scholarly journals A Distinctive DNA Damage Response in Human Hematopoietic Stem Cells Reveals an Apoptosis-Independent Role for p53 in Self-Renewal

2010 ◽  
Vol 7 (2) ◽  
pp. 186-197 ◽  
Author(s):  
Michael Milyavsky ◽  
Olga I. Gan ◽  
Magan Trottier ◽  
Martin Komosa ◽  
Ofer Tabach ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Dna Damage Response ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Damage Response

scholarly journals Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells

2014 ◽  
Vol 206 (4) ◽  
pp. 2064OIA143
Author(s):  
Cesare Lancini ◽  
Paul C.M. van den Berk ◽  
Joseph H.A. Vissers ◽  
Gaetano Gargiulo ◽  
Ji-Ying Song ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Dna Damage Response ◽  
Hematopoietic Stem ◽  
Functional Integrity ◽  
Damage Response

scholarly journals DNA-damage response gene GADD45A induces differentiation in hematopoietic stem cells without inhibiting cell cycle or survival

Stem Cells ◽  
2016 ◽  
Vol 34 (3) ◽  
pp. 699-710 ◽  
Author(s):  
Susanne Wingert ◽  
Frederic B. Thalheimer ◽  
Nadine Haetscher ◽  
Maike Rehage ◽  
Timm Schroeder ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Dna Damage Response ◽  
Response Gene ◽  
Hematopoietic Stem ◽  
Damage Response

Distinct Roles of CHK2 and Cell Death in Suppressing Oncogenic Potential of Tandem DNA Breaks in Human Hematopoietic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2659-2659
Author(s):  
Shahar Biechonski ◽  
Muhammad Yassin ◽  
Nasma Aqaqe ◽  
Leonid Olender ◽  
Melanie Rall ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Cell Death ◽  
Hematopoietic Stem Cells ◽  
Dna Damage Response ◽  
Parp Cleavage ◽  
Hematopoietic Stem ◽  
Damage Response ◽  
Induced Apoptosis ◽  
Dna Ends

Abstract DNA double strand breaks (DSBs) are the most dangerous genomic lesions that can be induced by endogenous and exogenous sources. DNA damage response determines cellular fate decisions following DSBs and can lead to cell death or cell survival. Incorrect DSB repair via canonical Non-Homologous End Joining (cNHEJ) or Alternative NHEJ (Alt-NHEJ) is the main source of oncogenic aberrations, including leukemogenic translocations, DNA sequence deletions and insertions. The long life span of Hematopoietic Stem Cells (HSC) and their practically unlimited potential for self-renewal requires efficient strategies to cope with DNA damage to eliminate erroneous genetic information inheritance to daughter cells. Although the critical importance of maintaining genome integrity for normal hematopoiesis and prevention of leukemogenesis has been established, definitive analysis of DNA damage response and its mutagenic outcomes in human HSC and Progenitors in response to DSBs is missing. Here we repot that human cord blood purified HSC (defined as CD34+CD38-CD45RA-) are exquisitely sensitive to irradiation (IR)-induced apoptosis in contrast to committed progenitors (defined as CD34+CD38+) as validated by PARP cleavage induction. Interestingly, pan-caspase inhibitor Z-VAD-FMK prevented, whereas CHK2 inhibitor (PV1019) failed in altering apoptosis onset of irradiated HSC. Strikingly, CHK2 inhibitor blocked IR-induced apoptosis in cycling HSC, suggesting differential wiring of DNA damage induced apoptosis in quiescent versus mitogenically stimulated HSC. To characterize cNHEJ repair pathway and its mutagenic potential in live primitive hematopoietic cells we analyzed I-SceI endonuclease induced tandem DSBs joining capacity using DNA repair reporter assay. We found that HSC exhibit inferior cNHEJ capacity as compared with committed progenitors. By decreasing DSBs persistence we revealed that progenitors utilize to the higher degree than HSC the mutagenic component of cNHEJ pathway that results in DNA deletions. We identified HSC-specific contribution of CHK2 kinase activity in limiting incorrect DNA ends joining. Blockade of apoptosis induction also led to the selective increase in mutagenic NHEJ in HSC. On the other hand, inhibition of DNA-PK led to increased oncogenic repair in progenitors only. Importantly, we revealed that HSC utilized mutagenic Alt-NHEJ pathway that depends on microhomologies search and extensive DNA ends processing less efficiently than Progenitors. Thus, our results indicate that oncogenic consequences of DSBs repair in HSC are distinctly minimized by the non-redundant cell death and CHK2 dependent mechanisms. More broadly, these findings will help to elucidate additional repair modifiers and the mechanism by which HSC contend with genotoxic stress. Disclosures No relevant conflicts of interest to declare.

