Alterations In the Bone Marrow Microenvironment Contribute to Oxidative Stress and DNA Damage In Hematopoietic Stem/Progenitors Carrying a Csf3r Truncation Mutation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 387-387
Author(s):  
Ghada M Kunter ◽  
Jill Woloszynek ◽  
Daniel C. Link
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Dna Damage ◽  
Single Dose ◽  
Bone Marrow Failure ◽  
Bone Marrow Microenvironment ◽  
Wild Type ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Increased Risk

Abstract Abstract 387 A shared feature of many bone marrow failure syndromes is their propensity to develop myelodysplasia (MDS) or acute myeloid leukemia (AML). The molecular mechanisms that underlie this susceptibility are largely unknown. Severe congenital neutropenia (SCN) is an inherited disorder of granulopoiesis that is associated with a marked increased risk of developing MDS/AML. Somatic mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), that truncate the carboxy-terminal tail are associated with the development of MDS/AML in SCN. Transgenic mice carrying a ‘knock-in’ mutation of their Csf3r (termed d715 G-CSFR) reproducing a mutation found in a patient with SCN have normal basal granulopoiesis but an exaggerated neutrophil response to G-CSF treatment. We previously reported that the d715 G-CSFR is able to cooperate with the PML-RARƒÑ oncogene to induce AML in mice. Herein, we summarize data supporting the hypothesis that alterations in the bone marrow microenvironment induced by G-CSF contribute to oxidative DNA damage in hematopoietic stem/progenitors cells (HSPCs) and possibly leukemic transformation. We previously showed that G-CSF treatment is associated with a marked loss of osteoblasts in the bone marrow, thereby potentially disrupting the osteoblast stem cell niche (Semerad, Blood 2005). Of note, patients with SCN chronically treated with G-CSF are prone to develop osteopenia, suggesting that osteoblast suppression by G-CSF also may occur in humans. We first asked whether the d715 G-CSFR was able to mediate this response. Wild-type or d715 G-CSFR were treated with G-CSF for 1–7 days and osteoblast activity in the bone marrow measured by expression of CXCL12 and osteocalcin. Consistent with previous reports, a decrease in osteocalcin and CXCL12 was not apparent until after 3 days of G-CSF treatment and reached a maximum after 7 days. Surprisingly, the magnitude of osteoblast suppression was greater in d715 G-CSFR compared with wild-type mice. The fold-decrease in osteocalcin mRNA from baseline in wild-type mice was 147 ± 70.1 versus 1,513 ± 1091 in d715 G-CSFR mice (p < 0.001). Likewise, a greater fold-decrease in CXCL12 mRNA was observed. We next assessed oxidative stress in c-KIT+ Sca+ lineage− (KSL) progenitors after G-CSF treatment. In both wild-type and d715 G-CSFR KSL cells no increase in reactive oxygen species (ROS) was observed at baseline or 12 hours after a single dose of G-CSF. However, after 7 days of G-CSF, a significant increase (3.4 ± 0.1 fold; p = 0.009) in ROS was observed in d715 G-CSFR but not wild-type KSL cells. To determine whether oxidative stress contributed to DNA damage, histone H2AX phosphorylation (pH2AX) was measured by flow cytometry. No increase in pH2AX was observed after short-term (less than 24 hour) G-CSF treatment. However, a modest but significant (1.9 ± 0.1 fold; p = 0.0007) increase in pH2AX was observed in d715 G-CSFR but not wild-type KSL cells after 7 days of G-CSF. To determine whether increased oxidative stress was casually linked to DNA damage, we co-administered the antioxidant N-acetyl cysteine (NAC) during G-CSF treatment. As expected, induction of ROS in KSL cells was markedly suppressed by NAC administration. Importantly, the increase in pH2AX levels in d715 G-CSFR KSL cells induced by G-CSF was completely blocked by NAC administration. Finally, to determine whether alterations in the bone marrow microenvironment, specifically decreased CXCL12 expression, contributed to DNA damage, we treated mice with AMD3100, a specific antagonist of CXCR4 (the major receptor for CXCL12). Treatment of wild-type or d715 G-CSFR mice with a single dose of G-CSF (3 hour time point) or with AMD3100 alone did not induce H2AXp. However, co-administration of AMD3100 with a single dose of G-CSF induced modest but significant H2AXp in d715 G-CSFR KSL cells (5.74 ± 1.06 fold; P<0.001). Collectively, these data suggest a model in which alterations in the bone marrow microenvironment induced by G-CSF may contribute to genetic instability in HSPCs and ultimately leukemic transformation. The mutant CSF3R may contribute to leukemogenesis through both increased ROS production in HSPCs and increased suppression of osteoblasts. Disclosures: No relevant conflicts of interest to declare.

