation/differentiation, senescence, and apoptosis in a dose-dependent manner, leading to their exhaustion. Therefore, the production of ROS in stem cells is tightly regulated to ensure that they have the ability to maintain tissue homeostasis and repair damaged tissues for the life span of an organism. In this chapter, we discuss how the production of ROS in normal stem cells is regulated by various intrinsic and extrinsic factors and how the fate of these cells is altered by the dysregulation of ROS production under various pathological conditions. In addition, the implications of the aberrant production of ROS by tumor stem cells for tumor progression and treatment are also discussed.
    Full-text · Article · Jun 2014 · Advances in Cancer Research
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    • "Up-regulation of the PI3K-Akt-mTOR (PI3K-Akt-mammalian target of rapamycin) pathway occurs via mutations in surface receptors like FLT3, c-Kit or by mutations in the genes encoding pathway constituents like PI3K, PTEN or Akt [7,8] . Akt is a serine/threonine protein kinase that exists in three conserved isoforms: Akt 1, 2 and 3. Of the three iso-forms present, Akt 1 and 2 are expressed to a higher extent in hematopoietic stem cells [9]. Akt is phosphorylated at Thr 308 by up-stream phosphoinositide-dependent protein kinase 1 (PDK-1) and at Ser 473 by mTOR complex 2 (mTORC2). "
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    ABSTRACT: Background Overall cure rates in acute myeloid leukemia (AML) continue to range between 60-65% with disease relapse being a major cause of mortality. The PI3K-Akt-mTOR kinase pathway plays a vital role in pro-survival signals within leukemic cells and inhibition of this pathway is being investigated to improve patient outcomes. Tracking activation of multiple signaling proteins simultaneously in patient samples can be challenging especially with limiting cell numbers within rare sub-populations. Methods The NanoPro 1000 system (ProteinSimple) is built on an automated, capillary-based immunoassay platform and enables a rapid and quantitative analysis of specific proteins and their phosphorylation states. We have utilized this nano-immunoassay to examine activation of Akt 1/2/3 and downstream mTOR target - eukaryotic initiation factor 4E-Binding Protein 1 (4EBP1). Results Assays for Akt 1/2/3 and 4EBP1 were standardized using AML cell lines (MV4-11, MOLM-14, OCI-AML3 and HL-60) prior to testing in patient samples. Target inhibition was studied using mTOR 1/2 inhibitor AZD-8055 and results were corroborated by Western blotting. The assay was able to quantify nanogram amounts of 4EBP1 and Akt 1/2/3 in AML cell lines and primary pediatric AML samples and results were quantifiable, consistent and reproducible. Conclusion Our data provides a strong basis for testing this platform on a larger scale and our long term aim is to utilize this nano-immunoassay prospectively in de-novo AML to be able to identify poor responders who might benefit from early introduction of targeted therapy.
    Full-text · Article · Jun 2014 · Journal of Translational Medicine
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    • "However, a significant alteration was found in the phosphorylation of the protein AKT, which was inhibited in the total HL-60 population (Figure 4C) but not in LSC (Figure 4E). This result is in agreement with previous studies showing that AKT is necessary for the survival of both normal [38] and tumor cells [39] from hematopoietic system. In addition, it is possible that that together with the maintenance of p-AKT level in LSC, other signaling proteins are playing a prosurvival role in these cells, e.g. the participation of p38MAPK which has been shown to be involved in the regulation of cell checkpoints for the promotion of cancer cells survival [40,41]. "
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    ABSTRACT: Over the last few years, studies have suggested that oxidative stress plays a role in the regulation of hematopoietic cell homeostasis. In particular, the effects of hydrogen peroxide (H2O2) range from hematopoietic cell proliferation to cell death, depending on its concentration in the intracellular milieu. In this work, we evaluated the effects of an oxidative environment on normal and leukemic hematopoietic cells by stimulating normal human (umbilical cord blood) and murine (bone marrow) hematopoietic cells, as well as human myeloid leukemic cells (HL-60 lineage), upon H2O2 stimulus. Total cell populations and primitive subsets were evaluated for each cell type. H2O2 stimulus induces HL-60 cell death, whereas the viability of human and murine normal cells was not affected. The effects of H2O2 stimulus on hematopoietic stem/progenitor cell subsets were examined and the normal primitive cells were found to be unaffected; however, the percentage of leukemic stem cells (LSC) increased in response to H2O2, while clonogenic ability of these cells to generate myeloid clones was inhibited. In addition, H2O2 stimulus caused a decrease in the levels of p-AKT in HL-60 cells, which most likely mediates the observed decrease of viability. In summary, we found that at low concentrations, H2O2 preferentially affects both the LSC subset and total HL-60 cells without damage normal cells.
    Full-text · Article · Dec 2013 · Cancer Cell International
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