Malignant Myeloid Stem Cells Bypass Oxidative-induced Cellular Senescence:An Experimental Study

PartⅠCD34+ cells from patients with myelodysplastic syndrome present different premature cellular senescenceObjectives:To investigate whether CD34+ cells from patients with myelodysplastic syndromes (MDS) undergo senescence via activation of the reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (p38) pathway.Methods:Bone marrow CD34+ cells were isolated from 47 patients with MDS, 14 with acute myeloid leukemia (AML), and 12 healthy controls. Cellular senescence in these cells was evaluated by senescence associatedβ-galactosidase (SA-β-gal) staining and quantification of p21C1lP1/WAF1 (p21) expression. Production of ROS,γ-H2AXand p38 activation in these cells were examined by flow cytometry, immunofluorescent staining and western blot, respectively.Results:The percentages of SA-β-gal positive senescent CD34+ cells increased in lower-risk MDS patients, but not in higher-risk MDS and AML patients, compared to that of healthy controls. The increases were associated with an elevated expression of p21 but not with an increased production of ROS and the activation of p38. In addition, the percentages of SA-β-gal positive senescent CD34+ cells were inversely correlated with the scores of the International Prognostic Scoring System (IPSS) in MDS patients.Conclusions:Our findings suggest that CD34+ cells undergo senescence in MDS patients independent of activation of the ROS-p38 pathway. PartⅡLower phosphorylation of p38MAPKa blocks the oxidative stress-induced senescence in myeloid leukemic CD34+ CD38- cellsObjectives:In this study we investigated the activated state of a pathway, in which ROS induces cellular senescence through DNA damage and the phophorylation of p38, in myeloid leukemic CD34+ CD38- cells.Methods:Bone marrow samples were obtained from patients with acute myeloid leukemia (AML, n=11) and chronic myeloid leukemia (CML, n=9). CD34+ CD38-cells were isolated from mononuclear cells from these bone marrow samples and K562 and KGla cells. Those from human cord blood served as control. Intracellular ROS level was detected by flow cytometry. Real-time RT-PCR was used to detect the expression of p21. Western blotting and immunofluorescence staining were employed to determine the p38 activation and the yH2AX level. The proliferation and apoptosis of CD34+CD38-cells were detected by MTT assay and flow cytometry.Results:Our results showed that ROS and DNA damage were substantially accumulated and p38 was less phosphorated in myeloid leukemic CD34+ CD38- cells as compared with hematopoietic stem cells (HSCs) and the H2O2-induced senescent HSCs. Furthermore, over-phosphorylation of p38 by anisomycin, a selective activator of p38, induced both the senescence-like growth arrest and apoptosis of CD34+ CD38-cells from K562 and KGla cell lines.Conclusions:These findings suggested that, although excessive accumulation of oxidative DNA damage was present in leukemia stem cells (LSCs), the relatively decreased phosphorylation of p38MAPK might help leukemic cells escape senescence and apoptosis. PartⅢMicroRNA-34c induces senescence-like growth arrest in myeloid leukemic CD34+CD38- cellsObjectives:In this study we investigated the expression, targets, and functional effects of miR-34c in myeloid leukemic CD34+CD38- cells; we also studied whether miR-34c could induce cellular senescence and whether miR-34c is regulated by ROS-p38 pathway.Methods:Bone marrow samples were obtained from patients with acute myeloid leukemia (AML, n=11) and chronic myeloid leukemia (CML, n=9). CD34+CD38-cells were isolated from mononuclear cells from these bone marrow samples and K562 and KGla cells. Those from human cord blood served as control. Real-time RT-PCR was used to detect the expression of miR-34c and p21. Western blotting was used to detect the expression of e2f3, cyclin-E, c-myc, CDK4. Cellular senescence was evaluated by SA-β-gal staining. KGla-AML-NOD/SCID mouse model was used to assess the effect of miR-34c in vivo.Results:The expression of miR-34c in myeloid leukemic CD34+CD38- cells was significantly decreased as compared with the counterpart of cord blood. Over-phosphorylation of p38 by anisomycin up-regulated the level of miR-34c in CD34+CD38- from KG1a and K562 cell lines. Transient transfection of miR-34c into CD34+CD38- from KG1a and K562 cell lines inhibited cell proliferation, cell cycle progression, and induced senescence-like growth arrest and apoptosis through down-regulation of several target protein expression (e2f3, cyclin-E, c-myc, CDK4). Moreover, miR-34c also suppressed in vivo growth of KG1a cells in mouse model.Conclusions:These findings suggested that miR-34c functions as a potent suppressor of cell proliferation through induction of senescence-like growth arrest and apoptosis and abrogation of miR-34c function could lead to AML development.