Increased ROS Stress and Alterations in the Bone Marrow Microenvironment Contribute to DNA Damage in Hematopoietic Stem/Progenitors Carrying a Csf3r Truncation Mutation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3630-3630
Author(s):  
Ghada M Kunter ◽  
Jill Woloszynek ◽  
Timothy Ley ◽  
Daniel C. Link
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Conflicts Of Interest ◽  
Bone Marrow Microenvironment ◽  
Short Term ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Osteoblast Activity ◽  
Increased Risk ◽  
Truncation Mutation

Abstract Abstract 3630 Poster Board III-566 Severe congenital neutropenia (SCN) is an inherited disorder of granulopoiesis that is associated with a markedly increased risk of developing MDS/AML. Somatic mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), are associated with the development of MDS/AML in SCN. These mutations invariably produce a truncated Csf3r that, though remaining ligand-dependent, transmits a hyperproliferative signal. Knock-in mice carrying a truncation mutation of the Csf3r (termed d715 G-CSFR) have normal basal granulopoiesis but an exaggerated neutrophil response to G-CSF treatment. We recently reported that expression of the d715 G-CSFR confers a strong clonal advantage at the hematopoietic stem cell level that is dependent upon exogenous G-CSF. Though not sufficient by itself, we previously reported that the d715 G-CSFR was able to cooperate with PML-RARa to induce AML in mice. Herein, we explore mechanisms by which CSF3R truncation mutations contribute to leukemic transformation. Specifically, we test the hypothesis that altered signaling by the d715 G-CSFR contributes to genetic instability through induction of reactive oxygen species (ROS) stress. We show that the basal level of ROS in c-KIT+ Sca+ lineage− (KSL) hematopoietic stem/progenitor cells (HSPCs) was similar in cells expressing wildtype and d715 G-CSFR. However, 24 hours after in vivo G-CSF stimulation, whereas the level of ROS was not significantly changed in wildtype KSL cells, it was induced 3.4 ± 0.1 fold in d715 G-CSFR cells (p = 0.009). To determine whether this increased ROS stress contributed to DNA damage, levels of phosphorylated histone gH2AX (pH2AX) were measured by flow cytometry. In wildtype mice, short-term (24 -hour) treatment with G-CSF had no affect on pH2AX levels in KSL cells. However, in d715 G-CSFR mice, G-CSF treatment was associated with a modest but significant increase in pH2AX levels (1.9 ± 0.1 fold; p = 0.0007). We and others previously showed that prolonged (more than 3-4 days), but not short-term (24 hour), treatment with G-CSF is associated with disruption of the osteoblast niche in the bone marrow. Since, most patients with SCN are treated chronically with G-CSF, we measured osteoblast activity in the bone marrow and H2AX phosphorylation in KSL cells after 7 days of G-CSF treatment. Notably, osteoblast activity, as measured by CXCL12 and osteocalcin expression, was reduced to a greater extent in d715 G-CSFR versus wild type mice. Interestingly, compared with short-term G-CSF treatment, pH2AX levels were significantly higher in d715 G-CSFR KSL cells after 7 day G-CSF treatment (2.6 ± 0.3 fold ± SEM; p = 0.006). To establish whether induction of ROS was responsible for the increase in pH2AX, we next co-administered the antioxidant N-acetyl cysteine (NAC) during the 7-day G-CSF treatment. As expected, induction of ROS was markedly suppressed in KSL cells by NAC administration. Moreover, the increase in pH2AX levels in d715 G-CSFR KSL cells by G-CSF was completely blocked by NAC administration. Collectively, these data suggest that both increased ROS stress and altered bone marrow microenvironment may contribute to genomic instability and leukemic transformation in patients with SCN carrying a CSF3R truncation mutation. Moreover, these data raise the possibility that anti-oxidant therapy may be an effective strategy to prevent MDS/AML in SCN. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Secondary CNL after SAA reveals insights in leukemic transformation of bone marrow failure syndromes