The Splicing Factor Heterogeneous Nuclear Ribonucleoprotein L (hnRNPL) Restricts p53 Dependent and p53 Independent Cell Death Pathways In Hematopoietic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2445-2445
Author(s):  
Marie-Claude Gaudreau ◽  
Damien Grapton ◽  
Florian Heyd ◽  
Charles Vadnais ◽  
Brian T Wilhelm ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alternative Splicing ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Effector Genes ◽  
Damage Response ◽  
Nuclear Ribonucleoprotein

Abstract Hematopoiesis is sustained by a pool of multipotent hematopoietic stem cells (HSCs) that have the capacity to differentiate into cells of all blood cell lineages. The pool of long-lived HSCs is maintained throughout life by the self-renewal ability of HSCs. New evidence suggests the process of alternative splicing is an important regulator of the maturation and activation of blood and immune effector cells. It is presently estimated that almost all multi-exon genes in human genome undergo alternative pre-mRNA splicing, and aberrant splicing has been linked to a variety of human pathologies. However, the role that pre-mRNA splicing may have for HSCs behaviour remains largely unexplored. Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is an RNA-binding protein that regulates alternative splicing by binding exonic splicing silencers elements (ESS) resulting in exon exclusion from the mature mRNA. RT-PCR analyses showed that hnRNPL is expressed in early stages of hematopoiesis including HSCs and lineage restricted hematopoietic progenitors. To test the role of hnRNPL in hematopoietic differentiation, we have generated conditional deficient mice, since a constitutive deletion of hnRNPL results in early embryonic lethality. Animals carrying two hnRNPL-floxed alleles (hnRNPLfl/fl) can be deleted at adult stage by the pIpC inducible MxCre transgene or by the VavCre transgene, which is expressed in all hematopoietic cells starting at embryonic stage E14. VavCre+hnRNPLfl/fl mice were not viable and did not progress further in their development than embryonic stage E17.5 and ablation of hnRNPL by pIpC injection caused a high rate of mortality in adult MxCre+hnRNPLfl/fl mice compared to control animals. Both the fetal liver (FL) of VavCre+hnRNPLfl/fl mice and the bone marrow (BM) of adult MxCre+hnRNPLfl/fl mice had a significantly reduced cellularity. Furthermore, flow cytometric analysis revealed in both FL and BM a significant reduction in frequency and absolute numbers of all mature blood cells, the lymphoid and myeloid precursors, CLPS, CMPs and GMPs and to a lesser extent the erythroid/megakaryocytic precursors (MEPs). Methylcellulose and both competitive and non-competitive transplantation assays demonstrated that HSCs lacking hnRNPL cannot generate lineage-committed progenitors and have lost their self-renewal capacity and reconstitution potential. A genome-wide analysis of mRNA expression and splicing through next-generation RNA sequencing of wild-type (WT) or VavCre+hnRNPLfl/fl E14.5 Lin- c-kit+ fetal liver cells (FLCs) revealed that hnRNPL deficiency affects not only alternative splicing but also gene expression levels in hematopoietic progenitors. In the absence of hnRNPL, genes implicated in regulating apoptosis, DNA damage response and cell division where found up-regulated in Lin- c-kit+ FLCs. Among those genes, many were p53 effector genes such as Cdkn1a, Ccng1, Trp53inp1, TrailR2, Bax and Zmat3. In addition genes that are known to be required for normal hematopoiesis and HSCs functions such as Gfi1, CD34, Csfr1, Egr1 and Runx1 were found down-regulated in those cells. Further analyses by qPCR and Western blots confirmed those findings and also showed that the level of p53 protein expression was upregulated in VavCre+hnRNPLfl/fl FLCs although the mRNA level is the same as in the WT cells suggesting that hnRNPL affects p53 mRNA translation efficiency. Similarly, several genes found differentially spliced are implicated in cell cycle progression or required for normal hematopoiesis in FL such as Bcl11a, Cdk4, Ccnd2 and TRP53bp1. These results together with an increased level of Reactive Oxygen Species (ROS) and elevated levels of phosphorylated histone H2AX (γ-H2AX, a sensor for double strand DNA breaks) suggest that hnRNPL regulates the activation of a p53 dependent DNA damage response pathway in hematopoietic stem cells. As a consequence loss of hnRNPL results in a loss of hematopoietic stem and progenitor cells. Our data also suggest that hnRNPL does not only regulate alternative splicing but also expression levels of a set of specific effector genes involved in HSC survival, proliferation, ultimately affecting self-renewal. Disclosures: No relevant conflicts of interest to declare.

