Inactivation of PUMA Protects Hematopoietic Stem Cells and Confers Long Term Survival in Response to High Dose γ-Irradiation

Radiation injury remains a significant health problem. New medical intervention to prevent or manage radiation damage is highly dependent on a deeper understanding of how radiation-induced cell death is accomplished in the irradiated tissue cells such as stem and progenitor cells. To date, relatively specific or untainted molecular mediators in apoptosis of tissue stem and progenitor cells upon radiation injury have not been clearly defined. The p53 pathway is known as a major molecular mechanism for cell apoptosis, upon the exposure of lethal radiation. Targeting p53 confers a radioprotective effect, but may increase tumorigenesis due to impaired cell cycle arrest for DNA repair. In our current study, we have examined the specific role of PUMA (p53 up-regulated mediator of apoptosis) in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). By quantitative RT PCR, we found that the level of PUMA mRNA was relatively low in the most primitive long-term repopulating hematopoietic stem cells (LT-HSC, isolated based on the immnunophenotype “CD34⁻LKS”) as compared to other hematopoietic cell populations from mice, but it was significantly elevated in response to γ-irradiation. In the mice lacking PUMA, while neither HSC number nor HSC function was altered under homeostatic conditions, the PUMA−/− HSCs appeared to be resistant to radiation damage in vivo as retrospectively quantified in a competitive HSC transplant model. Our further direct measurement with a single cell culture system for HSC growth in vitro, demonstrated that PUMA, but not p21 (the chief mediator of p53 in cell cycle arrest), is primarily responsible for the radiosensitivity of HSC in the p53 pathway (200 LT-HSCs analyzed for each cell type). Together, these data provide definitive evidence for PUMA as an essential mediator in radiation-induced apoptosis of tissue stem cells. We finally focused on the beneficial effects of targeting PUMA in HSCs and HPCs on the animal survival upon the exposure of lethal irradiation. Strikingly, the wild-type mice reconstituted with PUMA−/− hematopoietic cells exhibited a significant survival advantage after two rounds of 9-Gy γ-irradiation (18 Gy in total) as compared to the mice reconstituted with PUMA+/+ hematopoietic cells (95 % vs. 0 % survival in 20 days, n=21/each group; 50% vs. 0 % survival in 180 days, n=20 or 11/each group, respectively) as shown in the figure below. Moreover, unlike the p53−/− mice, those PUMA−/− reconstituted mice did not have an increased incidence of hematopoietic malignancies (n=20) within 180 days. Therefore, our current study establishes PUMA as an attractive molecular target for the development of therapeutic agents for the prevention and treatment of radiation injury.