2020 ◽  
Vol 4 (21) ◽  
pp. 5540-5546
Author(s):  
Laurent Schmied ◽  
Patricia A. Olofsen ◽  
Pontus Lundberg ◽  
Alexandar Tzankov ◽  
Martina Kleber ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Failure ◽  
Driver Mutations ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Proinflammatory Responses ◽  
Stem And Progenitor Cells

Abstract Acquired aplastic anemia and severe congenital neutropenia (SCN) are bone marrow (BM) failure syndromes of different origin, however, they share a common risk for secondary leukemic transformation. Here, we present a patient with severe aplastic anemia (SAA) evolving to secondary chronic neutrophilic leukemia (CNL; SAA-CNL). We show that SAA-CNL shares multiple somatic driver mutations in CSF3R, RUNX1, and EZH2/SUZ12 with cases of SCN that transformed to myelodysplastic syndrome or acute myeloid leukemia (AML). This molecular connection between SAA-CNL and SCN progressing to AML (SCN-AML) prompted us to perform a comparative transcriptome analysis on nonleukemic CD34high hematopoietic stem and progenitor cells, which showed transcriptional profiles that resemble indicative of interferon-driven proinflammatory responses. These findings provide further insights in the mechanisms underlying leukemic transformation in BM failure syndromes.

scholarly journals Bone Marrow Oxidative Stress and Acquired Lineage-Specific Genotoxicity in Hematopoietic Stem/Progenitor Cells Exposed to 1,4-Benzoquinone

2020 ◽  
Vol 17 (16) ◽  
pp. 5865
Author(s):  
Ramya Dewi Mathialagan ◽  
Zariyantey Abd Hamid ◽  
Qing Min Ng ◽  
Nor Fadilah Rajab ◽  
Salwati Shuib ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Dna Damage ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Protein Carbonyls ◽  
Mouse Bone Marrow ◽  
Tail Moment ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem

Hematopoietic stem/progenitor cells (HSPCs) are susceptible to benzene-induced genotoxicity. However, little is known about the mechanism of DNA damage response affecting lineage-committed progenitors for myeloid, erythroid, and lymphoid. Here, we investigated the genotoxicity of a benzene metabolite, 1,4-benzoquinone (1,4-BQ), in HSPCs using oxidative stress and lineage-directed approaches. Mouse bone marrow cells (BMCs) were exposed to 1,4-BQ (1.25–12 μM) for 24 h, followed by oxidative stress and genotoxicity assessments. Then, the genotoxicity of 1,4-BQ in lineage-committed progenitors was evaluated using colony forming cell assay following 7–14 days of culture. 1,4-BQ exposure causes significant decreases (p < 0.05) in glutathione level and superoxide dismutase activity, along with significant increases (p < 0.05) in levels of malondialdehyde and protein carbonyls. 1,4-BQ exposure induces DNA damage in BMCs by significantly (p < 0.05) increased percentages of DNA in tail at 7 and 12 μM and tail moment at 12 μM. We found crucial differences in genotoxic susceptibility based on percentages of DNA in tail between lineage-committed progenitors. Myeloid and pre-B lymphoid progenitors appeared to acquire significant DNA damage as compared with the control starting from a low concentration of 1,4-BQ exposure (2.5 µM). In contrast, the erythroid progenitor showed significant damage as compared with the control starting at 5 µM 1,4-BQ. Meanwhile, a significant (p < 0.05) increase in tail moment was only notable at 7 µM and 12 µM 1,4-BQ exposure for all progenitors. Benzene could mediate hematological disorders by promoting bone marrow oxidative stress and lineage-specific genotoxicity targeting HSPCs.