p53 Co-Activator Aspp1 Induces Apoptosis in Damaged Hematopoietic Stem Cells and Prevents Malignant Transformation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 603-603 ◽  
Author(s):  
Masayuki Yamashita ◽  
Eriko Nitta ◽  
Toshio Suda
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Hematological Malignancies ◽  
Aberrant Methylation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Apoptotic Genes

Abstract Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Mutation accumulation in HSCs precedes the development of leukemia and lymphoma, and these “pre-leukemic HSCs” can survive after chemotherapy, contributing to the relapse of the disease. Thus, understanding for the DNA damage response at a HSC level is a matter of critical importance for lifelong hematopoiesis, yet the protection mechanism for HSCs from DNA damage accumulation remains to be elucidated. During our study on the response of HSCs to ionizing radiation (IR), we have detected higher responsiveness of HSCs to DNA damage compared with committed progenitor cells: higher p53 activation was observed in HSC-enriched LSK (Lin-Sca1+cKit+) cells and LT-HSCs (CD150+CD41-CD48-LSK) than in myeloid progenitor-enriched LKS- cells. Of note, when treated with 4 Gy IR, LSK cells exhibited stronger upregulation of pro-apoptotic genes Bax, Noxa and Puma compared with LKS- cells, whereas upregulation of survival-contributing p21 and Mdm2 genes was comparable between the two populations. Corresponding to such characteristic behavior, we have identified apoptosis-stimulating protein of p53 1 (Aspp1) as a novel specific regulator of HSCs that provides HSCs with high sensitivity to apoptosis. We found that mRNA and protein of Aspp1 were specifically detected in LSK cells and LT-HSCs. To uncover the roles of Aspp1 in the regulation of HSCs, we evaluated HSCs of adult Aspp1 knockout (KO) mice. These mutant mice exhibited a major increase in the absolute number of LSK cells (1.5-fold; P<0.05) and LT-HSCs (2-fold; P<0.0005). Furthermore, self-renewal capacity of Aspp1-null HSCs was significantly enhanced as measured by serial competitive bone marrow (BM) transplantation assays (P<0.01). To assess the cause of enhanced self-renewal of Aspp1-null HSCs, we examined gene expression profile of Aspp1-null LSK cells before and after BM transplantation using multiplex quantitative RT-PCR array. Aspp1-null LSK cells showed higher expression of multiple quiescence-related genes including Tek, Mpl and Ndn. In line with this, Ki67 staining revealed that Aspp1-null LSK cells showed resistance to the loss of quiescence after serial BM transplantation (P<0.01), and Aspp1 KO mice showed accelerated recovery of peripheral blood and BM when treated with a single dose of 5-FU (P<0.05). Moreover, when serially transplanted or subjected to 4 Gy IR in vivo, Aspp1-null LSK cells exhibited higher resistance to apoptosis which was detected as decreased proportion of Annexin V-positive cells (P<0.05). Gene expression analysis consistently revealed that the induction of pro-apoptotic genes Bax, Noxa and Puma was impaired in irradiated Aspp1-null LSK cells. As a result of the reduced apoptosis, Aspp1-null LSK cells exhibited the tendency to retain persistent DNA damage after genotoxic stress as assessed by γH2AX and 53BP1 foci (chi-square test, P<0.05). Importantly, by breeding Aspp1 KO mice with Mx1-Cre mice and p53flox/flox mice, we verified that Aspp1 synergized with p53 to regulate self-renewal and genomic integrity of HSCs beyond its canonical p53-dependent function. Aspp1 loss further enhanced self-renewal capacity of HSCs in a p53-null background when assayed by serial BM transplantation (P<0.05). Likewise, Aspp1 deficiency further accentuated the accumulation of DNA damage after IR exposure in the absence of p53 (P<0.05). Consequently, whereas approximately half of the recipients receiving p53-null LSK cells died of thymic lymphoma, the recipient mice transplanted with LSK cells deficient for both Aspp1 and p53 were 100% lethal within 6 months after BM transplantation (log-rank test, P<0.01). These mice succumbed to hematological malignancies, mostly T-cell acute lymphoblastic lymphoma and leukemia (ALL) (88%) but also B-cell (6%) and myeloid (6%) malignancies. Taken together, our study demonstrates that Aspp1 attenuates HSC quiescence and induces apoptosis in damaged HSCs, in both p53-dependent and -independent manners, thereby inhibiting the development of leukemia and lymphoma in conjunction with p53 in HSCs. As loss of Aspp1 expression due to aberrant methylation of its promoter has already been proven to be an independent poor prognosis factor in ALL patients, Aspp1 may be a potential target for stem cell-directed therapy of leukemia and lymphoma. Disclosures No relevant conflicts of interest to declare.

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