Genetic instability, oxidative stress, and elevated p53 protein levels in Mysm1^tm1a/tm1a hematopoietic progenitors. (A) Increased frequency of micronucleated cells in the normochromatic erythrocyte (NCE) cell population of Mysm1^tm1a/tm1a mouse blood; representative flow cytometric plots and data quantification are presented. (B) Levels of DNA damage γH2AX marker and p53 tumor suppressor protein in the KLS (cKit⁺ lineage⁻ Sca1⁺) cells of Mysm1^tm1a/tm1a and control mouse BM. MFU indicates mean fluorescence units. (C) Increased levels of reactive oxygen species (ROS) in the progenitor cells of Mysm1^tm1a/tm1a BM, measured using carboxy-H₂DCFDA ROS-sensitive fluorescent dye. LT-SCs indicates long-term stem cells (KLS, Flt3⁻CD34⁻); ST-SCs, short-term stem cells (KLS, Flt3⁻CD34⁺); MPPs, multipotent progenitors (KLS, Flt3⁺CD34⁺). (D) Increased levels of ROS in Mysm1^tm1a/tm1a KLS cells in the BM chimeras. (E) Increase in apoptosis (annexin V⁺ 7AAD^+/−) and necrosis (annexin V⁻ 7AAD⁺) in Mysm1^tm1a/tm1a KLS cells in the BM chimeras. All bars represent means ± SEM; *P < .05, **P < .01, ***P < .001 using (A,B,D) t test or (C,E) ANOVA. (F) Model of the mechanisms of hematopoietic dysfunction in Mysm1^tm1a/tm1a mice: (i) ROS induce oxidative damage of DNA as well as other cellular components, and (ii) such damage leads to activation and stabilization of p53, (iii) which is a major inducer of cell apoptosis. (iv) p53 also induces production of ROS, and ROS are implicated in mediating cell-death downstream of p53. (v) Finally, DNA damage and oxidative stress may induce cell death via p53-independent mechanisms.