Apoptosis and the DNA Damage Response: The Role of the RB Tumor Suppressor Pathway in Oxidative Stress Responses in the Hematopoietic System.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. SCI-15-SCI-15
Author(s):  
Kay F. Macleod
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tumor Suppressor ◽  
Bone Marrow Failure ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Protein Levels ◽  
Replicative Stress

Abstract Abstract SCI-15 Exposure to pro-oxidants and defects in repair of oxidative base damage is associated with disease and aging and also contributes to the development of anemia, bone marrow failure and hematopoietic malignancies. Our work examines the role of the RB tumor suppressor pathway in the response of the hematopoietic system to oxidative stress and DNA damage. Evidence from mouse models has identified a role for the Rb protein (pRb) in the regulation of hematopoiesis through cell intrinsic functions in blood cell types but also through effects on the bone marrow microenvironment (Spike et al, 2004; Walkley et al, 2007; Daria et al, 2008). Such models have also demonstrated that pRb is required under stress conditions but not under conditions of steady state hematopoiesis (Spike et al, 2004; Spike et al, 2007; Daria et al, 2008). In particular, pRb was required to modulate the response of the hematopoietic system to replicative stress and hypoxia (Spike et al, 2007; Daria et al, 2008). To explain the mechanisms underlying these unique properties of pRb in hematopoiesis, we hypothesized that pRb protein levels are regulated by oxidative stress, including hypoxia and ROS generated as a consequence of stem cell location in the bone marrow niche or in response to replicative stress induced by agents such as 5-fluorouracil. Notably, hypoxia within the bone marrow niche has been reported to promote stem cell expansion and we postulated that this may be due to reduced pRb protein levels in response to hypoxia. We present evidence that pRb protein levels are regulated in wild-type bone marrow in response to replicative stress and that this in turn modulates expansion of stem cells and myeloid progenitors and also impacts end-stage differentiation in the erythroid lineage. Acetylation of pRb stabilized the protein in an active conformation while de-acetylation de-stabilized the protein and promoted pRb protein turnover and increased progenitor cell proliferation. We will present on-going studies that examine how hypoxia and/or ROS affects hematopoietic stem cell proliferation, self-renewal and differentiation in vivo as a function of pRb protein levels using conditional mouse models. The significance of our findings for bone marrow failure in human patients will be discussed. References Spike, B.T. et al. The Rb tumor suppressor is required for stress erythropoiesis. The EMBO J. 2004: 23, 4319-29. Spike, B.T., Dibling, B.C. & Macleod, K.F. Hypoxic stress underlies defects in erythroblast island formation in the Rb null mouse. Blood 2007; 110, 2173-81. Walkley, C.R., Shea, J.M., Sims, N.A., Purton, L.E. & Orkin, S.H. Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment. Cell 2007; 129, 1081-95. Daria, D. et al. The retinoblastoma tumor suppressor is a critical intrinsic regulator for hematopoietic stem and progenitor cells under stress. Blood 2008; 111, 1894-902. Funding: The author is grateful to the J.P. McCarthy Foundation, the Aplastic Anemia and MDS International Foundation and the National Heart Lung & Blood Institute (RO1 HL080262) for funding of work in her laboratory relating to oxidative stress, erythropoiesis and hematopoietic diseases. Disclosures No relevant conflicts of interest to declare.

Profilin 1 Is Essential for Retention and Metabolism of Hematopoietic Stem Cells in Bone Marrow

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1224-1224
Author(s):  
Junke Zheng ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Actin Binding ◽  
Wild Type ◽  
Cell Fates ◽  
Hematopoietic Stem ◽  
Multiple Cell

Abstract Abstract 1224 How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we show that, in a hematopoietic stem cell (HSC) -specific inducible knockout model, the cytoskeleton-modulating protein profilin 1 (pfn1) is essential for the maintenance of multiple cell fates and metabolism of HSCs. The deletion of pfn1 in HSCs led to bone marrow failure, loss of quiescence, increased apoptosis, and mobilization of HSCs in vivo. In reconstitution analyses, pfn1-deficient cells were selectively lost from mixed bone marrow chimeras. By contrast, pfn1 deletion did not significantly affect differentiation or homing of HSCs. When compared to wild-type cells, levels of expression of Hif-1a, EGR1, and MLL were lower and an earlier switch from glycolysis to mitochondrial respiration with increased ROS level was observed in pfn1-deficient HSCs. This switch preceded the detectable alteration of other cell fates. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that pfn1 maintained metabolism is required for the quiescence of HSCs. Furthermore, we demonstrated that expression of wild-type pfn1 but not the actin-binding deficient or poly-proline binding-deficient mutants of pfn1 rescued the defective phenotype of pfn1-deficient HSCs. This result indicates that actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Thus, pfn1 plays an essential role in regulating the retention and metabolism of HSCs in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Dlk-1 Regulates B Cell Development Through Altered Bone Marrow Stromal Microenvironment, Not by Affecting Hematopoitic Stem/Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3465-3465
Author(s):  
Edyta Pawelczyk ◽  
Heba A Degheidy ◽  
Allison L Branchaw ◽  
Kenn Holmbeck ◽  
Steven R Bauer
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Methyl Cellulose ◽  
Cell Development ◽  
B Cell Development ◽  
Bone Marrow Microenvironment ◽  
Wild Type ◽  
Mineral Density ◽  
Hematopoietic Stem

Abstract Abstract 3465 Introduction: DLK-1(delta-like 1) is a member of the EGF-like homeotic protein family whose expression is known to influence cell fate decisions through cell-cell interactions. It is also known to influence the differentiation of bone marrow stromal cells (BMSC) and hematopoietic stem cells (HSC) in bone marrow. Recently, we reported the essential role of DLK-1 in B cell development, which showed that the absence of DLK-1 led to accumulation of the earliest B cell progenitors (pre-pro B cells or Fraction A (Fr A)) in bone marrow, an altered pattern of B cell development in the spleen, and an altered humoral immune response. The objective of this study was to determine whether alterations in the HSC compartment or the BMSC microenvironment contributed to Fr A accumulation in mdlk1−/− mice. Methods: The mdlk1−/− and wild type bone marrow osteoblast and HSC compartments were analyzed by multicolor flow cytometry and in vitro methyl-cellulose colony forming cell assays. Bone marrow harvested from mdlk1−/− and wild type mice was assessed for BMSCs colony forming efficiency (CFU-F) and cultured. Supernatants from cultured BMSCs were analyzed by protein arrays. Since osteoblasts are an important component of the bone marrow microenvironment, OPN+CD45-TER119-ALP+ osteoblasts were identified in the bone marrow and quantified by flow cytometry. Finally, the femurs of mdlk1−/− and wild type mice were analyzed by micro-computed tomography (uCT) scanning. Results: Using flow cytometry, we observed no statistically significant changes in the HSC and progenitor populations in the absence of DLK-1 in mice at 4 and 16 weeks of age. The results of methyl-cellulose assay confirmed the findings of flow cytometry experiments and showed no statistically significant differences in the number of CFU-G, CFU-GM, and CFU-M of 4 and 16 week old mdlk1−/− mice as compared to wild-type control mice. However, significant alterations in the microenvironment of the mdlkl −/− were observed. CFU-F efficiency of mdlk1−/− bone marrow BMSC isolated from 4 week old mice was significantly decreased when compared to age-matched controls. Furthermore, the uCT scans showed the mineral density of the femoral bone significantly decreased in 4 week old mdlk1−/− mice and the number of osteoblast cells analyzed by flow cytometry was decreased by 10%. The analysis of BMSC supernatants revealed a striking down regulation of factors associated with osteoblast function and differentiation such as osteoactivin, PF-4, Follstatin-like 1, Frizzled-6, IGF-1, M-CSF, DKK-1 and others. Conclusions: Our results indicate that accumulation of the earliest B cell progenitors with DLK-1 ablation is the result of multiple defects in the bone marrow microenvironment including decreased CFU-F, decreased number of osteoblasts, decreased bone mineral density or alterations in factors important for osteoblast function but not from increase in numbers of hematopoietic stem or progenitors cells. Our laboratory is investigating this further. Disclosures: Pawelczyk: Baxter Inc.: currently employed by Baxter Inc. Other.

Premature Senescence in Hematopoietic Stem and Progenitor Cells in Ribosomopathy-Associated Bone Marrow Failure Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 298-298
Author(s):  
Hengjun Chao ◽  
Johnson M. Liu
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Ribosome Biogenesis ◽  
Bone Marrow Failure ◽  
Premature Senescence ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Bone Marrow Failure Syndromes ◽  
P53 Activation ◽  
Adult Bone

Abstract Introduction: Aged hematopoietic stem cells (HSCs) are known to functionally decline and are prone to development of myeloid malignancies. Recent work has highlighted the twin roles of replication stress and decreased ribosome biogenesis as drivers for the accumulation of DNA damage and senescence. Certain bone marrow failure syndromes, including Shwachman-Diamond syndrome (SDS), Diamond-Blackfan anemia (DBA), and the acquired 5q- syndrome, are characterized by defects in ribosome biogenesis. Furthermore, recent work has suggested a role for p53 activation, through the 5S ribonucleoprotein particle (RNP), in driving cells to senescence following perturbation of ribosome biogenesis. Methods and Results: Here, we have used multiplexing flow cytometry protocols to define, enumerate, and characterize hematopoietic cells of distinct differentiation stages and lineages in 2 DBA cord bloods and 4 adult bone marrows (2 SDS, 1 DBA, and 1 patient with a diminutive somatic deletion of 5q: ages 27, 32, 40, and 30, respectively), as compared with 4 normal cord bloods and 6 normal adult bone marrows. We included a patient with bona fide MDS (diminutive somatic deletion of 5q including RPS14 in a young adult) to compare with the SDS and DBA patients, who do not meet criteria for MDS. Our preliminary results revealed significant defects in the primitive HSC and multipotent progenitor (MPP) compartments in both DBA and SDS. Specifically, we found in DBA and SDS bone marrow and cord blood samples (compared to normal controls): significantly decreased numbers of primitive HSCs (Lin-CD34+CD133+CD38-CD45RA-CD49f+CD90+) and MPPs (Lin-CD34+CD133+CD38-CD45RA-CD49f-CD90-); increased levels of apoptosis and dysregulated proliferation; and G0-1/S cell cycle arrest. We also found significant increases in senescence-associated β-galactosidase staining and G0-1/S cell cycle arrest in Lin-CD34+ and Lin-CD34+CD38-CD133+ subpopulations in all 4 adult patient bone marrows, as compared with normal adult bone marrows processed in identical fashion [see Fig. 1 for representative data from Lin-CD34+CD133+ hematopoietic progenitor cells (HPCs) from one SDS patient]. Foci of the phosphorylated form of the variant histone H2AX (γH2AX) mark DNA damage, and γH2AX staining was similarly increased in comparison to controls (Fig. 1). The mechanism whereby disturbed ribosome biogenesis induces senescence has been suggested as involving 5S RNP-mediated p53 activation. However, our experiments did not demonstrate increased levels of p53 in the SDS patient marrows, as assessed by intracellular staining. Levels of p16, a well known marker of senescence, were markedly increased in the SDS patient samples, when compared to controls. Finally, in the 2 DBA cord bloods analyzed, there was increased senescence-associated β-galactosidase staining but to a lesser degree than in the adult bone marrow samples (as might be expected with temporal progression). Discussion: Taken together, our data suggest that ribosomopathies (which often present in childhood) are disorders of premature senescence. Consequent DNA damage accumulation and decreased repair and compensation may account for the development of MDS and acute myeloid leukemia, disorders seen in young ribosomopathy patients that ordinarily are rare in the general pediatric and young adult population. Disclosures No relevant conflicts of interest to declare.

Loss of Gfi1 Impedes Development of T-Cell Lymphoma upon Exposure to N-ethyl-N-nitrosourea but Predisposes to Severe Myleodysplastic Changes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2221-2221
Author(s):  
Cyrus Khandanpour ◽  
Ulrich Duehrsen ◽  
Tarik Möröy
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Cell Lymphoma ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Exogenous toxic substances often cause the initiation and development of leukemia and lymphoma by acting as mutagens. N-ethyl-N-nitrosourea (ENU) is a paradigmatic example for such a substance, which introduces point mutations in the genome through DNA damage and repair pathways. ENU is widely used to experimentally induce T-cell lymphomas in mice. We have used ENU to investigate whether the hematopoietic transcription factor Gfi1 is required for lymphomagenesis. The Gfi1 gene was originally discovered as a proviral target gene and a series of experiments with transgenic mice had suggested a role of Gfi1 as a dominant oncogene with the ability to cooperate with Myc and Pim genes in the generation of T-cell lymphoma. In addition, Gfi1 deficient mice showed a defect in T-cell maturation but also aberration in myeloid differentiation and an accumulation of myelomonocytic cells. ENU was administered i.p. once a week for three weeks with a total dose of 300mg/kg to wild type (wt) and Gfi1 null mice. Wild type mice (12/12) predominantly developed T-cell tumors and rarely acute myeloid leukemia, as expected. However, only 2/8 Gfi1 −/− mice succumbed to lymphoid neoplasia; they rather showed a severe dysplasia of the bone marrow that was more pronounced than in wt controls. These changes in Gfi1 null mice were accompanied by a dramatic decrease of the LSK (Lin-, Sca1- and c-Kit+) bone marrow fraction that contains hematopoietic stem cells and by a higher percentage (18%) of bone marrow cells, not expressing any lineage markers (CD4, CD 8, Ter 119, Mac1, Gr1, B220, CD3). In particular, we found that the LSK subpopulation of Gfi1 deficient mice showed a noticeable increase in cells undergoing apoptosis suggesting a role of Gfi1 in hematopoietic stem cell survival. In addition, Gfi1−/− bone marrow cells and thymic T-cells were more sensitive to DNA damage such as radiation and exposure to ENU than their wt counterparts pointing to a role of Gfi1 in DNA damage response. Our results indicate that Gfi1 is required for development of T-cell tumors and that a loss of Gfi1 may sensitize hematopoietic cells and possibly hematopoietic stem cells for programmed cell death. Further studies have to show whether interfering with Gfi1 expression or function might represent a tool in the therapy of leukemia.

Lentiviral FANCA Vectors Pseudotyped with a Modified Prototype Foamy Viral Envelope Corrects Murine Fanca−/− Hematopoietic Stem Cells with Reduced Toxicity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1677-1677
Author(s):  
Zejin Sun ◽  
Yanzhu Yang ◽  
Yan Li ◽  
Daisy Zeng ◽  
Jingling Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Bone Marrow Failure ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture

Abstract Fanconi anemia (FA) is a recessive DNA repair disorder characterized by congenital abnormalities, bone marrow failure, genomic instability, and a predisposition to malignancies. As the majority of FA patients ultimately acquires severe bone marrow failure, transplantation of stem cells from a normal donor is the only curative treatment to replace the malfunctioning hematopoietic system. Stem cell gene transfer technology aimed at re-introducing the missing gene is a potentially promising therapy, however, prolonged ex vivo culture of cells, that was utilized in clinical trials with gammaretroviruses, results in a high incidence of apoptosis and at least in mice predisposes the surviving reinfused cells to hematological malignancy. Consequently, gene delivery systems such as lentiviruses that allow a reduction in ex vivo culture time are highly desirable. Here, we constructed a lentiviral vector expressing the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSVG or a modified prototype foamyvirus (FV) envelope to correct Fanca−/− stem and progenitor cells in vitro and in vivo. In order to minimize genotoxic stress due to extended in vitro manipulations, an overnight transduction protocol was utilized where in the absence of prestimulation, murine Fanca−/− bone marrow cKit+ cells were co-cultured for 16h with FANCA lentivirus on the recombinant fibronectin fragment CH296. Transduction efficiency and transfer of lentivirally expressed FANCA was confirmed functionally in vitro by improved survival of consistently approximately 60% of clonogenic progenitors in serial concentrations of mitomycin C (MMC), irregardless of the envelope that was utilized to package the vector. Transduction of fibroblasts was also associated with complete correction of MMC-induced G2/M arrest and biochemically with the restoration of FancD2 mono-ubiquitination. Finally, to functionally determine whether gene delivery by the recombinant lentivirus during such a short transduction period is sufficient to correct Fanca−/− stem cell repopulation to wild-type levels, competitive repopulation experiments were conducted as previously described. Follow-up of up to 8 months demonstrated that the functional correction were also achieved in the hematopoietic stem cell compartment as evidenced by observations that the repopulating ability of Fanca−/− stem cells transduced with the recombinant lentivirus encoding hFANCA was equivalent to that of wild-type stem cells. Importantly, despite the fact that the gene transfer efficiency into cells surviving the transduction protocol were similar for both pseudotypes, VSVG was associated with a 4-fold higher toxicity to the c-kit+ cells than the FV envelope. Thus, when target cell numbers are limited as stem cells are in FA patients, the foamyviral envelope may facilitate overall greater survival of corrected stem cells. Collectively, these data indicate that the lentiviral construct can efficiently correct FA HSCs and progenitor cells in a short transduction protocol overnight without prestimulation and that the modified foamy envelope may have less cytotoxicity than the commonly used VSVG envelope.

Sign in / Sign up

Export Citation Format

Share Document